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INSTITUTION vt INOSHLINS S3ZIYVHYS!IT LIBRARIES SMITHSONIAN INSTITUTION Se NS § RA ve Bod \ 4 ie . SIHSON 5 fs & 3 ~~ vSON, 4 A Cas ‘> bs = ze > 4 = Vr pat SHLINS SAIYVYRAIT LIBRARIES JCUIITIAIe earuWwuaia ISHLINS SAIYVY 4SONIAN S, ISONIAN iSONIAN N111Sh> Y ISHLINS SJIYVYGIT LIBRARIES SMITHSONIAN Re SAX ZOOLOGICAL SCIENCE An International Journal VOLUME 4 1987 published by The Zoological Society of Japan ee yiys P87 iss aac tb J \ , i Smmnendy ys ) Seen wo o —— % nig , — a , Sy : - te ~ . Patel a rie | mek Ld Moh es “= oN ¥ aie e ~e pee, a v uf Sg 7 h eo lbsas ie i ‘ tT q i dee CONTENTS VOLUME 4 REVIEWS Katagiri, Ch.: Role of oviducal secretions in mediating gamete fusion in anuran amphib- Matsuno, A.: Ultrastructural classification of smooth muscle cells in invertebrates and VSL DIGGS 6 GL ore eee 15 Nagahama, Y.: Gonadotropin action on gametogenesis and steriodogenesis in teleost DETECS 2.50 SCR Oe eet mnie rinse 209 Price, D. A., N. W. Davies, K. E. Doble and M. J. Greenberg: The variety and distribu- tion of the FMRFamide-related peptides in SOC LUOSOS 5 ob eR ea eae ee 385) Asashima, M., T. Oinuma and V. B. Meyer- Rochow: Tumors in amphibia............ 411 Grunz,H.: The importance of inducing fac- tors for determination, differentiation and pattern formation in early amphibian de- “ELD CUS ee clic IA nt mn a Ure ne nn a eo rae 7/9 Uéno, S.-I.: The derivation of terrestrial cave SULEP ELS: 5 4 LS ie ai ae a RA aS AR 593 Terakado, K.: Fine structure of ascidian SUNT CUD TSC (CS Ae ane ar ee iil Keller, R.: Cell rearrangement in mor- (DO LEIS Gc, SiR ee ar rae 763 Loretz,C. A.: Rectal gland and crypts of Lieberkthn: is there a phylogenetic basis for ime Monalesimilanity? .....5...626 620660. 005. 935 Chiba, Y.: Insect circadian activity with spe- cial reference to the localization of the MACCMIARCIM sO N nIA ON oes oes ies Si weet es 945 ORIGINAL PAPERS Physiology Yamashita, S. and R.Tuji: Phototactic be- havior of the orb weaving spiders, Argiope amoena and Nephila clavata................ 23 Yamashita, S.: Dimming reaction of the orb weaving spider, Argiope amoena........... 31 Ando, M.: Regulation by intracellular ala- nine of water transport across the seawater CEMMMUCKSIMMRC re st ctercceiee ae oe cece cess iil Ando, M., Y. Furukawa and M. Kobayashi: Comparison of seawater and fresh-water eels for the effects of L-alanine on water transport ACKOSS MHCMMNESHING weet Pen. duc wee sess os: 45 Khan,M.M. and Y.K.Ip: Effect of host myo-inositol deficiency on Hymenolepis dimi- Wuta (Cestodaeere ree etre, ee ae 223 Tsukamoto, Y.: Morphometrical features of rod outer segments in relation to visual acuity and sensitivity in the retina of Rana cates- DEG ORR en Ne dt one eo ee 233 Kasukawa, H., N. Oshima and R. Fujii: Mechanism of light reflection in blue dam- Selitshenro tile pind OpnOke 7 ere near 243 Ohnishi, K.: Proposed tertiary olfactory path- ways in a teleost, Carassius auratus ........ 427 Takahashi, N.: Gonad response to j- aminobutyric acid in the sea urchin ........ 433 Takahashi, N. and M. Takahashi: Gonad re- sponse to calcium and a comparison of the effects of calcium, potassium, acetylcholine and y-aminobutyric acid on the sea urchin SOMAC Me rans oe eiee ee Ne oe oem ne eee 441 Hidoh, O. and J. Fukami: The mediation of cyclic AMP in octopaminergic modulation at neuromuscular junctions of the meal-worm, MERECDIIOUMOLMON Re ee ee ce ee 447 Kumazawa, T. and O. Suzuki: Diamine ox- idase activities in catfish tissues ............ 451 Oishi 1 EK: Lauber and? J Viniend: Ex- perimental myopia and glaucoma in chicks 455 Higuchi, T., H. Nakamura, K. Sawauchi and H. Okumura: Frequency block in the giant axons of a sabellid worm, Pseudopotamilla GQECHMT Ne ene Cairne Nae ee 607 Taneda, K.: Geotactic behavior in Para- mecium caudatum. 1. Geotaxis assay of individwalySpecimMente .....scae ee cs oe. oor 781 Taneda, K.: Geotactic behavior in Para- mecium caudatum. II. Geotaxis assay in a populationol the specimens: ..-.-.-4-- >... - 789 il Newland, P. L.: The structure and innerva- tion of a new muscle in the tailfan of the crayfish, Procambarus clarkii............... WS) Takei, Y. and I. Hatakeyama: Changes in blood volume after hemorrhage and injection of hypertonic saline in the conscious quail, (COMME? COMMOBS HDOMIGE Sacccaseacsceo0e. 803 Arii, N., K. Namai, F. Gomi and T. Nakazawa: Cryoprotection of medaka embryos during GCEVElOPMENE 5 5c. ore mee ann aah. 813 Hidaka, T. and T. Miyahara: Excitatory and inhibitory neuromuscular transmission in fish PEG MIMSCIS oe he eae eon see ere eae 819 Cell Biology Matsuno, A.: Ultrastructural studies on de- veloping oblique-striated muscle cells in the cuttlefish, Sepiella japonica Sasaki.......... 53 Iwasa, F., Y. Hasegawa, S. Ishijima, M. Oku- no, T.Mohri and H. Mohri: Effects of calmodulin antagonists on motility and acro- some reaction of sea urchin sperm ......... 61 Takagi, Y., T. Suzuki and C. Shimada: _Isola- tion of a Paramecium tetraurelia mutant with short clonal life-span and with novel life-cycle LE ALTE S ane ies cg ores ota ola na Steen) aa 73 Mesa, A. and B.Goni: Meiosis in the Japanese gryllacridid Anoplophilus acu- ticercus Karny, 1931 (Orthoptera Saltatoria, Rhaphidophoridae): Amphitelic orientation of the X and supernumerary chromosome(s) Itoh, T. J., H.Sato and A. Kobayashi: Ex- aminations of spindle structure modified by i eithe mitotic-anhesteliee. se eee 265 Takeuchi, S.: Cytochalasin B affects selec- tively the marginal cells of the epithelial sheet IPE CUUIUEC aac a vo Ceo ie ui he: 465 Seki, T., S. Fujishita, M. Azuma and T. Suzu- ki: Retinal and 3-dehydroretinal in the egg of the clawed toad, Xenopus laevis......... 475 Gomi, T., A. Kimura, H. Tsuchiya, T. Hashi- moto, K. Higashi and S. Sasa: Electron microscopic observations of the alveolar brush cellvonthe bullitopeeeseeene pre 613 Sawai, T.: Surface movement in the region of the cleavage furrow of amphibian eggs..... 825 Itoh, Y., D. H. Hu, K. Ohashi, S. Kimura and K. Maruyama: Lamprey connectin (COM- MUNICATION) 2.525 225.045) oe wb) Shimizu, K. and M. Hokano: Disappearance of immunoglobulin G in endodermal cells in non-immunized murine yolk sac placenta towards parturition (COMMUNICATION) Ogawa, M., K. Terakado and J. Okada: Ori- gin of binucleate cells in the neural gland of the ascidian Halocynthia roretzi (Drasche) (COMMUNICATION) ee eee 73M Genetics Kohno, S., M. Kuro-o, C. Ikebe, R. Katakura, Y. Izumisawa, T. Yamamoto, H. Y. Lee and S. Y. Yang: Banding karyotype of Korean salamander: Hynobius leechii Boulenger ... 81 Obara, Y.: Karyological differentiation be- tween two species of mustelids, Mustela erminea nippon and Meles meles anakuma_ 87 Saotome, K.: Chromosome numbers in 8 Japanese species of sea urchins ............ 483 Ikebe, C., K. Aokiand S. Kohno: Karyotype analysis of two Japanese salamanders, Hyno- bius nebulosus (Schlegel) and Hynobius dun- ni Tago, by means of C-banding ........... 621 Ota, H., M. Matsui, T. Hikida and T. Hidaka: Karyotype of a gekkonid lizard, Cosymbotus platyurus, from Sabah, Borneo, Malaysia (COMMUNICATION) 2. oeeeeee 385 Frankel, J.S.: Asynchronous expression of alleles at the alcohol dehydrogenase locus during Oryzias hybrid development (COM- MUNICATION) «ose sas Morse oe eee 735 Immunology Ahmad, R. A., B. L. James and A. B. Kamis: Acquired resistance against Microphallus pygmaeus in the laboratory mouse ......... 93 Kaiho, M. and I. Ishiyama: The distribution of A and B blood group antigens in tissues of the frog, Rana catesbeianay. 4.0). ) ape 627 Biochemistry Okai, Y., S. Ishizaka and U. Yamashita: A DNA synthesis inhibitory peptide from hu- man embryo fibroblasts — characterization of biologicalipropenrtics! ae. p eee Ene eeree 99 Kobayashi, K. and S. Horiuchi: Myofibril de- gradation by tail lysosomes from metamor- phosing bullfrog tadpoles: ... 2..escctues. ox: 107 Okai, Y.: Novel cytotoxic factors from tumor virus-transformed human embryo fibroblasts Shimada, K., H. Koyama and M. Asashima: Two-dimensional polyacrylamide gel analysis of papilloma and normal skin proteins in newt Takahashi, S. and K.Maruyama: Activity changes in myosin ATPase during meta- monpnosisrof fruitfly) calcu) he ee. 833 Asakura, A., Y.Nabeshima and K. Maruyama: Jn vitro synthesis of connectin in an extract of chicken embryo muscles CCOMMIUNTICATION):(.9...... o2sehee. vous oe Developmental Biology Nakajima, Y.: Localization of catechola- minergic nerves in larval echinoderms...... 293 Shimizu, T.: Jn vitro spermatids formation from diapausing pupal spermatogonia of the cabbage armyworm, Mamestra brassicae L. (Hepidoptera: Noctuidae)... 02.2.0. e0 0056. 301 Hazarika, L. K. and A. P. Gupta: Variations in hemocyte populations during various de- velopmental stages of Blattella germanica (L.) (Dictyoptera; Blattellidae) )...2. 2.002.220. 307 Kunieda, M. and M. Wakahara: Twin forma- tion in Xenopus laevis eggs centrifuged before HES ECLEAVADE shee Sen) oe ss JeaRlee PR Se le 489 Tsuneki, K.: A_ histological survey on the development of circumventricular organs in WAM OUSEV.ETUCOLALES) 25: sass). Sie eae wake: 497 Mizukami, S.: Further observations on divi- sion pattern of binucleate fish embryonic cells Suzuki, N., M. Kurita, K. Yoshino and M. Yamaguchi: Speract binds exclusively to sperm tails and causes an electrophoretic mobility shift in a major sperm tail protein of BEAMUECIINS: . 5.) VAlwr ie Wenn Pea tO4 1 Suzuki, N., M. Kurita, K. Yoshino, H. Ka- jiura, K. Nomura and M. Yamaguchi: Purification and structure of mosact and its derivatives from the egg jelly of the sea urchin GP CAStChHAPONICUS: edn risa a8. 03 -Ba tae 649 ill Tanimura, A. and H.Iwasawa: Germ cell kinetics in gonadal development in the toad BUSO JAPONICUS {OTINOSUS hs densi dy os eeayeer 657 Hori, R., V. P. E. Phang and T. J. Lam: Pre- liminary study on the pattern of gonadal development of the sea urchin, Diadema setosum, off the coast of Singapore ........ 665 Abé, S.-I. and S. Asakura: Meiotic divisions and early-mid-spermiogensis from cultured primary spermatocytes of Xenopus laevis ..839 Kato, S. and K. Kurihara: The intracellular supporting network in the Leydig cells of larval salamander skin (COMMUNICA- TION) yatectrs cations aie yaa Ses 187 Yoshizaki, N.: Isolation of spermatozoa, their ultrastructure, and their fertilizing capacity in two frogs, Rana japonica and Xenopus laevis (COMMUNICATION) .... 193 Akasaka, K., M. Taira, T.Shiroya and H. Shimada: Cloning of stage specific gene sequences from a cDNA library representing poly(A)* RNA of sea urchin prism embryos (COMMUNICATION) ................005. IBY Endocrinology Tanaka, S., M. Hattori and K. Wakabayashi: Steroidogenic activity of isoelectric gonado- tropin components in the pituitary of adult male newt, Cynops pyrrhogaster pyrrhogaster Cailliez, Z., J.-M. Danger, A. C. Andersen, J. M. Polak, G. Pelletier, K. Kawamura, S. Kikuyama and H. Vaudry: Neuropeptide Y (NPY)-like immunoreactive neurons in the brain and pituitary of the amphibian Rana CONCSO CLAN GER REN he: RA 123 Honda, H., T. Oishi and T. Konishi: Com- parison of reproductive activities between two Japanese quail lines selected with regard to photoperiodic gonadal response ......... 135 Hazarika, L. K. and A. P. Gupta: Structure, innervation, persistence and effects of juve- nile hormone on the prothoracic glands in adult Blattella germanica (L.) (Dictyoptera, Blattellidae) ret... .aiiayade dM. i lsas 3. eae 145 Ohnishi, E.: Growth and maturation of ova- ries in isolated abdomens of Bombyx mori: Response to ecdysteroids and other steroids Chen, R.-H., J.-Y. Lin, Y.-L. Yu and H. Y. Cheng: Annual changes in plasma and tes- ticular androgen in relation to reproductive cycle in a Japalura lizard in Taiwan........ 323 Uemura, H., A. Hattori, M. Wada and H. Kobayashi: Effects of intracranially im- planted cholecystokinin and substance P on serum concentrations of gonadotropins, pro- lactin and thyroid stimulating hormone in the PACA PORE, BE key GAELS Fee as S3ill Okawara, Y., T. Karakida, M. Aihara, K. Yamaguchi and H. Kobayashi: Involve- ment of angiotensin II in water intake in the Japanese eel, Anguilla japonica ............ 523 Chan, P. J.: Cyclic CMP alters squirrel mon- key (Saimiri sciureus) luteal cell structure via cyclic AMP-dependent mechanisms ........ SY) Jokura, Y. and A. Urano: Extrahypothala- mic projection of immunoreactive vasotocin fibers in the brain of the toad, Bufo japonicus Suzuki, S.: Plasma thyroid hormone levels in metamorphosing larvae and adults of a sala- mander, Hynobius nigrescens .............. 849 Takahashi, S. and S. Kawashima: Prolifera- tion of prolactin cells in the rat: Effects of estrogen and bromocryptine................ 855 Wheeler, C. M. and A. P. Gupta: Effects of two juvenile hormone analogs (R-20458, RO203600) and three juvenile hormones (JH1, JH2, JH3) on the external morphology and length of the spiculum copulatus (SC) in the male German cockroach, Blattella germa- nica (L.) (Dictyoptera: Blattellidae) ....... 861 Fukuda, M., Y. Nakano, K. Yamanouchi, Y. Arai and H. Furuya: Suppressive effect of right-side anterior hypothalamic lesion on ovarian compensatory hypertrophy in rats (COMMUNICATION) ..................55 197 Srivastav, S. P., K. Swarup and A. K. Srivas- tav: Prolactin cells of Clarias batrachus in response to corpuscles of Stannius extract administration (COMMUNICATION )..... 201 Yamada, C.andH. Kobayashi: Immunoreac- tive angiotensin II in the corpuscles of Stannius of the rainbow trout, Salmo gaird- neri (COMMUNICATION )................ 387 Hasan, N., S. Das, A. K. Srivastav and K. Swarup: Phosphocalcic response of Stan- nius corpuscles extract in the freshwater snake, Natrix piscator (COMMUNICA- TION) {2h Pv ie, SE ee 391 Ortiz, T., J. Pimero and R.Covenas: Met- enkephalin-like immunoreactivity in the nervous system of Helix aspersa (COM- MUNICATION) 0227.0 2228 DA ee 743 Morphology Win Win Yee and S. Kawashima: Sex differ- ence in the early histopathological changes of the kidney in Wistar/Tiw rats: 3.44) oe eee 867 Ishizeki, K.: Ultrastructural observations of the developing basophilic granulocytes in the loach kidney. ...73..5.0.05 7). eee 875 Yoshioka, E.: A method of measuring the volume of soft tissue (COMMUNICATION) Behavior Biology Machida, T., S. Iso and T. Noumura: Long- lasting effects of orchiectomy and its preced- ing procedures on open field behavior in male MICE... RIE ee JS Tomioka, K. and Y. Chiba: Entrainment of cricket circadian activity rhythm after 6-hour phase-shifts of light-dark cycle ............. 3)35) Chiba, A. and K. Aoki: Relationship be- tween daily variation of locomotor activity and that of plasma corticosterone levels in the newt, Cynops pyrrhogaster pyrrhogaster....543 Hayashi, S.: The effects of preputialectomy on aggression in male mice................. 551 Moriya, T. and Y. Miyashita: Body color and the preference for background color of the Siamese fighting fish, Betta splendens ...... 881 Oguma, Y., H. Kurokawa, S.M. Akai, H. Tamaki and J. Kajita: Interspecific differ- ences in some courtship behavioral properties among the four species belonging to the Drosophila auraria complex................ 889 Environmental Biology Endo, K. and M. Shibata: A circadian aspect of the photoperiodic time-measurement on the basis of the larval-ecdysis rhythm in the small copper butterfly, Lycaena phlaeas RUMEIMRSENEE GS ea 3k Scan ey seiees 683 Ecology Cheng, H. Y. andJ. Y. Lin: Annual ovarian, fat body and liver cycles of the grass lizard Takydromus stejnegeri in Taiwan........... 557 Kasuya, E., H.Shigehara and M. Hirota: Mating aggregation in the Japanese treefrog, Rhacophorus arboreus (Anura: Rhacophor- idae): a test of cooperation hypothesis ..... 693 Watanabe, M. and Y. Adachi: Number and size of eggs in the three emerald damselflies, Lestes sponsa, L. temporalis and L. japonicus (Odonata: Lestidae) (COMMUNICATION) Taxonomy Hasumi, M. and H.Iwasawa: Geographic variation in the tail of the Japanese salaman- der, Hynobius lichenatus, with special refer- Eee to taxonomic bearing ........-:......- 159 Hihara, F. and H. Kurokawa: The sperm length and the internal reproductive organs of Drosophila with special references to phy- Igecmcrc TelatiONships ......:..-.6+-..-+--- 167 Hirayama, A.: Two peculiar species of corophiid amphipods (Crustacea) from the Soiemiand Sea, Japan...:........-.---+--- 175 Sawada, I.andG. Kugi: Three new species of avian dilepidid cestodes from Oita Prefec- SLPS. SSDI IAs ele er ee 183 Matsuoka, N.: Biochemical study on the tax- Oonomic situation of the sea-urchin, ESCHAOCEHITOLUS AEPressSUS ..... 22.22 0++..5%- 339 Konishi, K. and R. Quintana: The larval stages of Pagurus brachiomastus (Thallwitz, 1892) (Crustacea: Anomura) reared in the 2 DUES CIs coll ee ee ree 349 Hayashi, K.-I. and Y. Ogawa: Spongicola levigata sp. now., a new shrimp associated with a hexactinellid sponge from the East China Sea (Decapoda, Stenopodidae)...... 367 Ehara,S. and T. Gotoh: Notes on the genus iV. Sasanychus Ehara, new status, with de- sciption of a new species from Hokkaido (CAcanma, letranyehidae). 22.2%... 22:60. Sis) Kuramoto, M.: Advertisement calls of two Taiwan microhylid frogs, Microhyla heymon- See Vis OFMGUE oS eae ea Seo 8 Selassie s 563 Hirayama, A.: Notes on the evolutionary sys- tematics of the genus Corophium .......... 569 Uchida, S. and H. Maruyama: What is Sco- pura longa Uéno, 1929 (Insecta, Plecoptera)? ACECVISOM:OL ENE; CENUS:. (05-5 5) so cee 699 Nagatomi, A.: Taxonomic notes on Coenomyiidae (Insecta: Diptera)........... 711 Sawada,I.: Further studies on cestodes of Japanese bats, with descriptions of three new species of the genus Vampirolepis (Cestoda: Fiymenolepididac) at. 2s... eee s,s. 1PA| Ohkubo, N.: A new species of Zetomimus (Acari: Oribatei) from Japan...3240.2 897 Baba, K., A. Nagatomi, H. Nagatomi and N. L. Evenhuis: Redescription of Villa myr- meleonstena (Insecta, Diptera, Bomby- liidae), a parasitoid of ant lion in Japan... .903 Ito, T.: Proposal of new terminology for the morphology of nauplius y (Crustacea: Maxil- lopoda: Facetotecta), with provisional de- signation of four naupliar types from Japan Hayashi, K.-I and T. Chiba: Rediscovery of Heptacarpus jordani (Rathbun) with notes on morphological variations (Decapoda, Caridea, Fippolytidac)yine e145 o02 nok 919 Others Proceedings of the 58th Annual Meeting of the Zoological. Society of Vapan’=.<- «22.22.22. 955 Abstracts of papers presented at the seminar on ‘Perspectives in marine biology—contribution to cell and developmental biology’ ....... 1120 BOOKSTEVIE WS ie ig oe a een sas 1138 AMNOUNCEHICIIS Hee) ee as ces tek oe ee ee 1141 Author indexer. oe ois ek es 8 1144 InsthictOnsyi@eANUtHOIs.. <2 5.) cee i oe es ce 205 ' . - t ~Bb Aire /Gdtede. wat owas VEE 44 > bial aos pen BSP Watt Ti ie enol beeai PE iinestate stk cient cork valvaphe acigtiel dap diag 7) boat, Vi, es, Suen T ead 2s be « : er ee ngmueod nahayoeht. sagembbihydansine aeeie'e Abs -S1,- adden ae See Ebi ee eres wed) on eto. Sik washed ois rend Ra le ty eR . phe $i saat ie aseyas bien es eo e's Hes :

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On A TEE ey ee bs hy _ aaa ees ih) via sien id anenduy sia. eee qeteT lpege hiv, aka \ goood a Sto ott dagen vt : pits wet oeiaubonpnts ; et ai aounay alent ey | ki Di (ey ~ ee on etaach is chery oh. sping ited sith preg (paytery a) ape be es Sree son edth er arate PIS ids ee ui ate eh ts vA)» e TEE ind ed ao td, wea iarenees A i poi edt) he epee ataays. ais al —— 2 with —_ Ong, sane’ heen mtd, An International Journal. ZOOLOGICAL SCIENCE The official Journal of the Zoological Society of Japan Editor-in-Chief: ve The Zoological Society of Japan: Hideshi Kobayashi (Funabashi) Toshin-building, Hongo 2-27-2, Bunkyo-ku, Managing Editor.” Jbson Tokyo 113, Japan. Tel. (03) 814-5675 Seiichiro Kawashima (Hiroshima) @lficers: Assistant Editors: Takeo Machida (Hiroshima) Sumio Takahashi (Hiroshima) President: Nobuo Egami (Tsukuba) Secretary: Yasuto Tonegawa (Urawa) Treasurer: Tadakazu Ohoka (Tokyo) Librarian: Shun-Ichi Uéno (Tokyo) Editorial Board: Howard A. Bern (Berkeley) Walter Bock (New York) Aubrey Gorbman (Seattle) Horst Grunz (Essen) Robert B. Hill (Kingston) Yukio Hiramoto (Tokyo) Susumu Ishii (Tokyo) Yukiaki Kuroda (Mishima) Koscak Maruyama (Chiba) Roger Milkman (Iowa) Hiromichi Morita (Fukuoka) Kazuo Moriwaki (Mishima) Tokindo S. Okada (Okazaki) | Andreas Oksche (Giessen) Hidemi Sato (Nagoya) Hiroshi Watanabe (Shimoda) Mayumi Yamada (Sapporo) Ryuzo Yanagimachi (Honolulu) ZOOLOGICAL SCIENCE is devoted to publication of original articles, reviews and communications in the broad field of Zoology. The journal was founded in 1984 as a result of unification of Zoological Magazine (1888-1983) and Annotationes Zoologicae Japonenses (1897-1983), the former official journals of the Zoological Society of Japan. ZOOLOGICAL SCIENCE appears bimonthly. An annual volume consists of six numbers more than 1000 pages including an issue containing abstracts of papers presented at the annual meeting of the Zoological Society of Japan. MANUSCRIPTS OFFERED FOR CONSIDERATION AND CORRESPONDENCE CONCERN- ING EDITORIAL MATTERS should be sent to: Dr. Seiichiro KAWASHIMA, Managing Editor, Zoological Science, Zoological Institute, Faculty of Science, Hiroshima University, 1-1-89 Higashisenda-machi, Naka-ku, Hiroshima 730, Japan, in accordance with the instructions to authors which appear in the first issue of each volume. Copies of instructions to authors will be sent upon request. SUBSCRIPTIONS. ZOOLOGICAL SCIENCE is distributed free of charge to the members, both domestic and foreign, of the Zoological Society of Japan. To non-member subscribers within Japan, it is distributed by Business Center for Academic Societies Japan, 6—16—3 Hongo, Bunkyo-ku, Tokyo 113. Subscriptions outside Japan should be ordered from the sole agent, VNU Science Press BV, Europalaan 93, 3526 KP Utrecht, (postal address; P.O. Box 2093, 3500 GB Utrecht), The Netherlands. Subscription rates will be provided on request to these agents. New subscriptions and renewals begin with the first issue of the current volume. All rights reserved. No part of this publication may be reproduced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1987, The Zoological Society of Japan Publication of Zoological Science has been supported in part by a Grant-in-Aid for Scientific Publication from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 4: 1-14 (1987) 987 Zo@logical Society of Japan Role of Oviducal Secretions in Mediating Gamete Fusion in Anuran Amphibians CHIAKI KATAGIRI Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan INTRODUCTION Mature unfertilized eggs of most animals are surrounded by extracellular coats or investments through which fertilizing sperm have to penetrate before fusing with the egg plasma membrane. These coats, composed mostly of glycoproteins with variable ultrastructures and molecular com- positions, notably include the vitelline envelope or vitelline coat and the jelly coat in many inverte- brates, and the zona pellucida in mammals. Stu- dies on gamete fusion, particularly in echinoderms and mammals, have documented that these egg- investments are not a simple barrier for a fertiliz- ing sperm but are obligatory participants in the process of sperm-egg fusion, playing such roles as specific binding sites for sperm or for inducing the acrosome reaction, etc. [1, 2]. In this context amphibians also provide a potentially good system, since a number of classical and recent studies have repeatedly shown that the jelly envelopes secreted by the oviduct are indispensable for sperm penetration into eggs [3, for earlier references]. However, the molecular bases for this jelly re- quirement in amphibian fertilization have been puzzling. In this paper, I will review first how this question has been challenged, and then I will summarize recent experiments, primarily from our laboratory, that have given a plausible explanation for a role of egg-jelly in anuran fertilization. Second, current studies implicating the functional significance of the non-jelly secreting portion of the oviduct will be discussed, to show how the Received October 3, 1986 oviduct of amphibians possesses an intriguing role in permitting sperm access to the egg. This discussion will be focussed mostly on anurans that have been studied more intensively than urodeles. References for studies on pertinent problems in urodeles can be found in other sources [4]. PRELIMINARIES TO FERTILIZATION The ovulated eggs of anuran amphibians, sur- rounded by the vitelline coat or vitelline envelope’ of several micrometers in thickness, are propelled towards the ostial openings of the oviduct by ciliary action and transported down the long reproductive tract to acquire several layers of jelly (Fig. 1). The eggs located in the expanded lowest portion of oviduct, 1.e., ovisac or uterus, are regarded as mature, since eggs taken from coelom are not fertilizable, but fully jellied uterine eggs are fertilizable in high frequencies and undergo normal development. It may be assumed, there- fore, that eggs acquire maturity during their passage down the oviduct. In fact, during trans- portation from ovary through coelom, oviduct to uterus, eggs undergo nuclear and cytoplasmic maturation. It has been shown, however, that the nuclear changes from germinal vesicle to second meiotic metaphase proceed quite independently from the location of eggs in the female reproduc- tive tract. Thus, after initiating the germinal vesicle breakdown (GVBD), eggs may attain " Designates the investment that is deposited around the oocytes during oogenesis, referred to as the vitelline membrane in the earlier literatures. 2 Cu. KATAGIRI ee Uterus (Ovisac) Fic. 1. The reproductive system of anuran female. The eggs released from the ovary to the coelom are moved by ciliary action towards the ostial opening of the oviduct, and pass through the non-jelly secreting pars recta (PR) and the jelly-secreting pars convolu- ta (PC) where they acquire several layers of jelly. The eggs located in the uterus are readily fertilizable upon insemination. J,—J, denote jelly layers in the case of Bufo japonicus. The vitelline coat surround- ing each egg is omitted. metaphase of the second meiotic division even if they are forced to remain in the coelom by ligating the oviduct [5]. These eggs also attain cytoplasmic maturity as evidenced by their ability to undergo cortical granule exocytosis as well as to elicit an activation potential [6,7] in response to artificial stimuli. Extensive studies on the mechanism of oocyte maturation during the past two decades have established that the entire maturational events from the initiation of GVBD to completion of meiosis can be effected in vitro [8]. It seems clear that the major role of the oviduct in anurans is to mediate or support the sperm-egg interaction during the fertilization process. EVENTS ASSOCIATED WITH SPERM ENTRY Under natural conditions, anuran eggs are ferti- lized as soon as they are spawned in fresh water. Thus, unlike most urodeles [9], fertilization in anurans is external. Perhaps as an adaptation to this natural mating habit, spermatozoa of anuran amphibians are not motile in high ionic strength solution but become motile when the ionic strength is lowered. They are not only motile but are kept viable for several hours in hypotonic solutions osmotically equivalent to fresh water (e.g., 0.1-0.05 De Boer’s or Ringer’s solution ; for composition of De Boer’s solution (DB), see legend for Fig.2). Hence, the usual method of 120 140 Total Concentration of Na. K*. Ca. &Mg” Fic. 2. Motility of Bufo japonicus sperm as a function of various cation concentrations in the surrounding media. Open circles, dilution of De Boer’s solution (DB) consisting of 110mM NaCl, 1.3mM KCl and 1.3mM CaCl; closed circles, dilutions of egg-jelly solubilized by UV-irradiation. Arrow indicates the total concentration in 0.5DB. Data by Hosono and Ishihara (unpublished). artificial fertilization in anurans consists of insemi- nating fully-jellied eggs freshly recovered from uterus with sperm suspended in 0.1—0.05 DB or Ringer. For the South African clawed frog Xenopus laevis, however, a slightly different method of artificial fertilization is adopted, be- cause of a unique salt requirement by spermato- zoa. The X. laevis sperm lose motility and fertilizing capacity rapidly in 0.05 DB, but are motile and remain viable in solutions with in- termediate ionic strengths (e.g., 0.5 DB). Although fertilization is possible by inseminating Oviducal Secretions and Gamete Fusion 3 with sperm suspended in 0.05 DB as originally developed by Wolf and Hedrick [10], insemination with sperm suspended in 0.5 DB is more effective in successfully fertilizing Xenopus eggs [11, 12]. Fertilizing spermatozoa pass through several layers of jelly envelopes and the vitelline coat (VC) before fusing with the egg plasma mem- brane. Usually, it is 3—8min after addition of sperm suspension that a fertilizing sperm is actual- ly incorporated in the egg. During this short period, the jelly envelopes hydrate and swell rapidly. Observations on fresh material gametes (unpublished) reveal that a sperm arriving at the surface of the VC stands still for a while showing a boring movements and is incorporated in almost perpendicular to the surface of egg, leaving a swollen VC and a local accumulation of cortical cytoplasm [13, 14] (Fig.3) at the site of sperm entry. Within seconds after the entry of sperm, the egg elicits a fertilization potential (or activation potential) including a rapid depolarization of the membrane potential, which is primarily due to the Fic. 3. Surface of the Hyla arborea egg 5min after insemination, showing an accumulation of the cor- tical cytoplasm (arrow) at the site of sperm entry and a local swelling of vitelline coat (V) above it. S, sperm heads (out of focus); J, jelly coat. opening of chloride channels [cf., 4]. There subse- quently ensues a wave of cortical granule exocyto- sis that spreads from the site of sperm entry over the entire surface of the egg [14]. The cortical granule exudate alters the VC to become im- penetrable by sperm, as most extensively studied in X. laevis [15]. Evidently these two events, the fertilization potential and the cortical granule exocytosis, constitute the fast and slow blocks to polyspermy in anurans. The problems related to the block to polyspermy in amphibians were recently reviewed by Elinson [4]. Regarding the reaction of sperm during the early phases of fertilization, two aspects are relevant to their passage through the egg envelopes. These include an acrosome reaction and a release of vitelline coat lysin. The acrosome reaction has so far been described only in two anurans, Leptodac- tylus chaquensis [16] and Bufo japonicus’ [17]. For L. chaquensis the exact pattern of “acrosome breakdown”, shown to be induced in a hypotonic medium, is not known. In B. japonicus the reaction involves the breakdown of the membrane- bounded acrosomal cap, thus exposing the inner membrane of the acrosomal cap which participates in the actual fusion with the egg plasma membrane (Fig.4). It should be emphasized that sperm do not undergo this reaction at any point during the b . | ; be : | : | Fic. 4. A schematic illustration of the intact acrosome (a) and the acrosome reaction (b-d) in Bufo japoni- cus, showing the fusion of outer acrosomal mem- brane (OA) with the overlying plasma membrane (M), resulting in a release of the contents of acro- somal cap (AC) and exposure of the inner acrosomal membrane (IA). (Yoshizaki and Katagiri [17]. Re- produced by permission from Alan R. Liss Inc.) passage through jelly, but those at the surface of or passing through the VC are definitely acrosome- reacted [16, 17]. The source of materials inducing the acrosome reaction will be discussed later. * Previously referred to as Bufo bufo japonicus or Bufo vulgaris formosus. See Matsui [18] for the new nomenclature of this species. 4 CH. KATAGIRI There is evidence for the acrosomal localization of protease(s) in a number of anuran species, as defined by the formation of a “halo” on an artificial substrate (gelatin film). Although direct evidence is lacking for their involvement in the VC lysis, specific inhibitor studies employing this assay system revealed either a tryptic (Rana pipiens, [19-21]) or chymotryptic (B. japonicus, [22]) type of acrosomal proteases. The proteolytic activity that digests the VC of unfertilized eggs, but not of fertilized eggs, has been obtained by extraction of sperm with hypotonic medium (B. arenarum,[23]) or Triton X—100 (B. japonicus, [22]). The VC lysin isolated from the latter has a MW of 54,000 with occasional larger, polymeric forms observed. Its enzymatic properties are like the serine-protease chymotrypsin. It is able to lyse the VCs of some heterologous anuran species: such lytic activity correlates well with the cross-fertilizability by Bufo sperm. The relatively low specificity of sperm VC lysin in terms of heterologous VC hydrolysis is in contrast with the high species-specificity of hatch- ing enzyme that is secreted by developing embryos to digest their own envelopes [24]. Most important in relation to the role of VC lysin in fertilization is that its release from individual sperm is highly correlated with the incidence of acrosome reac- tion, as discussed later. Besides the lysis of the VC, sperm may possibly possess other lytic activi- ties in order to accomplish fertilization. Among these lytic activities is the dissolution of a special jelly component (“bouchon” or animal plug) by Discoglossus pictus sperm, which was one of the first demonstrations of acrosome-associated lytic activity in any species of sperm [25]. At present, it has not been unequivocally shown if fertilizing sperm use jelly-digesting enzyme(s) during their passage through the jelly envelopes. ROLE OF EGG-JELLY Fertilizing sperm first have to pass through the jelly-envelopes before making a fusion with the egg. The necessity for a fertilizing sperm to pass through jelly envelopes was indicated as early as in 1919 when Bataillon [26] showed that coelomic eggs exposed to sperm suspension were not ferti- lized despite their full ability to respond to > parthenogenetic stimuli. Since then studies on a variety of species including both urodele and anuran amphibians confirmed that jelly-less eggs, either taken from the coelom or mechanically or chemically dejellied, cannot be fertilized when inseminated in water. Thus, just what role the egg-jelly plays has long been one of the major questions for understanding the mechanism of gamete-interactions in amphibians. Properties of Egg-jelly The jelly envelopes are made up of several layers of distinct composition and morphology which are highly variable according to the species or group of amphibians [27]. Chemical analyses have indicated their glycoprotein nature, with 60-70% carbohydrates and 30-40% proteins whose relative amount is variable according to species [28-30]. Their carbohydrate moiety is comprised of fucose, hexoses, a relatively large amount of hexosamines, and a small amount of sialic acid. Comprehensive analyses in Xenopus laevis revealed a total of 9 macromolecular compo- nents that are distributed differentially in 3 jelly layers [31]. Histochemical and analytical studies are available that implicate the difference in the glycoprotein composition of jelly layers [31-33], together with the specific localization of sulfated molecules in the innermost jelly layer [34]. Immunological analyses have demonstrated that jelly molecules include both species-specific con- figurations and components shared with other species or genera [35, 36]. Besides these, there are some antigenic components which are unique to particular layers of jelly [31, 35]. No studies, however, have elucidated the molecular nature of the antigenic determinants detected by conven- tional immunological techniques. In evaluating the chemical analyses described above, it should be noted that the egg-jelly contains both the mechanically stable structural materials and soluble molecules which can easily diffuse away into surrounding medium. The relative amount of insoluble and soluble jelly materials may be variable according to the species of anurans. It seems that relatively larger amounts in jelly are soluble in toads (e.g., Bufo arenarum,|37|; Bufo japonicus, {30]) than in frogs Oviducal Secretions and Gamete Fusion 5 (Ranas, [3, 30]). In an extreme case as found in B. arenarum, repeated washings of eggs with water render them unfertilizable apparently due to the loss of soluble materials (“diffusible factor”), suggesting their importance for fertilization [38]. Chemical analyses in B. japonicus [30] revealed that carbohydrate and protein composition of water-soluble jelly materials is essentially the same as insoluble materials. Egg-jelly as a Barrier for Sperm Penetration Upon immersion of uterine eggs in salt solu- tions, hydration of jelly promptly begins. The adhesiveness and the degree of swelling in indi- vidual layers of jelly vary depending on the tonicity and the kinds of ions in the surrounding media. Generally, the jelly is more swollen and more adherent in lower salt solutions than in isotonic solutions, e.g., De Boer’s or Ringer’s solution. In parallel with the demonstration of the necessity of jelly in the fertilization process, it has repeatedly been shown that upon hydration in lower salt solutions, the eggs become refractory to fertiliza- tion, although they retain responsiveness to activa- tion stimuli. The time required for the loss of egg-fertilizability after immersion in lower salt solutions is variable according to species of eggs, from 15—30 min (R. chensinensis, [39]; X. laevis, [10]) to 1hr or more (R. pipiens, [3]; B. bufo asiaticus, [40]). There is ample evidence that this loss of fertilizability is accompanied by physico- chemical changes in the particular layer of jelly envelopes that inhibit the migration of sperm through jelly. In R. chensinensis the blockade of sperm migration occurs both on the outermost and innermost layers, and these are ameliorated by treatment of jelly with trypsin [41]. A similar block in jelly of X. laevis [42] and Hyla arborea [43] is associated with an increased birefringence in a distinct middle layer of jelly, and this blockade in X. laevis is regained upon brief exposure to isotonic salt solution. It is emphasized that these changes of jelly accompanying the hydration in environmental conditions for normal fertilization decrease the actual number of sperm getting to the surface of VC, so that they function at least partly as a block to polyspermy. Other lines of evidence point to the possible involvement of egg-jelly in preventing cross- fertilization. Although a large number of inter- specific or intergeneric fertilizations are possible among amphibians [44], there are certain combina- tions where cross-fertilization does not occur. The block to heterologous sperm may occur at various steps preceding the actual gamete fusion, including the jelly layers, the VC, or the plasma membrane, depending on each combination. Of these the combinations between eggs of Bufo japonicus and sperm of Rana (R. chensinensis, R. japonica, and R. nigromaculata) are taken as examples where the jelly coat functions as a principal barrier for cross-fertilizability [36, 50]. The eggs of Ayla arborea are not fertilized by R. chensinensis sperm for the same reason [45]. Likewise, the eggs of Xenopus borealis are not fertilized by sperm of X. laevis because laevis sperm cannot penetrate the innermost jelly layer of borealis eggs. However, fertilization of borealis eggs with laevis sperm occurs if borealis eggs are enrobed with laevis jelly by transferring them to the oviduct of Jaevis. Alternatively, laevis eggs enrobed with borealis jelly cannot be fertilized with sperm of its own species but are still fertilized by borealis sperm [46]. Although the involvement of egg-jelly in pre- venting cross-fertilization is thus evident in certain crosses, the observed blockade of sperm migration in heterologous jelly cannot simply be ascribable to the lack of species-specific molecular interac- tions between sperm and the egg-jelly. Rather, in many cases the failure of sperm migration is more likely to be due to the incompatibility of the physical architecture of the egg-jelly. The R. pipiens eggs enrobed by R. clamitans jelly en- velopes are not fertilized by pipiens sperm, but are fertilized when dejellied eggs are inseminated in the presence of soluble components (“egg-water”) of clamitans egg-jelly [47]. Fertilizability of Oviducal Eggs A number of experiments have been conducted to determine the fertilizability of the eggs taken from different levels of the oviduct, in an attempt to define what layers of jelly envelopes are indispensable for fertilization. Although the re- sults in several species of amphibians favor the 6 CH. KATAGIRI general account that the fertilizability of eggs increases as they descend down the oviduct, it has not been easy to define an exactly specific jelly layer that is a prerequisite for fertilization. Experi- ments in Xenopus laevis indicated that the increase of fertilizability is gradual as the eggs travel down the oviduct, and there is no marked local rise correlated with a special jelly layer [48]. Other studies employing Bufo japonicus [49, 50], B. arenarum [51], and Rana pipiens [52] indicated that the rate of fertilization in eggs taken even from similar levels of the oviduct varied greatly according to the females used, the time after induction of ovulation, as well as the sperm concentration employed. Thus, in B. japonicus and B. arenarum, some batches of eggs taken from an upper portion of the oviduct may be fertilizable provided they are enrobed with a small amount of the inner second layer in addition to the innermost one. In other batches, however, eggs are not fertilizable until they are supplied with additional outer layers in the lower part of oviduct. Apparently one of possible reasons for these variances is the maturity of eggs, as exemplified by the fact that the nuclear maturity of eggs bears no direct relation to the location of oocytes in the female reproductive tract. Under the conditions where eggs are mature enough to be responsive to artificial stimuli, results obtained in Bufo [50, 51] and R. pipens [3] seem to agree in showing that the innermost gelatinous jelly layer is not sufficient to support fertilization. On the other hand, the eggs were fertilized well even when they were enrobed only with the outer layer of jelly by bypassing the upper levels of the oviduct [3, 50]. The importance of outer jelly layers (J; or J, in Bufo; V3 in R. pipiens) deserves attention because of their possi- ble relevance to retaining a diffusible factor that plays some essential role in supporting sperm entrance in eggs. The correlation of the loss of egg fertilizability with that of diffusible molecules has been particularly emphasized in studies with B. arenarum [53] and R. pipiens [3]. To summarize, studies on the fertilizability of eggs from various levels of oviduct have not given rise to conclusive results in understanding the role of jelly in fertilization. Re-evaluation of these studies should now be made in view of a more recent finding that — the oviduct has roles other than the secretion of jelly materials, as discussed later. Egg-jelly Factors Essential for Fertilization A more potent approach to the question of how the egg-jelly functions in fertilization consists of inseminating dejellied uterine eggs in the presence of various jelly preparations. This assay system has the advantage of using eggs with homogeneous maturity, as well as the ease of applying jelly preparations after diverse chemical or physical modifications. Eggs of Bufo japonicus, B. arenarum, B. amer- icanus, Hyla arborea, and Rana pipiens obtained from the uterus and dejellied completely by KCN or NaCN are not fertilized when inseminated in 0.1 Ringer or 0.05 DB, but have proved to be fertilizable when inseminated in the presence of appropriate jelly preparations. The jelly materials that had been dissolved by cyanide and dialyzed to remove the cyanide (“dialyzed jelly”) were highly effective in supporting fertilization of B. japonicus [54], B. arenarum [55], and Hyla arborea [45]. The nondialyzable fraction of jelly in Xenopus laevis which was solubilized by mercaptoethanol or UV-irradiation also supported fertilization of the eggs whose jelly was removed by sodium-sulfite to the extent that no fertilization occurred in 0.05 DB [56]. The other jelly material that supports fertilization of dejellied eggs of B. japonicus [30], B. arenarum [53], B. americanus [3], and R. pipiens [3] was obtained by shaking fully jellied uterine eggs in distilled water for 15—30min (“diffusible factor”: DF). The dialyzate but not the retentate after dialysis of DF retained a strong fertilization supporting activity in B. arenarum [53] and B. japonicus [30]. In R. pipiens, however, both dialyzate and retentate were required for insuring a high frequency of fertilization [3]. There is a possibility that a low activity of dialyzate in the R. pipiens DF was due to a relatively low concen- tration of the pertinent molecules. It was found additionally that in B. japonicus [30, 54, 57] synthetic polymers such as polyvinylpyrrolidone (PVP), dextran or Ficoll were effective in support- ing fertilization of cyanide-dejellied eggs. The possibility that the observed results were ascrib- able to a small amount of jelly materials remaining Oviducal Secretions and Gamete Fusion 7 undissolved may be eliminated by the fact that the Hyla arborea eggs from which the vitelline coat in addition to jelly had been removed regained fertilizability only when inseminated in dialyzed jelly or PVP [45]. Assays on the fertilization-supporting activity of jelly preparations with dejellied eggs made it possible to inquire into the nature of factor(s) involved in the jelly. First, the egg-jelly played its biological role less species-specifically. The di- alyzed jelly of B. japonicus and homogenized jelly of R. chensinensis were as effective as dialyzed jelly of the homologous species in supporting fertilization of dejellied Hyla eggs [45]. Dejellied eggs of B. japonicus were fertilized well in the presence of homogenized or trypsinized jellies from R. chensinensis or R. japonica [58], as well as of DF from R. japonica [30]. Likewise, DF from R. pipiens and R. clamitans were active in support- ing fertilization of dejellied eggs from B. america- nus and R. pipiens, respectively [3, 47]. Second, at least in Bufo, the active substances in jelly were regarded to be of small molecular weight since they were dialyzable, stable after boiling for 15 min [30], and remained active after extensive hydroly- sis by pronase [54]. Gel-filtration on Sephadex G-25 gave a molecular weight of less than 500 for active molecules [30]. These observations, together with the observed lack of correlation of immunological cross-reactivity in jellies with cross- fertilizability between pertinent species [36], argued against the proposal that specific molecular interactions operating between jelly and sperm surface underlie the essential role of egg-jelly in fertilization process [35]. Third, experiments in R. pipiens [3] and B. arenarum [53] indicated that preincubation with jelly materials did not promote the fertilizing capacity of sperm, but rather the jelly or jelly factors must be present at the moment of sperm entrance in eggs. In this respect the state of sperm affected by jelly cannot be paralleled with capacitation in mammalian sperm [59]. The exact molecular entity in egg-jelly essential for its biological activity was further studied with B. japonicus, because of the ease with which large amounts of diffusible jelly materials can be col- lected [60]. The dialyzate from UV-irradiated jelly (UVJD) supported fertilization of dejellied eggs in a dose-dependent manner in a certain range of concentrations, but decreased its activity as its concentrations became higher. Surprisingly the fertilization-supporting activity of UVJD was re- tained even after ashing at 600°C for 16hr, so that the involvement of organic substances was ex- cluded from the biological activity concerned. To inquire as to the role of inorganic salts contained in biologically active preparations, concentrations of inorganic salts in UVJD were determined and a reconstituted salt solution (RSS) was prepared according to the composition of UVJD. RSS comprising 3.4mM NaCl, 1.6mM KCl, 0.33mM CaCl,, and 0.48mM MgCl, proved to be equiva- lent to UVJD in insuring fertilization of dejellied eggs. Analyses of the relative role of individual Salts in RSS revealed that what is important for its biological activity is to contain at least 1mM Ca?~* and/or Mg**. Thus dejellied eggs are now ferti- lized in 0.05 DB supplemented with 1-5 mM CaCl, or MgCl, (0.05 DB contains CaCl, at 0.065 mM). The finding of Ca** and/or Mg’~ as essential factor(s) for supporting fertilization of dejellied eggs is compatible with all the properties of active jelly preparations that have so far been described. A question then arises as to how the relatively high concentration of divalent cations in RSS is related to the heretofore mentioned biological function of jelly envelopes in fertilization. The answer to this question came when the ionic conditions in egg- jelly at the time of fertilization were evaluated in conjunction with the motility of sperm, as follows [60]. First, both dialysis and gel-filtration studies indicated that jelly has a unique property of retaining divalent cations preferentially to monovalent cations. Second, anuran sperm are not motile unless total cation concentration is less than 20-25mM. Thus, the total cationic concen- tration in jelly envelopes of freshly laid eggs, measuring about 130mM, is too high for adequate sperm motility. In fact, it was found that uterine eggs become fertilizable after contact with water and hydration for 2—3 min, when the salinity within the jelly is low enough to assure the motility of sperm. However, a simple dilution of physiologi- cal saline apparently results in divalent cation concentration (0.065mM CaCl, in 0.05 DB) far below the level necessary for sperm-egg interac- 8 Cu. KaTAGIRI tions including the membrane fusion and acrosome reaction. An intriguing situation in jelly envelopes is that during the initial hydration process, both the dilution of total salinity and the maintenance of minimal essential divalent cations are effected, so that the environmental conditions necessary for successful sperm entrance into eggs are estab- lished. ROLE OF THE OVIDUCAL PARS RECTA (PR) Participation in Structural Alteration of VC A puzzling result emerging from the experi- ments with the dejellied uterine egg bioassay was that jelly-less coelomic eggs, even though phys- iologically mature, are not or hardly fertilized under the conditions which support fertilization of dejellied uterine eggs. It was provisionally reasoned that the oviduct serves some additional roles to make eggs accessible to fertilizing sperm. Two series of experiments in early 70s revealed that a major barrier to fertilization of coelomic eggs resides in the VC. Coelomic eggs from Rana pipiens were successfully fertilized under the same conditions as for dejellied uterine eggs provided the VC had been either altered by treatment with trypsin or cyanide, or mechanically torn with forceps [61]. Likewise, coelomic eggs from Bufo japonicus were fertilized when the VC had been removed by pronase or hatching enzyme [57]. Similar experiments were extended also to Xeno- pus laevis [62]. Thus, these studies strongly suggested a new role of oviduct as altering the VC so as to be penetrable by sperm, rather than inducing the receptivity of the egg itself to sperm. The evidence for the oviduct-induced alteration of the VC was first presented in X. laevis [63]. It was shown that the electron microscopically dis- tinct bundles or filaments seen in the VC of coelomic eggs become evenly dispersed in oviducal or uterine eggs, in parallel with the acquisition of penetrability of isolated VC by sperm. This alteration was ascribed to the first 1cm segment (1/20 of total length) of oviduct, since it occurred prior to addition of any jelly to the egg. Similar observations have been extended to the VC of other anurans, R. japonica [64], B. japonicus [65], and B. arenarum [66]. In R. japonica this fine structural alteration was accompanied by deposi- tion of oviduct-derived carbohydrate materials as detected by PA-CrA-methenamine method. All these effects on the VC were observed either in the eggs that had passed through the first 1/20th portion of oviduct (pars recta, PR) or those incubated in the extracts from this tissue. Electron microscopic observations of the PR cells in B. japonicus [65], R. japonica [64] and X. laevis [67] elucidated the unique epithelial cells that had been overlooked in a number of older observations on the amphibian oviduct [68, 69]. The non-ciliated epithelial cells in the PR possess electron-dense granules which are distinct from those in the jelly-secreting cells in more caudal levels of the oviduct, the pars convoluta (PC) (Fig.5). The granules in PR cells are small in number during the hibernation period, increase in number significantly upon hormonal stimulation for ovulation, and almost disappear after oviposi- tion. The acquisition in the VC of carbohydrate materials during passage of R. japonica [64] eggs through the PR is most probably ascribable to the secretion of PR granules. In B. japonicus [65] immunohistochemical studies showed a deposition of PR cell-specific antigen in the VC during passage of eggs through the PR. Thus, based on morphological criteria the effect of oviducal PR on the VC is two fold, viz., a partial degradation and/or disorganization of filament bundles and a deposition of PR-derived substances within the filament bundles. It should be added that in Xenopus laevis, the histologically defined PR comprises two distinct subportions, of which a more cephalic portion (PR1) is concerned with the structural alteration of the VC [67]. The caudal 1/5 portion (PR2) is involved in the formation of a fertilization laye (F-layer), as discussed later. i Molecular Basis for PR Function Analyses of how the PR functions in the fertilization process have been undertaken using crude extracts of PR homogenates (PRE) from ovulating females (Bufos, [65, 70]; R. japonica, [64]) or secretion fluids collected from ligated PR (B. arenarum, [70, 71]; X. laevis, [72]). In more Oviducal Secretions and Gamete Fusion 9 Fic. 5. TEM of non-ciliated epithelial cells in the pars recta of Rana japonica, before (a) and after (b) passage of several hundred eggs through this oviducal portion, showing the disappearance of electron-dense granules in (b). C, cilia. recent studies with B. japonicus [73, 74], fairly pure PR granules (PRG) isolated by Percoll density gradient centrifugation proved to possess all the functions of the PR. Most direct evidence for the involvement of PR in the fertilization process came from experiments in which coelomic eggs of B. arenarum [70,71] and B. japonicus |65, 74] pretreated with PRE or PRG were shown to be fertilized well in the presence of diffusible jelly materials or RSS. Although a 10 Cu. KATAGIRI preincubation time of 4hr with PRE or PRG in contrast to a presumed 10-15 min residence in PR in vivo was necessary to obtain a high fertilizability in B. japonicus, this success of fertilization of coelomic eggs provided strong support for the notion that PR secretions render the VC pene- trable by sperm. Besides affecting the VC, the PR substances possess a striking activity for inducing an acrosome reaction in sperm. Treatment of coelomic eggs with PRE followed by addition of sperm in physiological saline resulted in the lysis of the VC (B. japonicus, [65]; R. japonica, [64]), despite a failure of fertilization due to the lack of jelly or RSS. Likewise, the supernatant solution of the PRE or PRG preincubated with sperm for 30 min has a strong VC lytic activity, accompanied by the induction of the acrosome reaction and a loss of VC lysin activity extractable from sperm [65]. Most probably the contents of PRG deposited in the VC function to induce an acrosome reaction and a release of VC lysin from individual sperm. This scheme is compatible with the TEM observa- tion [17] that sperm passing through the jelly layers possess intact acrosomes, but those attaching to and passing through the VC are acrosome-reacted. The molecular basis for the PR-induced struc- tural transformation of VC has recently been the subject of intensive studies with Bufos and Xeno- pus. It was shown in B. arenarum [{71, 75] and B. japonicus [74] that the pretreatment of coelomic eggs with PRE or PRG renders the VC susceptible to the sperm lysin to the same degree as the VC of dejellied uterine eggs, as evidenced by a rapid dissolution of VC surrounding the eggs. Deter- minations of VC lysis in vitro indicated that the coelomic egg VC (CEVC) is not completely refractory to the sperm lysin as had been shown previously, but increases in susceptibility after treatment with PRE approximately 10 times [74]. A Ca**-dependent activity enhancing the CEVC susceptibility to sperm lysin has been partially purified from the PR of B. arenarum [75, 76]. The activity is of trypsin-like serine protease, as defined by its strong inhibition by SBTI and TLCK. These studies, combined with the observed increase in VC of both intrinsic fluorescence and the total number of 8 anilino-naphthalene binding sites, led © Miceli et al. [77] to propose that the PR-induced transformation of VC includes a dissociation of some superficial peptides so that the site for sperm lysin attack is increased. More recent studies with B. japonicus and X. laevis have provided new information as to how the PR affects the VC. SDS-PAGE analyses of Bufo VC protein revealed that in comparison with CEVC, uterine egg VC(UEVC) lacks 40-52K glycoproteins concomitant with an _ increased 52K | FP 36K a b c Fic. 6. SDS-PAGE analyses of vitelline coat proteins in Bufo japonicus, from coelomic egg (a), uterine egg (b), and coelomic egg treated with pars recta granule fraction (c). Numbers indicate molecular weights. stainability of a 39K glycoprotein and the appear- ance of a 36K glycoprotein [74] (Fig. 6). Similar changes of VC proteins occur in Xenopus, where a 43K glycoprotein in CEVC is converted to a 41 K in UEVC [78]. In both cases, the conversions can be induced upon incubation of coelomic eggs with PRG. Interestingly PRGs from Bufo and Xenopus share strong hydrolytic activities against synthetic substrates such as X-Arg-MCA and Val-Leu-Lys- MCA, and the incubation of coelomic eggs with heterologous PRGs between these two species results in specific glycoprotein changes in SDS- PAGE profiles of VC proteins in both reciprocal Oviducal Secretions and Gamete Fusion 11 combinations (unpublished observations). A pro- teolytic activity that induces a CEVC to UEVC type protein conversion has recently been partially purified from PRG of B. japonicus [91]. This activity, hydrolyzing preferentially Arg-MCA ter- minals in a highly Ca**-dependent manner, is inhibited by SBTI, leupeptin, PMSF and DFP, and thus shares properties in several respects with the trypsin-like enzyme obtained from B. arenarum [76]. It seems plausible that in the three species of anurans just discussed, the PR trypsin-like en- zyme(s) is functioning to induce a partial degrada- tion of VC proteins, so that the VC is accessible to sperm lysin. It is clear that the PR serves two distinct roles, i.e., the induction of acrosome reaction and the partial hydrolytic degradation of VC proteins. Of these, the proteolytic degradation of CEVC pro- teins is evidently prerequisite for establishment of egg fertilizability, since the inhibition of the PRE-induced CEVC to UEVC changes by incuba- tion of coelomic eggs with SBTI or leupeptin prevents the acquisition of egg fertilizability [74]. Once the VC glycoprotein changes have occurred, as in uterine eggs, treatment with trypsin inhibitors before insemination does not inhibit fertilization. It should be mentiond that the enzymatic partial hydrolysis of VC is not sufficient to ensure egg fertilizability, since no or only a low frequency of fertilization has been observed in the coelomic Bufo eggs whose VC had been altered by a partially purified PRG protease or the Xenopus PRG ({91]; unpublished observation). These last experiments are intriguing in that they suggest that the VC degrading and acrosome reaction inducing activities, both derived from PRG, can be studied as separate molecular entities. Finally, mention should be made of an another intriguing example of oviducal contribution to fertilization process which has been uniquely developed in Xenopus laevis. Upon fertilization or artificial activation, the vitelline envelope (VE) of unfertilized Xenopus eggs is transformed into the fertilization envelope (FE) by acquiring an elec- tron-dense layer (fertilization layer: F-layer) on the outer surface of the VE [79]. The F-layer is a protease- and mercaptoethanol-resistant precipi- tate that is formed as a Ca’*-dependent agglutina- tion involving both the cortical granule (CG)- derived lectin and its ligand of oviducal origin [15, 80]. Functionally, the F-layer provides a block to fertilizing sperm either mechanically or by masking sperm reactive sites on VE (a slow polyspermy block mechanism) [81, 82], as well as a molecular barrier to macromolecules released into peri- vitelline space from cortical granules [83, 84]. A CG lectin participating in the F-layer formation has been purified from CG exudates as a metallo- glycoprotein of more than 500K dalton that has a galactosyl sugar specificity for ligand binding [15, 84]. With regard to the ligand for CG lectin, the innermost jelly layer (J,;) has been implicated primarily based on its ability to undergo a pre- cipitation reaction with the CG lectin both in vivo and in vitro [15, 31]. Recent studies, however, raise the possibility that the CG ligand is present as a prefertilization layer (PF-layer) localized on the outer surface of VE, as a secretion product from the lower 1/5 portion of PR [85, 86]. This proposal is based on the electronmicroscopic and immuno- histochemical observations which indicate that molecules specific to the granules in PR2 epithelial cells are released and deposited on the VE. The exact origin of CG ligand, however, can be dual in view of the occurrence in an agarose plate of precipitation line between CG lectin and both PF-layer and J, materials, although in normal egg activation process the PF-layer occupies a poten- tially privileged position than the J, in reacting with the released CG lectin [84]. Whatever the exact source of CG ligand may be, PF-layer (PR2) or J,(p. convoluta), the F-layer formation in Xenopus provides a unique model in which the oviducal secretions evidently participate in the slow but efficient polyspermy block mechanism. In other anurans, the oviduct does not seem to be involved in the formation of FE or fertilization coat, since the VC of activated coelomic eggs becomes refractory to the sperm lysin [22, 87]. CONCLUDING REMARKS One of the basic concepts derived from studies on gamete interaction in amphibians is that the vitelline coat (VC) surrounding ovulated physiolo- gically mature eggs acts as a barrier for sperm. 12 Cu. KATAGIRI o Na, Kk ~ Ciel Mg 4 PRG- material COELOMIC Fic. 7. UTERINE Schematic illustration of alterations of the vitelline coat (VC) during passage of eggs through FERTILIZING oviduct and the role of egg-jelly (J) in mediating sperm entry through the vitelline coat, based on studies in Bufo japonicus. Figure 7 schematically illustrates how the secre- tions from the oviducal PR and PC in Bufo Japonicus function to overcome this barrier. Dur- ing the initial phase of fertilization, the unique nature of the jelly to hydrate and retain divalent cation assures both the motility and the minimal divalent cation requirement for gamete fusion. Sperm are acrosome-intact during passage through jelly, but undergo the acrosome reaction and release of VC lysin at the surface of the VC in response to the acrosome reaction-inducing sub- stance of PR origin. By this time, the VC has become highly susceptible to sperm lysin due to a partial hydrolytic degradation by the PR protease, so that sperm may find a path through the VC. According to this scheme, the role of jelly envelopes is simple enough to exclude the direct contribution of any macromolecules or organic substances and their localization in specific jelly layers. The scheme, however, does not rule out the possible involvement of the non-diffusible structural integrity of jelly, as emphasized in B. arenarum where the addition of non-diffusible jelly — molecules to a diffusible jelly fraction enhances the rate of fertilization [38]. Studies are needed to determine whether or not the function of jelly as proposed here can be extended to other anurans and urodeles, In this connection, it will be of interest to learn as to how a specially differentiated component of jelly (“animal plug”) in Discoglossus pictus eggs can restrict the site of sperm penetra- tion [88]. In contrast with jelly, possible function of PR secretions are multiple, including the induction of a conformational change of VC and the acrosome reaction, as well as the enhancement of VC lytic activity. Of these, a hydrolytic degradation of VC by a trypsin-like enzyme is evidently associated with the increased susceptibility of the VC to the sperm lysin. In this respect, the VC exhibits three different states with respect to susceptibility to sperm lysin: less susceptible in coelomic eggs, highly susceptible in uterine eggs, and completely refractory in fertilized eggs. Studies on the molecular basis of the differential suceptibilities of the VC towards sperm lysin should aid in under- Oviducal Secretions and Gamete Fusion 13 standing the mechanism of action of pertinent enzymes including the hatching enzyme [24, 89, 90]. Compared with the trypsin-like enzyme, little is known about the molecular nature of acrosome reaction-inducing activity from PRG. There is evidence suggesting the involvement of carbohy- drate moieties in fertilization process, as exem- plified by concanavalin A(Con A) inhibition of fertilization in Bufos [70, 74]. This observation is likely to be relevant to the inhibition by Con-A of the PRG-induced acrosome reaction (unpublished observation). Characterization studies of the PR- derived, acrosome reaction-inducing substance should be most promising. From a comparative view, the vitelline coat of amphibians is homologous to the zona pellucida of mammals and the vitelline envelope of echi- noderms, in the sense that it is deposited around the growing oocytes during oogenesis. However, in order to acquire the functional equivalence to mammalian or echinoderm counterparts, the amphibian vitelline coat has to be acted upon by secretory products of oviduct. Thus, the amphi- bian system provides a unique model to study the exact cellular source of molecules functioning in mediating gamete interactions, possibly together with their hormonal control. ACKNOWLEDGMENT I express my appreciation to Drs. J. Hosono, K. Ishihara, Y. Iwao, K. Takamune, and N. Yoshizaki, for their ingenious collaboration in pursuing studies with Bufo japonicus. My thanks are also due to Dr. J. L. Hedrick for his stimulating discussion and helpful com- ments to this article. REFERENCES 1 Glabe, C. and Vacquier, V. D. (1978) Proc. Natl. Acad. Sci. U.S.A., 75: 881-885. 2 Wasserman, P.M.,Florman, H.M. and Gerve, J. M. (1985) In “Biology of Fertilization”. Ed. by C.B.Metz and A. Monroy, Vol.2, Academic Press, New York, pp. 341-360. 3 Elinson, R. P. (1971) J. Exp. Zool., 176: 415-428. Elinson, R. P. (1986) Int. Rev. Cytol., 101: 59-100. 5 Aplington,H.W., Jr. (1957) Ohio J. Sci., 57: 91-99. GmeiwaoeY Ito, S~ and Katagim, Ch. (1981) Dev. Growth Differ., 23: 89-100. os 5 16 7) 18 19 20 21 22 23 24 25 26 75] 28 29 30 31 a2 33 34 35 36 Si Iwao, Y. (1982) Dev. Growth Differ., 24: 467-477. Masui, Y. and Clarke, H.J. (1979) Int. Rev. Cytol., 57: 185-282. McLaughlin, E.W. and Humphries, A.A., Jr. (1978) J. Morphol ., 158: 73-90. Wolf, D. P. and Hedrick, J. L. (1971) Dev. Biol., 25: 348-359. Moriya, M. (1976) J. Fac. Sci. Hokkaido Univ. Ser. VI, 20: 272-276. Hollinger, T. G. and Corton, G. L. (1981) Gamete Res., 3: 45-57. Elinson, R. P. and Manes, M. E. (1978) Dev. Biol., 63: 67-75. Picheral, B. and Charbonneau, M. (1982) J. Ultra- struct. Res., 81: 306-321. Wyrick, R.E., Nishihara, T. (1974) Proc. Natl. 2067-2071. Raisman, J.S. and Cabada, M. O. (1977) Growth Differ., 19: 227-232. Yoshizaki, N. and Katagiri, Ch. (1982) Res., 6: 343—352. Matsui, M. (1984) Contriv. Biol. Lab. Kyoto Univ., 26: 209-428. Penn, A. and Gredhill, B. L. (1972) Exp. Cell Res., 74: 285:288. Elinson, R. P. (1974) Biol. Reprod., 11: 406-412. Elinson, R. P. (1971) J. Exp. Zool., 177: 207-218. Iwao, Y. and Katagiri, Ch. (1982) J. Exp. Zool., 219: 87—9S5. Raisman, J.S. and Barbieri, F. D. (1969) Embryol. Exp., 1: 17-26. Katagiri, Ch. (1975) J. Exp. Zool., 1933: 109-118. Hibbard, H. (1928) Arch. Biol., 38: 251-326. Bataillon, E. (1919) Ann. Sci. Nat. Zool. Ser. 10, 3: 1-38. Salthe, S. N. (1963) J. Morphol., 113: 161-171. Folkes, B. F., Grant, R.A. and Jones, J. K.N. (1950) J. Chem. Soc.(440) part 3, 2136-2140. Bolognani, L., Bolognani, A. M., Lusignani, F. R. and Zonta, L. (1966) Experientia, 22: 601-603. Katagiri, Ch. (1973) Dev. Growth Differ., 15: 81-92. Yurewicz, E.C., Oliphant, G. and Hedrick, J. L. (1975) Biochemistry, 14: 3101-3107. Freeman, S. B. (1968) Biol. Bull., 135: 501-513. Steinke, J.H. and Benson, D.G., Jr. (1970) J. Morphol., 130: 57-66. Redncke lee Smith, Aud. | Yurewicz, k. ©. Oliphant, G. and Wolf, D. P. (1974) Biol. Reprod., 11: 534-542. Shaver, J.R. (1966) Am. Zool., 6: 75-87. Katagiri, Ch. (1968) SABCO J., 4: 33-43. Barbieri, F. D. and Raisman, J. S. (1969) and Hedrick, J. L. Acad: “Secret urS. Acs 71: Dev. Gamete Acta 14 38 39 40 41 42 43 44 45 46 47 48 49 50 SI 52 53 54 55 56 Sf) 58 59 60 61 62 63 64 65 66 CH. KATAGIRI Embryologia, 10: 363-372. Barbieri, F.D. and del Pino, E.G. (1975) Arch. Biol. (Bruxelles), 86: 311-321. Katagiri, Ch. (1961) J. Fac. Sci. Hokkaido Univ. Ser. VI, 14: 606-613. Tchou-Su and Wang, Y. L. (1956) Acta Exp. Biol. Sinica, 5: 75-122. Katagiri, Ch. (1962) Jpn. J. Zool., 8: 365-375. del Pino, E. G. (1963) J. Exp. Zool., 185: 121-132. Katagiri, Ch (1963) Zool. Mag., 72: 23-28. Moore, J. A. (1955) Adv. Genetics, 7: 139-182. Katagiri, Ch. (1966) J. Fac. Sci. Hokkaido Univ. Ser. VI, 16: 77-84. Brun, R. B. and Kobel, H. R. (1977) J. Exp. Zool., 201: 135-138. Elinson, R. P. (1974) J. Embryol. Exp. Morphol., 32: 325-335. Brun, R. P. (1974) Biol. Reprod., 11: 513-518. Kambara, S. (1953) Annot. Zool. Japon., 36: 78-84. Katagiri, Ch. (1965) J. Fac. Sci. Hokkaido Univ. Ser. VI, 15: 633-643. Barbieri, F.D. and Budeguer de Atenor, (1973) Arch. Biol. (Bruxelles), 84: 501-511. Glick, R. N. and Shaver, J. R. (1963) Exp. Cell Res., 32: 615-618. M. S. Barbieri, F.D. and Oterino, J.M. (1972) Dev. Growth Differ., 14: 107-117. Katagiri, Ch. (1966) Embryologia, 9: 159- NC2, Barbieri, F. D. and Embryologia, 10: 363-372. Wolf, D. F. and Hedrick, J. L. (1971) Dev. Biol., 25: 360-376. Katagiri, Ch. (1974) J. Embryol. Exp. Morphol., 31; 573-587. Katagiri, Ch. (1967) 67-73. Yanagimachi, R. (1978) Curr. Top. Dev. Biol., 12: 83-105. Ishihara, K., Hosono, J., Kanatani, H. and Kata- giri, Ch. (1984) Dev. Biol., 105: 435-442. Elinson, R. P. (1973) J. Exp. Zool., 183: 291-302. Stewart-Savage, J. and Grey, R. D. (1984) Exp. Cell Res., 154: 639-642. Grey, R.D., Working, P.K. and Hedrick, J. L. (1977) J. Exp. Zool., 201: 73-84. Yoshizaki, N. and Katagiri, Ch. (1981) Growth Differ., 23: 495-506. Katagiri, Ch., Iwao, Y. and Yoshizaki, N. (1982) Dev. Biol., 94:1-10. Mariano, M.I., de Martin, M.G. and Pisano, A. (1984) Dev. Growth Differ., 26: 33-42. Raisman, J. S. (1969) Annot. Zool. Japon., 40: Dev. 67 68 69 70 71 G2 73 74 75 76 77 78 79 80 81 82 Yoshizaki, N. (1985) J. Morphol., 184: 155-169. Lee, P. A. (1967) J. Exp. Zool., 166: 107-120. Boisseau, C. (1973) J. Microsc., 18: 341-358. Cabada, M. D., Mariano, M.I. and Raisman, J. S. (1978) J. Exp. Zool., 204: 409-416. Miceli, D. C., Fernandez, S. N., Raisman, J. S. and Barbieri, F. D. (1978) J. Embryol. Exp. Morphol., 48: 79-91. Bakos, M. and Hedrick, J. L. (1986) Zool. Sci., 2: 934. Katagiri, Ch. (1986) In “ The Molecular and Cellu- lar Biology of Fertilization : Gamete Interaction”. Ed. by J. L. Hedrick, Plenum Press, New York, in press. Takamune, K., Yoshizaki, N. and Katagiri, Ch. (1986) Gamete Res., 14: 215-224. Miceli, D. C., Fernandez, S. N. and del Pino, E. J. (1982) Biochem. Biophys. Acta, 526: 289-292. Miceli, D. C. and Fernandez, S. N. (1982) J. Exp. Zool., 221: 357-364. Miceli, D. C., Fernandez, S. N. and Moreno, R. D. (1980) Dev. Growth Differ., 22: 639-643. Gerton, G.L. and Hedrick, J. L. (1986) J. Cell. Biochem. 30: 341-350. Grey, R. D., Wolf, D. P. and Hedrick, J. L. (1974) Dev. Biol., 36: 44-61. Wolf, D. P., Nishihara, T., Wert, D. M., Wyrick, R.E. and Hedrick, J. L. (1976) Biochemistry, 15: 3671-3678. Schmell, E.D., Gulyas, B.J. and Hedrick, J. L. (1983) In “Mechanism and Control of Animal Fertilization”. Ed. by. J. F. Hartman, Academic Press, New York, pp. 365-413. Grey, R.D., Working, P. K. (1976) Dev. Biol., 54: 52-60. Nishihara, T. and Hedrick, J. L. (1977) Proc. Fed. Am. Soc. Exp. Biol., 36: 811. Yoshizaki, N. (1986) Dev. Growth Differ., 28: 275-283. Yoshizaki, N. and Katagiri, Ch. (1984) Zool. Sci., 1: 255-264. Yoshizaki, N. (1984) Dev. Growth Differ., 26: 191-195. Miceli; DEC; ~del Pino, Ey J., Barbieri, F.D., Mariano, M..I. and Raisman, J. S. (1977) Dev. Biol., 59:101-110. Campanella, C. (1975) Biol. Reprod., 12: 439-447. Urch, U. A. and Hedrick, J. L. (1981) J. Supramol. Struct. Cell. Biochem., 15: 111-117. and Hedrick, J. L. Urch, U. A. and _ Hedrick, J.L. (1981) = Arch. Biochem. Biophys., 206: 424-431. Takamune, K. and Katagiri, Ch. (1987) Dev. Growth Differ., 29: in press. ZOOLOGICAL SCIENCE 4: 15-22 (1987) © 1987 Zoological Society of Japan REVIEW Ultrastructural Classification of Smooth Muscle Cells in Invertebrates and Vertebrates AKIRA MATSUNO Department of Biology, Faculty of Science, Shimane University, Matsue 690, Japan INTRODUCTION Ultrastructural studies on smooth muscle cells were begun in the 1960s, and have revealed characteristic structures in various organs and tissues of vertebrates as well as of invertebrates. These morphological studies have shown that there are several types of smooth muscle cells in each organ and tissue of the same animal [1-6], and that similar organs and tissues in different species of animals have different types of cells [7-10]. In this paper, I have attempted to classify the various smooth muscle cells by their ultra- structural characteristics. The relationship be- tween the ultrastructure and function in various organs or tissues is also discussed. CLASSIFICATION OF SMOOTH MUSCLE CELLS It is possible to classify smooth muscle cells by size and the arrangement of myofilaments which are scattered in the main part of the cells. Smooth muscle cells were grouped into four types by the following markers: i) the diameter of the thick myofilament, ii) density of an arrangement of dense bodies, iii) the size of the cell, iv) other characteristics (structure of sacrcoplasmic reticu- lum (S.R.) systems, mitochondria, etc.). These markers made it possible to distinguish ultra- structural differences in the four types of smooth muscle cells. Ultrastructural characteristics of the four types of cells and their distributions in organs Received September 22, 1986 or tissues of several species of animals are listed in Table 1. The characteristics are also presented schematically in Figure 1. Fic. 1. Schematic representation of four types of smooth muscle cells. Note differences in diameter of thick myofilaments, number of dense bodies and their arrangements in the cell. 1) A-type smooth muscle cell The A-type cells have thick myofilaments about 14 nm in diameter and thin myofilaments about 7 16 A. MATSUNO Table 1. Four types of cells with distribution in tissue or organ Diameter of | Dense bod . thick myo- and its Sar op le Animal Tissue or organ Cell type filament arrangement A-type Stephanoscyphus tentacle [18] Atorella tentacle [19] Tamanovalva adductor [17] ne ts Spondylus adductor [7] Z ae ee eee sea cucumber body wall [4] ; Te echinoid spine [1] few in number __ distributed Pee ti ; -14nm at echinoid ovarian wall [2] Bees: periphery frog intestine [13] a oa chicken gizzard [12] mouse tunica [16] guinea pig taenia coli [14] cat urinary bladder [15] B-type Atorella oral disc [19] : small size : large size snail body wall [6] about 40nm rn opisthobranchiate sheathed muscle [23] : distributed disordered at Dolabella body wall [26] periphery Mytilus byssus retractor [24, 25] C-type Stephanoscyphus _ taeniol [18] small size Atorella taeniol [19] AD aay Atrina adductor [10] sak, A , distributed Astarte adductor [27] few in at number periphery Meretrix adductor [9] tellin adductor [8] Fragum adductor D-type cuttlefish intestine [30] small size : ; ; slightly oligochaeta intestine [32] sometimes 14-40 nm regular ee Biers Phoronis large vessel [31] arrangement P sea cucumber intestine [5] echinoid ampulla [3] nm in diameter. The cells are generally small in size, and show an elongated spindle-shape. A nucleus is positioned in the central region and cell organelles in the periphery are not well developed. Thick myofilaments sometimes disappear when the conditions of fixation are not suitable. Thus, they were not recognized in early investigations [11]. In chicken gizzard muscle cells, the thick myofilaments were observed only under conditions of low pH (6.6) during fixation [12, 13]. However, they were recognized in smooth muscle cells of guinea pig taenia coli even under usual conditions" of fixation [14]. Now, thick myofilaments in this type of cell are universally observed under an electron microscope (Fig. 2). Thin myofilaments consist of actin-and tropomyosin. Dense bodies are sometimes found connecting thin myofila- ments. They appear in an elongated oval shape, and are small in size and number. The A-type cell is also found in the urinary bladder muscle of the cat [15], and tunica muscle of the mouse [16]. In invertebrates, the A-type cell was reported in many papers concerning the adductor muscle of Spondylus cruentus [7] and Tamanovalva limax Classification of Smooth Muscle Cells 17 Fic. 2. A longitudinal-section of an A-type cell in the tentacle of Atorella polyp [19]. The epithelio-muscular cell (left) has a myofibril (my) near the mesoglea (me). The myofibril bears thick myofilaments small in size and thin ones running in parallel. Dense bodies are not shown in this figure. 27,000, scale bar in the figure represents 0.5 pm. [17]. The cells are also present in the spines and ovarian walls of an echinoid [1, 2], longitudinal muscles of the sea cucumber [4] and tentacle muscle of coelenterates, Stephanoscyphus [18] and Atorella [19]. Organs or tissues having the A-type cell are usually wall muscles of ducts in vertebrates which show repeated contraction-relaxations in a slow but long-lasting manner. On the other hand, in invertebrates, this type of cell is present in motive organs, exerting flexible and long-distanced con- traction-relaxations. The A-type cell is a rather primitive and inefficient type of smooth muscle cell for the following reasons : 1) the arrangement of thick and thin myofilaments is not ordered, ii) dense bodies are small in number and their arrangement is not regular, and iii) the S. R. system in the cell is not well-developed. 2) B-type smooth muscle cell The B-type cells have medium size (about 40 nm in diameter) thick myofilaments and thin myofila- ments of 7nm in diameter (Figs. 3 and 4). This type of cell is elongated in shape and tapers at both ends. It bears a nucleus in the center, and has cell organelles in the peripheral region showing a similar grade of development to the A-type cell. Dense bodies are large in size, but small in number. A thick myofilament similar in diameter in the oyster is shown to consist of a core of paramyosin with myosin at the surface [20], and it bears a regular periodicity of 13-17 nm intervals at its surface [21, 22]. Reports on this type of cell are mainly con- cerned with invertebrates and rarely with verte- brates. The B-type cell is frequently observed in invertebrate organs or tissues such as sheathed muscles of an opisthobranchiate mollusc [23], body wall muscles in the pond snail [6], anterior byssus retractor muscles of Mytilus [24, 25], longitudinal body wall muscles of Dolabella [26], and circular muscle in the oral disc of a coelenterate [19]. The B-type cell is the most popular type in motive organs of molluscs and echinoderms and is used to move or support their bodies. Upon 18 A. MATSUNO Fic. 3. A longitudinal-section of a B-type cell in the sheathed muscle of Placobranchiate [23]. Thick myofilaments (about 40 nm in diameter) run in parallel. d: dense body. 39,200, scale bar represents 0.5 um. Fic. 4. A cross-section of a B-type cell in the oral disc of Atorella [19]. Thick myofilaments do not show a regular arrangement. me: mesoglea. x 46,300, scale bar represents 0.5 um. Classification of Smooth Muscle Cells 19 investigation of these motive tissues or organs, some of them showed “catch” contractions. The cell, therefore, seems to have two functions though they appear to be structurally similar. One of the functions is the “catch” contraction that is present in motive organs of gastropods or retractor mus- cles of bivalves. “Catch” contraction, however, is not so remarkble. The muscles are not very large in size and do not show specialized structures. The other function is the slow but mighty contraction that is observed in the most popular motive organs or tissues in molluscs and echinoderms. 3) C-type smooth muscle cell The C-type cells have very thick (60-120nm in diameter) myofilaments and thin myofilaments of 7 nm in diameter. The cell is large in diameter and long in length, and has a nucleus in its central region. Dense bodies are large in size, but small in number and are arranged in a disordered way. Tubules and S. R. systems are not fully developed. The very thick myofilaments bear a regular periodicity of paramyosin. Their length must be a few micra although they can not be traced from end to end. They taper at both ends. An array of thick myofilaments is not regular in cross-section, but run parallel to each other. Thick myofilaments sometimes appear in various sizes in a cross- section [27]. Their appearance may be a result of the disordered arrangement of thick myofilaments, that is, the thick ones may be cross-sections of the central portion of a thick myofilament and thin ones may be cross-sections of the terminal portion of the same thick myofilament. The thick myofila- ment shows a cored feature but does not show a tubular one (Figs. 5 and 6). The C-type cells mainly investigated in adduc- tors of bivalves, and are not reported in verte- brates. The exceptions are taeniols of coelenterate polyps of Stephanoscyphus [18] and Atorella [19]. A number of investigations were carried out in adductor muscles of a telline [8], Meretrix [9], Atrina [10] and Fragum. The very thick myofilaments may be associated with “catch” contractions, as they are usually present in “catch” muscles [26, 27]. It has been discussed that the “catch” contraction might have some relation to paramyosin in the very thick myofilaments [28, 29]. The cell is a rather primitive type, as in the A- and B-type cells, since it has similar characteristics in its profile and intracellular structures except for its very thick myofilaments. The C-type cell must have de- veloped a specialized course resulting in its mighty contraction and “catch”-mechanism for the follow- ing reasons: 1) similar cells that show large profiles and have very thick myofilaments, are gathered in parallel in an adductor muscle, ii) the direction of cell contraction is limited by its very thick myo- filaments, and ii) their very thick myofilaments are very long. 4) D-type smooth muscle cell The D-type cells have thick myofilaments of various size (14—40 nm in diameter), thin myofila- ments of 7 nm in diameter and many dense bodies. The cell generally appears small in cross-section. The cell is characterized by its dense distributions of dense bodies. Dense bodies connecting each thin myofilament measure to be about 0.2 um in length [5]. They are generally arranged in an ordered way. Intervals between dense bodies are short. Cell organelles such as mitochondria or S. R. systems are more developed than those in A-, B- and C-types of cells (Figs. 7 and 8). The D-type cell is reported only in muscles in invertebrates. The representative D-type cells are present in the intestinal walls of the sea cucumber [5] and cuttlefish [30], in an echinoid ampulla [3], in the large vessel of Phoronis [31], and in the longitudinal muscle of the intestine in Branchiura [32]. The D-type cell is the most developed and differentiated of the smooth muscle cells, and resembles the oblique-striated muscle cells. However, it is distinguishable by the following features: 1) the arrangement of the dense bodies in the D-type cell is not as ordered as in oblique- striated muscle cells, 11) cell organelles, especially its S.R. systems, are not so well-developed. This type of a cell, however, facilitates speedy and repeated movements of organs and tissues of invertebrates. The pattern of movement may be related to the short intervals between dense bodies. The D-type cells must be employed in invertebrates instead of cross- or oblique-striated muscle cells to move quickly. 20 A. MATSUNO 5 x 5. magica SG eg Bed eee s ae, ; 2 2 Re tng = Pos * os Fic. 5. A cross-section of a C-type cell in the adductor of Meretrix. Very thick myofilaments are shown. They appear as somewhat irregular circles in cross-section. mi: mitochondrion, sr: sarcoplasmic reticulum. 15,800, scale bar represents 0.5 um. Fic. 6. A longitudinal-section of thick myofilaments of a C-type cell in the adductor of Fragum. Very thick myofilaments bear regular periodicities, about 14.5nm in interval. <50,600, scale bar represents 0.5 ym. Classification of Smooth Muscle Cells 21 Fic.7. A longitudinal-section of a D-type cell in the intestine of sea cucumber [5]. Many dense bodies (d) are scattered among myofilaments. Thick myofilaments are not large in size. 15,800, scale bar represents 1 ~m. Fic. 8. A longitudinal-section of a D-type cell in the intestine of Brachiura [32]. Dense bodies (d) are shown in somewhat regular array. Sarcoplasmic reticular systems (sr) appear to be slightly developed. 20,900, scale bar represents 1 um. 22 A. MATSUNO COMMENTS I should mention a special type of smooth muscle cell not listed in Table 1 . Smooth muscle cells in ascidians have ultrastructures similar to the A-type cell [33-35], but the cells show thoroughly different characteristics. They contain thin myofilament-linked regulatory systems of contrac- tion in the same way as cross-striated muscle cells e3)) I have tried to classify smooth muscle cells from an ultrastuctural standpoint, and it is not clear whether the classification coincides with the one from the physiological and biochemical stand- points. I expect another classifications from other views. Very probably, they are not so different from the present classification. ACKNOWLEDGMENTS The author expresses his sincere thanks to Prof. K. Maruyama of Chiba University for his warm en- couragement and critical advice on this manuscript. I am also indebted to Dr. S. Kawaguti, Prof. Emeritus of Okayama University for his critical advice during this work. REFERENCES 1 Kawaguti,S. and Kamishima, Y. (1965) Biol. J. Okayama Univ.,11(1-2): 31-40 2 Kawaguti, S. (1965) Biol. J. Okayama Univ., 11 (3-4): 66-74. 3 Kawaguti, S. (1965) 11(3—4): 75-86. 4 Kawaguti,S. and Ikemoto, N. (1965) Molecular Biology of Muscular Contraction, Igaku Shoin Ltd., pp. 610-613. 5 Kawaguti, S. (1964) Biol. J. Okayama Univ., 10 (1-2): 39-50. 6 Plesch, B. (1977) Cell Tissue. Res., 180: 317—340. Biol. J. Okayama Univ., 7 Kawaguti,S. and Ikemoto, N. (1959) Biol. J. Okayama Univ., 5 (1-2): 73-87. 8 Kawaguti,S. and Ikemoto,N. (1961) Biol. J. Okayama Univ., 7 (1-2): 17-29. 9 Kawaguti,S. and Ikemoto, N. (1960) Biol. J. 10 11 12 13 14 IS) 16 17 18 19 20 21 jij 23 24 Ma) 26 Di 28 29 30 31 3 333} Okayama Univ.,6 (1-2): 1-18. Kawaguti,S. and Ikemoto, N. (1957) Okayama Univ., 3 (4): 248-268. Panner, B. J. and Honig, C. R. (1967) J. Cell Biol., 35: 303-321. Kelly, R. E. and Rice, R. V. (1968) J. Cell Biol., 37: 105-116. Rosenbluth, J. (1971) J. Cell Biol., 48: 174-188. Nonomura, Y. (1968) J. Cell Biol., 39: 741-745. Kawaguti,S. and Ikemoto, N. (1957) Biol. J. Okayama Univ., 3 (3): 159-168. Lane, B. P. (1965) J. Cell Biol., 27: 199-213. Kawaguti,S. and Yamasu, T. (1960) Biol. J. Okayama Univ., 6(1-2): 61-70. Kawaguti, S. and Yoshimoto, F. (1973) Biol. J. Okayama Univ., 16(3—4): 47-66. Matsuno, A. (1981) Annot. Zool. 171-181. Elliott, A. (1974) Proc. R. Soc. Lond., B, 186: 53-66. Nonomura, Y. (1974) J. Mol. Biol., 88: 445—455. Elliott, A. (1979) J. Mol. Biol. ,132: 323-341. Kawaguti, S. (1968) Biol. J. Okayama Univ., 14(1-2): 13-20. Biol. J. Japon., 54: Kawaguti,S. and Ikemoto, N. (1957) Biol. J. Okayama Univ., 3 (3): 107-122. Sobieszek, A. (1973) J. Ultrastruct. Res., 43: 313-343. Sugi, H. and Suzuki, S. (1978) J. Cell Biol., 79: 454-466. Morrison, C.M. and Odense, P.H. (1974) J. Ultrastruct. Res., 49: 228-251. Ruegg, J.C. (1971) Rhysiol. Rev., 51, Suppl. 1: 201-248. Johnson, W. H.., Kahn, J. S. and Szent- Gyorgyi, A, G. (1959) Science, 130: 160-161. Kawaguti, S. (1964) Biol. J. Okayama Univ., 10 (3-4): 93-103. Kawaguti, S. and Nakamichi, A. (1973) Biol. J. Okayama Univ., 16 (3-4): 73-82. Naitoh, T. and Matsuno, A. (1985) Experientia, 41: 370-372. Toyota, N., Obinata, T. and Terakado. K. (1979) Comp. Biochem. Physiol., 62B: 433-441. Shinohara, Y. and Konishi, K. (1982) Zool., 221: 137-142. Terakado, K. and Obinata, T. (1986) Cell Tissue Res., in press. JawExp: ZOOLOGICAL SCIENCE 4: 23-30 (1987) © 1987 Zoological Society of Japan Phototactic Behavior of the Orb Weaving Spiders, Argiope amoena and Nephila clavata SHIGEKI YAMASHITA! and RIEKO TUII Department of Biology, Faculty of Science, Kyushu University, Fukuoka &12, Japan ABSTRACT—Phototactic behavior of tethered orb weaving spiders which walked on a y-maze globe was investigated under open-loop conditions. On a dark background, the spiders tended to turn away from a test light given to the eyes (negative phototaxis). On a light background, however, the spiders tended to turn towards the test light (positive phototaxis). When a small light spot for background illumination was presented to only a portion of the eyes, the spiders showed a negative phototaxis to the test light. On the other hand, when the brain was illuminated through the cuticle covering it, the spiders showed a positive phototaxis to a test light given to the eyes. It is concluded that extraocular photoreceptors which should be present in the brain control the phototactic behavior of these spiders. INTRODUCTION Orb weaving spiders have four pairs of simple eyes arranged in two rows on the frontal part of the prosoma. The eyes of the orb weaving spiders, Argiope, have been studied morphologically [1, 2] and physiologically [3-6]. The present study was undertaken to examine the phototactic behavior of the orb weaving spiders and also to examine that phototactic behavior may be significantly affected by a kind of extraocular photoreceptor. MATERIALS AND METHODS Animals used in this study were female orb weaving spiders, Argiope amoena and Nephila clavata, which were collected in open fields. Individuals of both species ranged from 20-25 mm in body length, and from 1.2-1.7 g in weight. The spider was held rigidly in space by a glass tube of about 1.5mm diameter, waxed to the posterior part of the prosoma (Fig. 1A), or by a light guide of about 2mm diameter waxed to the central part of the prosoma which was just above Accepted June 26, 1986 Received May 23, 1986 * Present address: Biological Laboratory, Kyushu Insti- tute of Design, Shiobaru, Fukuoka 815, Japan. the brain (Fig. 1B). Then, the spider was given a y-maze globe with slightly lighter weight than that of the spider itself (about 0.8-1.2g) to hold. Usually, 7-10 spiders were prepared at the same time, and only those spiders which showed spon- taneous walking activity in a dim room were used for the experiments. From a total of about 250 spiders prepared, about 40 showed sufficient walk- ing activity to enable them to be used in the experiments. These spiders made turns at y-arms one per every 2-10 seconds. However, most of them continued to show walking activity for less than two hours. We continued to make experi- ments as long as the spiders showed walking activity. For test stimuli, two lights emitted from two 6-8 V tungsten filament lamps were passed through heat-absorbing filters and calibrated neutral densi- ty filters, and focused onto the tips of two light guides, respectively. One light guide was aimed horizontally at the frontal part of the prosoma from an angle of 45° clockwise to the body axis, and the other anticlockwise to the body axis. Thus, each test light illuminated the right or left eyes preferentially. The test light subtended a visual angle of about 2° at the spider eyes. The intensity of the test light without neutral density filters was referred to as unit intensity, and had a value of about 30lux at the preparation. 24 S. YAMASHITA AND R. TuJI (Or A Fic. 1. LG to brain Diagram of the experimental apparatus. The spider is fixed in the center of a 28cm drum by a glass tube (A), or by a light guide (B). See text. TL, tungsten lamp; BGL, tungsten lamp for background light; LG, light guide; GT, glass tube; L, lens; ND, neutral density filter; HF, heat absorbing filter. In most cases, spiders made 100-120 turns at y-arms for each test (each point on figures), half of them during illumination of the right eyes, plus a half during illumination of the left eyes. Successive illumination of the right and left eyes were performed in order to compensate for side prefer- ences independent of visual stimulation. Prior to the start of each test run, spiders were adapted to each test light or background light for 5-10 min. The turning reaction was defined as P—N/P-+N, where P is the number of turns at y-arms directed towards the test light, and N is the number of turns directed away from the test light. For background illumination, scattered light of a tungsten lamp placed over the spider was used (Fig. 1A). The intensity was controlled by chang- ing the voltage of an electrical power source. For illumination of only a portion of the eyes or the brain, the light emitted from a tungsten lamp was passed through a heat-absorbing filter, calibrated neutral density filters and wedges, and then focused onto the tip of a light guide. The other tip of the light guide (about 2 mm in tip diameter) was positioned almost in contact with the eyes (Fig. 1A), or was touched onto the central part of the prosoma just above the brain (Fig. 1B). The light could illuminate the eyes or the brain, although it also illuminated the vicinity of the eyes or the brain. Blue light (440nm peak wavelength) and green light (536nm) were produced with interfer- ence filters. The half bandwidths of the blue filter was 38nm, and that of the green was 14nm. To observe the movements of the y-maze globe in the dark, dim red light (edge filter, 50% value at 650 nm) was focused onto the globe. The red light was illuminated throughout the experiments. RESULTS Turning tendency in the absence of the test light When the illumination intensity of background light was below 600lux, spiders which walked on a y-maze globe made choices of y-arms at the rate of one time per every about 2-10 sec. The Phototaxis of Orb Weaving Spiders 25 relationship between choice frequency and back- ground illumination could not be determined. However, when the background light intensity was over 600—1,000 lux, the choice frequency became very low, i.e. most spiders showed little walking activity. Turning tendencies of spiders, to which no test light was presented, were observed under various background light intensities (Fig. 2). In this experi- ment, turning reaction was defined, for conven- ience, as R-L/R+L, where R is the number of turns towards right direction and L is that of left direction. As can be seen in Figure 2, both on the dark- and light-backgrounds, the value of the turning reaction for each spider was almost con- stant, although it varied from spider to spider. This observation shows that background light intensity has little effect on the spontaneous turning tendencies of spiders. Changes in the phototactic behavior by background lightness In Figure 3, the turning reactions of five spiders on the dark-background are plotted against the +04 ‘ss ° 2 Oo oe) @ _ D £ c _ 5 -e =0:2 -0.4 0 200 relative intensity of the test light. The spiders tend to turn away from the test light and this tendency becomes stronger as the stimulus intensity in- creases. It is obvious that the spiders show negative phototaxis on a dark-backgroud. The negative phototaxis on a dark-background changed into positive phototaxis on a light- background. Figure 4 shows the responses of eleven spiders under various background light intensities. In this and following experiments, the intensity of the test light was restricted to —0.3 log units. On the dark-background, all spiders showed negative phototaxis. The tendency for a negative phototactic response decreased as the background became lighter, and at about 100—300lux back- ground, spiders did not show any phototaxis. When the background became still lighter than 100—300 lux, the spiders showed a positive photo- taxis and this tendency was augmented as the background lightened. To examine the role of each pair of eyes on the phototaxis, only one or two pairs of the eyes were left intact; all the other pairs were destroyed surgically and then covered with black paint. The amoend: @ clavata:ad00 A. N. a 400 600 Background illumination, lux Fic.2. Turning tendencies of one Argiope and three Nephila under various background light intensities. Each symbol represents a single spider. Turning reaction S. YAMASHITA AND R. TuJI Turning reaction 707! @) | Ssc5— Sng a a 8 ee q © =0,.2 A.amoenda: =0.4 N.clavata: ©@00 SY: © a < -0.6 29 = @) Test light, log! Fic. 3. Intensity-turning reaction curves for individual spiders on a dark-background. ® +0.4 . aGmoend : .clavatad: geQq00 ®@®®OO0 IZ I> aO.8'54i ! ee eee ee Seen eee Sh 0 200 400 600 Background illumination, lux Fic. 4. Effects of background illumination on the phototactic response. Phototaxis of Orb Weaving Spiders oF TABLE 1. light (600 lux) Eyes n All eyes AM eyes PM eyes AL and PL eyes No eyes RP WN NY wo Dark-background Role of each pair of eyes on the phototactic responses on a dark-background and in Turning reaction Light-background — 0.47 + 0.12 = ial 27 en al 0 8 = 58 + 0.29 = (8% + 0.20 — 0.48 ame = 0:03 Se. AM, anterior median; PM, posterior median; AL, anterior lateral; PL, posterior lateral. n= number of spiders tested. Mean (+ S.D.) of turning reactions obtained from 1-9 spiders. In the experiments for the AM, PM, and AL and PL eyes, light guides which provided test stimuli were aimed at the AM, PM, and AL and PL eyes, respectively. results are summarized in Table 1, where turning reactions obtained on the dark-background and on the light-background of 600lux are shown. With one or two pairs of eyes, the spiders showed similar phototactic behavior to that of normal spiders, i.e. negative phototaxis on a dark- background and positive phototaxis on a light- background. However, when all of the eyes were blinded, no phototactic behavior was observed. +0.3 N. clavata E ses (a) | ee = 0 © _ D & c 5 E=0:3 Illumination of eyes, Fic. 5. These observations show that the phototactic behavior of the spiders is induced by signals from the eyes, and that there are no significant differ- ences in function among the four pairs of eyes with respect to phototactic behavior. If the changes in phototaxis from negative to positive which are dependent on the background lightness is mediated by visual signals, then back- ground illumination presented to only the eyes *K@ (600 lux background) d) log | Effects of illumination of the eyes on the phototactic response. The spider showed a positive phototaxis during illumination of the whole spider at 600lux background (* @), but a negative phototaxis during illumination of the eyes. Both ocular illumination of —0.8 log units (arrow) and whole illumination of 600 lux elicited similar ERGs in an AM eye. 28 S. YAMASHITA AND R. Tuli should also change the phototaxis. To test this, a small light spot for background illumination was presented to a portion of the eyes. The light intensity of background illumination presented only to the eyes, and that presented to the whole spider were compared by recording ERGs from the AM eye under conditions similar to those used for the behavioral experiments. Both ocular illumination of —0.8log units and whole illumina- tion of 600 lux elicited similar ERGs in the AM eye. However, the effects of these two kinds of illumination on phototaxis were quite different. An example is shown in Figure5. The spider showed positive phototaxis during illumination of the whole spider. However, it shows negative phototaxis during illumination of the eyes. These observations suggest that extraocular photorecep- tors control the phototactic behavior of spiders. Extraocular photoreceptors and phototaxis Extraocular photoreceptors which take part in the phototaxis may be present in the brain. If this is true, spiders should show positive phototaxis during lighter illumination of the brain. The effects of illumination of the brain on the photo- +04 cE ie) Se 0 @) © oO) & a =0n4 0.4 tactic response are shown in Figure6. On a dark-background, all spiders showed a negative phototaxis. As the intensity of cerebral illumina- tion was increased, these spiders became positively phototactic. The light given to the brain diffused within the prosoma and part of the light reached the eyes, but it was small; ERGs elicited by cerebral illumination of intensity of 1.0 (Fig. 6) were smaller than those elicited by ocular illumina- tion of about —1.6log units (Fig.5). It is, therefore, difficult to consider that the diffused light which reaches the eyes has direct effects on the changes in phototaxis from negative to posi- tive. These observations show that extraocular photo- receptors, presumably located in the brain, control the phototactic behavior of spiders. The effects of blue, green and white light of equal energy on the brain are compared in Table 2. The intensities of light were corresponded to about one-tenth of the white light used in Figure 6. It was clear that blue light had a marked effect on the phototactic response. In contrast, green light had little effect. White light had an intermediate effect between that of blue and breen. These observa- .amoend: ov0e®6 .clavatad: oo A N 0.6 Illumination of brain, intensity Fic. 6. Effects of illumination of the brain on the phototactic response. Phototaxis of Orb Weaving Spiders 29 TABLE 2. Effects of blue (440nm), green (536nm) and white light on the brain Light to brain None Turning reaction —()39 Blue Green White +0.18 == Or oll +0.04 Mean of turning reactions was obtained from two spiders. tions suggest that the extraocular photoreceptors have a maximum sensitivity in the blue region. DISCUSSION In the present study, the brain was illuminated through the dorsal cuticle via a light guide. A dorsal view of the prosoma of Argiope and Nephila is shown in Figure 7. In both species, the central A. amoena N. clavata Fic. 7. Drawings of the prosoma of A. amoena and N.clavata. All hairs covering the prosoma are re- moved. part of the dorsal cuticle contains little pigments, although the area of the non-pigmented cuticle varied from spider to spider. The brain of the spider is located beneath the non-pigmented cuti- cle which transmits light. Yamashita and Tateda [5] reported that efferent neurons in the brain of Argiope are directly excitable by light. They have observed that illumination of the brain through the central part of the dorsal cuticle via a light guide increases the discharge rate of the cerebral photo- sensitive neurons which are recorded from the eye in the intact animal. It is, therefore, apparent that the light delivered by the light guide reaches the brain without significant diminution in intensity, although the light also illuminates other regions of the brain at the same time. We assume that extraocular photoreceptors are activated by light passing through the transparent area of dorsal cuticle. The cerebral photosensitive neurons reported by Yamashita and Tateda [5] have a maximum sensitivity at about 420-—440nm. On the other hand, the eyes have maximum sensitivities at about 480-540nm in the visible and at about 360nm in the ultraviolet [3,6]. As shown in the present study, blue light (440nm_peak- wavelength) had a marked effect on the phototac- tic response, but green light (536nm) had little effect. These observations support the idea that extraocular photoreceptors control the phototactic behavior of the spiders, and suggest that the extraocular photoreceptors in the present study may be the cerebral photosensitive neurons re- ported by Yamashita and Tateda [5]. If this is true, the cerebral photosensitive neurons may be more directly involved in the dimming reaction than in phototaxis, since they respond markdly to a diminution in light intensity [5]. We have in fact observed that when the background light is turned off, the spiders tend to turn transiently towards the test light. ACKNOWLEDGMENTS This research was supported in part by grants from the Ministry of Education of Japan. The authors are deeply indebted to Dr. D. R. Stokes (Emory University, Atlan- ta, Georgia, U.S.A.) for his helpful discussion and comments on this manuscript, and also to Prof. H. Tateda (Kyushu University) for his helpful discussion throughout this work. REFERENCES 1 Uehara, A., Toh, Y. and Tateda, H. (1977) Fine structure of the eyes of orb-weavers, Argiope amoena L. Koch (Aranea: Argiopidae). 1. The anteromedial eyes. Cell Tissue Res., 182: 81:91. 2 Uehara, A., Toh, Y. and Tateda, H. (1978) Fine structure of the eyes of orb weavers, Argiope amoena 30 S. YAMASHITA AND R. Tut L. Koch (Aranea: Argiopidae). 2. The anterolateral, posterolateral and posteromedial eyes. Cell Tissue Res., 186: 435-452. Yamashita, S. and Tateda, H. (1978) Spectral sensi- tivities of the anterior median eyes of the orb web spiders, Argiope bruennichii and A. amoena. J. Exp. Biol., 74: 47-57. Yamashita, S. and Tateda, H. (1981) Efferent neural control in the eyes of orb weaving spiders. J. Comp. Physiol., 143: 477-483. 5 Yamashita,S. and Tateda, H. (1983) Cerebral photosensitive neurons in the orb weaving spiders, Argiope bruennichii and A. amoena. J. Comp. Physiol., 150: 467-472. Yamashita, S. (1985) Photoreceptor cells in the spi- der eye: spectral sensitivity and efferent control. In “Neurobiology of Arachnids”. Ed. by F.G. Barth, Springer-Verlag, Berlin-Heidelberg-New York- Tokyo, pp. 103-117. ZOOLOGICAL SCIENCE 4: 31-35 (1987) © 1987 Zoological Society of Japan Dimming Reaction of the Orb Weaving Spider, Argiope amoena SHIGEKI YAMASHITA! Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan ABSTRACT — The reaction to the dimming of light in the tethered orb weaving spider which walked on a y-maze globe was investigated under open-loop conditions. The spiders tended to turn transiently towards the lighter direction after the dimming of light. It is suggested that cerebral photosensitive neurons play a role in the dimming reaction. INTRODUCTION Extraocular neural photoreceptors have been reported for many animals representing various invertebrate phyla and are reviewed by Yoshida [1]. Yamashita and Tateda [2] showed that the efferent neurons in the brain of orb weaving spiders, Argiope amoena and A. bruennichii, whose signals control the responses of the eyes, are directly sensitive to light. The discharge rate of the cerebral photosensitive neurons (the efferent neurons) increases transiently following the dim- ming of illumination of the eyes (the dimming reaction). Interaction of the cerebral photosensi- tive neurons and the eyes plays a role in increasing the dimming reaction [2, 3]. Prominent responses to the turning off and the dimming of light have been shown for the second order cells (I-cells) and the third order cells (A-cells) in the barnacle visual pathways [4, 5] and for the pallial nerve of Spisula solidissima [6|. The impulses generated in the A-cells of barnacles in response to dimming cause a withdrawal of the cirri and closure of the opercular plates [7-9]; impulses in the afferent axon of the pallial nerve of Spisula cause withdrawal of the siphon [6]. In orb-weaving spiders, impulses in the cerebral photosensitive neurons produced as a result of the interaction of the cerebral photosensitive neurons and the eyes may play a role in dimming reaction Accepted July 8, 1986 Received May 23, 1986 " Present address: Biological Laboratory, Kyushu Insti- tute of Design, Shiobaru, Fukuoka 815, Japan. behavior. In the present study, reactions to the dimming of light in orb weaving spiders have been examined behaviorally. MATERIALS AND METHODS Female orb weaving spiders, Argiope amoena, collected in open fields were used throughout this study. Methods were similar to those described previously [10]. The spider was held rigidly in space and was given a ring or a y-maze globe to hold. For test stimuli, a couple of light emitted from two 6-8V tungsten filament lamps was passed through heat-absorbing filters and focused onto the tips of two light guides, respectively. Initially, test light of similar intensity was simul- taneously presented to the right and left eyes from two light sources of 45° clockwise and 45° anti- clockwise to the body axis. The intensity of each test light was about 35lux at the preparation. Reductions in light intensity were achieved by simultaneous introduction of neutral density filters into the two test light paths or were achieved by turning off one of the two test light; the other light was continued to illuminate the right or left eyes. In the latter case, the right test light and the left test light were turned off alternately in order to compensate for side preferences independent of visual stimulation. The duration of illumination was controlled by a mechanical shutter. Duration of the lighter period (illumination by two test light) was 2-5 min; the darker period (illumination by one test light), 2-4 min. The illumination cycle was repeated as long as each spider showed 32 S. YAMASHITA walking activity. However, most spiders showed walking activity only for less than 3—4hr in a day. Each spider was tested for two or three days. The turning reaction was defined as P—N/P+N, where P is the number of turns at y-arms directed towards a test light which was continued to illuminate the eyes after dimming, and N is the number of turns away from the test light. The turning reaction was averaged every 10 or 20 seconds. In most cases, spiders made, at least, 40 turns for each 10 or 20 seconds. RESULTS Walking activity following dimming The changes in walking activity following diminution of the light intensity were examined for five spontaneous walking spiders. A typical exam- ple is shown in Figure 1. The spider walked on a ring at a speed of about 2.5cm/sec before the dimming. Due to diminution of the light intensity, the spider stopped for a brief period, especially when the diminution was great. But then walking speed of the spider increased beyond the predim- 6 A logI=1.0 Walking speed, cm/sec 0.5 0 0.5 ming level for less than 2-4 min. The increase was augmented as the diminution increased. In this spider, the maximum walking speed was about 6cm/sec and occurred for 10—20sec after a decre- ment of 1.0 log unit in intensity. The average value of the maximum walking speed for five spiders was 5.7 cm/sec, or 1.9 times faster than that before the dimming. The increase in walking activity was also observed when only a portion of the eyes was exposed to a dimming light. However, illumination of the brain through the dorsal cuticle had little effect on walking activity [cf. 10]. It is unlikely, therefore, that cerebral photosensitive neurons play a direct role in controlling walking activity. Turning reaction to dimming The turning reaction following a diminution of light intensity was examined for eight spontaneous walking spiders. All of them tended to turn transiently towards the light (positive reaction) follwing a diminution of light intensity. Three examples are shown in figrure 2. The reaction was the most positive just after dimming and then it became weaker with time. Finally, all spiders 1 NS 2 Time, min Fic. 1. Walking activity following dimming. Walking speed of one spider is plotted against the time after the beginning of 4 different decrements of light intensity. Decrement of lihgt intensity (Alog I) is indicated for each curve in log units. Dimming Reaction of Orb Weaving Spiders 33 Turning reaction Time, min Fic. 2. Turning reactions to the dimming of light for three spiders. The reaction of each spider was tested for three days independently of time of day, when the spider showed spontaneous walking activity. Each curve represents one spider. Turning reaction is plotted against the time after the beginning of a decrement of light intensity. +04 16:00-20:00 +02 Turning reaction 9:00-12:00 -0.4 r----2 =0:6 @) (OS 1 1s) 2 Time, min Fic. 3. Turning reaction of one spider to a dimming of light in the morning (9: 00-12: 00) and in the evening (16: 00-20: 00) for two days of the experiment. 1, first day; 2, second day. 34 S. YAMASHITA tended to turn away from the light (negative reaction) [cf. 10]. As can be seen in Figure 2 , however, the maximum value of the positive reaction varied from spider to spider. For exam- ple, the spider which showed the largest positive reaction among the eight spiders examined (solid line in Fig. 2), achieved the maximum value of about 0.4 and the period of the positive reaction lasted at least two minutes. The spider did not show a clear negative reaction even within four minutes after dimming. However, when the turning reaction was examined five or more minutes after the dimming, this spider showed a negative reaction. On the other hand, one spider showed a smaller positive reaction (broken line in Fig. 2), with the maximum value of about 0.09 and a period of less than twenty seconds. In one of eight spiders, it was observed that the dimming reaction varied with time of day (Fig. 3). The reaction of this spider was tested in the morning from 9:00 to 12:00 and in the evening from 16:00 to 22:00. For initial two days of the experiment, the spider showed a strong, positive reaction in the evening, but a weaker reaction in the morning. Unfortunately, this spider greatly reduced its walking activity on the third day. None of the other spiders showed sufficient walking activity both in the morning and in the evening. In the morning, an average value of the turning reaction for these seven spiders (P,—N,/P;+N,), obtained in the first twenty seconds after dimming was 0.19, and in the evening it was 0.28; where P, and N, are the total numbers of P and N made by the seven spiders. However, whether the orb weaving spiders show a diurnal periodicity of the dimming reaction could not be demonstrated. DISCUSSION Yamashita and Tuji [10] reported that the orb weaving spiders, Argiope amoena and Nephila clavata, which walked on a y-maze globe on a dark-background, tend to turn away froma light shown to the eyes (negative phototaxis), whereas they tend to turn towards the light during illumina- tion of the brain (positive phototaxis). They concluded that extraocular photoreceptors which are likely to be present in the brain control the ' phototactic behavior, and suggested that the ex- traocular photoreceptors are the cerebral photo- sensitive neurons (the efferent neurons) reported by Yamashita and Tateda [2]. The cerebral photosensitive neurons responded markedly to a diminution in light intensity [2]. As shown in the present study, the spiders showed a prominent reaction to the dimming. The observation sup- ports the idea that the extraocular photoreceptors reported by Yamashita and Tuji [10] are the cerebral photosensitive neurons reported by Yamashita and Tateda [2]. The cerebral photo- sensitive neurons showed a circadian oscillation in spike frequency with a period of approximately 22 hr under constant darkness [11]. As a result, the anterior median eye also showed a circadian oscillation of sensitivity with a period of approx- imately 22 hr [11, 12].. The maximun frequency of the efferent spikes in the optic nerve is seen in early period of the circadian “ night” state. Later in the “night”, efferent spike frequency becomes very low. Little or no efferent activity is present during the whole “day”. If the cerebral photo- sensitive neurons take part in the dimming reac- tion, one would expect that spiders show a circadian oscillation of the dimming reaction as shown in Figure 3. ACKNOWLEDGMENTS This research was supported in part by grants from the Ministry of Education of Japan. The author wish to express his gratitude to Dr. D. R. Stokes (Emory Univ. USA) for his helpful discussion and comments on this manuscript. Gratitude is also extended to Prof. H. Tateda (Kyushu Univ.) for his helpful discussion. REFERENCES 1 Yoshida, M. (1979) Extraocular photoreception. In‘“Handbook of Sensory Physiology, Vol. VII/6A: Comparative Physiology and Evolution of Vision in Invertebrates”. Ed. by H.Autrum, Springer- Verlag, Berlin-Heidelberg-New York, pp. 581-640. 2 Yamashita,S. and Tateda, H. (1983) Cerebral photosensitive neurons in the orb weaving spiders, Argiope bruennichii and A. amoena. J. Comp. physiol., 150: 467-472. 3 Yamashita, S. (1985) Photoreceptor cells in the spider eye: spectral sensitivity and efferent control. In “Neurobiology of Arachnids”. Ed. by F. G. Dimming Reaction of Orb Weaving Spiders 35 Barth, Springer-Verlag, York-Tokyo, pp. 103-117. Stuart, A. E. and Oertel, D. (1978) Neuronal prop- erties underlying processing of visual information in the barnacle. Nature, 275: 287-290. Oertel, D. and Stuart, A. E. (1981) Transformation of signals by interneurons in the barnacle’s visual pathway. J. Physiol., 311: 127-146. Kennedy, D. (1960) Neural photoreception in a lamellibranch mollusc. J. Gen. Physiol., 44: 277-299. Gwilliam, G. F. (1963) The mechanism of the shad- ow reflex in Cirripedia. I. Electrical activity in the supraesophageal ganglion and ocellar nerve. Biol. Bull., 125: 470-485. Gwilliam, G. F. (1965) The mechanism of the shad- ow reflex in Cuirripedia. IJ. Photoreceptor cell Berlin-Heidelberg-New 10 11 12 response, second-order responses, and motor cell output. Biol Bull., 129: 244-456. Gwilliam, G. F. (1976) The mechanism of the shad- ow reflex in Cirripedia. III. Rhythmical patterned activity in central neurons and its modulation by shadows. Biol. Bull., 151: 141-160. Yamashita, S. and Tuji, R. (1987) Phototactic be- havior of the orb weaving spiders, Argiope amoena and Nephila clavata. Zool. Sci., 4: 23-30. Yamashita, S. and Tateda, H. (1981) Efferent neu- ral control in the eyes of orb weaving spiders. J. Comp. Physiol. 143: 477-483. Yamashita, S. and Tateda, H. (1978) Spectral sensi- tivities of the anterior median eyes of the orb web spider, Argiope bruennichii and A. amoena. J. Exp. Biol., 74: 47-57. a npelcagieae iepresanentas a ats pik rate 4 ; Fa LY U labia » Nae eb A wey ‘cha! Riess He TSS IGE NE ve eh Bde eck oil uk Aarne nithceen gael ceeabaoneey aie inher onpscongal GG, Loatvagens. Rie Eanes sails ror | eae ae ) as Ate « fhe. wien as. iL yerreion: Mi Br Ke y gi ; POPES ; score bee Bh. ae he ; t - cs ~ " \s i, : t } t s al sl 7“ ig ps + ciao j pleat ay cf eh’ 4 > he = a ( 17 2 Pea Py ar, A oa tgs at ‘ NAD? a eae bP Was si iv 5 é f eo oy) Tye ha) Searing wd F 2 i ’ f Mah | ea And eo i Mt i he Ue ‘ ra te is , ‘ SURF he et k ; bi Ge PS Ss ; } ‘3 Pepa — > - « rt —. ‘ " e 0 af é ‘or : Pi ees te NS hie SCT Ys ] rTLaF hy Peay Rea ates ty eS ga CY he UO RE eat htn pity ie phi ’ _ - P , } 4 MA + 7% iz x oe ia i holy $ ‘ j —— i ' ut ‘ rey ‘ #4 ‘ 4 , ‘ te & r 3 ¥ o f ) t yi ii S 4 “477 ' mal ii 7 id ~ es f 14,2 ' 1 eat 4 vee ts ¥ ty Cd a oe on tts < } a. | s r \ ; PRE. oe eae | . i .f F \ L rie a es ae 4 \ a} ty ay ; re A. og ait ¥ Say t A P ry Vira Pe FUSS ; he : ae. 7 ’ i i : ‘ fee) 4 v et a yey oi Pear snk paot i ? Pros a) iy 4 . nate ate a wen! ar " i at 8 mast eee Tey 4 rey Lis phe reed. 2 ee ") Swede i ; : ae ee 4 preven’ SRS ra ee oe CM See ea es dh ee 2) Oe eae Ih) wid a Vac “ we ‘PQO,; 24.9 NaHCO; (pH 7.3 on bubbled with 38 M. ANDO 95% O-5% CO, gas mixture). Furosemide (Tokyokasei Co., Tokyo, Japan) or various amino acids (Katayama chemical Co., Osaka, Japan ) was added to the mucosal side usually. At the end of the experiments, the intestine was cut longitudinal- ly and spread on a graph paper; the surface area of the intestine was measured using a planimeter (Ushikata, 220L). RESULTS Effects of .-alanine in standard Ringer solution Figure 1 illustrates typical effect of L-alanine on the PD and the net water flux. When 5 mM L-alanine was added into the serosal perfusate solution, the serosa-negative PD was enhanced gradually, after latent period of 20min, and attained to a steady level after 50 min. The net water flux from mucosa to serosa was also acceler- ated in parallel with the enhancement of the serosa-negativity in the PD. After washing out the serosal alanine, the PD and the net water flux decreased gradually. When L-alanine (5mM) was (ul/ern®10 min) ) flux water | 5 mM Ala (S) Net 100 Time Fic. 1. 200 added into the mucosal fluid, the PD was initially depolarized only slightly (by 0.3+0.0mV, N=8), and then began to increase in serosa-negativity gradually. The net water flux started to increase after the initiation of the increment in the serosa- negative PD; the latent period being 20—30 min. Both the PD and the net water flux continued to increase over 100 min. Further addition of L- alanine (5 mM) into the serosal fluid did not add to these increases. Therefore, it can be concluded that alanine is more effective from the mucosal side than from the serosal side. Relationship between the mucosal t-alanine concentration and the PD or the net water flux is shown in Figure 2. After the PD reached a steady level in standard Ringer solutions, 0.1, 1.0, 5.0 and 10.0mM alanine was added stepwise into the mucosal fluid. Until the concentration was raised to 5.0mM, no enhancements of the PD and the net water flux were observed. When 5mM t-alanine was added into the mucosal fluid, these two parameters increased gradually as cited above. Further addition of L-alanine (10mM) into the mucosal or serosal fluid did not add to these 400 300 (min) Sidedness of the effects of L-alanine on the PD (©) and the net water flux (@). After bathing the intestine with standard Ringer solutions, 5mM L-alanine was added into the serosal fluid (S); first arrow. At the second arrow, the serosal alanine was removed, and 5mM t-alanine was added into the mucosal fluid (M) at the third arrow. Finally, both sides were applied with 5 mM t-alanine (fourth arrow). Alanine and Water Transport 39 increases. Therefore, following experiments were all made by 5mM t-alanine. To determine which ion flux contributes to the enhanced water flux, effects of 5mM t-alanine on the net Nat, K* and Cl” fluxes were examined. As shown in Table 1, mucosal addition of L-alanine enhanced the net Na* and Cl” fluxes by the same amount, and tended to increase the net K* flux only slightly. Since NaCl absorption across the seawater eel intestine is due to a coupled Na‘t-— K*—-Cl~ transport [1-3], it is likely that L-alanine enhances the Na*-K*-Cl~ transport and secon- darily the water transport. Following results also Support such an idea. Effects of L-alanine in the presence of furosemide Since the Na‘-K*-Cl~ transport in the sea- water eel intestine is known to be inhibited by mucosal application of furosemide [3], in the next experiments, effects of alanine were examined after abolishing the Na*-K*—Cl~ transport by furosemide (Fig. 3). When 0.01mM furosemide was added into the mucosal fluid, the serosa- negative PD and the net water flux were dimin- E pip i NS S iS as -6 § 10 o S = =. one ae Gn: he Pa: : 10 mM 10 mM | Ala Ala (S) x< -2 an) 0.1 mM = Ala i 8 yas pa 8 0 <5) m is auc Ala = a | E 10 -3 5 o 0 ® 0 S 5 @ Z 0 50 100 150 200 Time (min) Fic. 3. A typical effect of L-alanine on the PD (C) and the net water flux (@) in the presence of furosemide. After bathing the intestine with standard Ringer solution, 0.01mM furosemide was added into the mucosal fluid. In the presence of furosemide (open column), 5mM t-alanine was added into the mucosal fluid (first arrow). At the second arrow, both of the mucosal alanine and furosemide were removed by rinsing the intestine twice with the standard Ringer solution. A trace of the PD immediately after addition of alanine is also shown in the inserted dotted column. : Wash ) ( plicm”10m in) oO flux oO water Net (@) —1___J 0 60 120 180 290 Time (min) Fic. 4. After-effect of L-alanine on net water flux. After treatment with 0.01 mM furosemide, 5mM L-alanine was added into the mucosal medium at 40 (@), 60 ((_), 80 (A) or 100 min (MM); arrow heads. At 120min, both of the mucosal alanine and furosemide were removed by rinsing the intestine with the standard Rinder solution. As a control (©), furosemide was removed after treatment for 120 min without addition of alanine, and 5mM t-alanine was applied into the mucosal fluid at 190 min. Each point represents the mean value. The number of preparations is indicated in the parentheses. Alanine and Water Transport 4] ished immediately to lower steady levels (around —2mvV and 4.0 l/cm?-10 min respectively). In the presence of furosemide, no enhancement in the net water flux were induced by the addition of 5mM t-alanine, whereas only the initial depolar- ization in the PD (0.3+0.1mV, N=6) was observed as in the absence of furosemide. When furosemide and alanine were removed by rinsing the intestine twice with the standard Ringer solution (alanine-free), both the PD and the net water flux were enhanced transiently. Since these two parameteres, especially in case of the net water flux, continue to decrease with time even in the initial control periods, these enhancements appear significant. Figure 4 shows effects of pretreatment with L-alanine on the net water flux. With prolongation of the treatment time, higher net water flux was observed in spite of the absence of alanine in the bathing media. The maximal after-effect was obtained after the pretreatment with L-alanine for 60-80 min. The serosa-negative PD was also enhanced with prolongation of the pretreatment with L-alanine (Fig. 5), and the maximal after-effect was obtained when the loading time with alanine was 60—80 min, similarly as in case of the net water flux. Relationship between the loading time and the peak responses of the after-effect in the net water flux or the PD is shown in Figure6. With prolongation of the loading time, the net water flux increased almost linearly. On the other hand, when 5 mM t-alanine was added into the mucosal fluid under the standard condition, thereafter the mucosal fluid containing L-alanine, the net water flux increased also linearly with time after latent period of 20 min (Fig.6A, dotted line). Since these relations between the loading time and the net water flux were closely similar, irresperctive of the presence or absence of alanine in the medium, it is unlikely that L-alanine stimulates water trans- port via acting from outside of the cells. The peak after-effect of the serosa-negative PD also in- creased with prolongation of the loading time, and the relation was almost parallel to that obtained after addition of L-alanine to the mucosal side Wash , -6 Q eer: 5mM Ala =—_7 Q v Vv v O -3 O Y\ Aire Q _—— = a5 R J ala O < ¥ —— SS a) 5mM Ala 0.01 mM Furosemide | 0 ee 0 60 120 180 290 Time (min) Fic.5. After-effect of L-alanine on the PD. In the presence of 0.01mM furosemide (open column), 5 mM t-alanine was added into the mucosal medium at 40 (@), 60 (LJ), 80 (A) or 100 min (HH); arrow heads. At 120 min, both of the mucosal alanine and furosemide were removed. In control (©), 5mM t-alanine was added into the mucosal fluid at 190min. Each point represents mean value. The number of preparations is indicated in the parentheses. 42 (Fig.6B, dotted line), whereas the serosa- negativity was lower in the presence of alanine. Effects of other amino acids Table 2 shows effects of various amino acids on the PD and the water flux. Among various amino acids examined, only L- and p-alanine, L-glutamine and L-glutamic acid enhanced the net water flux. In case of other amino acids, the net water flux continued to decrease as in the initial control period without such amino acids, thus they are represented by negative signs in Table 2. Even after treatment with these non-effective amino acids, a significant enhancement of the net water flux was induced by 5mM 1-alanine. The effects on the PD seem to be composed of — on (yl/ern210 min) flux water > Net (0) 50 100 Loading Fic. 6. M. ANDO two phases, initial and final. An initial slight depolarization of the serosa-negative PD was observed immediately after addition of L-alanine (APD=0.3+0.0mV, N=8), p-alanine (0.1+0.0 mV, N=4), t-serine (0.2 mV), or L-glutamine (0.1 +0.0mV, N=3). Relatively large depolarization was observed after L-cysteine (1.8mV), L-glutamic acid (1.0+0.5mV, N=4) or p-glutamic acid (2.6 +0.1mV, N=3). On the other hand, an initial slight hyperpolarization was observed in case of t-leucine (4PD=—0.5+0.2mV, N=3), p- glutamine (—0.9+0.2mV, N=3) or L-histidine (—0.6+0.2 mV, N=3). Mucosal addition of man- nitol (10mM), as a control, showed neither initial depolarization nor hyperpolarization. After 100 min, however, only L- and p-alanine, L-glutamine -8 4 (6) = Bee fey > ' ' ! (3 ' ' 4 (8) a a B 0 @) 50 100 time (min) Relations between the peak after-effects and the loading time. A. Peak after-effect of the net water flux as functions of the loading time. The peak after-effect (@) was obtained after removing both furosemide and alanine by rinsing with alanine-free Ringer solution in Fig. 4. For comparison, the time course of the net water flux after addition of alanine was shown as open circles, where 5 mM L-alanine was applied into the mucosal fluid at time zero, thereafter the mucosal medium containing L-alanine. B. Peak after-effect of the PD as functions of the loading time. The peak after-effect (@) was obtained after removing both furosemide and alanine in Fig. 5. Dotted line shows the time course of the PD after addition of 5mM t-alanine into the mucosal fluid at time zero((), thereafter the mucosal medium containing alanine. Each point and vertical bar indicate the mean and S.E., respectively. The number of preparations is indicated in the parentheses. Alanine and Water Transport 43 TABLE 2. Effects of various amino acids on the transepithelial potential difference (PD) and the net water flux (Ji2° Amino acids An a Glycine 10 L-alanine 5 D-alanine 10 L-valine 10 L-leucine 10 L-norvaline 10 L-serine 10 L-cysteine 10 L-methionine 10 L-tryptophan 10 L-proline 10 L-asparagine 10 L-glutamine 10 p-glutamine 10 L-glutamic acid 10 D-glutamic acid 10 L-aspartic acid 10 L-lysine 10 L-ornithine 10 L-histidine 10 f8-alanine 10 y-aminobutyric acid 10 N APD Ape (mV) (ul/cm? - 10 min) 3 (Pe ere (0).22 == 4). 3) ace) 8 == 70) ey Dor} 31o(8) ae (08) 4 Sid taney (OES) 4.2+ 0.6 5 (DS), Se 0)3) —2.4+ 0.4 3 2) ae. WaT) Sate) 4 1 —0.4 —2.8 iL —(0.5 —3.0 1 bos) —4.6 1 0.8 = 3.7 il 0.8 il 1 0.5 —3.1 3) 0.0 + 0.4 =.2) ge 2 3 = 6y.) ae (0,3) QO. ae Ie 3 0.7 + 0.4 = 6).3) ae ile 4 —3.0 + 0.9 3).3) ae I. 3 2.2 + 04 =410 ae ley 4 O).3) ae W,7 —1.0+0.4 3 Om aa028 —3.4+4+ 1.4 3 OO —2.4+ 0.4 3 O.5) ae (0); Il 47), ae WY 1 —().3 —2.1 1 0.0 —2.9 After bathing the intestine with standard Ringer solution, various amino acids was added into the mucosal fluid and the PD and the net water flux were measured every 10 min for 100 min. Results are given as mean+S.E. Each value is presented as a difference (4) between the values before and 100 min after the addition of amino acid. Positive values represent depolarization in the PD and increase in the net water flux. Negative values represent hyperpolarization and decrease, respectively. and L-glutamic acid enhanced the serosa-negativity (final phase), accompanied by the enhancement in the water flux. DISCUSSION The present study demonstrates that L-alanine stimulates the net Na‘, Cl” and water fluxes under the standard condition (Table 1). Since the NaCl transport in the sea-water eel intestine is due to a coupled Na*-K*—-Cl~ transport which contributes to the water absorption [1-3] these results suggest that L-alanine stimulates the Na*-K*-Cl~ trans- port, and secondarily water transport. This sug- gestion is supported by a finding that the enhance- ment of the net water flux induced by t-alanine is completely blocked by treatment with furosemide (Fig. 3), an inhibitor of the Na*-K*-Cl~ trans- port in the eel [3] or flounder [6] intestine. The enhancement of the serosa-negative PD can also be explained by the stimulated Na*-K*-Cl7 transport, because this cotransport system en- hances intracellular accumulation of K* and Cl~ thus enhances K* efflux across the brushborder membrane and Cl” efflux across the basolateral membrane, as explained in the flounder intestine [7]. 44 M. ANDO After removing both furosemide and alanine, however, the effect of alanine (after-effect) were evoked (Figs. 4-6), suggesting that L-alanine or its metabolite(s) act(s) within the cells. This sugges- tion is further supported by such findings as serosal alanine also enhances the serosa-negative PD and the net water flux, whereas the enhancements are much smaller than those by mucosal alanine, and as a long latent period is required before the effects of mucosal alanine appear. An initial depolarization of the PD was always observed immediately after addition of L-alanine into mucosal fluid, but its magnitude was too small (ca. 0.3mV) to be analysed. Although a large depolarization has been demonstrated by L-leucine in the flounder intestine bathed with Cl -free (replaced with gluconate) Ringer solution [8], such a large depolarization by t-leucine or L-alanine was not observed in the seawater eel intestine bathed with gluconate-Ringer solutions (unpub- lished observation). If the small depolarization in this study reflects the Na“ -alanine cotransport, the cotransport system seems to be refractory to furosemide, because a similar small depolarization is observed in the presence of furosemide and the after-effects of L-alanine are induced rapidly after removing furosemide (Figs.3,4 and 5B). The latter phenomenon indicates that L-alanine is already taken up into the cells. A parallel shift of the PD by the presence of alanine (Fig. 6B) may also be explained by the alanine-evoked potential. ACKNOWLEDGMENT This research was supported in part by Grant-in-Aid No. 58370047 from the Ministry of Education, Sciences. and Culture, Japan. REFERENCES 1. Ando, M. (1983) Potassium-dependent chloride and water transport across the seawater eel intestine. J. Membrane Biol., 73: 125-130. 2 Ando, M. (1985) Relationship between coupled Na* -K*-Cl~ transport and water absorption across the seawater eel intestine. J. Comp. Physiol., 155: 311-317. 3 Ando., M. and Utida, S. (1986) Effects of diuretics on sodium, potassium, chloride and water transport across the seawater eel intestine. Zool. Sci., 3: 605-612. 4 Ando, M., Sasaki, H. and Huang, K. C. (1986) A new technique for measuring water transport across the seawater eel intestine. J. Exp. Biol., 122: 257-268. 5 Ando, M. and Kobayashi, M. (1978) Effects of strip- ping off the outer layers of the eel intestine on salt and water transport. Comp. Biochem. Physiol., 61A: 497-501. 6 Frizzell, R.A., Smith, P.L., Vosburgh,E. and Field, M. (1979) Coupled sodium-chloride influx across brush border of flounder intestine. J. Mem- brane Biol., 46: 27-39. 7 Halm, D.R., Krasny, E.J. Jr. and Frizzell, R. A. (1985) Electrophysiology of flounder intestinal mu- cosa. I. Conductance properties of the cellular and paracellular pathways. J. Gen. Physiol., 85: 843-864. 8 Thompson, K. A. and Kleinzeller, A. (1985) Glu- cose transport in intestinal epithelia of winter floun- der. Am. J. Physiol., 248: R573—R577. ZOOLOGICAL SCIENCE 4: 45-52 (1987) © 1987 Zoological Society of Japan Comparison of Seawater and Fresh-water Eels for the Effects of L-alanine on Water Transport across the Intestine MASAAKI ANDO, YASUO FURUKAWA and MAKoTo KOBAYASHI Laboratory of Physiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan ABSTRACT— Using a new perfusion system, effects of L-alanine on the net water and ion fluxes across the intestine were examined in vitro in the seawater and fresh-water eels. In the seawater eel intestine, mucosal addition of 5mM t-alanine stimulated the net water flux and the serosa-negative potential difference (PD), accompanied by an enhancement in the net Na‘ and Cl” fluxes under the standard condition. On the other hand, in the fresh-water eel intestine the net water and ion fluxes were not affected significantly by the addition of alanine, indicating that alanine-sensitivity is enhanced by seawater adaptation of the eels. After injecting cortisol into the fresh-water eels, the alanine-sensitivity was enhanced to near the seawater levels, indicating that the alanine-sensitivity is regulated by cortisol. Furthermore, from the difference in the time course of the PD after setting each intestine isolated from seawater or fresh-water eels to the system, roles of alanine and cortisol in seawater adaptation of the eels are discussed. INTRODUCTION In the previous study, we developed a new system for measuring water transport across the intestine and found that L-alanine enhanced water absorption across the seawater eel intestine [1]. Furthermore, it has recently been demonstrated that the effect of L-alanine results from acting within the cells [2]: intracellular alanine stimulates water absorption in the seawater eel intestine. Although it is well established in invertebrates that intracellular amino acids increase when they adapt to a hypertonic saline [3-5], in euryhaline teleosts it is still controversial whether intracellular amino acids increase [6,7] or not [8] during seawater adaptation. Therefore, in the present study, the effects of L-alanine on the net water and ion fluxes across the intestine were compared between the seawater and fresh-water eels. The results obtained demonstrate that the seawater eel intestine is more sensitive to alanine than the fresh-water eel intestine, and that such an incre- ment in the alanine-sensitivity is induced by cortisol. Accepted September 4, 1986 Received August 19, 1986 MATERIALS AND METHODS Japanese cultured eels, Anguilla japonica weighing about 200g were obtained from a com- mercial supplier and kept in a fresh-water tank at 20°C for more than a week before use. Half of them were adapted to seawater (20°C) for more than a week. For assaying the effect of cortisol, cortisol (Schering) was injected intraperitoneally into the fresh-water eels at a dose of 0.5 mg/100g body weight and the fish were sacrificed 24 hr after the injection. These eels were decapitated and the intestine was excised. The outer muscle layers of the intestine were stripped off following our previous method [9]. After everting the intestine, a cylindrical polyester mesh was inserted into the middle part of the intestinal tube. These opera- tions were done on a cooled plate placed on ice: it took about 40min. The serosal side of the intestine was perfused with the standard Ringer solution at a constant rate (ca. 173 l/min), and the effluent was collected every 10 min. Details of the apparatus for simultaneous measurement of net water flux and transepithelial potential difference (PD) are described previously [1]. The net water flux was calculated by subtracting the perfusion rate from the rate of effluent flow. 46 M. ANbDo, Y. FURUKAWA AND M. KoBAYASHI Net ion fluxes were calculated from the difference between the products of ionic concentration and collected volume in each of the perfusate and the effluent as described previously [2, 10]. Sodium and K* concentration was measured with flame photometry (Hiranuma, FPF—2A) and Cl” con- centration was determined with a chloride counter (Hiranuma, CL—-5M). The standard Ringer solution contained (mM/l): 118.5 NaCl; 4.7 KCl; 3.0 CaCl); 1.2 MgSO,; 1.2 KH,PO,; 24.9 NaHCO; (pH7.3 on bubbled with 95% Or-5% CO, gas mixture). For nutrient Ringer solution, 5mM t-alanine or pD-glucose (Katayama Chemical Co., Japan) was added to the standard Ringer solution. At the end of the experiments, the intestine was cut longitudinally and spread on a graph paper: surface area of the intestine was measured using a planimeter (Ushi- kata, 220L). All data are expressed as mean+S.E. (18) NFR -8 (7) Gilc+ Ala S E -4 (13) Gle (=) al SW 0 0 40 80 Time Fic. 1. RESULTS Effects of nutrients Figure 1 shows time course of the PD across the seawater and fresh-water eel intestine. In the seawater eels, higher serosa-negative PD (ca. —7mV) was observed immediately after bathing the intestine with the standard Ringer solutions, but thereafter the serosa-negativity decreased gradually during perfusing the serosal side, sug- gesting that some essential substance(s) for main- taining the high level is/are lost from the seawater intestine during the perfusion. Glucose (5mM) had no effect on the decline of the PD. When 5mM t-alanine was added to the glucose Ringer solutions, however, the PD returned to the high level and remained at that high level. On the other hand, in the fresh-water eels the PD was less than —1mV initially and then increased its negativity during the perfusion. The presence of glucose did -8 (6) Gilc+Ala -4 (ONFR FW 0 0 40 80 (min) Time course of the transepithelial potential difference (PD) across the seawater (SW) and fresh-water (FW) eel intestine. At time zero, the intestine was set to the apparatus and bathed with various Ringer solutions, such as nutrient-free (NFR), 5mM p-glucose containing (Glc) or 5mM p-glucose+5mM t-alanine containing (Glc+Ala) Ringer solutions, and then the serosal fluid started to be perfused. The number of preparations is indicated in the parentheses. Role of Alanine in Seawater Adaptation of the Eel TABLE 1. and the net water flux ( fresh-water (FW) eels yrRo net Effects of D-glucose and L-alanine on the transepithelial potential difference (PD) ) across the intestine isolated from seawater (SW) or Nutrient (mM) eed eg p 108 Glucose Alanine NS. (l/cm? - hr) SW 0 0 — ef ote Se 39:9 74.0 5 0 —4.1+0.5 69.5 = 9.0 5 5 —7.4 + 0.4* Sies = 2.67 FW 0 0 —3.3+ 0.2 Dont cE 3 5 0 —3.6 + 0.6 36.4 + 5.8 5 > —7.0 + 0.5* 39.0 + 6.1 Each value was obtained under a nearly-steady with each experimental solution. Difference from control values under nutrient-free condition, penacan--S.E. (Ni —6): 47 state, more than | hr after bathing the preparation * P<0.001 (Student ¢-test). TaBLE2. The effects L-alanine on the transepithelial potential difference (PD), net ion fluxes (Inet » jx, JS) and net water flux (JE2°) across the seawater (SW), fresh-water (FW) or cortisol-injected fresh-water (FW+Cortisol) eel intestine Adaptation Condition N 2 Use net ie Tne, P (mV) uct (ueq/cm? - hr) Bet (ul/cm? -hr) SW Control 9 —4.0+0.3 9.5+0.9 0.0+0.1 8.0+0.5 56.1+4.1 L-alanine Otay peg el S-Satetk Ls t 0.1+0.1 1247220 Oye eadeoet es FW Control 10 —3.6+0.3 3 4:35.38 0.0+0.1 ui vasi).7/ AEG ine 88 L-alanine Ges a O07, OnEEO? 5.6+0.9 33.7+6.6 FW Control 9 —5.1+0.6 oar 12 —0.4+0.1 7.6+0.8 45.9+4.8 + Cortisol | alanine POA ems tle 9 0.5402) Melek; 65.5+5.6* After bathing the intestine with standard Ringer mucosal fluid (L-alanine). reached a nearly-steady level. Difference from control values, * P<0.05, not enhance the increment of the PD significantly, but alanine enhanced it gradually to near —7 mV. Under these three experimental conditions, the steady state values of the PD were closely similar to those in the seawater eels, respectively. Table 1 summarizes the steady state values of the PD and the net water flux under various experimental conditions. In nutrient-free Ringer (NFR) solutions, the net water flux across the seawater eel intestine was almost twice as high as that in the fresh-water eels. The presence of alanine enhanced the net water flux in the seawater eels, but not in the fresh-water eels, whereas it enhanced the PD by the same amount in both the seawater and fresh-water eels. Glucose had no Each value (Mean+S. SP <0. OF solutions (control), 5mM t-alanine was added to the E.) was obtained after the PD and the net water flux *** P<(0.001 (paired f-test). significant effects on these two parameters in both eels. Effects of alanine Since even 10mM glucose has no effect on the PD and the net water flux [1], following experi- ments were restricted to clarify the effects of L-alanine. Figure2 demonstrates that mucosal alanine plays a major role in the increment of the PD on both seawater and fresh-water eels. When alanine was added to the mucosal fluid after bathing the intestine with standard Ringer solu- tions, the PD depolarized initially by 0.2+0.0 and 0.4+0.1mV (N=8) in the seawater and fresh- water eels respectively, thereafter the serosa- 48 M. ANbo, Y. FURUKAWA AND M. KoBAYASHI -8 A B 5mM Alanine r (8) = (8) (5) -~4 (5) Alanine-free (a) ae 0 0 50 Time 100 0) 50 (min) Fic. 2. The effects of L-alanine on the transepithelial potential difference (PD) across the seawater (@) or fresh-water (©) eel intestine. A. After bathing the intestine with standard Ringer solutions (nutrient-free), 5mM t-alanine was added to the mucosal fluid at time zero. B. After bathing the intestine with glucose-alanine Ringer solutions which contain 5mM p-glucose and 5mM t-alanine, mucosal L-alanine alone was removed at time zero, while the serosal fluid still contained L-alanine and both fluids contained p-glucose. The number of preparations is shown in the parentheses. negativity increased gradually. On the other hand, when mucosal alanine was omitted after bathing the intestine with glucose-alanine Ringer solu- tions, the PD hyperpolarized initially by 0.8+0.3 and 0.8+0.2mV (N=5) in the seawater and fresh-water eels respectively, thereafter the serosa- negativity decreased gradually (Fig. 2B). After all, the time courses of the PD were almost identical in both the seawater and fresh-water eels. The effects of mucosal alanine on the net water flux are shown in Figure 3. When 5mM 1-alanine was added to the mucosal fluid of the seawater eel intestine, the net water flux started to increase gradually after latent period of 20min. When mucosal alanine was removed after bathing the intestine with glucose-alanine Ringer solutions, the net water flux increased initially but thereafter declined gradually (Fig.3B). These time courses of the net water flux across the seawater eel intestine were in parallel to those in the PD. In the fresh-water eel intestine, in contrast, neither addi- tion nor omission of alanine had no significant effect on the net water flux. Effects of cortisol Since it is well known that cortisol alters the fresh-water eel intestine to a seawater type [11, 12], in the next experiment, effects of alanine were examined in the cortisol injected fresh-water eels. Figure 4 shows the time course of the PD across the cortisol-treated fresh-water eel intestine. Im- mediately after bathing the intestine with Ringer solutions, about —3mV was observed; an in- termediate PD between fresh-water and seawater eels (cf. Fig. 1). Then the PD increased rapidly within 10min. These initial increments in the serosa-negative PD were closely similar to those in the intact fresh-water eels, but the later phases were similar to those in the seawater eels. Although alanine had no effects on the water Role of Alanine in Seawater Adaptation of the Eel 49 A 15 ia 5mM Alanine E © a= = 10 x< 2 & 5 .@) > Net Time (8) | Alanine-free | (5) ek eee ee if 100 0 50 (min) Fic. 3. The effects of L-alanine on the net water flux across the seawater (@) or fresh-water (©) eel intestine. A. After bathing the intestine with standard Ringer solutions, 5 mM t-alanine was added to the mucosal fluid (at time=0). B. After bathing the intestine with glucose-alanine Ringer solutions, mucosal alanine alone was omitted (at ttme=0). The number of preparations is indicated in the parentheses. transport across the intact fresh-water eel intes- tine, it enhanced the net water flux after treatment with cortisol (Fig. 5). When 5mM t-alanine was added to the mucosal standard Ringer solution, the net water flux increased gradually after latent period of 20min and remained at the high level thereafter. On the other hand, omission of the mucosal L-alanine after bathing the intestine with glucose-alanine Ringer solutions, resulted in a decrease of the net water flux (Fig. 5B). These effects of alanine were essentially similar to those in the seawater eels, whereas the magnitude was slightly smaller. Table 2 shows ion fluxes across the intestine isolated from seawater, fresh-water or cortisol- injected fresh-water eels, and also demonstrates the effects of alanine on their ion fluxes. In these three kinds of intestine, Na* and Cl” were absorbed mainly from mucosa to serosa under the standard condition (control). Although a signif- icant K~ secretion from serosa to mucosa (P< 0.05) was observed in the cortisol-injected fresh- water eels, it was negligibly small compared to the Na* and Cl” fluxes. The NaCl absorption was two-fold higher in the seawater eels than that in the fresh-water eels, and cortisol-treatment mim- icked the seawater adaptaion. Although the ion and water absorption across the cortisol-treated fresh-water eel intestine were slightly lower than those in the seawater eels, these differences were not statistically significant. When 5mM t-alanine was applied, the NaCl absorption was enhanced significantly in the seawater and cortisol-injected fresh-water eels, but not significantly enhanced in the intact fresh-water eels, whereas it tended to increase. DISCUSSION The present study demonstrates that alanine- sensitivity in the water absoption across the intes- tine is enhanced by seawater adaptation of the eels. In the seawater eels, the net water flux was accelerated by mucosal L-alanine (Fig. 3), accom- 50 M. ANbDo, Y. FURUKAWA AND M. KOBAYASHI (5) i: GicsAla > £ (6) -4 NFR Q o. Cant Z0 80 Time (min) Fic. 4. Time course of the PD across the intestine isolated from cortisol-injected fresh-water eels. At time zero, the intestine was set to the apparatus and bathed with standard (NFR) or glucose-alanine (Glc + Ala) Ringer solutions, and then the serosal fluid started to be perfused. The number of preparations is indicated in the parentheses. panied by an enhancement of net Na* and Cl~ fluxes (Table 2). On the other hand, in the fresh-water eel intestine alanine did not significant- ly enhance the net Na*, Cl” and water ‘fluxes. Since the net Na‘, Cl” and water fluxes in the seawater eel intestine are regulated by an intra- cellular alanine [2], the lack of alanine-effect in the fresh-water eels may indicate that in the fresh- water eels alanine content within the enterocyte can not reach to a seawater level. However, it is also possible that the ion transport systems are not developed enough to respond to an increment in the intracellular alanine in the fresh-water eels, even if the alanine content is identical to that in the seawater eels. Which possibility is plausible can not be determined from the present study; direct measurements of the amino acid content within the tissue are necessary. Although the reasons why the effects of alanine are different between the seawater and fresh-water eels are not clear yet as cited above, the present results clearly indicate that the alanine-sensitivity of the eel intestine is regulated by cortisol: the alanine-effects in the fresh-water eels were en- hanced to near the seawater level after treatment with cortisol (Fig. 5 and Table 2). Although the serosa-negative PD in the fresh- water eels was also influenced by alanine similarly as in the seawater eels (Fig. 2 and Table 2), this potential change may be due to their high tissue resistance, because the tissue resistance in the fresh-water eel intestine is higher than that in the seawater eels [13], thus even a small current (= total net ion fluxes) can make a large PD across the fresh-water eel intestine. In fact, a slight incre- ment of the net Na* and Cl” fluxes was obtained after application of alanine to the fresh-water eel intestine, though not statistically significant (Table DD) Perfusing the seawater eel intestine with stand- ard Ringer solutions (NFR), the net water flux decreased gradually with time accompanied by a diminution of the PD (Fig. 1). This phenomenon can be explained by a loss of alanine or its metabolite(s) from the enterocytes, since addition of alanine restored these two parameters to their initial levels and omission of alanine diminished them (Figs.2 and 3). On the analogy of the seawater eels, some essential substance(s) might be lost from the fresh-water eel intestine during perfusion, since the PD increased after the perfu- sion (Fig. 1) and the steady state value of the net water flux was six-fold higher (Table 1) than that in the sac preparation [9]. In addition, the net Na and Cl” fluxes in the fresh-water eels were also much higher (Table 2) than those described pre- viously in the sac preparation [14, 15], whereas these previous measurements were performed in the intact intestine with muscle layers (not strip- ped). Although prolactin is well known to be an essential hormone for fresh-water adaptation of the teleost fish [16, 17], mammalian prolactin (0.5-1.0 U/ml) did not inhibit the PD and the net - water flux in the fresh-water eel intestine in vitro Role of Alanine in Seawater Adaptation of the Eel 51 iS S5mM Alani E m anine = a = £10 -10 3 (5) =~ > je x< tl 2 ra ) a5) w Alanine-free 1o) 3 a A B @ Zz 0 0 0 Botan: 100 0 7 50 Time (min) Fic. 5. The effects of L-alanine on the net water flux (@) and the PD (() across the intestine isolated from the cortisol-injected fresh-water eels. After bathing the intestine with standard Ringer solution (NFR), 5mM L-alanine was added to the mucosal fluid at time zero. B. After bathing the intestine with glucose-alanine Ringer solutions, mucosal alanine alone was omitted at time zero. The number of preparations is indicated in the parentheses. (unpulished observation). The initial rapid increase in the PD was also observed in the cortisol-treated fresh-water eels (Fig. 4). Although cortisol is well known to enhance the active Cl” tranport [18], which contributes to the serosa-negative PD across the eel intestine, and to enhance the water absorption to the seawater levels [11, 12, 15], it must be noticed that these measurements are performed under a steady state condition, after enough equilibration period. The present result demon- strates that the effect of cortisol is not fully developed, at least immediately after isolating the intestine from the fresh-water living eels, but is induced in 10 min after bathing the intestine with salt solutions. To determine whether this induc- tion is caused from a loss of an essential substance in the fresh-water eels or from a direct action of salt(s) to the luminal membrane of the enterocyte, further experiments are necessary. ACKNOWLEDGMENT We are grateful to Professor Tetsuya Hirano, Ocean Research Institite, University of Tokyo, for stimulating discussions. This research was supported in part by Gant-in-Aid No. 5870047 from the Ministry of Educa- tion, Sciences and Culture, Japan. REFERENCES 1 Ando, M., Sasaki, H. and Huang, K. C. (1986) A new technique for measuring water transport across the seawater eel intestine. J. Exp. Buol., 122: 257-268. 2 Ando., M. (1986) Regulation by intracellular ala- nine of water transport across the seawater eel intestine . Zool. Sci., 4: 37-44. 3 Florkin, M. and Schoffeniels, E. (1969) Molecular Approaches to Ecology, Academic Press, New York. 4 Schoffeniels, E. and Gilles, R. (1970) Osmoregula- tion in aquatic arthropods. In “Chemical Zoology, Volts foe ede by ae Virshlonrkingandieb alnScheen: Academic Press, New York, pp. 255-286. 5 Gilles, R. (1975) | Mechanisms of iono and osmoregulation. In “Marine Ecology, Vol. 2”. Ed. by Kinne, Wiley Interscience, New York, pp. 259-347. 6 Huggins, A. K. and Colley, L. (1971) The changes in the non-protein nitrogenous constituent of muscle during the adaptation of the eel Anguilla anguilla L. from fresh water to seawater. Comp. Biochem. Physiol., 38B: 537-541. 7 Venkatachari, S.A. T. (1974) Effect of salinity 10 11 12 SV adaptation on nitrogen metabolism in the fresh water fish Tilapia mossambica. 1. Tissue protein and amino acid levels. Marine Biol. ,24: 57-63. Goldstein, L. and Forster, R. P. (1970) Nitrogen metabolism in fishes. In “Comparative Biochemistry of Nitrogen Metabolism, Vol. 2 ”. Ed. by J.W. Campbell, Academic Press, New York, pp. 495-518. Ando, M. and Kobayashi, M. (1978) Effects of stripping off the outer layers of the eel intestine on salt and water transport. Comp. Biochem. Physiol., 61A: 497-501. Ando, M. (1983) Potassium-dependent chloride and water transport across the seawater eel intes- tine. J. Membrane Biol., 73: 125-130. Hirano, T. and Utida, S. (1968) Effects of ACTH and cortisol on water movement in isolated intestine of the eel, Anguilla japonica. Gen. Comp. Endocri- nol., 11: 373-380. Ando, M. (1974) Effects of cortisol on water trans- port across the eel intestine. Endocrinol. Jpn., 21: 539-546. 13 14 15 16 17/ 18 M. ANDO, Y. FURUKAWA AND M. KoBAYASHI Ando, M., Utida, S. and Nagahama, H. (1975) Ac- tive transport of chloride in eel intestine with special reference to sea water adaptation. Comp. Biochem. Physiol., 51A: 27-32. Utida, S., Hirano, T., Oide, H., Ando, M., John- son, D. W. and Bern, H. A. (1972) Hormonal con- trol of the intestine and urinary bladder in teleost osmoregulation. Gen. Comp. Endocrinol., Suppl. 3: 317-327. Hirano, T., Morisawa, M., Ando, M. and Utida, S. (1976) Adaptive changes in ion and water transport mechanism in the eel intestine. In “Intestinal Ion Transport”. Ed. by J.W.L. Robinson, MTP Press, Lancaster, pp. 301-317. Bern, H. A. (1975) Prolactin and osmoregulation. Am. Zool., 15: 937-948. Bern, H. A. (1977) Some possible contributions of comparative endocrinology to mammalian and hu- man endocrinology. Zool. Mag., 86: 1-9. Chen, T.S. T., Ando, M., Utida, S. and Huang, K. C. (1975) Ion fluxes and permeability studies of Japanese cultured eel inteatine. Fed. Proc., 34: 470. ZOOLOGICAL SCIENCE 4: 53-59 (1987) Ultrastructural Studies on Developing Oblique-Striated Muscle Cells in the Cuttlefish, Sepiella japonica Sasaki AKIRA MATSUNO Department of Biology, Faculty of Science, Shimane University, Matsue 690, Japan ABSTRACT—Developmental changes in muscle cells located in the mantle of cuttlefish (Sepiella japonica) were investigated by electron microscopy. The circular muscle cells in the mantle, designated oblique-striated muscle cells, have peculiar myofilamental arrangements. Muscle cells observed in this work were taken from six stages (between stage 31 and 40) and larvae 48hr after hatch. The differentiation of muscle cells can be divided into three phases: stages 31—36 (1st phase), stage 38 (2nd phase) and 24—48 hr after hatching (3rd phase). The ultrastructural characteristics in each phase are as follows. In the first phase, myofilaments are organized in the cytoplasm surrounded by endoplasmic reticular (E.R.) systems, and bundles constituted of thick and thin myofilaments appear. In the second phase, myofilamental bundles gradually become striated. Formation of the Z-body seems to be lead by tubular E.R. from E.R. systems under the cell membranes. In the last phase, bundles having A- and I-bands fuse into one thick bundle. The fusion was accompanied by the process which forms the © 1987 Zoological Society of Japan Z-bodies into a line, and muscle cells are completed in this phase. INTRODUCTION The cuttlefish shows rather quick movements and they have well-developed muscles in the body wall or mantles. The well-developed muscles are classified as the oblique-striated muscle. They have regular myofilamental arrangements, alter- nating A- and I-bands in a direction towards the longitudinal axis of the cell, as seen in myofibrils of cross-striated muscle cells. In a rectangle section taken in that direction of the cell, sets of thick and thin myofilaments are arranged in an oblique pattern shifting their array among each other at a definite distance apart, thus the designation, ob- lique-striated muscle. In a cross-section, sets of myofilaments appear in a regular pattern as the myofibrils of cross-striated muscles [1]. Differentiations of cross-striated muscle cells have been already investigated by Obinata et al. [2] in chick embryos from the ultrastructural and biochemical standpoints. Fischman [3] and Shima- da et al. [4] had investigated closely by electron microscope the processes of the formation of A- Accepted August 18, 1986 Received June 23, 1986 and I-bands in embryonic or cultured chick muscle cells. Another cross-striated type of epitherio- muscular cells in Aurelia showed somewhat pecul- lar processes as compared to the chick cross- striated muscle cells [5]. These reports have suggested that thin bundles of myofilaments having the A-band are formed first, then I-bands and Z-lines gradually appear. I have attempted to reveal those processes in an oblique-striated muscle cell under the electron microscope as well as how and when the character- istic arrays of myofilaments appear. As a result, some differences in the developmental process between oblique-striated and cross-striated muscle cells have been observed. MATERIALS AND METHODS Embryos and larvae of the cuttlefish (Sepiella japonica) were used in this study. Embryos at stages 31, 33, 36, 38 and larvae 24 and 48 hr after hatching were successively fixed for electron mi- croscopy. The stage of each embryo was estimated according to the “Table of Normal Embryonic Stage” [6] and in some cases upon advice from Dr. M. Yamamoto of the Marine Biological Labora- 54 A. MATSUNO tory of Okayama University. Six stages of embryos and larvae were prefixed in a solution of 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) at room temperature. After prefixation, they were rinsed with the buffer solution and arms and shells in the mantles were removed if necessary. They were then postfixed in a solution of 1% osmium tetroxide in 0.1M phosphate buffer (pH 7.3) at 4°C. Fixed specimes were dehydrated in a series of ethanol and embedded in epoxy resin through n-butyl-glycidyl-ether. Embedded specimens were cut into thin sections with glass or diamond knives. Sections were stained with saturated aqueous uranyl acetate and lead citrate. They were observed under a JEM 100 C-type electron microscope at magnifications rang- ing from 2,000-—20,000. RESULTS Specimens in each stage were observed at the left side and middle portion of the mantle, where the muscle layer was thickest. The mantle of the cuttlefish consists of three muscular systems, the longitudinal muscle which runs_ longitudinally along the long axis of the mantle, the circular muscle, and the radial muscle, which runs trans- versely from the outer surface to the inner surface, crossing the circular one. Ultrastructural observa- tions were carried out on the circular muscle cells in the mantle, and their ultrastructural charac- teristics are described separately at each stage. Observed muscle cells were the most well- developed ones in each stage, as cells did not develop simultaneously. Stage 31: Muscle cells showed cell differentia- tion, and possessed a nucleus, scanty cytoplasm and undeveloped, scattered cell-organelles (Fig. 1). In this stage, a nascent myofilamental bundle was already observed. It was short in length and thin in diameter. The myofilamental bundle contained 3-7 thick myofilaments and showed no cross-striations, but thick and thin myofilaments were arranged roughly parallel in the bundle. The bundle was always surrounded by endoplasmic reticular (E.R.) systems (Fig. 1), suggesting that the myofilamental bundle might be organized in the cytoplasm surrounded by E.R. In this stage, one bundle was observed in a cell, but many were recognized in the advanced stages. Stage 33: The general appearance of a cell in this stage was not so well-developed, but cell- organelles seemed somewhat advanced compared with those of the previous stage. The myofilamental bundle increased in diameter by 2-3 times compared with that of the previous stage (Fig.2). Myofilaments in the bundle were also increased in number, and their directions were not so regular but roughly parallel (Fig.2) . The bundle was also surrounded by E. R. systems. Stage 36: A muscle cell of this stage gradually was developing its cell-organelles. The myofil- amental bundle was thicker than the one at the previous stage. The arrangement of myofilaments in the bundle was not so regular, that is, there were no A- or I-bands. Myofilaments ran parallel in the bundle (Fig.3). The bundle showing an oval section of about 0.8m in major axis, were situated at a peripheral portion and ran along the long axis of the cell (Fig. 4). Stage 38: A cell in this stage showed the most dynamic structural changes of all the stages ex- amined. A longitudinal section of the mantle showed cross-sections of circular muscle cells and longitudinal sections of thin longitudinal and radial cells (Fig. 5). Longitudinal muscle cells ran below the inner and outer surfaces of the mantle in a longitudinal direction at definite intervals. Radial ones penetrated circular muscular layers from inside and outside of the mantle, and connected outer longitudinal muscles to inner longitudinal ones (Fig. 5). Almost all the cells in the circular muscle had myofilamental bundles in the peripher- al regions. A nucleus in the central region, about 9 ym in diameter, was tapered toward the both ends. Myofilamental bundles were arranged in a disorderly way (Fig.5). At this stage, a cross- striation-like structure was first clearly observed in a myofilamental bundle in longitudinal section (Fig.6). As shown in Figure 6, tubular E.R. attached to both ends of the A-bands. They extended to the interspaces of the A-band and the neighboring one. The tubules ran in a zigzag line and contained electron dense materials in them. - They originated from E.R. systems situated just Ultrastructure of Developing Muscle 55 Fic. 1. A longitudinal section of muscle cells at stage 31. A nascent myofilamental bundle (nm) is shown near a nucleus. It is surrounded by E.R. systems (small arrows). 16,700; scale bar, 1 um. Fic. 2. A longitudinal section of muscle cells at stage 33. Two nascent myofilamental bundles (nm) are shown. Bundles grow in size as compared to the one at the prvious stage. Myofilaments are arrayed in a disorderly way. They are surrounded by E.R. systems as at stage 31. x 10,200; scale bar, 1 um. Fic. 3. A longitudinal section of muscle cells at stage 36. Myofilamental bundles increase in length, but do not grow much in thickness. 7,700; scale bar, 1 um. Fic. 4. A cross-section of muscle cells at stage 36. Myofilamental bundles surrounded by E.R. systems (arrows) are situated at the periphery of a cell. Thick myofilaments show a disordered arrangement. 18,600; scale bar, 1 ym. 56 A. Matsuno Ultrastructure of Developing Muscle 57 under the cell membranes, and the tubular por- tions extended to the myofilamental bundles. The tubules usually penetrated into the bundles, but in some cases, they reached other E.R. systems situated on the opposite side (Fig.6). Thick myofilaments in the bundle measured about 24- 35 nm in diameter, while thin ones measured about 7nm. Thick and thin myofilaments in the bundle ran parallel to each other. Larvae began hatching at this stage and the next stage. Larva 24hr after hatching: Some myofilamental bundles having cross-striations gathered into one thick bundle at this stage. As shown in Figure 7, two thin bundles with A- and I-bands were present together at the one end of the cell. Because the two bundles were not simply united with each other and the connecting Z-bodies were fused into one line, the line of Z-bodies first appeared in a zigzag pattern (Fig.7). Before the gathering began, E.R. systems surrounding the thin bundles had disappeared, and the newly reconstructed bundles became thick and ordered. Larva 48hr after hatching: Muscle cells were developed completely at this stage. In cross- section, cells appeared to be filled with myofila- ments, and the cytoplasm disappeared except for the central region where there were a few mitochondria and other cell-organelles. Myofila- ments rearranged to form regular units that stood one behind another along the long axis of the cell (Fig. 8). The regular units were surrounded by tubules and composed of 60-70 thick myofilaments (Fig. 8). In a longitudinal section, cells usually showed a regular oblique-striated pattern of myofilaments (Fig. 9). A-bands in the contracted state measured about 1.4m, and Z-bodies appeared alternately at a Z-line with a cross- section of tubules. These characteristic features were the same as the ones of an adult sepia (Fig. 9). DISCUSSION In the present study, the developmental course of the oblique-striated muscle cell has been traced succesively to determine how and when the char- ateristic array of myofilaments is started and formed. From observation, the process may be divided into three phases. The first phase is stages 31-36, the second 38 and the third 24—48 hr after hatching. In the first phase, a thin and short myofilamental bundle is constructed at a peripheral region of the cell. The bundle includes two kinds of myofila- ments that run roughly parallel to each other. The two kinds of myofilaments may be constructed by actin and myosin, respectively, judging from their diameters. Profiles of the bundle resemble pre- viously reported nascent myofibrils of developing cross-striated muscle cells [2—4, 7, 9-12], and those of the lymph-heart [13]. However, the bundle seems rather thin in size as compared to the nascent myofibrils. It is inappropriate to regard the myofilamental bundle in oblique-striated mus- cle cells as a nascent “myofibril”, because the bundle never grows into a myofibril as shown in cross-striated muscle cells. Myofilamental units in an oblique-striated muscle cell resemble a myofib- ril in cross-section, but they are not exactly true myofibrils. Fic. 5. A cross-section of muscle cells at stage 38. Circular muscle cells show cross-sections, and longitudinal (Im) and radial muscle cells (rm) appear in longitudinal sections. Muscle cells (circular ones) are generally filled with myofilaments. 3,000; scale bar, 5 um. Fic. 6. A longitudinal section of muscle cells at stage 38. Three myofilamental bundles are shown. They show striations divided by tubular E.R. (arrows). These striations become a line of sarcomeres. X 12,800; scale bar, 1 um. Fic. 7. A longitudinal section of muscle cells in a larva 24 hr after hatching. Two myofilamental bundles in a central cell are gathered into one thick bundle. Z-lines of the two bundles are arranged in a zigzag line in the thick bundle (arrows). 7,700; scale bar, 1 um. Fic. 8. A cross-section of muscle cells in a larva 48hr after hatching. Many myofilamental units appear in cells enclosed by E.R. systems. They appear regularly crossing cells at the short axis. x 13,800; scale bar, 1 um. Fic.9. A longitudinal section of muscle cells in a larva 48 hr after hatching. Cells become fully developed and show structures completely similar to adults. Clear oblique-striated lines appear. Z-bodies and tubular E.R. appear alternately in the line (arrows). 15,400; scale bar, 1 um. 58 A. MATSUNO It is not clear which myofilaments, thick or thin, are formed first. However, it is believed that filament formation occurs in a limited area sur- rounded by E.R. systems as shown in Figure 1. This ia a very characteristic feature in the process of developing nascent myofilamental bundles of oblique-striated muscle cells. The bundle gets thicker and the number of myofilaments increases to about 60-70 thick myofilaments. These features resemble those of a nascent myofibril in myofilamental arrangements, that is, two kinds of myofilaments run parallel to each other but show no striated pattern, as reported previously [5]. In the second phase, a myofilamental bundle become cross-striated after the bundle increases in size. The procedure of cross-striation resembles that of cross-striated muscle cells; tubules from E.R. systems attach to the sites of myofibrils at lengths that correspond to an A-band. Allen and Pepe [7] suggested in regard to formation of the cross-striated pattern of developing chick embryos, that the transverse tubule was the first indication of a banding pattern. Similar investiga- tions were reported on chick skeletal muscle in vitro [8]. On the contrary, Z-disk formations in stress-fiber were triggered by tubules during its formation [14]. Tubules originated from E.R. systems just under the cell membrane, and con- tained electron dense materials. They ran in zigzag lines to transverse the bundle. It is not clear whether or not the electron dense materials were secreted from the tubules. It appears that the materials may have some relation to the organiza- tion of Z-bodies. The Z-band (Z-disk) of cross-striated muscle cells originated from dense-body-like structures which connect several thin myofilaments and resemble that of smooth muscle cells. The dense- bodies appeared side by side, in order to complete a Z-band [2-4]. In another case, Z-bands of a nascent myofibril of cross-striated muscle cells appeared in a complete structure at an early stage [3]. Epitherio-muscular cells in coelenterates have cross-striated myofibrils which possess Z-bands that remain at lines of dense bodies, but are not complete Z-disks as shown in cross-striated muscle cells [5]. The Z-bodies in cuttlefish muscle cells in the present study are arranged similar to the epitherio-muscular cell type, that is, the Z-band is constructed by a line of dense-bodies. Thus, the process where by the formation of Z-bodies is lead by tubular E.R. is remarkable in a nascent myofilamental bundle in oblique-striated muscle cells. A- and I - bands are organized completely in this phase. In the last phase, muscle cells become complete, and the mantle constituted from these complete muscle cells show rhythmical pulsating contrac- tions. Myofilamental bundles with A- and I-bands fuse into a thick bundle in the cell. Fusion pro- gresses first from coupling the two bundles and then putting Z-bodies into a line with each other. E.R. systems of the two bundles disappear at this stage, and only tubular systems remain in the fused bundle. Bundles having different sizes of sarco- meres are also fused. The newly constructed thick myofilamental bundle shows a zigzag line of Z-bodies in longitudinal section. The zigzag lines are gradually rearranged into an oblique line, and the muscle cells are completed. These processes have not been previously reported in either ob- lique or cross-striated muscle cells. ACKNOWLEDGMENTS My sincere thanks to Prof. K. Maruyama of Chiba University for his helpful advice and critical reading of this manuscript. I am also indebted to Prof. M. Yoshida of the Marine Biological Labortatory of Okayama University for his encouragement and kindness in sup- plying the cuttlefish embryos and larvae. REFERENCES 1 Kawaguti, S. (1963) Electron microscopy on the heart muscle of the cuttlefish. Biol. J. Okayama Univ., 9: 27—40. 2 Obinata, T., Yamamoto, M. and Maruyama, K. (1966) The identification of randomly formed thin filaments in differentiating muscle cells of the chick embryo. Dev. Biol., 14: 192-213. 3. Fischman, D. A. (1967) An electron microscope study of myofibril formation in embryonic skeletal muscle. J. Cell Biol., 32: 557-575. 4 Shimada, Y., Fischman, D. A. and Moscona, A. A. (1967) The fine structure of embryonic chick skeletal muscle cells differentiated in vitro. J. Cell Biol., 35: 445-453. 5 Matsuno, A. (1983) An electron microscopic study Ultrastructure of Developing Muscle 59 on the development of cross-striated muscles in ephyrae of Aurelia aurita. Zool. Mag. Tokyo, 92: 416-422. Yamamoto, M. (1982) Normal stages in the de- velopment of the cuttlefish, Sepiella Japonica SASAKI. Zool. Mag. Tokyo, 91: 146-157. (In Japanese) Allen, E. R. and Pepe, F. A. (1965) Ultrastructure of developing muscle cells in the chick embryo. Am. J. Anat., 116: 115-148. Ezerman,E.B. and _ Ishikawa, H. (1967) Dif- ferentiation of the sarcoplasmic reticulum and T system in developing chick skeletal muscle in vitro. J. Cell Biol., 35: 405—420. Obinata, T., Yamamoto, M. and Maruyama, K. (1967) Morphological and biochemical studies on myofibrillar formation in developing chick embryo. Sci. Papers College General Educ. Univ. Tokyo, 17: 95-120. 10 11 12 13 14 Shimada, Y. (1971) Electron microscope observa- tions on the fusion of chick myoblasts in vitro. J. Cell Biol., 48: 128-142. Mair, W. G. P. (1981) Ultrastructure of developing human muscle. Biol. Neonate., 40: 276-294. Peng, H.B., Wolsosewick, J.J. and Cheng, P.C. (1981) The development of myofibrils in cultured muscle cells: A whole-mount and thin-section elec- tron microscopic study. Dev. Biol., 88: 121-136. Markozashvili, M. I. and Rumyantsev, P. P. (1984) Ultrastructure of muscle fibers and cells synthesizing DNA in lymph heart of developing frogs and chick embryos. Cell Tissue Res., 238: 369-379. Dlugosz, A. A., Antin. P. B., Nachmias, V. T. and Holtzer, H. (1984) The relationship between stress fiber-like structures and nascent myofibrils in cul- tured cardiac myocytes. J. Cell Biol. , 99: 2268-2278. ~ Ke og oauerrekinn) Teo fd £ nba cn belief Pe bt gh PEE vagie de nolent 6h) AO bat Shi. ook Be, Jong a, pe : gel ash 6 en Y Suuvsslan st ) (iaer' yal: Wy n 4 AAR Save me Sse pee” ‘oP Bute - (BAe oe hae) ae aE oe RE ORR: Ae {inder hievaaeestt ie cin ey ja Seed een Te SRRRD VRP. 3 en Cet beards ive oe enti oe PARA SR Acar AN wey aT ee Hii ior A zeling, .. ae Se wy i erty at me em bas. a Pi detia ne mae ress op Be 2peporser Wink now ee Dror aie saat et hon weak) B: ff Chae) 4 hearer hae J Weidiedeseo tanh Et meet ey res es Te ghiriieiivys ules hen Sty ato eee r : RRS ie oer at voy Bhan eet) ve | RE Ss ocean: He eon tebe cs Riad, Vaart, Shitty WEEE nine Amand: Mf " El. os aa erent encase Ved tioning, out Peer) Ah aston A ernarinee be “yD i iar win Set anoint ‘Bae ey HiT e aiioay “te ates Weclne CORT A ad ni Ry ae ee eR ree. POD sits: cs wate Se . ie 7 Pe TGs pe it3 7% nto t eh Peay as 3 ‘eit 5 b's tere } need * wr wh i 7 oe aa" , p Rs | 1 , és ‘ ™ ~ Wich, gars na ca ' wi LSE NE ag WE + Rot paints a eke aah wees phe : ca: 3 wl pophcta kay this ( Yedite: treehos, int Rex : A ee 3 } Ce =A ae ie oe Tete A : o ot mie ‘ any ii 7 A ot \J 4, mae i<4ts ; ae i A ZOOLOGICAL SCIENCE 4: 61-72 (1987) Effects of Calmodulin Antagonists on Motility and Acrosome Reaction of Sea Urchin Sperm Fuyuxi Iwasa!, YuKo Hasecawa!, Sumio Isayima', Makoto OKuNo, Tosutko Mouri and Hipeco Mouri’ Department of Biology, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan ABSTRACT—FEffects of calmodulin antagonists on intact and demembranated sea urchin sperm were examined to elucidate possible functions of calmodulin in sperm motility and the acrosome reaction. Calmodulin antagonist W-7 immobilized intact sperm in artificial sea water (ASW), but did not affect motility of Nonidet-demembranated sperm. When sperm was exposed to W-7, oxygen consumption rate was reduced to 3.3% of control level and flagellar bending became non-propagating to the apical end. The acrosome reaction induced by egg jelly was inhibited concomitant with the loss of motility. W-7 also caused depolarization of sperm membrane. Such phenomena were dependent on amounts of the drug and occurred rather slowly. These effects of W-7 were reversible and canceled by exogenously added calmodulin. Theophilline partially canceled the immobilizing effect of W-7. In Na‘-free ASW, W-7 showed different effects, i.e. it reinitiated motility of immobilized sperm. Target(s) of W-7 and functions of calmodulin were considered based on the above results. If W-7 antagonized calmodulin, it seems that calmodulin has multiple functions in motility and the acrosome reaction of sea urchin sperm. © 1987 Zoological Society of Japan INTRODUCTION Calcium ions (Ca**) function as an intracellular mediator of acrosome reaction and motility in sea urchin sperm [1]. The acrosome reaction, i.e. exocytosis of acrosomal vesicle and elongation of actin filaments (acrosomal process) is a prereq- uisite for fertilization in sea urchins. Egg jelly, the natural inducer of the acrosome reaction, is replaceable by Ca** ionophore A23187 [2], in- dicating that the reaction is triggered by an increase in intracellular Ca?t concentration. Another important function of sperm is flagellar movement. In sea urchin sperm, patterns of flagellar bending of _ reactivated Triton- demembranated models change depending on Ca** concentration at demembranation and reac- tivation [3]. Accepted August 18, 1986 Received June 26, 1986 ' Present address: Suntory Institute for Biomedical Re- search for F. Iwasa, Life Science Laboratory, Showa Denko K.K. for Y. Hasegawa and National Institute for Basic Biology for S. Ishijima. * To whom reprint requests should be addressed. On the other hand, initiation or maintenance of motility requires cAMP-dependent phosphoryla- tion of certain protein(s) in sperm of rainbow trout [4], sea urchin [5], dog [6], etc. There seems to be close relationship among regulations by cAMP and Ca** [7]. Presence of calmodulin in sea urchin sperm has been demonstrated [8]. A calmodulin antagonist, W-7 (N-(6-aminohexyl)-5-chloronaph- thalenesulfonamide), inhibits the acrosome reac- tion of sea urchin sperm induced by egg jelly [9] and also immobilizes sea urchin sperm [10]. Another calmodulin antagonist, trifluoperazine (TFP), changes swimming pattern of dog sperm from straightforward to circular [6]. According to studies on effects of [7H]-W-7 on CHO-K, cells, W-7 passes through the plasma membrane and distributes into the entire cyto- plasm [11]. Although W-7 remains at low concen- trations in the plasma membrane, it does not accumulate in the membrane or in the nucleus. When cells are replaced into media without W-7, the incorporated W-7 is released from the cells with time. One mole of calmodulin binds 3 moles of W-7 and its Kd is 11 4M, whereas affinity of W-5 (N-(6-aminohexyl)-naphthalenesulfonamide) for 62 F. Iwasa, Y. HASEGAWA et al. calmodulin is one eighth of that of W-7 [12]. In this study, we tried to elucidate possible functions of calmodulin in motility and the acro- some reaction of sea urchin sperm examining effects of calmodulin antagonists on live and Nonidet-treated sperm. We mainly used W-7 as a calmodulin antagonist and W-S as its control. MATERIALS AND METHODS Materials Sea urchin sperm was collected by injecting 0.5M KCl into the coelomic cavity. Hemicentrotus pulcherrimus sperm were used for observation of the acrosome reaction, measurement of oxygen consumption, membrane potential and intracellu- lar concentration of Ca*+. Sperm of H. pulcherri- mus, Anthocidaris crassispina and Strongylocentro- tus nudus were used for observation of motility. Artificial sea water Compositions of artificial sea waters, ASW, Ca’*t-free ASW (CFASW), Ca’t-free ASW con- taining EGTA (CF(G)ASW), Na*-free ASW (NaFASW), Na‘, Ca’t-free ASW (NaCFASW), Na*, Ca’t-free ASW containing EGTA (NaCF- (G)ASW) are listed in Table 1. Reagents Calmodulin antagonists, W-7 and W-5 pur- chased from Rikaken Ltd., were dissolved in distilled deionized water (DDW) at 5mM and aliquots were stored at —20°C until use. TFP was a gift from Yoshitomi Pharmaceutical Industries Ltd. T1233 (N?-dancyl-L-arginine-4-t-piperazine amide) was a gift from Dr. M. Maruyama of Mitsubishi-Kasei Institute of Life Sciences. Theophilline was dissolved in DDW at 1M. Calmodulin was purified from porcine brain by the method of Teo et al. [13]. BSA (bovine serum albumin) was a commercial Fraction V (Reheis Chemical Co., Phenix, USA). DiO-C,-(5) (3,3 diethyloxadicarbocyanine iodide) was dissolved in DDW and filtered through 0.45 ~m nitrocellulose membrane before use. The concentration of the dye was determined by the absorption at 575 nm. Quin-2 tetramethyl ester was purchased from Amersham, dissolved in dimethylsulfoxide at 3 mM and stored at —20°C. CCCP (carbonyl cyanide m-chlorophenylhydrazone) was dissolved in ethanol and stored at —20°C. Observation of flagellar movement One yl of semen was suspended in 200 pl of ASW, and after 30 sec 10 ul of the suspension was diluted into 5001 of ASW containing various reagents. Observations were made using a phase contrast microscope (200) without coverslip. In some cases, motility was recorded on video tapes. Sperm most actively swimming just beneath the Open space were observed. Sperm motility was scored at 4 grades, i.e. swimming fast (++), swimming slowly (+), motile but not swimming (+) and immotile (—). Observation of acrosome reaction Semen was diluted 500 times with ASW, and W-7, W-5 or other reagent was added 30sec after dilution. The acrosome reaction of sea urchin TABLE 1. Compositions (mM) of artificial sea waters ASW CFASW CF(G)ASW NaFASW NaCFASW NaCF(G)ASW NaCl 458 474 458 — == = Choline Cl == = = 458 458 418 KCl 9.6 9.6 9.6 9.6 9.6 9.6 MgCl, 48.5 48.5 48.5 48.5 48.5 48.5 CaCl, 10.4 — — 10.4 — — EGTA = = 10.0 a = 10.0 EPESs 10.0 10.0 10.0 10.0 10.0 10.0 pH (Jo 8.2 8.2 8.2 8.2 8.2 4 N-(2-hydroxyethyl)-piperazine-N -3-propanesulfonic acid. Effects of Calmodulin Antagonists on Sperm 63 sperm was induced by adding equal volume of “egg sea water” [9] to sperm suspension. One minute after the addition of “egg sea water”, sperm were fixed with 3% glutaraldehyde in ASW. Fixed sperm were mounted on grids for electron micros- copy, negatively stained with 1% uranyl acetate and observed under the Hitachi HS-9 electron microscope. Sperm were classified into 3 groups, that is, acrosome-reacted, not reacted and not distinct because of destruction or aggregation. Demembranated sperm models Nonidet models of sea urchin sperm were prepared by the method of Okuno and Brokaw [3]. Demembranation solution and reactivation solu- tion were modified as follows. Nonidet demem- branation solution: 0.15M KCl, 20mM_ Tris (Tris(hydroxymethyl)aminomethane)-HCl, 1mM DTT (dithiothreitol), 0.04% (w/v) Nonidet P-40, 2mM MgCl, 0mM (for potentially asymmetrical model) or 5 mM (for potentially symmetrical mod- el) CaCl, pH8.2. Reactivation solution: 0.2M KCl, 20mM Tris-HCl, 2mM MgCl, 1.8mM EGTA (ethyleneglycol bis(2-aminoethy] ether)tet- raacetic acid), 0-1.9mM CaCl, 0.22mM ATP, l1mM DTT, 2% (w/v) polyethyleneglycol 6,000, pH 8.2. Measurement of oxygen consumption Oxygen consumption of sea urchin sperm was measured using a Clark type oxygen electrodes. Sperm concentration was approximately 1 x 10°/ml and 50 u«M W-7 or 10 ~#M KCN was added 2 min after the onset of measurement. At various intervals, small portions were withdrawn to ex- amine sperm motility under the microscope. Measurement of membrane potential Relative values of membrane potential of sea urchin sperm were estimated using a fluorescent probe, 3,3 diethyloxadicarbocyanine iodide (diO- C,-(5)). DiO-C,-(5) is one of cyanine dyes, which are used for measurement of membrane potentials [14]. Cyanine dyes bind to biomembrane depend- ing on its potential, and reduce their fluorescence intensity [15]. Five «| of semen was diluted in 1 ml of ASW and mixed with 1ml of diO-C,-(5) in ASW at certain intervals after semen dilution. Fluorescence intensity was measured using a Shi- madzu fluorescence spectrophotometer RF-540 at excitation wavelength of 574nm and emission wavelength of 595 nm. Measurement of intracellular concentration of Ca** Relative values of intracellular concentration of Ca**in sea urchin sperm were measured using Quin-2 by the method developed by Tsien [16]. Semen (0.2ml) was mixed with 0.8ml of ASW containing 0.1mM CaCl, and 10y1 of 3mM Quin-2 tetramethyl ester, and incubated for 30 min at room temperature. After the addition of 9 ml of ASW containing 0.1mM CaCl, the mixture was further incubated for 30min at 0°C. Sperm was then pelleted by centrifugation at 4,000rpm with Tomy No.3 angle rotor for 5min and again incubated for 30min in 10ml of ASW containing 0.1mM CaCl, at 0°C. The last step was repeated once more. Measurement of fluorescence intensity was carried out by diluting pelleted sperm 200 times and at excitation wavelength of 339nm and emission wavelength of 492nm. Measurement of intracellular concentration of Ca** in Na*-free ASW was done with the same procedure in NaFASW or NaCFASW instead of ASW contain- ing 0.1mM CaCl, or CFASW. RESULTS Effects of calmodulin antagonists on sperm motility and acrosome reaction Calmodulin antagonists, W-7, T1233 and TFP at the concentrations of 1-5x10~°M, arrested flagellar beating of sea urchin sperm, whereas W-5 had no effect at the same concentration. Figure 1 shows changes in flagellar movement of A. crassis- pina sperm diluted 210* times in ASW and exposed to 254M W-7. Flagellar bending was gradually becoming non-propagating and observ- able only at proximal region of flagellum. Beat frequency dropped at that time, whereas ampli- tude did not change for longer period. Finally sperm became immotile with straightened flagellum. W-7 also inhibited the acrosome reaction in- duced by egg jelly as previously reported [9]. In 64 F. Iwasa, Y. HAseGawa et al. 6.25 1.33 Fic. 1. Changes of flagellar movement of A. crassispina sperm exposed to W-7. Semen was diluted 2 x 10* times in ASW containing 25 »M W-7. Flagellar movement was recorded on a video tape. Numerals indicate min and sec after dilution. 100 Motility (%o) Ome diy 24S G51 Cea am OM Time (min) (b) 100 ae oF — 80 6 60 © = Soe w E 40 a oO 2 < 20 < 0 Y Gi 2 ee Sen ee Time (min) Fic. 2. Effects of W-7, W-5 and KCN on motility (a) and acrosome reaction (b) of H. pulcherrimus sperm. Semen was diluted 500 times in ASW containing various reagents. @, 20 uM W-7; &, 20 uM W-7+ 20 uM calmodulin; ©, 20 uM W-5; x , 50 «M KCN; [_], none. the present experiment, the time dependency of the inhibition and its relationship to inhibition of motility were examined. Figure 2 shows results of a typical experiment. H. pulcherrimus sperm were diluted 500 times in ASW containing 20 «M W-7, 20 uM W-5 or 504M KCN, and percentage of motile sperm (Fig. 2a) and percentage of acro- some-reacted sperm (Fig. 2b) were determined. Concentration of sperm was 7X10//ml before - the addition of “egg sea water”. The effect of W-7 on the acrosome reaction was canceled by the addition of 20 ~M calmodulin. W-5 was much less effective than W-7 in inhibiting the acrosome reaction. KCN inhibited motility, but not the acrosome reaction. The results indicate that targets of W-7 and KCN are different and that there is no direct relationship between motility and the acrosome reaction. Effects of Calmodulin Antagonists on Sperm 65 (a) 100 80 60 40 °*lo Of motile sperm 20 0 O SB 8 i WW We ils time (min) Ore 2 > ac4 Fic. 3. iL) IS ZO) 22 Oa (b) oO 28x10! w 55x10! @ 1.4x108 Ss 8 10 time (min) Effect of W-7 on motility of H. pulcherrimus sperm. Percent motile sperm was counted (a) when semen was diluted to 4.7 x 10’/ml at various concentrations of W-7 in ASW and (b) when H. pulcherrimus semen was diluted to various concentrations in ASW containing 20 uM W-7. Calmodulin antagonists, W-7 and TFP at 50 uM, neither stopped flagellar movement nor changed the bending pattern of Nonidet-demembranated models of sea urchin sperm. It was the case for both potentially symmetrical and potentially asym- metrical models. The results show that W-7 does not affect motile systems in the axoneme. Inhibition of the motility of H. pulcherrimus sperm in ASW by W-7 was dependent on concen- tration of W-7 and of sperm as shown as typical results in Figure 3. As will be described in the following section, an autonomous recovery of motility was observed at low concentrations of W-7. When the drug concentration was kept constant, the motility-inhibiting effect of W-7 became less prominent with increasing concentra- tion of sperm. This was also true for KCN. Recovery from the effects of calmodulin antagonists Inhibition of flagellar bending by W-7 occurred independently of exogenous Ca**. H. pulcherri- mus semen was diluted 2,000 times in ASW or CFASW containing 10mM EGTA (CF(G)ASW). Sperm showed symmetrical bending in both media. When each suspension was diluted 10 times in corresponding ASW containing 50 uM W-7, sperm stopped motility in either case. Ten minutes after, S50 ~.M calmodulin was added to each suspension. Then sperm in ASW reinitiated motility, but sperm in CF(G)ASW did not. This result suggests that W-7 bound to intracellular calmodulin after the drug passed through the plasma membrane. The recovery by calmodulin from the effect of W-7 required exogenous Ca**. This indicates that intracellular concentration of W-7 decreased as calmodulin bound to W-7 extracellularly. Figure 4a is a record on video tape of the recovery process of flagellar bending from inhibi- tory effect of W-7 by calmodulin. As calmodulin diffused, bending reinitiated at the proximal end of flagellum and propagated to the apical end. At that time, flagellar bending was symmetrical but beat frequency was too low to cause forward swimming. As time went on, so-called Ca’t quiescent sperm [17] were frequently observed. Then beat frequency increased with asymmetrical bending. Some sperm became to show symmetri- cal bending with time, but others remained still asymmetrical. Thus the inhibition of motility by W-7 was reversible. Theophilline is known to be an inhibitor of cyclic nucleotide phosphodiesterase and is effective on phosphodiesterase in sea urchin sperm (Dr. K. Ishida, personal communication). The exposure of A. crassispina sperm, immobilized by W-7, to 2 mM theophilline caused reinitiation of flagellar bending and in some sperm the bending prop- 66 F. Iwasa, Y. HaseGaAwa et al. (a) 13.37.88 13.37.34 1B 3Tee : y « om ee Fic. 4. 14.21.94 he co 8 2s 299 14.22.01 (a) Recovery of flagellar beating of A. crassispina sperm from inhibitory effect of W-7 by calmodulin. A. crassispina semen was diluted 2 10* times in ASW containing 50 uM W-7. After the sperm ceased movement, calmodulin was added to the sperm suspension. Top figure represents a sperm immobilized by 50 ~M W-7. Time (min, sec, 10~*sec) after the addition of calmodulin is indicated. (b) Incomplete recovery of flagellar beating of A. crassispina sperm from inhibitory effect of W-7 by theophilline. Numerals are min and sec after the addition of theophilline. agated. Loci of the reinitiation of bending were not always at the proximal end (Fig. 4b). The recovery by theophilline was incomplete because sperm did not show forward swimming and only continued slow bending for at least 20min. The effect of theophilline was independent of exoge- nous Cat. The recovery of motility by theophil- line was not seen in S. nudus sperm. Recovery or canceling of the effect of W-7 on the acrosome reaction by theophilline was not observed. Sperm immobilized by W-7 recovered their motility by merely diluting the sperm suspension with ASW. Figures 2a and 3 show that when the drug concentration was low, sperm reinitiated motility as time elapsed without any further treatments. It may be because the cells expel W-7 [11]. Nonidet-demembranated models prepared from the sperm immobilized by W-7 showed - normal swimming after reactivation by ATP. Effects of W-7 on sperm in Na* -free ASW Sea urchin sperm requires exogenous Na‘ for maintaining motility to eliminate H* produced by respiration in mitochondrion. NH,Cl or NH; temporally elevates intracellular pH (pHj) of immotile sperm and recovers motility in NaFASW [18]. When A. crassispina semen was diluted 1 x 10* times in NaFASW with or without 50 uM W-7, sperm were immotile in both cases. Ten mM NH,Cl reinitiated motility of both sperm. Dura- tion of motility was about 3 min in the absence of W-7 and about 30 sec in the presence of W-7. After the temporary movement ceased, 10mM NH, Cl was further added. Sperm in ASW without W-7 started to swim as before and third addition of Effects of Calmodulin Antagonists on Sperm 67 NH, Cl was also effective, whereas sperm in ASW with W-7 did not move this time. On the other hand, NH,C! did not reinitiate motility of sperm immobilized by W-7 in ASW. Therefore, W-7 does not seem to stop flagellar movement by preventing elevation or lowering of pH;. Effects of W-7 on sperm in NaFASW were studied in more detail using S$. nudus sperm. Sperm diluted 1Xx10* times were immotile with straightened flagella. When W-7 or W-5 was added 1 min after dilution, they had no effect. However, W-7 added 4 min after dilution reiniti- ated motility which lasted for 3-5 min. The pattern of immobilization in NaFASW with W-7 was different from that in ASW with W-7. Bending continued to propagate along the whole flagellum until it stopped. The pattern was similar in the case of NH,Cl, although the patterns of initiation by W-7 and by NH,Cl differed from each other in the following three points. 1) Recovery by W-7 occurred after some time had elapsed since sperm stopped motility in NaFASW, whereas this was not the case with NH,Cl. 2) NH,Cl reinitiated motility immediately, but W-7 had about 1 min lag time until reinitiation. 3) Although recovery by NH,Cl was repeatable, recovery by W-7 occurred only once. In NaFASW, duration of motility was 3-5 min with W-7, 10 min with NH,Cl, but only about 2 min with both W-7 and NH,Cl. When NH,Cl was added to sperm immobilized in W-7, a small O> (uM) 314 sperm ay portion of sperm reinitiated motility which con- tinued for about 1 min. Further addition of NH,Cl had no effect. W-5 had no reinitiation effect in contrast to W-7. However, about equimolar concentration of cal- modulin did not cancel the effect of W-7. Con- trary, calmodulin even prolonged the duration of motility. BSA in place of calmodulin in NaFASW containing W-7 also prolonged the swimming time. Immobilized sperm in Na*, Ca**-free ASW con- tainng 10mM EGTA (NaCF(G)ASW) also reinitiated motility when W-7 was added. The effect was more remarkable than in NaFASW. Calmodulin prolonged the duration of swimming, although calmodulin does not seem to bind to W-7 extracellularly in this case. Next we examined what happened after swim- ming in ASW. Semen was diluted 200 times in ASW and pelleted by centrifugation. When the pelleted sperm was diluted 1X10* times in NaFASW, sperm were immotile. NH,Cl reiniti- ated motility reversibly, but W-7 scarcely recov- ered motility. In summary, W-7 has two effects on sperm motility in NaFASW. Namely, W-7 stopped NH,Cl-induced motility of sperm, and reinitiated motility of immotile sperm. Oxygen consumption of sperm exposed to W-7 Oxygen consumption rates of sperm exposed to 50 uM W-7 or 10 ~M KCN were measured using 0 5 Fic. 5. (a a pi pre eg a a pe ea ee none 10 15 (min) Effects of W-7 and KCN on oxygen consumption rate of H. pulcherrimus sperm. Oxygen consumption was measured using suspensions of approximately 1 x 10° sperm/ml. Two min after the addition of sperm, 50 uM W-7 or 10 uM KCN was added. Motility is indicated as (—), (+) or (++). 68 F. Iwasa, Y. HASEGAWA et al. oxygen electrodes. As shown in Figure 5, only 3.3% in the presence of W-7 and 1.8% in the presence of KCN of oxygen consumption as compared with control value were detected. Sperm exposed to W-7 became immotile about 5min after the addition of W-7, whereas sperm in the presence of KCN swam actively in spite of the reduction in oxygen consumption (cf.[19]), although higher concentration of KCN such as 50 HM inhibited motility as described above. This means that the inhibition of respiration of sea urchin sperm does not necessarily lead to an inhibition of motility. The reduction of oxygen consumption by W-7 seems to be caused by the inhibition of motility. Changes in membrane potential (Vm) Relative values of membrane potential of sperm were estimated using a fluorescent probe, diO-C,- (5). Our previous work [20] which measured membrane potential of sea urchin sperm using the dye gave the following conclusions. 1) Membrane 4O NO O O Ded Time (min) De Onan OO 0! a0 potential of sea urchin sperm largely depends on concentration of Cl’. 2) Depolarization is de- tected by adding “egg sea water” or A23187, which is observed as an increase in fluorescence intensity. H.pulcherrimus sperm diluted 1X 10* times in ASW became immotile 2-3 min after exposure to diO-C,-(5). Duration of swimming was shorter as the dye concentration was higher. The measure- ment was completed within 30sec after adding diO-C,-(5) unless otherwise stated. Fluorescence intensity was settled as the dye was transferred from the solution to the sperm membrane. Membrane potential of H. pulcherrimus sperm - was measured in ASW, CF(G)ASW and NaFASW (Table 2). In these ASWs, increase of fluorescence intensity was detected when W-7 was added, indicating the depolarization of sperm membrane. Na*, which also reinitiates sperm motility in NaFASW as NH,Cl does, increased the fluores- cence intensity to the level of that in ASW. Depolarization of sea urchin sperm membrane in ASW caused by W-7 was further studied for 100 0) lOO 1502 200 W-7 or KCN Conc.(juM) Fic. 6. Effects of calmodulin antagonists and KCN on fluorescence intensities of diO-C,-(5) in sperm suspension. (a) Changes of fluorescence intensities after diluting H. pulcherrimus semen in ASW. Twenty yl of H. pulcherrimus semen was diluted in 4ml of ASW with or without W-7. Fluorescence was measured 30sec, 2min, 5min and 7 min after dilution by mixing 1 ml of each sperm suspension and equal volume of 0.1 uM diO-C,-(5) in ASW. Fluorescence intensity at zero time was measured by diluting semen 400 times in 2 ml of 0.05 pM diO-C,-(5) in ASW with or without W-7. @, 50 uM W-7; ©, 50 uM W-5; (J, none. (b) Fluorescence intensities after 5-min incubation of H. pulcherrimus sperm suspension. Semen of H. pulcherrimus was diluted 200 times in ASW containing 0, 5, 10, 15, 25, 35 or 50 uM W-7, and after 5 min incubation fluorescence intensities were measured by mixing equal volume of W-7 and 0.1 uM diO-C,-(5) in ASW. Effects of Calmodulin Antagonists on Sperm 69 TABLE2. Relative fluorescence intensities (%) of diO-C,-(5) in sea urchin sperm suspen- sions Reagent ASW CF(G)ASW NaFASW None 32° < 30 <30 50 uM W-7 95 97 76 50 uM W-S5 61 59 40 50 uM KCN 60 ON) neds 10mM NaCl — — 55 H. pulcherrimus semen was diluted 200 times in each ASW containing various reagents and mixed with equal volume of 0.2 ~M diO-C,-(5) in each ASW 4 min after dilution. Sperm suspensions diluted 200 times in NaFASW became immotile within 2 min. * Fluorescence intensity in each ASW without sperm was taken as 100%. > Not determined. (a) none y, fluorescence intensit (arbitrary unit) fluorescence intensity (arbitrary unit) ZS Lethe Gio] SB time (min) time dependency and dose dependency. Com- pared with control experiment, of which fluores- cence intensity was nearly constant after dilution, W-7 gradually increased the fluorescene intensity. With W-S, on the other hand, values at zero time and 30sec were somewhat higher than control, but almost the same as control thereafter (Fig. 6a). Figure 6b shows that the fluorescence changed a little up to 25 ~M of W-7 and remarkably increased above 25 uM. KCN did not increase the fluores- cence at 25 uM and brought about 2-fold increase at 200 ~«M. Thus depolarization effect was greater with W-7 as compared with the effect of KCN. Similar depolarization to that caused by W-7 was observed when sperm suspension was exposed to CCCP, an effective uncoupler of oxidative phos- phorylation, in ASW. (b) NH,Cl (d) CCCP Oo eZ = SrA. 6 7 8 9 time (min) Fic. 7. Effects of NH,Cl, W-7 and CCCP on membrane potential and motility of H. pulcherrimus sperm in ASW. At first, fluorescence of 0.05 ~M diO-C,-(5) in ASW was measured. Following reagents were contained in the above solution. (a) none, (b) 10mM NH, Cl, (c) 50 uM W-7, (d) 1x10~’M CCCP. Next, changes in fluorescence intensities were recorded continuously after 400 times dilution of semen into each ASW. Motility of the same sperm suspension was evaluated as —, +, + and ++. 70 F. Iwasa, Y. HASEGAWA et al. After measuring the fluorescence intensities of 0.1 uM diO-C,-(5) in ASW or NaFASW contain- ing 10mM NH,Cl, 504M W-7 or 1—2X10° ’M CCCP, H. pulcherrimus sperm were diluted in each solution by 400 times (ASW) or 2,000 times (NaFASW) to record changes of fluorescence continuously (Figs. 7 and 8). Sperm motility was also observed. In ASW, NH,Cl did not change membrane potential (Vm) and motility so much (Fig. 7b), but in NaFASW it raised Vm to the level of that in ASW concomitant with activation of motility (Fig. 8b). W-7 raised Vm and reduced motility in ASW (Fig. 7c). As mentioned previ- ously, W-7 caused temporary movement in NaFASW. A rise in Vm was observed after such a movement (Fig. 8c). CCCP increased Vm as W-7 did in ASW, but the inhibition of motility by CCCP occurred more slowly than that by W-7 (Fig. 7d). On the other hand, a rise in Vm by CCCP in NaFASW was less than that by W-7, and reinitiation of motility was not observed (Fig. 8d). Intracellular concentration of calcium (Ca** ;) Finally the question as to whether W-7 changes Ca’t,; of sperm was examined. H. pulcherrimus (a) none (b) NH,Cl fluorescence intensity (arbitrary unit) OM) BRS Gh Ona! time (min) DS & time (min) sperm treated with Quin-2 were diluted 200 times in CFASW and fluorescence intensity was meas- ured. Emission peak was shifted from 435nm to 470 nm by hydrolysis of Quin-2 tetramethyl ester. Sperm were motile during the measurement. Fluorescence intensity of sperm suspension in CFASW (200 times diluted) changed little by the addition of calmodulin antagonists (data not shown). We also measured Ca’; of sperm in NaFASW. H. pulcherrimus sperm treated with Quin-2 in NaFASW and diluted 200 times in this ASW were immotile, but reinitiated motility by 20mM NH,Cl. Emission peaks were found at 470 nm and 435nm. The sperm was diluted in NaCFASW and the fluorescence intensity was measured. NH,Cl reinitiated motility temporally, but did not change Ca’t;. Although W-7 also reinitiated motility in NaFASW, no change was found in Ca**; (data not shown). In conclusion, W-7 does not seem to change Ca’**,. We could not find any parallel change of Ca’t; to those of motility, the acrosome reaction or membrane potential. (c) W-7 (a) (CGE P liye Sn 2 time (min) Oia eee Sha time (min) Fic. 8. Effects of NH,Cl, W-7 and CCCP on membrane potential and motility of H. pulcherrimus sperm in NaFASW. Measurements were made as in Figure 7, except that dilution of semen was 2,000 times and that concentrations of diO-C,-(5) and of CCCP were 0.1 4M and 2x107~’7M, respectively. Effects of Calmodulin Antagonists on Sperm Wa DISCUSSION When sea urchin sperm were exposed to a calmodulin antagonist, W-7, in ASW, flagellar movement was arrested, the acrosome reaction failed to occur and sperm membrane was depo- larized, although we could not really know which memberane was depolarized to what extent (see below). Inhibition of flagellar movement was also observed with other calmodulin antagonists, T1233 and TFP. These three phenomena were dependent on the amount of W-7, and occurred relatively slowly, that is, not instantaneously. It appears to take some time before W-7 reaches its targets. In contrast, no remarkable change was detected in Ca**.. Therefore, W-7 does not seem to inhibit flagellar movement by raising Ca** concentration in the flagellum. Nor W-7 inhibits the acrosome reaction by preventing the increase of Ca** concentration in the sperm head. It should be noted, however, that only the average level of Ca’t in sperm was estimated by using Quin-2. The obtained results do not exclude the possibility of local fluctuation of Ca** concentration within the cells. Membrane potential measured may reflect mainly that of the inner mitochondrial membrane, because CCCP raised Vm in ASW. However, effects of NH,Cl, W-7 and CCCP on Vm and sperm motility in ASW and NaFASW were dif- ferent from each other. Therefore, it is not possible that W-7 functions only as an uncoupler of oxidative phoshorylation in mitochondria. Unlike W-7, CCCP had no inhibitory effect on repeated reinitiation of motility by NH,Cl in NaFASW. Concerning the effects of W-7 in ASW and NaFASW, the following two observations were made: W-7 stopped motility of sperm which had optimal pH, and were motile; W-7 gave a tempo- rary motility to sperm which had low pH; and were immotile. Targets of W-7 in these two effects seem to be different. The effect of W-7 in NaFASW was not canceled by equimolar concentration of calmo- dulin, raising a question as to whether the effect came from the inhibition of sperm calmodulin by W-7. Such an effect was also observed in NaCF- (G)ASW. In our preliminary experiments, changes in flagellar and ciliary movement caused by W-7 were also observed in golden hamster sperm and cilia of sea urchin embryos. W-7 affected only the princi- pal bend of golden hamster sperm from the cauda epididymis leaving the reverse bend unaffected (cf. [21]), whereas W-5 had no such an effect. H. pulcherrimus embryos at mid-gastrula stage which show straightforward swimming with rotation were exposed to W-7, W-7 plus calmodulin or W-S. Among them, only W-7 changed motility pattern to either rotation or straight swimming only. The results so far obtained suggest that W-7 has some targets in sperm concerning their flagellar movement. No direct evidence, however, has been obtained to indicate that they are calmodulin. Theophilline, a phosphodiesterase inhibitor, par- tially released sea urchin sperm from the inhibitory effect of W-7. If calmodulin is main target, the observation indicates involvement of calmodulin in regulation of cAMP content, because continuation of sea urchin sperm motility requires cAMP- mediated phosphorylation [5]. In sea urchin sperm, creatine phosphokinase seems to have a role to shuttle to mediate high energy phosphate between mitochondrion and tail [22]. If the enzyme is inhibited by dinitro- fluorobenzene, flagellar bending is restricted to the proximal end near the mitochondrion and respira- tion rate is reduced to one third [22]. In the presence of W-7 in ASW, sea urchin sperm exhibited a similar restricted bending at the pro- ximal region of the flagellum before the final arrest, although oxygen consumption was much reduced to 3.3% of the control. The result suggests the possibility that calmodulin partici- pates in energy supply throughout the flagellum. Measurement of ATP and creatine phosphate content in sperm exposed and not exposed to W-7 revealed no significant differences between these two groups [23]. Furthermore, estimation of stiffness of the flagellum immobilized by W-7 according to the method of Okuno and Hiramoto [24] indicated that they are in a relaxed state [23]. Since lack of ATP results in a rigor state of the flagellum [25], the above result would mean that there remained the sufficient amount of ATP in the immotile flagellum. V2 ACKNOWLEDGMENTS The authors wish to thank Dr. T. Kobayashi for teaching us measurements of membrane potential using fluorescent probe and for supplying the reagent and Ms. N. Niitsu-Hosoya for her help in measuring oxygen consumption. We also thank Drs. I. Nishida and M. Miyao for their helpful discussion. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan (Nos. 58340041 and 59480020) to H. M. 10 11 REFERENCES Epel, D. (1978) Mechanism of activation of sperm and egg during fertilization of sea urchin gametes. In “Current Topics in Developmental Biology”. Vol. 12. Ed. by A. A. Moscona and A. Monroy, Academic Press, Inc., New York, pp. 185-246. Summers, R. G., Talbot, P., Keough, E. M., Hylander, B.L. and Franklin, L. E.(1976) Iono- phore A23187 induces acrosome reactions in sea urchin and guinea pig spermatozoa (1). J. Exp. Zool., 196: 381-385. Okuno, M. and Brokaw, C.J. (1981) Effects of Triton-extraction conditions on beat symmetry of sea urchin sperm flagella. Cell Motility, 1: 363-370. Morisawa, M. and Okuno, M. (1982) Cyclic AMP induces maturation of trout sperm axoneme to initiate motility. Nature (London), 295: 703-704. Ishiguro, K., Murofushi, H. and Sakai, H. (1982) Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of Triton X-100 models of sea urchin and starfish spermatozoa. J. Cell Biol., 92: 777-782. Tash, J.S. and Means, A. .R. (1982) Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium. Biol. Reprod., 26: 745-763. Tash, J.S. and Means, A. R. (1983) Cyclic adeno- sine 3,5’ monophosphate, calcium and _ protein phosphorylation in flagellar motility. Biol. Reprod., 28: 75-104. Jones, H. P., Bradford, M. M., McRorie, R. A. and Cormier, M. J. (1978) High levels of a calcium- dependent modulator protein in spermatozoa and its similarity to brain modulator protein. Biochem. Biophys. Res. Commun., 82: 1264-1272. Sano, K. (1983) Inhibition of the acrosome reaction of sea urchin spermatozoa by a calmodulin antago- nist, N-(6-aminohexyl)-5-chloro-1-naphthalene-sul- fonamide (W-7). J. Exp. Zool., 226: 471-473. Sano, K. (1982) Ca requirement and effect of calmodulin inhibitor W-7 on sea urchin fertilization. Dev. Growth Differ., 24: 406 (Abstract). Oono,S. and Hidaka, H. (1981) Calmodulin 12 13 14 15 16 7 18 19 20 21 pip ig) 24 25 F. Iwasa, Y. HAsEGAwa et al. sogaizai—Saibounai bunpu. In “Calmodulin”. Ed. by H. Hidaka and S. Kakiuchi, Kodansha, Tokyo, pp. 137-139 (in Japanese). Hidaka, H., Yamaki, T., Naka, M., Tanaka, T., Hayashi, H. and Kobayashi, R. (1980) Calcium- regulated modulator protein interacting agents in- hibit smooth muscle calcium-stimulated protein kinase and ATPase. Mol. Pharmacol., 17: 66—72. Teo, T., Wang,T.H. and Wang, J. H. (1973) Purification and properties of the protein activator of bovine heart adenosine 3’, 5’-monophosphate phosphodiesterase. J. Biol. Chem., 248: 588-595. Hoffman, J. F.and Laris, P. C. (1974) Determina- tion of membrane potentials in human and Amphiuma red blood cells by means of fluorescent probe. J. Physiol., 239: 519-552. Sims, P.J., Waggoner, A.S., Wang, C.-H. and Hoffman, J. F. (1974) Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochem., 13: 3315-3330. Tsien, R. Y. (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature (London), 290: 527-528. Gibbons, B. H. and Gibbons, I. R. (1980) Calcium- induced quiescence in reactivated sea urchin sperm. J. Cell Biol., 84: 13-27. Bibring, T., Baxandall, J. and Harter, C. C. (1984) Sodium-dependent pH regulation in active sea urchin sperm. Dev. Biol., 101: 425—435. Mohri, H. (1956) Studies on the respiration of sea-urchin spermatozoa I. The effect of 2,4- dinitrophenol and sodium azide. J. Exp. Biol., 33: 73-81. Kobayashi, T., Iwasa, F. and Mohri, H. (1981) Membrane potential change during acrosome reac- tion of sea urchin sperm. Zool. Mag., 90: 416 (Abstract). Ishijima, S. and Mohri, H. (1985) A quantitative description of flagellar movement in golden hamster spermatozoa. J. Exp. Biol., 114: 463—475. Thmbes, R. M. and Shapiro, B. M. (1985) Metabo- lite channeling: A phoshorylcreatine shuttle to mediate high energy phosphate transport between sperm mitochondrion and tail. Cell., 41: 325-334. Iwasa, F. (1985) Studies on functions of calmodulin in echinoderm eggs and sperm. D. Sc. Thesis, The University of Tokyo. Okuno,M. and _ Hiramoto, Y. (1979) Direct measurements of the stiffness of echinoderm sperm flagella. J. Exp. Biol., 79: 235-243. Gibbons, B. H. and Gibbons, I. R. (1974) Prop- erties of flagellar “rigor waves” formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm. J. Cell Biol., 63: 970-9835. ZOOLOGICAL SCIENCE 4: 73-80 (1987) © 1987 Zoological Society of Japan Isolation of a Paramecium tetraurelia Mutant with Short Clonal Life-span and with Novel Life-cycle Features YOSHIOMI TAKAGI, TAKAKO SUZUKI and CHIAKI SHIMADA Department of Biology, Nara Women’s University, Nara 630, Japan ABSTRACT— We have isolated a mutant of Paramecium tetraurelia with a short clonal life-span, about one tenth that of the wild type stock. The short clonal life-span was coupled with senescent characteristics such as a low fission rate, a tendency to undergo autogamy even with excess food, and an intraclonal heterogeneity of division potential. However, the short clonal life-span of the mutant was coupled also with presenescent characteristics such as a high rate of food vacuole formation and excretion, high viability after autogamy, and the presence of autogamy-immaturity stage. It was normal also in its abilities to swim backward, to discharge trichocysts, and to divide the macronucleus properly. This mutant is, therefore, not a consistent senescence mutant but a mutant with conflicting life-cycle features in which presenescent and senescent characteristics are mingled. We can now expect to isolate additional mutants with altered life-cycle features, which will make it possible to use this protozoan species to study the genetic control of aging and life-span. INTRODUCTION Paramecium cells age and die as normal diploid cells from multicellular organisms such as humans, chickens and hamsters cultured in vitro age and die [1-5]. The maximum limit of the division potential is, in either case, species-specific and predictable; for example, it is about 250 fissions for P. tetraurelia [6] and about 600-700 fissions for P. caudatum [7-9] when conjugation or autogamy marks time zero. It is very likely, therefore, that the process of limiting the cellular or clonal life-span is somehow programmed, not being completely subject to stochastic events. One of the most useful ways to understand the programmed events of the complicated process may be the isolation of mutants involved in the events and the dissection of the process by breeding analysis. Although mutants with altered life-spans have been reported in several organisms [10-15], the advantage of the genetic analysis has not been fully exploited. In fruit flies or nematodes, which are suitable for genetic crosses, the organismic life- span, not the cellular life-span, can be the target of Accepted August 9, 1986 Received July 7, 1986 the analysis because of the postmitotic nature of their aging cells. In humans or chickens where cellular aging is evident, genetic crosses are im- possible or difficult. In mice or rats where genetic crosses are easier, cells readily transform to immortal cell lines when transferred to in vitro culture so that cellular aging becomes elusive [16, 17]. In some fungi, which include mutants that have various division potentials or those that “senesce” [18], the life is basically immortal and thus appears to be irrelevant to cellular aging. Paramecium has been a candidate for genetic analysis of aging and life-span [19], as Paramecium and related ciliates have been used for genetic analysis of clonal development [20-25]. But no mutants with short life-spans had been known, until some previously established mutants were found recently to be associated with short life- spans [26]. We wish to report here a newly isolated mutant that was screened for short life-span after chemical mutagenesis. The association revealed by Aufderheide and Schneller [26] was not seen in this mutant. This mutant expressed young and old characteristics simultaneously. In P. tetraurelia, the nuclear and cytoplasmic genetic background can be kept invariable through autogamies and the cells showing short life-span can be used for 74 Y. TAKAGI, T. SUZUKI AND C. SHIMADA genetic crosses. Thus, finding a short life-span mutant may open the way for genetic studies of aging and life-span. MATERIALS AND METHODS Cells Wild-type stock 51 of Paramecium tetraurelia was used for mutagenesis and as a control culture. KL-DD6, a trichocyst non-discharging mutant carrying nd242 gene [27], was provided by Dr. D. Nyberg of University of Illinois and used as another control culture. Culture conditions Culture medium was prepared after Sonneborn [28] with a modification of Cerophyl concentra- tion; 10g/l instead of 2.5 g/l was used. Klebsiella pneumoniae was inoculated 1—2 days before use. Cultures were maintained in serial isolation lines with a single cell transfer daily or on alternate days. Ages of lines were given in logon where n is the number of fission products. Plastic hemagglu- tination plates with 80 holes (Tomy Seiko, T-2) were used as culture vessels. The plates were sterilized before use by exposure to UV-light and covered with Saran wrap [29]. Cultures were kept at about 25°C unless other- wise specified. Mutagenesis and screening Cells in a logarithmic growth-phase were treated with 80 ug/ml N-methyl-N -nitro-N-nitroso- guanidine for 1 hr. Then they were washed three times with fresh culture medium and suspended in small drops each including about 50 cells. The size of the culture drops was controlled to allow the cells to undergo one cell division. The additional cell division would be necessary to induce auto- gamy [30] but further divisions were avoided to minimize producing cells with the same genotype. After ascertaining autogamy in more than 95% of the sampled cells from each drop, the remaining cells were isolated into the holes of culture plates containing 0.5ml culture medium. They were cultivated in isolation lines (primary lines) at 27-30°C, while the plate with the surplus cultures remaining after the first isolation was stored at 10-20°C. When some primary lines died before 50 fissions, secondary lines were reinitiated from the corresponding surplus cultures. Since the clonal life-span is coupled with the number of fissions rather than days [31], they should have remained younger. Therefore, we screened the secondary lines to determine if they might die again before 50 fissions. RESULTS Isolation of a mutant with short clonal life-span Among 80 mutagenized lines, 69 died before the age of 50 fissions. Among the 69 early death lines, 23 remained alive in the corresponding surplus cultures in the stored plate. Among 23 secondary lines reinitiated, four lines died again before the age of 50 fissions. One of the four lines, however, named d4-SL4, escaped clonal extinction by means of autogamy. Figure 1 shows the survival curve of a d4-SL4 clone in contrast with that of a wild type stock 51 clone. For each clone, 20 lines were initiated from 20 cells of 100% autogamy cultures and cultivated in serial isolations without replacements. The average clonal life-span of the 20 d4-SL4 lines was 4 fissions and the maximum was 12 fissions; the stock 51 average was 150 fissions and the maximum 210 fissions. The extremely short clonal life-span of d4-SL4 was inherited through four succeeding autogamies (Fig. 2). Profiles of the survival curve were similar for the autogamous progeny of four generations, with life-spans of the fourth autogamous progeny being most variable ranging from 0 to 19. Besides these data, we have repeatedly ascertained the short clonal life-span of d4-SL4. Occasionally, however, some lines managed to live more than 50 fissions. From such lines, we initiated new isola- tion lines from a 100%-autogamy culture and maintained them with a complete check of auto- gamy: all but one of the daily fission products were sacrificed to examine if they had fragmented macronuclei. The maximal life-span of d4-SL4 so far examined in isolation cultures was 22 fissions, if the lines showing evidence of autogamy were ~ excluded. Thus, d4-SL4 terminates its clonal A Mutant with Short Clonal Life-span 75 100 i > > — = 50 W °o 0 0 50 Clonal age Fic. 1. 150 200 in fiSSIONS Survival curves of mutantstock d4—SL4 (closed circles) and wild-type stock 51 (open circles). Clones were cultivated at 25+2°C in 20 isolation lines without replacements. life-span either in early death or in autogamy. In spite of the short life, the progeny viability of 50 oO S (S) 100 oO (=) Sri vanvel (@) On (2) *lo (2) © iD) > Oy) (oe) NOMA Ze OD joe) ie) Oo Clonal age in fissions d4-SL4 was very high as shown in Figure 2, where most of the progeny showed an initial survival of 100%. Accompanying characteristics of d4-SL4 d4-SL4 is a slow divider. This is obvious in isolation cultures where fission rates were scored daily or on alternate days. In case of two clones in Figure 1, for example, the mean daily fission rate of stock 51 and d4-SL4 was 3.7 and 0.7, respect- ively during the first three days after autogamy, and 3.9 and 0.4, respectively during the next three day-interval. d4-SL4 is prone to undergo autogamy. In isolation lines of wild-type stocks, autogamy is seldom seen in logarithmically growing surplus cultures, until they reach their final life-spans [32, Takagi and Nobuoka, unpublished ]. In isolation lines of d4-SL4, however, cells with fragmented macronuclei indicative of autogamy were often detected in well-fed surplus cultures. d4-SL4 is heterogeneous in division potential. All of the cells from a young wild-type culture are able to divide (homogeneous division potential) to produce the same number of cells not deviating Fic. 2. Survival curves of four succeeding autogamous progeny of d4—SL4 from top to bottom. The num- ber of isolation lines was 10, 30, 50, and 20 for each of four autogamous progenies, respectively. The fourth progeny were studied in 3 different exauto- gamous cultures with 20 isolation lines each. 716 Y. TAKAGI, T. SUZUKI AND C. SHIMADA Q \ Fic. 3. 3 Y Zz GG Cd e Cd Intraclonal heterogeneity of division potential in d4—-SL4. A single cell of 3 fissions old (1) was allowed to divide for 12 days, during which all of the fission products were isolated every 3 days to follow the cell fate. Part of the 12th day-products were further isolated and allowed to divide for 3 more days. The number of cells produced on the 12th and 15th day was indicated in the relative length of the arch. For 12 days or during 4 isolations, there occurred heterogenous cell lineages; died (2), grew fastest (3), grew slowest (4), for example. from 2" where n is the number of cell divisions less than 4 (synchronous division) [7]. d4-SL4 cells often produced division products deviating from 2", including n=O as exemplified in Figure 3. Starting with a single cell of three fissions old (1 in Fig. 3), we isolated all of the division products produced for three days; we repeated this proce- dure three times or, as for some products, four times. During four isolations or 12 days, the fastest- (3 in Fig. 3) and slowest-grown line (4 in Fig. 3) underwent 11.7 and 7.6 fissions, respective- ly. Some lines died during this interval (2 in Fig. 3, for example). Even in some 12th day products that had experienced relatively uniform divisions, in-— traclonal heterogeneity of division potential be- came evident on the Sth isolation or on the 15th day. Characteristics sharing with wild-type stock d4-SL4 is morphologically normal at a dissecting microscopic level. d4-SL4 is normal in its capacity to discharge trichocysts. This was ascertained by dropping a saturated solution of picric acid on mutant cells. They discharged trichocysts regardless of the cell-cycle and life-cycle stages. d4-SL4 is normal in its capacity to swim back- ward. This was ascertained by introducing mutant A Mutant with Short Clonal Life-span ii) cells into Ba** test-solution [33]. d4-SL4 can distribute the macronucleus proper- ly to the daughter cells. Dividers were taken from log-phase cultures, stained with Dippell’s solution [34], and examined under a compound micro- scope. The number of cells with improperly dividing macronuclei were two among 53 dividers in d4-SL4, and one among 55 dividers in stock 51. This difference can be regarded negligible from the standard of macronuclear misdividers [26]. d4-SL4 has a normal rate of food-vacuole formation. This was ascertained by counting black food vacuoles after 0.06% (final concentration) Rotring ink was introduced to log-phase cultures. The average rate of food-vacuole formation in six experiments was compared among three stocks, i.e., d4-SL4 of unidentified clonal ages, stock 51 of the clonal ages younger than 50 fissions, and KL-DD6 younger than 15 fissions (Fig. 4). There were no Statistical differences among three stocks at any time (t-test, p<0.01). e d4-SL4 © Stock 51 ASAE DDS W) v S 10 UO fae} > 18} oO oO O Nee ES) <3) EQ) & = Ze 0 10 20 30 Minutes in label Fic.4. Rate of the food-vacuole formation into 3 stocks. Cells were introduced in 0.06% Rotring ink and the number of black food vacuoles were scored every Smin. Mean+SD of 6 experiments are shown. d4-S14 has a normal rate of food-vacuole excretion. This was ascertained by counting black food-vacuoles after the log-phase culture was first colored and then washed three hours later. Figure 5 shows the results of six experiments. The difference among three stocks at any time was insignificant (t-test, p<0.05). d4-S14 has an autogamy immaturity period. The autogamy immaturity period during which autogamy cannot be induced is the first stage of the clonal life-cycle following autogamy [1, 35]. In order to test a hypothesis that d4-SL4 may be a permanent senescence mutant truncating the early part of the life-cycle stages, cells of known ages were induced to undergo autogamy. In wild-type stock 51, no autogamy could be induced before 14 fissions, and 100% autogamy was possible after 21 fissions. In d4-SL4, we were able to induce autogamy in cells at the age of 7 fissions or more but failed in cells at the ages of 5 and 6 fissions. oe) G425u4 if Oo Stoek 5! = Ke DDS By elO uv Oo = O ce > so) ) < i) © ra) E | FE 6) 10 20 30 Minutes in unlabeled medium Fic.5. Rate of the food-vacuole excretion in 3 stocks. Cells were preincubated in culture medium contain- ing 0.06% Rotring ink for 3hr to stain the food vacuoles black. After cells were washed three times by mild centrifugation, the number of food vacuoles was scored every 5min. Mean+SD of 6 experi- ments are shown. 78 Y. TAKAGI, T. SUZUKI AND C. SHIMADA DISCUSSION This is the first report on a mutant with a short clonal life-span that was isolated following mutagenesis. Clones with extremely short life- spans are known to occur commonly after conjuga- tion or autogamy especially of aged cells; but they are all lethal and never established as a short life-span stock [35]. Short life-spans could result from any defective mutations in macronuclear genes which are essential to the clonal life of Paramecium. Since the macronucleus develops from mitotic products of the fertilization nucleus during autogamy and since all of the genetic loci become homozygous after autogamy [35], any defective changes even of the recessive type in the micronucleus would be expressed in the macronu- cleus after autogamy. The consequence should be a production of lethal clones with extremely short clonal life-spans. The present stock d4-SL4 was able to repeat the short clonal life-cycles starting with the autogamy-immaturity stage and with somewhat restored fission rates. Therefore, we can exclude the possibility that mutations in d4-SL4 are in the loci controlling key metabolic processes. This study shows that the clonal life-span can be extremely shortened keeping normal the micronuclear function to produce fertile progeny and also keeping normal the macronuclear functions to take food, excrete, swim, and respond to stimuli. Many parameters characterize the senescent stage of the clonal life-cycle of Paramecium [4]. Although they do not necessarily come to an expression at the same time in all of the members of the clone, their expressions are more ‘or less coordinated [36]. Low fission rate [1], low progeny viability after autogamy [29], intraclonal heter- ogeneity of division potential [7], a proneness to undergo autogamy in excess food [32], and the reduced rates of the food vacuole formation and excretion [32] are all characteristics of the senes- cent stage. Truly senescent cells expressing some of these characteristics would be expected to express also the others. But this was not true of d4-SL4. d4-SL4 might be considered young in the high progeny viability after autogamy and in the high rate of the food vacuole formation and excretion, while it might be considered old in its low fission rate, in its intraclonal heterogeneity of division potential, and in its autogamy-prone nature in excess food. d4-SL4 might be considered senescing early in that it has a short interval of autogamy immaturity and in that its low fission rate declines further in six days after autogamy. d4-SL4 is, therefore, not a uniformly senescent mutant but a mutant with conflicting life-cycle features in which presenescent and senescent characteristics are mingled. Recently, Aufderheide and Schneller [26] found several stocks with short clonal life-spans among previously isolated nd (trichocysts non-dis- charging) mutants of P. tetraurelia. They further demonstrated that the short clonal life-spans were not associated with the inability to discharge trichocysts but rather were associated with the inability to divide the macronucleus properly. Unequal distribution of the macronucleus to the daughter cells is a senescent characteristic [36]. Perpetual unequal distribution could be a sufficient condition for the short clonal life-span, as dis- cussed by Aufderheide and Schneller [26]; but it cannot be a necessary condition because no asso- ciation between them was found in d4-SL4. Similarly, the factor that is responsible for the low fission rate could be responsible for the short clonal life-span in d4-SL4, although their associa- tion might not be found in other mutants. By isolating various life-cycle mutants, we may under- stand if two factors are dissectible and what is a necessary and sufficient condition for the short life-span. If we could find a short life-span mutant without any accompanying senescent characteris- tics, we might identify the genetic locus that controls the clonal life-span directly. Breeding analysis on d4—-SL4 is under investiga- tion. The low fission rate is used as a tentative marker of the short life-span, because the deter- mination of the life-span difference is a laborious work and because the short life-span of d4-SL4 is often perturbed by autogamy. Preliminary results suggest that the short life-span of d4-SL4 is inherited through the nucleus, not the cytoplasm: No segregation of the marker phenotype was found in F, progeny after conjugation between d4-SL4 and a wild-type stock, and segregation was A Mutant with Short Clonal Life-span 79 found in F, progeny after autogamy, with a marker gene indicating normal conjugation and autogamy. Although it is still a long way off to determine if the short life-span is dissectible from the low fission rate and how many genes are involved, we can now expect to isolate other mutants with altered life-cycle features and to perform the breeding analysis with a hope that we will have significant insights on how life-span is limited. ACKNOWLEDGMENTS This work was supported in part by a grant from the Itoh Science Foundation. We are grateful to Dr. P. Abhayavardhani for improving the manuscript. 10 11 REFERENCES Sonneborn, T. M. (1954) The relation of autogamy to senescence and rejuvenescence in Paramecium aurelia. J. Protozool., 1: 38—53. Hayflick, L. and Moorhead, P. S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Res., 25: 585-621. Hayflick, L. (1977) The cellular basis for biological aging. In “Handbook of the Biology of Aging”. Ed. by C.E.Finch and L. Hayflick, Van Nostrand Reinhold, New York, pp. 159-186. Smith-Sonneborn, J. (1981) Genetics and aging in Protozoa. Int. Rev. Cytol., 73: 319-354. Raes, M. and Remacle, J. (1983) Ageing of ham- ster embryo fibroblasts as the result of both dif- ferentiation and _ stochastic mechanisms. Exp. Gerontol., 18: 223-240. Smith-Sonneborn, J. (1985) Aging in unicellular organisms. In “Handbook of the Biology of Aging”, 2nd ed. Ed. by C.E.Finch and E.L.Schneider, Van Nostrand Reinhold, New York, pp.79-104. Takagi, Y. and Yoshida, M. (1980) Clonal death associated with the number of fissions in Para- mecium caudatum. J. Cell Sci., 41: 177-191. Karino, S. and Hiwatashi, K. (1981) Analysis of germinal aging in Paramecium caudatum by micro- nuclear transplantation. Exp. Cell Res., 136: 407-415. Karino, S. and Hiwatashi, K. (1984) Resistance of germinal nucleus to aging in Paramecium: Evidence obtained by micronuclear transplantation. Mech. Ageing Dev., 26: 51-66. Smith, J. R. (1978) Genetics of aging in lower organisms. In “The Genetics of Aging”. Ed. by E. L. Schneider, Plenum, New York, pp.137-149. ; Takeda, T., Hosokawa, M., Takeshita, S., Irino, M., Higuchi, K., Matsushita, T., Tomita, Y., Yasu- 12 13 14 15 16 17 18 19 20 21 22 Zz, 24 75) 26 27 hira, K., Hamamoto, H., Shimizu, K., Ishii, M. and Yamamuro, T. (1981) A new murine model of accelerated senescence. Mech. Ageing Dev., 17: 183-194. Dick, J. E. and Wright, J. A. (1984) Human diploid fibroblasts with alteration in ribonucleotide reduc- tase activity, deoxyribonucleotide pools and in vitro lifespan. Mech. Ageing Dev., 26: 37-49. Brown, W. T. (1985) Genetics of human aging. Rev. Biol. Res. Aging, 2: 105-114. Johnson, T. E. (1985) Aging in Caenorhabditis ele- gans: Update 1984. Rev. Biol. Res. Aging, 2: 45-60. Lints, F. A. and Bourgois, M. (1985) Aging and lifespan in insects with special regard to Drosophila: Review 1982-1984. Rev. Biol. Res. Aging, 2: 61-84. Todaro, G.J. and Green, H. (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol., 17: 299-313. Meek, R.L., Bowman, P.D. and Daniel, C. W. (1980) Establishment of rat embryonic cells in vitro. Relationship of DNA synthesis, senescence, and acquisition of unlimited growth potential. Exp. Cell Res., 127: 127-132. Bertrand, H., Chan, B. S-S. and Griffiths, A. J. F. (1985) Insertion of a foreign nucleotide sequence into mitochondrial DNA causes senescence in Neurospora intermedia. Cell, 41: 877-884. Aufderheide, K. J. (1984) Cellular aging: An over- view. In “Cellular Ageing”. Ed. by H. W. Sauer, Karger, Basel, pp. 2-8. Siegel, R.W. (1961) Nuclear differentiation and transitional cellular phenotypes in the life cycle of Paramecium. Exp. Cell Res., 24: 6-20. Siegel, R. W. (1967) Genetics of ageing and the life cycle in ciliates. Symp. Soc. Exp. Biol., 21: 127-148. Bleyman, L. K. and Simon, E. M. (1967) Genetic control of maturity in Tetrahymena pyriformis. Genet. Res. Camb., 10: 319-321. Bleyman, L. K. (1971) Temporal patterns in the ciliated protozoa. In “Developmental Aspects of the Cell Cycle”. Ed. by I.L.Cameron, G.M.Padilla and A. M. Zimmer, Academic Press, New York, 9 OVI. Nanney, D. L. and Meyer, E. B. (1977) Traumatic induction of early maturity in Tetrahymena. Ge- netics, 86: 103-112. Myohara, K. and Hiwatashi, K. (1978) Mutants of sexual maturity in Paramecium caudatum selected by erythromycin resistance. Genetics, 90: 227-241. Aufderheide, K. J. and Schneller, M. (1985) Phe- notypes associated with early clonal death in Para- mecium tetraurelia. Mech. Ageing Dev., 32: 299-309. Nyberg, D. (1978) Genetic analysis of trichocyst discharge of the wild stocks of Paramecium tetraure- 28 29 30 oi 80 lia. J. Protozool., 25: 107-112. Sonneborn, T. M. (1970) Methods in Paramecium research. In “Methods of Cell Physiology, Vol.4”. Ed. by D. M. Prescott, Academic Press, New York, pp. 241-339. Rodermel, S.R. and Smith-Sonneborn, J. (1977) Age-correlated changes in expression of micronu- clear damage and repair in Paramecium tetraurelia. Genetics, 87: 259-274. Mikami, K. and Koizumi, S. (1983) Microsurgical analysis of the clonal age and the cell-cycle stage required for the onset of autogamy in Paramecium tetraurelia. Dev. Biol., 100: 127-132. Smith-Sonneborn, J. and Reed, J. C. (1976) Calen- dar life-span versus fission life-span of Paramecium aurelia. J. Gerontol., 31: 2-7. 34 33 34 35 36 Y. TaKaai, T. SUZUKI AND C. SHIMADA Smith-Sonneborn, J. and Rodermel, S. R. (1976) Loss of endocytic capacity in aging Paramecium. J. Cell Biol., 71: 575-588. Chang, S-Y. and Kung, C. (1973) Temperature- sensitive pawns: Conditional behavioral mutants of Paramecium aurelia. Science, 180: 1197-1199. Dippell, R. V. (1955) A temporary stain for Para- mecium and other ciliate protozoa. Stain Technol., 30: 69-71. Sonneborn, T. M. (1974) Paramecium aurelia. In “Handbook of Genetics, Vol.2”. Ed. by R.C. King, Plenum, New York, pp. 469-594. Takagi, Y. and Kanazawa, N. (1982) Age-asso- ciated change in macronuclear DNA content in Paramecium caudatum. J. Cell Sci., 54: 137-147. ZOOLOGICAL SCIENCE 4: 81-86 (1987) Banding Karyotype of Korean Salamander: Hynobius leechii Boulenger' SEI-ICHI KoHNo”*, MASAKI Kuro-07, CHIKAKO IKEBE*, REIKO KATAKURA2, YASUHIRO IZUMISAWA*, TADAO YAMAMOTO’, HEI YUNG LEE? and SuH YUNG YANG? * Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan, 3Department of Biology, Faculty of General Education, Toho University, Funabashi, Chiba 274, Japan, 4Buso High School, Yokohama, Kanagawa 222, Japan and °Department of Biology, Inha University, Inchon, Korea ABSTRACT— The chromosome constitutions of 56 embryos of Hynobius leechii from eight localities of Korea were analyzed by Giemsa staining, C-banding and R-banding. Banding patterns of 18 out of 28 chromosome pairs were identified by the analyses. The short arms of No. 10 chromosomes, each forming one negative R-band, showed intra-specific variations in size; The short arms of No. 10 chromosomes of the specimens from three northern localities (Sogumgang, Kangnung and Yangju) were larger than those from four southern localities (Kyeryongsan, Chupungryong, Kyonju and Chindo). The specimens from one locality (Chongson) between the northern and southern localities showed No. 10 chromosomes with large short arms and also those with small ones. The difference in the arm ratio of No. 10 chromosome between the specimens from three northern (large short arm) and four southern localities (small short arm) was statistically significant at <0.01 level. The size of the terminal band of the long arm of No.2 chromosome in H. leechii (positive in C-banding and negative in R-banding) was larger than that in H. nigrescens. Except for these 2 bands and other minor intra-specific chromosome aberrations, no difference could be detected between H. leechii and H. nigrescens in the © 1987 Zoological Society of Japan banding patterns of their 18 chromosome pairs identified. INTRODUCTION The genus Hynobius which is distributed in several countries of east Asia differentiates into many species, especially in Japan [1] and there are several studies available on karyotaxonomy of Japanese Hynobius [2-12]. These studies have reported that the diploid chromosome numbers of this genus are 40 (H. retardatus), 56 (H. nebulosus and other eight species) and 58 (H. naevius and other three). The karyotypes of the latter two (those with 56 and 58 chromosomes) are shown to be almost the same except for one additional small chromosome pairs in the species with 58 chromo- Accepted July 15, 1986 Received May 7, 1986 " This paper corresponds to “Cytogenetic studies of Hynobiidae (Urodela).V”. © To whom reprints should be requested. 7 Deceased on November 2, 1985. somes. The chromosome number of most of the pond type species is 2n=56 [3-7, 9, 10, 14] whereas that of the mountain-brook type is 2n=58 [12]. In order to consider the process of differentia- tion of the genus Hynobius, the karyotype analysis of the Korean pond type salamander, Hynobius leechii, seemed to be indispensable to the search for the original karyotype of Japanese Hynobius. The chromosomes of H. leechii was studied by sectioning and Hematoxylin staining procedure by Makino in 1934, and the chromosome number of this species was reported to be 56 [13]. The Giemsa stained karyotype of this species was given by Chon [14]. However, there has been no report on the banding karyotype of this species. In this paper, we would like to present the results of the karyotype analyses of this Korean Hynobius, H. leechii, by the newly improved chromosome banding methods for Urodela (C- 82 S. Konno, M. Kuro-o et al. banding and R-banding) [8, 11]. MATERIALS AND METHODS The embryos in egg-capsules of Hynobius leechii were collected from eight localities in Korea: Sogumgang, Kangnung, Yangju, Chongson, Kyeryongsan, Chupungryong, Kyongju and Chin- do as shown in Figure 1. The detailed information on the number of the specimens collected from the eight localities and of the metaphases analyzed is summarized in Table 1. The chromosome preparations were made according to the improved method of Ikebe and Kohno [4]; using whole embryos, 24hr of 0.5% colchicine pretreatment, 30-40 min of hypotonic treatment in 15 times diluted Amphibian Ringer’s after breaking embryos to pieces with a Pasteur pipette, fixation by fixative consisting of three parts of methanol and one part of acetic acid and air drying. The C-banding slides were made according to the method by Sumner [15] with slight modifica- tions. The R-banding patterns were obtained by the method described by Kuro-o ef al. [11]. The embryonal cells were cultured with BrdU (400 yg/ml) for 33 hr and the chromosome preparations were made by ordinary air drying method after the colchicine treatment. The slides were stained by FPG (Fluorescent plus Giemsa) staining proce- dure. a o @, 1@ 2 Ss, ®, 4 "6 6 % de 7 ee o° Ay é | v ae he Fic. 1. Eight localities where the Hynobius leechii embryos were collected. 1. Sogumgamg, 2. Kangnung, 3. Yangju, 4. Chongson 5. Kyeryong- san, 6. Chupungryong, 7. Kyongju 8. Chindo. RESULTS Karyotype analyses were made in a total of 201 metaphases from 56 specimens in 20 egg capsules TABLE 1. Numbers of the metaphases analyzed and of the specimens collected from eight localities in Korea Collection Date of nego No. of metaphases / No. of embryos sigs collecHon capsules Giemsa C-banding R-banding Total 1 Sogumgang Apr. ‘84 2 6/2 — V2 13/4 2 Kangnung Apr. 85 1 2) 3/2 4/2 12/4 3 Yangju Mar. 84 3 10/4 15/6 PAK) 47/19 4 Chongson Apr. ‘85 3 11/3 8/2 6/4 D5 7 5 _Kyeryongsan Apr. 85 D 10/3 5/1 7/3 22/6 6 Chupungryong Mar. ‘85 3 10/3 5/2 7/4 MDT) 7 Kyongju Mar. ‘85 3 11/3 18/2 DYN 31/4 8 Chindo Apr. 85 3 13/3 13/1 3/2 29/5 Total 20 76/23 67/16 58/27 201/56 Banding Karyotype of Hynobius leechii 83 collected from eight localities as shown in Table 1: 76 metaphases from eight localities by the conven- tional Giemsa staining, 67 metaphases from seven localities by C-banding and 58 metaphases from eight localities by R-banding. The analyses of the conventional Giemsa stained karyotypes revealed the polymorphism of No. 10 chromosome (Fig. 2). The karyotypes which showed No. 10 chromosome pair with large short arms were found in the specimens from three out of eight localities, Sogumgang, Kangnung and Yangju. Those having No. 10 chromosome pair with small short arms were found in four localities; Kyeryongsan, Chu- pungryong, Kyongju and Chindo. The former specimens having No. 10 chromosome pair with large short arms were distributed in the northern part of Korea and the latter were found in the southern part of Korea. In the remaining one locality, Chongson, which was located between the northern and the southern parts, No. 10 chromo- somes of both types were observed in the speci- mens collected. Three pairs of No. 10 chromo- somes of the specimens from Yangju (large short arm) and another three pairs from Kyeryongsan 1} CHUS( ai 0 it hei ni a aa BG AA KR Xx Wm ne .= Ab Aor aa — Ra Am ew AH HH 2" nh bab AL b NC if ae rT cr bi uk Aa AA MK AR NE BE ee on aa no ah @h GA wc «eu eee eee oy 95 Fic. 2. Two conventional Giemsa staining karyotypes of Hynobius leechii collected from two localities (Yangju: a, Chindo: b). The arrows point out No. 10 chromosome pairs which show polymorphism in their short arms. 84 S. KoHno, M. Kuro-o et al. (small short arm), each pair being stained by the conventional Giemsa, C-banding and R-banding, respectively, were shown in Figure 3. The mean arm ratios of No. 10 chromosomes in the speci- mens from the eight localities are summarized in Table 2. The measurement of the arm ratios of No. 10 chromosomes, which have morphological Bei; B Fic. 3. No. 10 chromosome pairs of Hynobius leechii collected from 2 localities (Yangju: A, Kyeryong- san: B). The three pairs each stained by convention- al Giemsa, C-banding and R-banding, respectively, are shown. TABLE 2. similarities to Nos. 11 and 12, was made using R-banded chromosomes chosen at random. It may be necessary to note that the arm ratios of R-banded No. 10 chromosomes are somewhat smaller than those of Giemsa-stained ones because of the enlargement of their short arms due to the extreme uptake of BrdU. As shown in Table 2, the difference in the mean arm ratio of No. 10 chromosomes between the specimens from the three northern localities (Sogumgang, Kangnung and Yangju) and those from the four southern localities (Kyeryongsan, Chupungryong, Kyongju and Chindo) was statisti- cally significant according to “t” test (p<0.01). The data from Chongson which located between the two groups mentioned above showed the medium value between those from the two areas. Based on the R-banding and C-banding pat- terns, 18 out of 28 pairs of the chromosomes of this species could be identified. They were all of the large sized (Nos. 1-9) and medium-sized (Nos. 10-13) and 5 out of 15 small-sized (Nos. 14, 15, 20, 21 and 22) chromosome pairs (Fig.4). Their banding patterns were identical to those reported on Hynobius nigrescens by Kuro-o et al. [11] except for the terminal band on the long arm of No. 2 chromosome; the band of H. leechii being larger than that of H. nigrescens. In two out of 56 specimens from the eight localities, one numerical and two _ structural chromosome aberrations were observed: One specimen from Kangnung had 57 chromosomes due to trisomy of No. 13 chromosome. The other from Chindo had two chromosome aberrations, Mean arm ratios (long arm/short arm) of No. 10 chromosomes in Hynobius leechii collected from eight localities in Korea Collection No. of measured Mean acme ratios HASU DY sites chromosomes 1 Sogumgang 7 AOD ae O27 2 Kangnung 6 esis) az (0) I14/ 2b) ac W.71l 3 Yangju 20 LO) a2 O22 4 Chongson A 2 Jal ae (ARS 5 Kyeryongsan 21 2.44 + 0.26 6 Chupungryong 19 Des), ae VAY 7 Kyongju 8 2.46 + 0.21 2.48 + 0.22 8 Chindo 25 2.49 + 0.21 Banding Karyotype of Hynobius leechii 85 i Di oh ane a i 2? qt ) 7” aa% PR B88 2 13 14 15 a An * 2% * us * 16 — 19 @e7 kh @ Ba * 20 21 22 aa a @e ~ “ia * an ? “a * em * 23 26 Fic. 4. R-banding karyotype of Hynobius leechii from Yangju, with a C-banded chromosome being placed on the right side of each pair. namely, a No. 21 chromosome with a large short arm, and a No.1 chromosome with an atypical C-banding positive band on the long arm close to the centromere. The position of this band corre- sponded to that of a secondary constriction on the chromosome which was detected by the conven- tional Giemsa staining (Fig. 2). DISUSSION In the Korean Peninsula (the area being about 2/3 of Japan), only one species of the genus Hynobius, H. leechii, is found [1], whereas in Japan 14 species of the genus Hynobius, differenti- ated into pond type and mountain-brook type, are found [1, 10, 12]. In our chromosome observation of the specimens in the southern half area of the Korean Peninsula, the banding patterns of H. leechii were the same except for one band on the short arm of No. 10 chromosome. This band was positive in C-banding and negative in R-banding (Fig. 3), suggesting that this region (band) contains constitutive heterochromatin and scarcely includes structural genes. The terminal band on the long arm of No. 2 in H. leechii was also constitutive heterochromatin (Fig. 4) and was larger than those in H. nigrescens and H. tokyoensis reported by Kuro-o et al. [11] and Kohno et al. [8]. The intra- and inter-specific karyotypic variations in H. leechii we observed were only in heterochromatic ones, except for three chromosomal aberrations shown in the specimens from two localities. The sizes of the short arms of the No. 10 chromosomes in the specimens from the southern part of this peninsula are supposed to have been reduced. In Japanese Hynobius, the sizes of the short arms of No. 10 chromosomes vary from none (H. nebulosus) [4, 7] to the size as large as the smaller ones in H. leechii (H. tokyoensis and others) [3-5, 7, 8, 10-12, 16]. The problem of 86 S. KoHNo, M. Kuro-o et al. differentiation and karyotype evolution of this genus is going to be discussed elsewhere in connection with the morphological variations of the short arms of No. 10 chromosomes (Ikebe et al., unpublished data). As to the embryo from Kangnung, showing aneuploid karyotype (2n=57), it is not possible to state whether the embryo would have grown to be a normal adult or not, even though it did not show external morphological anomalies at the time of the observation. In our experience in karyotype analyses of Hynobius embryos, some hyperdiploid and hypodiploid embryos could be observed in other species, for example, in H. tsuensis (Ikebe et al., unpublished data). In order to study the presence of the aneuploidy in H. leechii, the screening of the karyotypes of adult populations should be carried out. ACKNOWLEDGMENTS The authors are grateful to Dr. T. Ishihara, National Institute of Radiological Sciences, Chiba, for his correct- ing and improving the manuscript. The authors are also grateful to Mr. Park, C.S. and Miss Lee, H. S. in Inha University for their assistance in collecting our materials. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 58540484). REFERENCES 1 Sato, I. (1943) A Monograph on the Japanese Urodeles. Nippon Shuppan-sha Osaka, pp. 21-24, 27-40, 438-440. (In Japanese) 2 Makino, S. (1956) A review of the chromosome numbers in animals, Hokuryukan, Tokyo, pp. 143-144. (In Japanese) 3. Azumi, J. and Sasaki, M. (1971) Karyotypes of Hynobius retardatus Dunn and Hynobius nigrescens Stejneger. Chromosome Information Service, 12: 31-32. 4 Ikebe, C. and Kohno, S. (1979) Cytogenetic studies of Hynobiidae (Urodela). I. Karyotypes of Hyno- bius nebulosus nebulosus (Schlegel) and Hynobius nebulosus tokyoensis Tago. Proc. Jpn. Acad., 55 10 fel 12 13 14 US 16 B(9): 436-440. Ikebe, C. and Kohno, S. (1979) Karyotypes of Hynobius nigrescens Stejneger and Hynobius liche- natus Boulenger. Chromosome Information Service, 27: 13-15. Morescalchi, A., Odierna, G. and Olmo, E. (1979) Karyology of the primitive salamanders, family Hynobiidae. Experientia, 35:1434-1435. Seto, T., Utsunomiya, Y. and Utsunomiya, T. (1983) Karyotypes of two representative species of Hynobiid salamanders, Hynobius nebulosus (Schlegel) and Hynobius naevius (Schlegel). Proc. Jpn. Acad., 59 B(7): 231-235. Kohno, S., Ohhashi,T. and _ Ikebe, C. (1983) Cytogenetic studies of Hynobiidae (Urodela). II. Banding karyotype of Hynobius tokyoensis Tago. Proc. Jpn. Acad., 59 B(8): 271-275. Seto, T. and Matsui, M. (1984) Karyotype of the Japanese salamander, Hynobius abei. Experientia, 40:874. Matsui, M., Seto, T. and Miyazaki, K. (1985) The karyotype of Hynobius takedai M. Matsui et Miyazaki, 1984, with comments on the karyotypic relationships among Japanese salamanders of the genus Hynobius. Jpn. J. Genet., 60: 119-123. Kuro-o, M., Ikebe,C. and _ Kohno, S. (1986) Cytogenetic studies of Hynobiidae (Urodela). IV. DNA replication bands (R-banding) for the genus Hynobius and the banding karyotype of Hynobius nigrescens Stejneger. Cytogenet. Cell Genet., (In press) Ikebe, C., Yamamoto, T. and Kohno, S. (1986) Karyotypes of Japanese Hynobiid salamanders, Hynobius kimurae Dunn and Hynobius boulengeri (Thompson). Zool. Sci., 3: 109-113. Makino, S. (1934) The chromosomes of Hynobius leechii and H. nebulosus. Trans. Sapporo Nat. Hist. Soc., 13: 351-354. Chon, S. K. (1982) Studies on the karyotypes of the Korean Amphibians. Thesis of Master of Science, Inha University, Korea (In Korean with English Abstract). pp. 1-27. Sumner, A. T. (1972) A simple technique for de- monstrating centromeric heterochromatin. Exp. Cell Res., 75: 304-306. Ikebe, C., Kuro-o, M., Katakura, R., Kusada, S. and Kohno, S. (1984) Morphological variation of No. 10 chromosome in 9 pond type Hynobius from Korea and Japan. Zool. Sci., 1: 899. ZOOLOGICAL SCIENCE 4: 87-92 (1987) Karyological Differentiation between Two Species of Mustelids, Mustela erminea nippon and Meles meles anakuma YOSHITAKA OBARA Department of Biology, Faculty of Science, Hirosaki University, Hirosaki 036, Japan ABSTRACT—The karyotypes of the Japanese badger, Meles meles anakuma, were presented using conventional and differential staining. Its diploid number was 44, in accordance with Tsuchiya’s observation. The sex determining system was XX for the female and XY for the male. Two of the five specimens studied herein carried a heteromorphic autosomal pair (B6) which was considered to have been produced by a deletion of a G-positive block. The banded chromosomes of the badger were compared with those of the Japanese ermine, Mustela erminea nippon, with the aim of defining karyological differentiation between these two remotely related mustelids which belong to different subfamilies, Melinae or Mustelinae, respectively. A low grade of G-band homology was found between them, in agreement with a marked dissimilarity in their chromosome constitution. Over one-third of the total chromosome length hardly showed any G-band homology between the species. The phylogenetic relationships of the Japanese mustelids were briefly discussed, introducing © 1987 Zoological Society of Japan karyosystematic evaluation into the former taxonomic system. INTRODUCTION The karyotypes of the ermine, Mustela erminea, have been studied by several workers with the use of both conventional [1-3] and differential [4-6] staining. The Japanese ermine, M. erminea nip- pon, is almost identical with the continental taxa in the G- and C-banding patterns as well as in the diploid number and chromosome constitution [7-9]. On the other hand, the chromosomes of the Eurasian badger, Meles meles, have been studied only by conventional staining [1, 10-13]. Up to the present, no attempt has been made to compare the badger karyotypes with those of other mustelid species, even in conventionally stained materials. As to the Japanese taxon, M. meles anakuma, neither conventional nor banded karyotypes have been presented as yet, though its diploid number has been determined to be 44 by Tsuchiya [14]. In this paper the chromosomes of the Japanese mustelids, Mustela erminea nippon and Meles Accepted August 26, 1986 Received July 4, 1986 meles anakuma, were compared, making use of G- and C-banding methods, to estimate the in- tergeneric conservatism and/or variation of G- and C-banding patterns. . MATERIALS AND METHODS Four adult specimens (one female and three males) of the Japanese badger, Meles meles ana- kuma, were captured alive near Mt. Nishimata, south of Hirosaki, Aomori Prefecture. The female specimen was pregnant when caught, and one male was retained from the pregnant specimen as chromosome material. Chromosome preparations were obtained from either bone marrow cells of the adult specimens or liver cells of the fetus. The two adult specimens of the Japanese ermine, Mustela erminea nippon, studied here are the same as those used in previous works [8-9]. The sampling procedure is almost the same as those previously reported [7, 15]. For G- and C-banding the ASG [16] and BSG [17] methods were adopted. Two hundred and fifty two badger metaphases and 337 ermine ones were examined under the 88 Y. OBARA hi fh AN 14 SA “a AR 19 20 21 Fic. 1. microscope to ascertain their diploid number of chromosomes. Twenty six conventionally stained, 16 G-banded and 17 C-banded metaphases were karyotyped in the former species and 30, 41 and 24 ones in the latter species. RESULTS The chromosome number was found to be 2n= Ho On 4b AG an oo 2 A) 20 21 X.Y Hh AK u een | (} arene AR fa 4 15 16 17 18 @) Conventional karyotypes of a female Japanese ermine (A) and a male Japanese badger (B). 44 in both species, but their chromosome con- stitutions differed greatly (Fig. 1). No. 17 of the ermine chromosomes (E17) carried a_ typical secondary constriction in the proximal region of its long arm (arrow head). Nos. 14 and 18 of the badger chromosomes (B14 and B18) carried very small satellites terminally in the short arm. The sex chromosomes, X and Y, were medium-sized metacentric and the smallest acrocentric in both Karyotypes of Two Species of Mustelids 89 5 xx a Ny 3 if ‘tA wo RA ie in q Aii ai Hb da 10 1 fiy Ri: avy 15 16 17 18 A 4 te 04 allied (A) 19 20 21 * » ts * 2 * ’ @ 1 2 3 x ¥ ~ es @. : ; 5 6 7 10-— 11 cd ¢ ® « @ a a ® e oe ee 12 13 14 16 17 18 ee # . - * “os 19 20 21 \8) Fic. 2. G-banded (A) and C-banded (B) karyotypes of a male Japanese badger. species. The short arms of the B6 homologue were apparently heteromorphic (arrow); the large one is submetacentric (SM) and the small one sub- telocentric (ST). This heteromorphism was con- sidered to be substantial in two (an adult female and its male fetus) of the five specimens, since it was detected in all the metaphases analysed, so far as these two specimens were concerned. The B6 homologue of the remaining three specimens was homologous for the SM element. G- and C-banded karyotypes of the badger are shown in Figure 2. All the chromosomes, includ- ing morphologically similar ones, could be precise- ly identified by their G-banding patterns. The satellites were G- and C-negative. While the B6-SM element carried a large amount of C- heterochromatin (C-positive block) on the proxi- mal half of its short arm, the B6-ST element carried no such large C-positive block on its short arm, just containing a small amount of centromeric C-heterochromatin. The C-positive block region was faintly stained as a G-negative segment when G-banded (arrows). The badger Y chromosome, forming more than one-third of its X chromosome (38.63+0.68%) in length, seemed to be totally heterochromatic, thus being stained darkly by C- 90 Y. OBARA Sem @ , yn &e >* 15 "2 1 hs ah ig " 10 47 5 6 oc, ae mG aie. 12 13 14 : Qe ee a 20 21 20 21 FIG? 3) e A, Wf 14 10 ne nx 1 15 16 17 18 Pair-matching of G-banded chromosomes between the Japanese ermine (right element of each pair) and the Japanese badger (left element of each pair). Unpaired ermine chromosomes were put together in the largest rectangle of the lowest right and seven badger chromosomes enclosed in the remaining small rectangles were left alone unpaired. banding and faintly by G-banding. The ermine Y was also C-positive along its entire arm, but in size it was almost one-fifth of its X chromosome (20.46 +0.91%) in length. A small interstitial C-band could always be detected on the short arm of B1, though it was liable to go unnoticed due to its small size. In addition to these non-centromeric C- bands, varying sizes of centromeric C-bands were observed in all of the chromosomes from deeply- stained large ones to faint small ones. Figure 3 summarizes the pair-matching analysis, by side-by-side arrangement of the ermine and badger chromosomes which was made according to G-band homology. Nine pairs, including the X chromosome, showed one-to-one correspondence between species, and six badger chromosomes could only be paired partially with six ermine chromosomes mainly in the long arms. Arrow heads indicate unpaired segments. The remaining seven chromosomes hardly showed any G-band homology between species. These unpaired non- homologous chromosomes and chromosome seg- ments amounted to about 36% of the haploid length of the ermine chromosomes. No informa- tion was available with respect to the interrelation of these unpaired parts. The subtelocentric pair No. 11 of the ermine (E11) is known to have a large portion of C-heterochromatin on its short arm which is quite similar in its appearance to the C-positive block of B6 [8-9]. In view of this fact, the conventionally stained, and C-banded chromosome of E11 and B6 were compared in detail (Fig. 4). Their long arms share a common origin, as evident from their Karyotypes of Two Species of Mustelids 9] ST SM cy thi B6 Fic. 4. Comparison of E11 and B6 chromosomes. Top: Conventinonally stained, Middle: G-banded, Bot- tom: C-banded. Arrow heads: Euchromatic seg- ments on the short arms. G-band homology. On the other hand, the short arms of E11 and B6—ST may be different in their origin in spite of their morphological similarity; the entire short arm of E11 consisted of C-hetero- chromatin, and in reverse the short arm of B6-ST was totally euchromatic. Based on the similarity in the size and location of their C-positive blocks, the short arm of E11 itself may correspond to the proximal half of the short arm of B6-SM. DISCUSSION In general, the mustelines show extensive inter- specific variations of karyotypes [5, 18]. The Japanese mustelines, the Japanese ermine, the least weasel (LW) and the Japanese weasel (W), also show marked interspecific variation in number and structure of chromosomes [7-9]. Their karyological relationships could be explained by Robertsonian fusion and quantitative alteration of C-heterochromatin. These findings strongly sug- gest that G-banding patterns of these musteline species have been conserved within the genus in spite of their marked differentiation of karyotypes. The highly conservative nature of G-banding pattern was also detected between the ermine and the marten (M) which belong to different genera, Mustela and Martes, respectively (unpublished data). Their karyological relationship could be explained by three Robertsonian rearrangements, where the combination patterns of the chromo- somes involved apparently differed from those found in the ermine and the least weasel, or the ermine and the Japanese weasel [8—9]. Such a high degree of G-band homology between Mustela and Martes may in itself imply a closeness of their phylogenetic relationship, though they can be regarded as remote relatives from their conven- tional karyotypes as well as from usual taxonomic criteria. The badger chromosomes differed greatly from the ermine ones in both the chromosome con- stitution and the G-banding pattern. No G-band homology was found between these species in over a third of the haploid length of chromosomes. Such reduced G-band homology may directly reflect the remoteness of their phylogenetic rela- tionship. According to the usual taxonomic criteria, the ermine is considered to be one of the more primitive froms and the badger is one of the more advanced ones among the mustelids. Based on this point of view, E15, E10 and E14 might have been involved, most probably through the Robertsonian fusion system, in the production of B1, BS and B10, respectively, and by contraries the interrelation of B17 and E1 could be ascribed to the presence or absence of the short arm of E1, most probably being explicable by the Robert- sonian fission system. It is also reasonable to consider that the B6-SM might have been formed by translocation of a euchromatic segment of unknown origin into the C-positive block, and the B6-ST by deletion of the C-positive block from the B6-SM. The heteromorphic pair B7 and ES could be explained by a paracentric inversion in the short arm of ES. The three Robertsonian fusions detected in the badger did not correspond in the combination of chromosomes involved to any of 7 Robertsonian fusions of three other mustelid species so far examined. Taking notice of E10 involved in the Robertsonian rearrangements, E10 and E16 cor- responded to the long arm and short arm of LW1 in their G-banding pattern [9], E10 and E13 to 92 Y. OBARA those of W1 [8], E10 and E21 to those of M9 (unpublished data) and E10 and an unknown element to those of B5. In the light of these findings, it is likely that these mustelid species have differentiated independently with one another from the ancestral ermine-like stock through dif- ferent chromosome rearrangements. Among them, the badger has undergone the most intensive alteration of chromosomes, so that more than one-third of its genome length lacked G-band homology to any of the ermine chromosomes. These findings strongly suggest involvement of intricate and complicated rearrangements such as repeated inversions, compound translocations and their combinations during the course of their karyological differentiation. A high degree of karyological differentiation between the badger and the ermine is now well established based not only on the conventional staining but also on differential staining. Thus, the findings obtained here may support the appropriateness of the former taxonomic system in which the badger belongs to the subfamily Melinae and the other four species mentioned above to the subfamily Mustelinae. To understand the exact interrelation between these unpaired parts of chromosomes, closely related species such as hog-badger, stink badger, ferret badger, American badger and hon- ey badger, all of which belong to the Melinae or Mellivorinae that is close to the Melinae, should be examined in detail, paying special attention to their G-band homology. ACKNOWLEDGMENTS The author wishes to express his gratitude to Professor Kazuo Saitoh, Faculty of Science, Hirosaki University, for his helpful advice and suggestions. Sincere thanks are also due to Professor Karl Fredga, Department of Genetics, University of Uppsala, for reading and revising the manuscript with expert criticism. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 58540479). REFERENCES 1 Fredga, K. (1967) Comparative chromosome studies of the family Mustelidae (Carnivora— Mammalia). Hereditas (Lund), 57: 295. 2 Meylan, A. (1967) Les chromosomes de Mustela erminea_ cicognanii BONAPARTE (Mamm.— 10 11 1 13 14 15 16 17 as Carnivora). Can. J. Genet. Cytol., 9: 569-574. Hsu, T. C. and Benirschke, K. (1968) An atlas of mammalian chromosomes. Vol2. Folio 80, Springer-Verlag New York Inc. Grafodatsky, A.S., Volovuev, V.T., Ternovsky, D.V. and Radjabli, S. I. (1976) G-banding of the chromosomes in seven species of Mustelidae (Car- nivora). Zool. J., 55: 1704-1709 (in Russian with English summary). Grafodatsky, A. S., Ternovsky, D. V., Isaenko, A. A. and Radjabli, S.I. (1977) Constitutive heter- ochromatin and DNA content in some mustelids (Mustelidae, Carnivora). Geneticka, 13: 2123-2128 (In Russian with English summary). Mandahl, N. and Fredga, K. (1980) A comparative chromosome study by means of G-, C- and NOR- bandings of the weasel, the pygmy weasel and the stoat (Mustela, Carnivora, Mammalia). Hereditas, 93: 75-83. Obara, Y. (1982) Comparative analysis of kar- yotypes in the Japanese mustelids, Mustela nivalis namiyei and M. erminea nippon. J. Mammal. Soc. Jpn., 9: 59-69. Obara, Y. (1985) G-band homology and C-band variation in the Japanese mustelids, Mustela erminea nippon and M. sibirica itatsi. Genetica, 68: 59-64. Obara, Y. (1985) Karyological relationship be- tween two species of mustelids, the Japanese ermine and the least weasel. Jpn. J. Genet., 60: 157-160. Muldal, S. (1950) A list of vertebrates observed at Bayfordbury, 1949-1950. John Innes Hort. Inst., 41 (Ann. Rep.): 39-41. Fredga, K. (1966) Chromosome studies in six spe- cies of Mustelidae and one of Procyonidae. Mam- mal. Chrom. Newsl., 21: 145. Omodeo, P. and Renzoni, A. (1966) The karyotype of some Mustelidae. Caryologia, 19: 219-226. Wurster, D.H. and Benirschke, K. (1968) Com- parative cytogenetic studies in the order Carnivora. Chromosoma (Berl.), 24: 336-382. Tsuchiya, K. (1979) A contribution to the chromo- some study in Japanese mammals. Proc. Jpn. Acad., 55 B (4): 191-195. Obara, Y. and Miyai, T. (1981) A_ preliminary study on the sex chromosome variation in the Ryukyu house shrew, Suncus murinus riukiuanus. Jpn. J. Genet., 56: 365-371. Sumner, A. T., Evans, H. J. and Buckland, R. A. (1971) New technique for distinguishing between human chromosomes. Nature (Lond.) New Biol., 232: 31-32. Sumner, A. T. (1972) A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res., 75: 304-306. Bengtsson, B. O. (1980) Rates of karyotype evolu- tion in placental mammals. Hereditas. 92: 34-47. ZOOLOGICAL SCIENCE 4: 93-97 (1987) © 1987 Zoological Society of Japan Acquired Resistance against Microphallus pygmaeus in the Laboratory Mouse RoHANI A. AHMAD, BRIAN L. James! and Attias B. Kamis~ Jabatan Zoologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia, and ‘Department of Zoology, University College of Swansea, SA2 8PP, United Kingdom ABSTRACT— Variations in worm burden indicate that Swiss albino and DBA/2 mice are more susceptible to infection by Microphallus pygmaeus than TO or C57BL/10 mice. The worm burden in outbred Swiss albino mice, however, has a much lower coefficient of variability than in the inbred DBA/2 strain. Susceptibility to infection decreases with age in all strains of mice. The worm burden is significantly lower in Swiss albino and C57BL/10 mice immunized by an oral primary infection, and in Swiss albino mice immunized by an intraperitoneal primary infection. Protection by oral immunization and by intraperitoneal injection persist for at least 12 days. INTRODUCTION Most literature covering study of acquired im- munity in Digenea is concerned with parasites of human importance, namely Schistosoma spp. [1, 2] and of agricultural importance, namely Fasiola hepatica [3-6]. It is to be expected that these long-lived parasites which undergo migration through tissues of the mammalian hosts stimulate responses which induce a measure of protection against subsequent infection. The relative in- efficiency of these responses in inducing protection may be attributed to the ability of the parasite to combat host defenses. In particular, all flukes are protected by the glycocalyx secreted by the syncy- tial tegument [7-9]. In contrast, it seemed unlikely that a short-lived gut parasite without a tissue stage in development, such as Microphallus pygmaeus would stimulate sufficient responses to induce protection against subsequent infection. Nevertheless, earlier work [11, 12] suggests that suppression of the host immune responses increases the worm burden of M. pygmaeus in mice. It seemed appropriate therefore to examine if Accepted September 24, 1986 Received July 24, 1986 * To whom reprints should be requested. acquired immunity to M. pygmaeus as measured by a reduction in worm burden in the intestine could be induced in the laboratory mouse. Two methods of primary infection were employed, namely (a) a single oral infection of 2,000 metacercariae and (b) intraperitoneal injection of 2,000 metacercariae. METERIALS AND METHODS Experimental host Mice used in the present investigations were maintained under conventional condition in the animal facility. The mice were housed in plastic cages (6 per cage), measuring 31 <12x11cm in a room at 18-22°C with 12hr of flourescent lighting (between 22:30 and 10:30hr). Food and water were available ad libitum. Parasite The metacercariae of M. pygmaeus were obtained from daughter sporocysts in the haemocoel of the digestive gland of the primary molluscan host, Littorina saxatilis tenebrosa and kept for a short period in artificial sea water before being orally inoculated into mice. Recovery of adult worm from mice _ After the required period postinoculation, the mice were killed, the whole of the small intestine quickly removed and placed in vial containing mammalian saline. The intestine was split opened lengthwise under saline and then incubated at 37°C for 94 R. A. AHMAD, B. L. JAMES AND A. B. Kamis 25-30 min. The parasite migrated from the intes- tine into vial. Adult worms were counted with the aid of a microscope. Statistics A student’s ¢ test or one way analysis of variance was done as appropriate on each set of data. RESULTS The susceptibility of four strains of mice to M. pygmaeus A total of 72 male mice aged between 5 and 18 weeks were used, with 6 animals in each category. The four strains include 2 outbred strains, Swiss albino and TO and 2 inbred strains, DBA/2 and C57BL/10. The mice were infected with 1,000 metacercariae in 0.2 ml artificial sea water and the adult worms recovered 2 days post-inoculation and counted. Swiss albino and DBA/2 mice are clearly more susceptible (Table 1) to infection by Microphallus pygmaeus than TO or CS57BL/10 mice but the differences are significant (P<0.05) only with the former. The worm burden in outbred Swiss albino mice has a much lower coefficient of variability (Table 1) than in inbred DBA/2 mice and are thus more suitable for experimental purposes. The susceptibility to infection decreases with age in all strain of mice. Acquired resistance against M. pygmaeus in 5 and 10 week old male Swiss albino and C57BL/10 mice induced by a primary oral infection of metacercariae Twenty 5 week old and twenty 10 week old mice from each strain were given an oral primary TABLE 1. infection of 2,000 metacercariae in 0.2 ml artificial sea water but 10 of each with 0.2 ml artificial sea water only. At 2 days post-primary infection (PPI), ten infected mice from each strain and age group were killed and the worm burden counted to confirm the existence of a viable primary infection. On the 12th day PPI when the primary infection was presumed to have terminated, 20 mice from each strain and age category, 10 preinfected with metacercariae and 10 only with sea water, were challenged with 1,000 metacercariae, the mice 14 [ee non - immunized 12 immunized nao = 16 +13) Pia OuO| 8Fr L re* ” S= =) 6 So J OF in 0 . S week group 10 week group Fic. 1. Percentage (+SE) of adult Microphallus pyg- maeus recovered 2 days post-challenged infection (PCI) from non-immunized and immunized 5 and 10 week old Swiss albino mice (n=10) given an oral primary infection of 2,000 metacercariae and chal- lenged orally with 1,000 metacercaiae 12 days post- primary infection (PPI). Mean number (+SD) and coefficient of variability (C. V.) of adult Microphallus pygmaeus recovered two days post-inoculation from four strains of five to eighteen week old male mice (n=6) infected orally with 1,000 metacercariae Sarnins 5 weeks old 10 weeks old 18 weeks old Mean+S.D. C.V.(%) Mean+S.D. C.V.(%) Mean+S.D. C.V.(%) Swiss UO.) ae VOLS. ANS 6825 == IStSeOrs M3 32 (30) S4h8) TO 38.2 + 30.8 80.6 S/o) ae S80. etsats) M2ef se WD teil DBA/2 84.2 + 36.2 43.0 O19 se ALO | Sil 64.0 + 40.3 63.0 CS57BL/10 Sar Ss” S59) 46.3 + 23.1 49.9 38.8+417.2 44.4 Acquired Resistance against M. pygmaeus 95 fe] non - immunized E immunized +s: Pi20:01 adults % 5 week group \O week group Fic.2. Percentage (+SE) of adult Microphallus pyg- maeus recovered 2 days post-challenged infection non-immunized and immunized 5 and 10 week old C57BL/10 mice (n=10) given a primary oral infec- tion of 2,000 metacercariae and challenged orally with 1,000 metacercariae, 12 days post-primary in- fection (PPI). were killed 2 days post-challenged infection and the worms counted in the usual manner. The worm burden is significantly lower (P< 0.01) in 5 and 10 week old outbred male Swiss albino mice immunized by an oral primary infec- tion of metacercariae than in unimmunized mice (Fig. 1). Similarly, the worm burden is lower (P< 0.01) in orally immunized than in unimmunized 5—10 week old male inbred C57BL/10 mice (Fig. zy. Acquired resistance against M. pygmaeus in 5-6 week old male Swiss albino mice induced by intraperitoneal inoculation of metacercariae Of the 40 mice used, 20 controls were injected intraperitoneally (IP) with 0.2ml artificial sea water and 20 test animals with 2,000 living metacercariae in 0.2 ml artificial sea water using a 23 gauge needle. On the 6th day post intraperitoneal injection, ten of the control groups and 10 of the rest animals were challenged orally with 1,000 metacercariae in 0.2 ml sea water. The mice were killed 2 days post challenge infection (PCI) and the adult Digenea counted. On the 12th day the remaining mice were challenged with 1,000 metacercariae and examined for parasites as above. The worm burden is lower (P<0.01) in in- traperitoneally immunized than in unimmunized 5 week old male Swiss albino mice. The extent of protection is not significantly altered from 6 days to 12 days (Fig. 3) after intraperitoneal injection. = : : non- immunized | immunized a x Br 0-05 200 i6o L = 3s 120 8 * a [ine | °o - 80 3 E = Cc 40 e) 6 days PPI 12 days PPI Fic.3. Mean number (+SE) of adult Microphallus pygmaeus recovered 2 days PCI from _ non- immunized and immunized 5 week old Swiss albino mice (n=10) given an intraperitoneal primary infec- tion with 2,000 metacercariae and challenged orally with 1,000 metacercariae 6 or 12 days post-primary infection (PPI). DISUSSION Variations in susceptibility to infection amongst various strains of mice may be attributed to genetic differences [13]. The relatively high mean recov- ery and coefficient of variability indicates that the outbred Swiss albino mouse is a suitable ex- 06 R. A. AHMAD, B. L. JAMES AND A. B. KAmIS perimental host for M. pygmaeus. The induction of a degree of persistent protec- tion to a challenge infection by a relatively low single primary infection of healthy metacercariae is perhaps surprising for such a short-lived gut parasite which does no damage to the host. This result is, however, consistent with the earlier work [11, 14] which suggests that this parasite is respon- sive to the host immune system. The protection afforded by the intraperitoneal injection of metacercariae is particularly relevant in this respect. The metacercariae degenerate and die within 24hr, being surrounded by adhering macrophages [15]. It is significant that initially most macrophages adhere to the parasite in the neighbourhood of the oral and excretory orifices where most antigens are present. Similarly, Werheim and Hamada [16] observed that the infected larvae of Nippostrongylus brasiliensis, injected intraperitoneally into naive rats, were coated with adhering macrophages. Perez and Smithers [17] also found macrophages adhering to the surface membrane of Schistosoma mansoni and correlated this with protective immunity in the rat. In contrast, Engelkirt et al. [18] showed that mostly mast cells and eosinophils adhere to Taenia taeniaeformis in the peritoneal cavity of rats. Previous workers have tried to induce immunity by injecting parasites, with a tissue phase in development, intraperitoneally, for example Mesocestoides corti [19] and Fasciola hepatica [20, 21]. According to White et al [19], the degree of resistance induced in C57BL/6 mice is independ- ent of the route of inoculation. Doy and Hughes [20] reported a high degree of resistance in rats previously infected and challenged intraperi- toneally. However, Rajasekariah and Howell [21] failed to find any significant difference in worm recovery between intraperitoneally challenged rats and their respective control. Further work is required before it is possible to deduce the mechanism of resistance to, or to identify the specific antibodies involved, in intestinal M. pyg- maeus infection. REFERENCES 1 Maddison, S. E., Slemenda, S. B., Chandler, F. M. 10 iL 12 13 14 15 16 and Kagan, I. G. (1981) Acquired immunity in B cell-deficient mice to challenge exposure following primary infection with Schistosoma mansoni. Am. J. Trop. Med. Hyg., 30: 609-615. Mahmoud, A. A. F., Warren, K. S. and Peters, P. A. (1975) A role for the eosinophil in acquired resistance to Schistosoma mansoni infection as determined by anti-eosinophil serum. J. Exp. Med., 142: 805-813. Doy, T. G., Hughes, D. L. and Harness, E. (1981) Hypersensitivity in rats infected with Fasciola hepa- tica: possible role in protection against a challenge infection. Res. Vet. Sci., 30: 360-363. Lang, B. Z. (1974) Host-parasite relationships of Fasciola hepatica in the white mouse. V. Age of fluke responsible for the induction of acquired immunity. J. Parasitol., 60: 90-92. Rajasekariah, G. R. and Howell, M. J. (1978) Ac- quired immunity to the trematode Fasciola hepatica in rats. Aust. J. Exp. Biol. Med. Sci., 56: 747-756. Smithers, S. R. (1976) Immunity to Trematoda in- fections with special reference to schistosomiasis and fascioliasis. In “Immunology of Parasitic Infec- tions”. Ed. by S. Cohen and E. H. Sadun, Blackwell Scientific Publications, Oxford, pp. 296-332. Erasmus, D. A. (1972) The Biology of Trematodes, Edward Arnold, London. Eramus, D. A. (1977) The host-parasite interface of trematodes. Adv. Parasitol., 15: 201-242. Lumsden, R. D. (1975) Surface ultrastructure and cytochemistry of parasitic helminths. Exp. Para- sitol., 37: 267-339. Robinson, R.D. and Halton, D. W. (1983) Func- tional morphology of the tegument of Corrigia vitta (Trematoda: Dicrocoeliidae). Z. Parasitenkd., 69: 319-333. Ahmad, R.A., James,B.L. and Kamis, A.B. (1986) Retention and egg production of Microphal- lus pygmaeus in mice: the influence of the adrenal cortex. Z. Parasitenkd., 72: 479-485. Mills, A. R., Brain, P. E. and James, B. L. (1973) Microphallus pygmaeus: Effect of long-acting ACTH preparation on establishment and retention in alimentary canal of the mose. Exp. Parasitol., 34: 251-256. Wakelin, D. (1978) Genetic control of susceptibility and resistance to parasitic infection. Adv. Para- sitol., 16: 219-308. Hameed, M. Z. (1971) Studies on larval Digenea from marine molluscs. Ph. D. thesis, University of Wales. Ahmad, R. A. (1984) Studies on Marine Digenea in Experimental Hosts: Microphallus pygmaeus (Levinsen 1881) in the laboratory mouse. Ph. D. thesis, University of Wales. Wertheim, G. and Hamada, G. (1980) Nippostron- 17 18 Acquired Resistance against M. pygmaeus 97 gylus brasiliensis: quantitative and qualitative aspects of larval destruction in the peritioneum of the rat. Isr. J. Med. Sci., 16: 560-561. Perez, H. A. and Smithers, S. R. (1977) Schistoso- ma mansoni in the rat: The adherence of mac- rophages to schistosomula in vitro after sensitization with immune serum. Int. J. Parasitol., 7:315—-320. Engelkirk, P.G., Williams, J. F. and Signs, M. M. (1981) Interactions between Taenia Taeniaeformis and host cells in vitro: Rapid adherence of peri- toneal cells to strobilocerci. Int. J. Parasitol., 11: 463-474. 19 20 21 White, T.R., Thompson, R.C. A. and Penhale, W. J. (1982) A comparative study of the susceptibil- ity of inbred strains of mice to infection with Mesocestoides corti. Int. J. Parasitol.,12: 29-33. Doy, T. G. and Hughes, D. L. (1982) Evidence for two distinct mechanisms of resistance in the rat to reinfection with Fasciola hepatica. Int. J. Parasitol., 12: 357-361. Rajasekariah, G. R. and Howell, M. J. (1977) The fate of Fasciola hepatica metacercariae following challenge infection of immune rats. J. Helminthol., 51: 289-294. 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EY~(7978] Clagehe tte bay ee pine Inet ta Pek r — go i to) Sn etaee Ww Eee aca (AN ADE. pat: ‘terauksy hy Uaveniy ZOOLOGICAL SCIENCE 4: 99-105 (1987) © 1987 Zoological Society of Japan A DNA Synthesis Inhibitory Peptide from Human Embryo Fibroblasts—Characterization of Biological Properties Yasuyl Oxar’*, SHIGEAKI ISHIZAKA* and UKi YAMASHITA® 'Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Machida, Tokyo 194, ?Third Department of Internal Medicine, Nara Medical University, Kashiwara, Nara 634, and *Department of Immunology, University of Occupational and Environmental Health, Medical School, Fukuoka 807, Japan ABSTRACT—Low-molecular-weight inhibitory factors for lymphocyte DNA synthesis are known to be released from fibroblasts or fibroblast-like cells. Amongst these factors, 5 kDa peptide from human embyo fibroblasts strongly suppressed lymphocyte DNA synthesis when added at early periods after the lectin stimulation without causing a significat change in the lymphocyte viability. This peptide inhibited the lymphocyte DNA synthesis induced by interleukin 1, 2 or some mitogens. It also suppressed both the LPS-dependent polyclonal antibody (Ig M) production by B cells and the induction of hapten- reactive cytotoxic T cells. These results indicate that the 5 kDa peptide from human embryo fibrobalasts has a potential ability for the regulation of lymphocyte proliferation and functions. INTRODUCTION Lymphocyte reaction in immune responses are regulated by some soluble factors, which exhibit positive and negative effects on lymphocyte growth and functions. Previously, one of the authors (Y.O.) reported that some mammalian and insect cultured cells released low-molecular- weight peptides inhibitory to lymphocyte DNA synthesis [1-4]. However, the biological prop- erties of these factors have not yet been elucidated extensively. Amongst these factors, the authors analyzed here the biological properties of the 5 kDa peptide derived from human embryo fibro- blasts. This peptide inhibited the lymphocyte proliferative responses induced by interleukin 1, 2 or other mitogens, and suppressed both the cyto- toxic T cell function and the antibody production by B cells. Accepted July 22, 1986 Received May 8, 1986 * Present address and reprint requests: Laboratory of Molecular Oncology, Tsukuba Life Science Center, Yatabe, Tsukuba, Ibaragi 305, Japan. MATERIALS AND METHODS Cell culture Human embryo fibroblast cell line was kindly donated from Dr. S. Gotoh (Department of Biochemistry, University of Occupational and Environmental Health, School of Medicine, Kita- kyushu). The description of this cell line was found elsewhere [5]. The cells were grown in Eagle’s minimum essential medium (MEM, Nissui Seiyaku Co., Tokyo) supplemented with 10% fetal calf serum (FCS, Gibco, Grand Island Biological Lab., New York) in 5% CO, 95% air at 37'C. The cells became confluent in 4 days after a 1:4 splitting. The confluent cells were further cultured in a serum-free medium for 2 days to remove serum-derived materials. The serum-free medium was changed in every other day for two weeks and the pooled culture supernatant was served as a starting material to be examined. During this culture, the cell viability was not changed sig- nificantly. Preparation of DNA synthesis inhibitory peptide Two volumes of saturated ammonium sulphate solution were added to the culture medium (250 ml) and centrifuged at 12,000 xg at 15min. The 100 Y. Oxal, S. ISHIZAKA AND U. YAMASHITA precipitate was dissolved into 5ml of 10mM phosphate-buffered saline (PBS, pH 7.2), applied on a Sephadex G-100 column (Pharmacia, 1.8 x 48cm) and eluted with 10mM phosphate buffer (pH7.2) containing 50mM NaCl at Sml per fraction. Some portion of the active fraction at less than 10 kDa of Sephadex G—100 chromatography was applied on CM Sephadex column (Pharmacia, 15cm) and eluted with 10mM phosphate buffer (pH7.2) containing various NaCl concentrations. The active fraction of CM Sephadex chromatogra- phy was applied on a Sephadex G—25 column (Pharmacia, 1.4x90cm) and eluted with PBS at 2.5 ml per fraction. The 5 kDa peptide was clearly separated at this purification step. Assay for lymphocyte DNA synthesis Thymocytes and splenocytes DNA syntheses were assayed according to the previous method [1, 2]. Thymocytes or splenocytes (510°) from C3H/He mice (Shizuoka Experimental Animal Lab., Shizuoka) were cultured in 0.1 ml of RPMI 1640 medium (Nissui Seiyaku Co.) supplemented with 10% FCS in the presence of Concanavalin A (Con A, Sigma, 5 g/ml) for 2 days. The cells were then labeled with 0.5 ~Ci of [7H|TdR (5 Ci/mmol, Amersham, England) during the last one day. At the end of culture, trichloroacetic acid (TCA) was added at 10% in final concentration. The TCA- insoluble fraction was harvested on GF/C mem- brane filter (Whatman) and the radioactivity of the membrane was counted. Interleukin 1 (IL 1) was obtained from the culture medium conditioned by C3H/He mouse macrophages and _ partially purified by Sephadex G-100 and DEAE Sephadex A-25 columns (Pharmacia). Interleukin 2 (IL 2) was obtained from the culture medium con- ditioned by Con A-stimulated C3H/He mouse splenocytes and partially purified by Sephadex G-100 and DEAE Cellulose column chromatog- raphies. Induction and assay system of hapten-reactive cytotoxic T cells Induction and assay were performed by the previous method [6]. Stimulator T cells were prepared by passaging C3H/He mouse spleen cells through a Nylon column. They were treated with mitomycin C (50 ug/ml, Kyowa Hakko Kogyo Co., Tokyo) at 37°C for 1hr and then treated with 10mM trinitrobenzen sulfonate for trinitrophenol (TNP)-coupling at 37°C for 10 min. Responder T cells were cultured with TNP- modified or unmodified stimulator T cells (2 x 10°) in the presence or absence of spleen macrophages (210°) in 2ml of Peck-Click culture medium containing 10% FCS at 37°C for 5% CO, and 95% air for 5 days. In the *'Cr release assay by cytotoxic T cells, X5563 plasmacytoma cells from C3H/He mice were used as target cells. In vitro sensitized T cells (510°) were mixed with >!Cr- labeled X5563 cells (1 x 10*), which were modified with TNP at effector cell to target cell ratio of 50:1 in 0.2ml of RPMI 1640 medium containing 10% FCS. After the mixed cell culture at 37°C for Shr in 5% CQO, and 95% air, the culture super- natant was harvested with the aid of a Titertek collection system (Flow Labs, England) and the radioactivity on the filter was counted in a y- counter. The percentage of specific lysis of target cells was calculated as: % specific lysis=100 experimental release —control release maximal release — control release the maximal release was obtained by incubating >!Cr-labeled target cells in 1% Nonidet P40 (Sigma Chemical Co.) and the control release was obtained by incubating target cells with unsensi- tized T cells. , where Assay for antibody production by mouse lympho- cytes Assay for antibody production by mouse lym- phocytes was performed according to the previous method [7]. Splenocytes (110°) from DBA /2CrSlc mice (Shizuoka Experimental animal Lab.) were cultured for 4 days in 0.2 ml of RPMI 1640 medium supplemented with 10% FCS in the absence or presence of lipopolysaccharide (Sigma, 100 ug/ml) at different concentrations of 5 kDa inhibitory peptide. The number of IgM antibody- forming cells was determined by the plaque assay with protein A (Sigma). In brief, 25 sl of lympho- cyte suspension mentioned above, 25 wl of a 1:8 suspension of protein A-coupled sheep red blood cells (SRBC), 251 of guinea pig complement (Boelinger) diluted at 1:6 were added to 0.5% agar (Difco)(0.2ml) containing 0.05% DEAE _ dextran (Pharmacia). The cell mixture was plated Inhibitory Peptide for Lymphocyte DNA Synjthesis 101 on a Petri dish, covered with a coverglass, incu- bated at 37°C for 4 hr and plaque forming cells (PFC) were counted. RESULTS The authors prepared 5 kDa peptide inhibitory to lymphocyte DNA synthesis from the serum-free culture medium conditioned by human embryo fibroblasts. The low-molecular-weight fraction at less than 10 kDa was separated from the con- ditioned medium by Sephadex G-—100 column chromatography and this fraction was applied on a CM sephadex column. As shown in Figure 1, the major inhibitory activity was eluted at 500mM NaCl. When the active fraction from CM Sephadex chromatography was further applied on a Sephadex G—25 column, the several inhibitory activities were observed (Fig. 2). Amongst these 50 mM 250 mM 500 mM _ eaaeee (2H) TaR INCORPORATION (CPM x 104) be 10 20 FRACTION NUMBER Fic. 1. Elution profile of the inhibitory activities for lymphocyte DNA synthesis of the culture medium in CM Sephadex column chromatography. The inhibi- tory activities at less than 10 kDa from Sephadex G—100 column chromatography were applied on a CM Sephadex column and eluted with 10 mM phos- phate buffer containing various NaCl concentrations as shown in the figure. To remove the effect of the salts in the fractions on the assay for DNA synthesis, each fraction was diluted with 10mM _ phosphate buffer to 5-fold and its activity was assayed, activities, the present study was focused on the major activity at 5kDa. This fraction was charac- terized to be a heat-stable peptide as described in the previous report [3]. Although this peptide exhibited a strong inhibition on lymphocyte DNA synthesis, it did not change the viability of lympho- cytes significantly (Table 1) suggesting that the 5 kDa peptide was not toxic for lymphocytes. When the 5 kDa peptide was added to the lymphocyte culture at 0-S hr after the lectin sag i epg in) [°H]TaR INCORPORATION (CPM x Lo) 10 20 30 FRACTION NUMBER Fic. 2. Separation of 5 kDa peptide from other inhibi- tory activities by CM Sephadex G—25 column chro- matography. The inhibitory activities of fraction 14-17 in Fig. 1 were concentated and applied on a Sephadex G—25 column and eluted with PBS as described in materials and methods. Arrows in the figure show the elution positions of molecular weight markers; cytochrome C (13 kDa), insulin (5.7 kDa), Bacitracin (1.5 kDa) and NaCl from the left to the right. The smallest activities at fraction 23-26 are derived from the contaminant salts. 102 Y. Oxal, S. ISHIZAKA AND U. YAMASHITA stimulation, a remarkable inhibition of DNA synthesis was observed (Table2). When the interval between the lectin-stimulation and peptide addition was prolonged to 10hr, the inhibitory effect was slightly decreased and no significant suppression was detected any more when added at 30 hr after lectin-stimulation (Table 2). To analyze the action of this peptide on the cell membrane function, the authors examined the effect of this peptide on uridine and UTP incorporation in the activated lymphocytes. The incorporation during early periods after the addition of the peptide was decreased as compared with the activated lympho- cytes without the peptide (data not shown). These findings are the same as those in 1 kDa inhibitory peptide reported previously [1]. Immune responses are regulated by some solu- ble factors. Amongst those, IL 1 and IL 2 have been known as typical stimulatory factors for lymphocyte proliferation and functions [8, 9]. The authors studied the effect of 5 kDa peptide on the lymphocyte DNA synthesis induced by IL 1, 2 or other mitogens. As can be seen in Figure 3, IL 1- TaBLe 1. Effect of 5 kDa peptide on lymphocyte viability Viable cell numbers (x 10°) Control + Peptide Experiment 1 MeN) 2.16 Experiment 2 2.26 2.18 EE SE aR as Ed Splenocytes (510°) from C3H/He mice were cultured for 2 days in the absence or presence of the peptide (25 wl of fraction 13-15 in Fig. 2). The value in the table is an average of duplicate dye exclusion tests. TABLE 2. times after the lectin-stimulation Added at (hr) [7H]TdR incorporation (CPM) or 2-induced thymocyte DNA synthesis in the presence of Con A was considerably suppressed by the addition of 5 kDa peptide. This peptide also suppressed the thymocyte DNA synthesis induced by Con A alone. In another experiment (data not shown), it could also suppress the LPS-induced DNA synthesis by B lymphocytes of a nude mouse. These results indicate that the 5 kDa peptide from human embryo fibroblasts exhibit the nonspecific suppressive effects on T and B lympho- cyte DNA syntheses induced by IL 1, 2 and other mitogens. Next, the authors studied the effect of this peptide on the biological functions of lymphocytes. The result on the antibody production by murine B lymphocytes was shown in Figure 4. A remarkable IgM production was induced by a polyclonal activator, LPS (100 ~g/ml) and this IgM produc- tion was inhibited by the addition of the peptide in a dose-dependent manner (Fig. 4). At last, the effect of the peptide on the hapten-reactive cytotoxic T cell function was analyzed. As indicated in Table 3, Nylon column- purified T cells alone did not show the cytotoxic T cell activity to TNP-coupled stimulator cells (1.2%). When the macrophages as accessory cells were added to the culture, a remarkable cytotoxic T cell activity was evidenced as the lysis of TNP-coupled tumor cells (X5563). The addition of the peptide to the culture considerably reduced this cytotoxic T cell activity induced by the macrophages. These findings indicate that the 5 kDa peptide from human embryo fibroblasts can repress both the activation of polyclonal antibody production by B cells and the induction of hapten- Effect of 5 kDa peptide on lymphocyte DNA synthesis when added at different Relative activity (7%) untreated 13,260 0 598 + 5) WY) xc 10 2,634 + 30 LO3892 +: ++ 035 100.0 16 4.5 Ds) 6.0 581 19.8 873 82.1 Splenocytes (5 X 10°) from C3H/He mice were cultured in RPMI 1640 medium—10% FCS with Con A. The 5 kDa peptide (50 wl of fraction 13-15 in Fig. 2) was added at different times as indicated in table. The activity is expressed as the mean and standard error of triplicate assays. Inhibitory Peptide for Lymphocyte DNA Synjthesis 103 10 ag (=) ao x = a Oo Z, Enis & a fag oO A fea re) 1S) Z, H ~ xe B ar} (oo 0 None Con A Con A Con A Con A Con A Con A = - “ + + Peptide IL 1 IIL; db TG 2 I) 2 + + Peptide Peptide Fic. 3. Effects of 5 kDa peptide on the lymphocyte activation by IL 1, 2 and mitogen. C3H/He mouse thymocytes (510°) were cultured in RPMI 1640-10% FCS in the absence or presence of 5 kDa peptide (251 of fraction 13-15 in Fig.2) under the different experimental conditions as shown in the figure. The column and bar represent the mean and standard error of triplicate assays. TABLE 3. Effect of 5 kDa peptide on the induc- tion of hapten-reactive cytotoxic T cells = °'Cr Release CCG! (% Specific release) A none kL 2e Od peptide —1.4+0.9 | macrophages 80.0 + 0.9 macrophages + peptide 43.4 + 1.2 Nylon column-purified T cells (510°) were cul- tured with TNP-modified T cells (210°) in the absence or presence of 5 kDa peptide (25% in the culture mixture) and macrophages (2 x 10°) at 37°C for 5 days. The induced cytotoxic T cell activity was detected by culturing with °'Cr-labeled TNP- NUMBERS OF PFC (x 102 / CULTURE) Loe} None LPS LPS LPS LPS (PBS) + + + Peptide Peptide Peptide (25 pl) (50 pl) (100 hl) Fic. 4. Effect of 5 kDa peptide on IgM production by murine B lymphocytes. Splenocytes (110°) from DBA mice were cultured for 4 days in agar culture system with various amounts of 5 kDa peptide coupled X5563 cells as target. The value is express- ed as the mean and standard error of triplicate assays. (fraction 13-15 in Fig. 1) and IgM positive cells were counted by a protein A-PFC assay. The column and bar represent the activity of the mean and standard error of triplicate assays. 104 Y. Oxal, S. ISHIZAKA AND U. YAMASHITA reactive cytotoxic T cell function. Judging from the results mentioned above, 5 kDa peptide from human embryo fibroblasts seems to suppress the overactivation of lympho- cyte proliferation and functions and may be associated with the lymphocyte regulation in im- mune responses in vivo. DISCUSSION In this section, the authors discuss the biological significance of the peptide reported in this paper. First of all, fibroblast infiltrate into the inflamma- tory regions induced by the injury or infection together with neutrophils, macrophages and lym- phocytes. The activated macrophages and lym- phocytes release the soluble factors which stimu- late fibroblast chemotaxis and _ proliferation [10-12]. In contrast, the authors previously eluci- dated that fibroblasts produce thymocyte- activating factors stimulating lymphocyte prolif- eration and functions [13-16]. In the present paper, the authors describes that fibroblasts re- lease a low-molecular-weight peptide inhibitory to lymphocyte proliferation and function. These findings altogether suggest that fibroblasts have a potential ability for the regulation of lymphocyte proliferation and functions in the inflammatory reactions in vivo. The similar peptides are also found in the culture medium of human and murine fibroblasts, insect fibroblastic cells and murine splenocytes: They have been evidenced to posess the similar biological and biochemical properties to those of the peptide in this paper [1-4]. These peptides can inhibit DNA synthesis of the producing cells themselves without affecting their viability, and the largest amount of the peptide is released into the culture medium at the confluent cell stage (data not shown). Thus it seems reasonable to infer that these peptides may play an important role in the growth regulation of fibroblasts or lymphocytes in an autocrine manner. Another function of these peptides is a bacteri- cidal activity: They have been known to cause lysis in some Gram-negative bacteria such as E. coli or P. maltophilia {1, 2]. Since the similar peptides are also found in mammalian serum and insect — hemolymph [17-19], they seem to play a defense role against invading bacteria in the circulating system of the animal body. In conclusion, the release of these peptides from fibroblasts seems to raise an interesting problem in the field of immunological regulation in verte- brates and invertebrates. REFERENCES 1 Okai, Y. (1984) 3T3 fibroblasts release a low- molecular-weight inhibitory peptide of lymphocyte DNA synthesis into serum-free culture medium. Biochim. Biophys. Acta, 802: 287-291. 2 Okai, Y. (1985) Insect fibroblast-like cells release inhibitory peptides of lymphocyte DNA synthesis into serum-free culture medium. Insect Biochem., 15: 483-487. 3. Okai, Y. (1986) Heterogeneous molecular weight inhibitory factors for lymphocyte DNA synthesis from human embryo fibroblasts—two conversion pathways by trypsin-like protease(s). Zool. Sci., 3: 271-276. 4 Okai, Y. (1984) Murine splenocytes release an inhibitory peptide of lymphocyte DNA synthesis into serum-free culture medium. Mol. Biol. Rep., 10: 57-62. 5 Yanagisawa, K., Suenaga, Y., Nishio, K. and Gotoh, S. (1983) Establishment of a human diploid cell strain. J. Univ. Occup. Environ. Health (Kitakyushu), 5: 49-54. 6 Yamashita, U. and Hamaoka, T. (1979) The re- quirement of Ia-positive accessory cells for the induction of hapten-reactive cytotoxic T lympho- cytes in vitro. J. Immunol., 123: 2637-2643. 7 Ishizaka, S. and Moller, G. (1982) Litium chloride induces partial responsiveness to LPS in nonre- sponder B cells. Nature, 299: 365-366. 8 Mizel, S.B., Oppenheim, J.J. and Rosenstreich, D.L. (1978) Characterization of lymphocyte- activating factor (LAF) produced by a macrophage cell line, P388D, II. Biochemical characterization of LAF induced by activated T cells and LPS. J. Immunol., 120: 1504-1508. 9 Mier, J. W. and Gallo, R. C. (1982) T cell growth factor. Limphokines, 6: 137-163. 10 Leibovich, S. J. and Ross, R. (1975) The role of the macrophage in wound repair—a study with hydro- cortisone and antimacrophage serum. Am. J. Pathol., 78: 71-100. 11 Leibovich, S.J. and Ross, R. (1976) A_ mac- rophage-dependent factor that stimulates the prolif- eration of fibroblasts in vitro. Am. J. Pathol., 84: 501-514. 12 13 14 15 Inhibitory Peptide for Lymphocyte DNA Synjthesis Wahl, S. M. and Wahl, L. M. (1980) Modulation of fibroblast growth and functions by monokines and lymphokines. Lymphokines, 2: 179-201. Okai, Y., Tashiro, H. and Yamashita, U. (1982) 3T3 fibroblasts are stimulated by 12—O-tetra- decanoyl-phorbol-13-acetate to produce thymocyte- activating factors. FEBS Lett., 142: 93-95. Okai, Y., Gotoh,S. and Yamashita, U. (1985) Thymocyte-activating factors from SV40-trans- formed human embryo fibroblasts. Immunol. Lett., 9: 153-159. Okai, Y. (1985) A large-molecular-weight thymo- cyte-activation factors in extracellular matrix from SV40-transformed human embryo fibroblasts. Im- 16 17 18 19 105 munol. Lett., 11: 63-68. Okai, Y. and Yamashita, U. (1986) Human embryo fibroblast cell line produces factors to induce cyto- toxic T lymphocytes. Zool. Sci., 3: 765-771. Okai, Y. (1985) DNA synthesis inhibitory peptides in mammalian serum and insect hemolymph. Mol. Biol. Rep., 10: 123-128. Okai, Y. (1985) Dual functions of DNA synthesis inhibitory peptide in fetal calf serum. Immunol. Lett., 9: 229-223. Okai, Y. (1985) A bactericidal substance in insect hemolymph strongly suppresses lectin-induced mammalian lymphocyte DNA synthesis. Zool. Sci., 2: 663-669 PA eigen i edd Se ee se Testa Filho Bal “ ps amr A | dak, nna eed Bay sm ‘tn aieseitire AWE ae nous awl (2aet)” bi igi wah FS | Jounnl vere Meo hes nt ebtignq eda Knee eVR snort zronost “gener 4 POGOe Had »ttod locaneeyt stentdirdi — vognn! mu aonoterine ie sawp eener) ¥ sat PE os Pe is ER fyo beds hertitareel preston ABABA, felt a3 ou) iigiewete ae sot. dows, 2tegdlignge ARG. anoodany! ererbigrisn cheek abr pevwrrinar Vas ate er ae eae oes ici ie = i 2. CORI Oi several: Sek te ia ORES AA eee os LES art Bhi cru : “ or - aS ‘ . tie er Sud 4% Petey niin th Sea ae Ey. TE eee bdo vee r Wi an tall Liste ve wy J . i ri : ; a) PAR ® i ; fis “ oo » “s 2 y al TS (a = ‘ jd ee ta CE OE, PVP er eee i ee We , me : ‘ ‘ rhs. ti i. aa i= 4 ahs ress Ld ‘ q 1) ONES a: a9 a “ ; ie bal a: Gee etiam Soa Pe ek EEG i ie ia ae Megat rts ee aes Dey . # : ; : 5 ti 3) i ‘ She Cine i hee c 1a Ae t 4 146 : t a p ine rey iN : i 4 vie 5 . i . n ¥ ’ a Be PARR, LEO UE ie DAC VTE ee Paine PR ae Bese ‘ios f ; Fe FLT RAN ES bie ivehony! I risieaa ery ‘ eile (gas Alb acne 11S Sh branes” m4 ‘cin si we fs tees Five he pe sth BP ee erty iat a hentia wes Ml ol yim oyii a Tere bed oy . Panta tity 3 jms Fs j * ae ae Bocaeeu ‘i re : ¢ Aine “vr and Kas ay / f S4phes 4 } ry ieeriat ow total i ey J + i h imc td Sj ans , rue ttre 7 ‘ y , ‘ eh i ¥ = ie, as 4, ay 5 4 uy iq/' 48 7 ¥ }, e * ! : ti i thes , . ‘ AS } Hl 6 id i a ; . | if? ‘i z ; ‘ { t 4 i mais he t € ' oa i i Pot hoa Tarts w facie 7 ne ner j " ot - lan Oink i a ae ; ph. € ~ 4 } ra OED b a e—-_~8e =» -§@ eS — 170 000 6.8 electrophoresis (Fig.1). Myofibrillar proteins were hydrolyzed most strongly at pH 3.4. Myosin heavy chains and actin, which have molecular weights of 180,000 and 45,000 respectively, and other myofibrillar components having lower molecular weights were hydrolyzed almost com- 97 400 95 400 36 500 20 100 M. W. 170 000 97 400 95 400 36 500 v2 Fic. 1. Hydrolysis of myofibrillar proteins. Myofibrils were incubated in the presence (+) and absence (—) of a lysosomal fraction for 3 hr and then subjected to SDS-polyacrylamide gel electrophoresis as described in Materials and Methods. Incubation buffers used were 50 mM sodium citrate-citric acid buffer (pH2.9-5.9) and 50mM Na,HPO,-NaH,PO, buffer (pH 5.7-7.4). The values of right margin indicate molecular weights of five marker proteins, which migrate to indicated positions. Arrowheads indicate the degrading products of myosin heavy chains. 110 K. KOBAYASHI AND S. HorIucuHi pletely at this pH. The hydrolysis products of myosin heavy chains having molecular weights of 150,000, 97,000 and 90,000 were observed. At pH 2.9 and pH 3.4, a small amount of the hydroly- sis products of myosin heavy chains was detected even in the absence of a lysosomal fraction. This hydrolysis was partly suppressed by leupeptin (data not shown), suggesting contamination of myofibrillar preparations by a thiol proteinase(s). In the range of pH 4.0 to 5.4, myofibrils were less degraded than at more acidic pH: myosin heavy chains and actin were hydrolyzed in part and the number of hydrolysis products on SDS- polyacrylamide gels decreased. In the range of pH 5.9 to 7.4, myosin heavy chains degraded into fragments with molecular weights of 135,000, 125,000, 93,000 and 89,000. On the other hand, actin did not show any sign of degradation. Most of other myofibrillar components disappeared even at neutral pH during incubation. Figure 1 also shows that the amounts of proteins in control samples decreased remarkably between pH2.9 and 4.4. This was because myofibrils tended to form insoluble aggregates under these conditions. After incubation without a lysosomal fraction 31% of myofibrillar proteins was recov- ered in solubilizing buffer at pH2.9, 43% at pH3.4, 28% at pH4.0 and 32% at pH 4.4 (the mean of two experiments). In the range of pH 5.0 oor’ inital L aan Pea ar aes oes ee er o tee cD) £ Th) ce) > ~ 200 =| (©) | 2 5 100} =| 0 <{ ali, penstatil. and bone ! S| Jt 7 8 pH Fic. 2. Effect of pH on myofibril degrading activity. Myofibrils were incubated with a lysososmal fraction for 3hr, and then TCA-soluble products were meas- ured fluorometrically as described in Materials and Methods. Incubation buffers used were the same as those in Fig. 1. . and 7.4, the values increased and reached into almost 100% above pH 6.4. Myofibril degrading activity in a lysosomal fraction was quantified by measuring the amount of TCA-soluble products. As shown in Figure 2, the highest activity was observed at pH3.4. This result agreed with that from SDS-polyacrylamide gel electrophoresis. The second peak of the activity was observed at pH 5.4, and the activity decreased gradually as pH increased. Morphological observation Myofibrils dis- appeared immediately after mixing with acidic buffers (pH 2.9 and pH3.4), suggesting dissocia- Fic. 3. Phase contrast micrograph of myofibrils. Myofibrils were incubated for 3hr at pH5.4 in the presence (B) and absence (C) of a lysosomal frac- tion. Zero time control (A) is also shown. Scale: 20 pm. Myofibril Degradation by Lysosomes 111 tion of myofibrillar components. Therefore mor- phological changes of myofibrils by a lysosomal fraction were observed at pH5.4. As shown in Figure 3, myofibrils were cut into fragments after 3 hr incubation with a lysosomal fraction (Fig. 3B). On the contrary, myofibrils without a lysosomal . | | | | fraction were not degraded but aggregated during incubation (Fig. 3C). Fragmentation of myofibrils was also observed at pH 6.8, but more slowly. Effect of inhibitors A myofibrillar preparation was incubated with a lysosomal fraction in the presence of inhibitors and the effect of inhibitors on myofibril degradation was estimated by SDS- polyacrylamide gel electrophoresis (Fig. 4). At pH 3.4, leupeptin, antipain and chymostatin, inhibitors of thiol proteinases, suppressed marked- ly the hydrolysis of myofibrillar proteins including myosin heavy chain and actin (Fig.4A). And unusual products such as a peptide fragment below the band of actin appeared. On the other hand, pepstatin, an inhibitor of carboxyl proteinases, exhibited no inhibitory effect. Neither DIFP, an inhibitor of serine proteinases, nor bestatin, an inhibitor of aminopeptidases, were effective for the inhibition of myofibril degradation. These results showed that only thiol proteinase inhibitors could inhibit myofibril degrading activity when each inhibitor was used alone. However, pepstatin was shown to inhibit the activity which remained in the presence of leupeptin, because in the presence of both leupeptin and pepstatin myofibrillar pro- teins were hardly hydrolyzed. At pH5.4, the hydrolysis of myofibrillar pro- teins was inhibited by leupeptin, antipain and chymostatin, but not by pepstatin, DIFP and bestatin as well as at pH 3.4 (Fig. 4B). However, myofibril degradation which occurred in the pres- ence of leupeptin at this pH was little suppressed by pepstatin and different from leupeptin- insensitive degradation at pH 3.4. Further inhibi- tion of leupeptin-insensitive degradation was not achieved by either an increased amount of pepsta- tin (up to 12.5 ug/ml) or 0.1 mM DIFP (data not shown). At pH 6.8, leupeptin, antipain and chymostatin Fic. 4. Effect of inhibitors on hydrolysis of myofibrillar proteins at pH3.4 (A), pH5.4 (B) and pH6.8 (C). Myofibrils were incubated for 3hr with (lanes 2-9) or without (lane 1) a lysosomal fraction. Inhibitors added to incubation mixtures were as follows: no inhibitor (lane 2), leupeptin (lane 3), antipain (lane 4), chymostatin (lane 5), pepstatin (lane 6), leupep- tin and pepstatin (lane 7), bestatin (lane 8) and DIFP (lane 9). Arrowheads indicate the degrading products of myofibrillar proteins. 112 K. KOBAYASHI AND S. HorrucHi inhibited myofibril degradation almost completely (Fig. 4C). Pepstatin did not show any effect in the presence and absence of leupeptin. DIFP and bestatin were not effective as well as at pH 3.4 and pH 5.4. The effects of inhibitors on myofibril degrada- tion was examined by measuring TCA-soluble products. Leupeptin suppressed 87% of myofibril degradation at both pH 3.4 and pH5.4 (the mean of two experiments). Suppression by pepstatin was 8% at pH 3.4 and 3% at pH 5.4. In the presence of the two inhibitors 96% of myofibril degradation was suppressed at pH3.4 and 92% at pH5.4. These results confirmed that thiol proteinase in- hibitors are potent inhibitors of myofibril deg- radation by lysosomes. Effect of cysteine The above results suggested that a thiol proteinase(s) plays an important role in the degradation of myofibrils by a lysosomal fraction. Therefore, cysteine which had been known as an activator of a thiol proteinase in tadpole tails [11] was removed from incubation mixtures and the extent of myofibril degradation was compared to that in the presence of cysteine (Fig. 5). Stimulating effect of cysteine on the hydrolysis of myofibrillar proteins was remarkable 5.4 PH 3-4 6-8 Fic.5. Effect of cysteine on hydrolysis of myofibrillar proteins. Myofibrils were incubated for 3hr in the presence (+) and absence (—) of 5mM cysteine and a lysosomal fraction at pH3.4, pH5.4 and pH6.8. — C, cysteine; L, a lysosomal fraction. at pH 6.8, moderate at pH 5.4 and slight at pH 3.4. DISCUSSION Myofibrils were degraded by a lysosomal frac- tion from regressing tadpole tails especially at acidic pH (Fig. 1). Measurement of TCA-soluble products showed that myofibril degrading activity had two optimum pHs: the highest one was observed at pH3.4 and the second highest was observed at pH5.4 (Fig. 2). Myofibrils formed insoluble aggregates during incubation under acidic conditions, especially below pH 4.4. The decreased amount of myofibrils available as subs- trate may affect the activity of a lysosomal pro- teinase(s). Therefore it is not clear whether or not two acid proteinases with different pH optimum exist in a lysosomal fraction. Localization of myofibril degrading activity in lysosomes was confirmed by the fact that a lysosomal fraction showed the highest specific activity among subcellular fractions (data not shown). A preliminary experiment showed that a lysosomal fraction isolated from the tails of pro- metamophic tadpoles degraded myofibrils as well as tail lysosomes from metamorphosing tadpoles, suggesting the presence of a similar proteinase in both growing and regressing tails. Almost all of the myofibrillar proteins including myosin heavy chains and actin could be hydrolyzed by a myofibril degrading enzyme in a lysosomal fraction (Fig. 1). However, sensitivity to the attack of the proteinase was not identical among myofibrillar proteins. Myosin heavy chains seem to be more susceptible to the proteinase than actin, because myosin heavy chains were hydrolyzed at any pH examined but actin was hydrolyzed only at acidic pH. Yoshizato and Nakajima [15] reported that the tail extract of metamorphosing bullfrog tadpoles contains a thiol proteinase, an actin-degrading enzyme. Recently the actin-degrading enzyme was purified and characterized [16, 17]. This enzyme hydrolyzed actin at pH 6.0 and formed a degrading product with molecular weight of 28,000. When the enzyme was incubated with myofibrils, myosin heavy chains were hydrolyzed in addition to actin. It is not clear now whether a thiol proteinase in Myofibril Degradation by Lysosomes 113 lysosomes is identical to the actin-degrading en- zyme or not. However, actin was not hydrolyzed at around pH 6.0 in our present study. These two proteinases seem to be different from each other, but purification and characterization of the lyso- somal enzyme is needed to clarify it. A series of inhibitor experiments indicated that a thiol proteinase must be a major myofibril degrading enzyme in lysosomes. The stimulating effect of cysteine on myofibril degrading activity further supports that the activity is related to a thiol proteinase. Unusual peptide fragments appeared in the presence of thiol proteinase inhibitors at pH 3.4 (Fig. 4A). And pepstatin inhibited the appearance of these products. Therefore this leupeptin- insensitive activity could be attributed to a car- boxyl proteinase which has different substrate specificity from that of a thiol proteinase. When pepstatin was used alone, the result obtained from SDS-polyacrylamide gel electrophoresis was in- consistent with that from the measurement of TCA-soluble products: pepstatin did not affect the profile of peptide fragments on SDS-poly- acrylamide gels but inhibited partly the formation of TCA-soluble products. The reasons why diffe- rent results were obtained are not clear. In any case, a carboxyl proteinase is possibly a minor myofibril degrading enzyme in lysosomes. Komukai ef al. [6] recently observed that au- tolysis of tail muscle homogenates under acidic conditions was inhibited by leupeptin in metamor- phosing bullfrog tadpoles. Pepstatin inhibited the autolysis only when both pepstatin and leupeptin were used together. These results correspond to the present results. A lysosomal fraction contains two acid pro- teinases, a thiol proteinase and cathepsin D. A thiol proteinase which hydrolyzes myofibrillar pro- teins is probably identical to the thiol proteinase described by Kobayashi and Horiuchi [11] with respect to the optimum pHs of their activities and the effects of inhibitors and of cysteine. A carboxyl proteinase which hydrolyzed myofibrillar proteins is possibly cathepsin D [18]. Both activi- ties of a thiol proteinase and cathepsin D increase in tails during metamorphosis [11, 19]. But physiological function of the two proteinases may be different from each other. Seshimo et al. [20] showed that triiodothyronine-induced tail fin re- gression in vitro was inhibited by pepstatin but not by leupeptin, antipain and chymostatin. This indicates an important role of cathepsin D in tail fin regression. On tail muscle degeneration, however, a thiol proteinase probably plays more important role than cathepsin D as discussed above. Physiological function of lysosomal thiol pro- teinases on muscle degradation was suggested by Ishiura et al. [21], who proposed two-step mecha- nism of plasmocid-induced muscle degradation in rats. Plasmocid, a myotoxic agent, rapidly causes selective loss of @-actinin in muscle cells: this may be a non-lysosomal process. Subsequently mac- rophages invade into muscle tissues and phagocy- tose degraded muscle cells. Degradation of myofibrils in macrophages may be performed by cathepsins B and L, because macrophages were shown to contain a large amount of these thiol proteinases. These processes seem to resemble those of muscle degeneration in tadpole tails. Ultrastructural observations showed that muscle cells, in contrast to macrophages, contained only a little number of lysosomes even at metamorphic climax [2, 3, 5]. It is likely that a thiol proteinase in macrophages, which were shown to invade into muscle tissues and phagocytose sarcolytes in re- gressing tails [1-5], participates in the myofibril degradation. ACKNOWLEDGMENT This work was supported by Grant-in-Aid 58740359 from the Ministry of Education, Science and Culture, Japan and by The Science Research Promotion Fund from Japan Private School Promotion Foundation. REFERENCES 1 Weber, R. (1964) Ultrastructural changes in regressing tail muscles of Xenopus larvae at metamorphosis. J. Cell Biol., 22: 481-487. 2 Fox, H. (1972) Muscle degeneration in the tail of Rana temporaria \arvae at metamorphic climax: an electron microscopic study. Arch. Biol. (Liége), 83: 407-417. 3. Fox, H. (1975) Aspects of tail muscle ultrastructure and its degeneration in Rana temporaria. J. 10 11 12 114 Embryol. Exp. Morphol., 34: 191-207. Kerr, J.F.R., Harmon, B. and Searle, J. (1974) An electron-microscope study of cell deletion in the anuran tadpole tail during spontaneous metamor- phosis with special reference to apoptosis of striated muscle fibres. J. Cell Sci. ,14: 571-585. Watanabe, K. and Sasaki, F. (1974) Ultrastructural changes in the tail muscles of anuran tadpoles during metamorphosis. Cell Tissue Res., 155: 321-336. Komukai, M., Kobayashi, K. and Horiuchi, S. (1986) Autolysis in the tail muscles of metamor- phosing tadpoles. Comp. Biochem. Physiol., 85B: 55-59. Kobayashi, K., Hara, M. and Horiuchi, S. (1985) Isolation of lysosomes from the tail of metamor- phosing bullfrog tadpole. Comp. Biochem. Physiol., 81B: 603-607. Taylor, A. C. and Kollros, J. J. (1946) Stages in the normal development of Rana pipiens larvae. Anat. Rec., 94: 7-23. Perry, S. V. and Grey, T. C. (1956) A study of the effects of substrate concentration and certain relax- ing factors on the magnesium-activated myofibrillar adenosine triphosphatase. Biochem. J., 64: 184-192. Read, S.M. and Northcote, D. H. (1981) Mini- mization of variation in the response to different proteins of the coomassie blue G dye-binding assay for protein. Anal. Biochem., 116: 53-64. Kobayashi, K. and Horiuchi, S. (1983) Lysosomal thiol proteinase in the tadpole tail of Rana cates- beiana: some properties and changes in the activity during metamorphosis. Zool. Mag., 92: 130-137. Umezawa, H. and Aoyagi, T. (1977) Activities of proteinase inhibitors of microbial origin. In “Pro- teinases in Mammalian Cells and Tissues”. Ed. by A. J. Barrett, Elsevier/ North-Holland Publishing 13 14 15 16 17 18 19 20 21 K. KOBAYASHI AND S. HorIucui Co., Amsterdam, pp. 637-662. Laemmli, U.K. and Favre, M. (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol., 80: 575-599. Bohlen, P., Stein, S., Dairman, W. and Uden- friend, S. (1973) Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys., 155: 213-220. Yoshizato, K. and Nakajima, Y. (1982) Actin de- gradation in the metamorphosing bullfrog tadpole tail. Dev. Growth Differ., 24: 553-562. Motobayashi, N. Y. and Yoshizato, K. (1986) Par- tial purification and characterization of an actin- degrading proteinase from the tail of metamorphos- ing tadpoles. Zool. Sci., 3: 83-89. Motobayashi, N. Y., Horiguchi, T. and Yoshizato, K. (1986) Mode of degradation of myofibrils and muscle tissues by the actin-degrading enzyme. Zool. Sci., 3: 91-96. Sakai, J. and Horiuchi, S. (1979) Characterization of cathepsin D in the regressing tadpole tail of bullfrog, Rana catesbeiana. Comp. Biochem. Phys- iol., 62B: 269-273. Sakai, J. and Horiuchi, S. (1979) Changes in cathepsin D activity in the tail of Rana catesbeiana larvae during spontaneous metamorphosis. Zool. Mag., 88: 116-121. Seshimo, H., Ryuzaki,M. and Yoshizato, K. (1977) Specific inhibition of triiodothyronine- induced tadpole tail-fin regression by cathepsin D-inhibitor pepstatin. Dev. Biol., 59: 96-100. Ishiura, S., Nonaka, I., Nakase, H., Tada, A. and Sugita, H (1984) Two-step mechanism of myofibril- lar protein degradation in acute plasmocid-induced muscle necrosis. Biochim. Biophys. Acta, 798: 333-342. ZOOLOGICAL SCIENCE 4: 115-122 (1987) Steroidogenic Activity of Isoelectric Gonadotropin Components in the Pituitary of Adult Male Newt, Cynops pyrrhogaster pyrrhogaster SHIGEYASU TANAKA, MASA-AKI Hatrori) and KatsuMI WAKABAYASHI! Department of Morphology and 'Hormone Assay Center, Institute of Endocrinology, Gunma University, Maebashi 371, Japan ABSTRACT— Biological activities of isoelectric GTH components in newt pituitary were studied using in vitro testosterone or cyclic AMP production by minced mature zone of newt testes. Seven isoelectric GTH components were prepared from adult male newt pituitaries by isoelectric focusing using pH 3.5-10 ampholites. These components showed receptor-binding activity to the Anolis testis. Among them, pIs of component F and G seemed to agree with those of Ambystoma FSH when estimated by its chromatographic behavior. These isoelectric GITH components were incubated with the minced testicular tissue at different tempeature (8, 20 or 35°C) for 3hr in order to see their biological activity. The component F/G with pI 5.24 and 4.98 was effective in stimulating testosterone production similarly to the other five components which have alkaline to neutral pIs. When testes were incubated at 20°C, maximal steroidogenic activity was 7-17 times greater than basal activity without GTH, and a dose-response relationship was observed with GTH components. Furthermore, testosterone production increased as temperature increased. In contrast, regrardless of temperature, almost all the components stimulated cyclic AMP production at the highest doses tested (2-5 times over control). The relationship © 1987 Zoological Society of Japan between steroidogenic activity and temperature was discussed. INTRODUCTION Bullfrog Rana catesbeiana pituitary has two chemically distinct gonadotropins (GTH) which show similar behavior to mammalian LH and FSH in ion-exchange chromatography, respectively [1-7]. Recently, available information on biolog- ical activities of these two GTHs has been accumulated [6-12]. However, with respect to other amphibians, especially in urodele species, many problems related to their biochemical and biological properties remain unsolved. We have previously examined isoelectric focusing (IEF) profiles of newt pituitary GTH activity in different reproductive states, employing Xenopus and Ano- lis radioreceptor assay (RRA), and found seasonal difference in the IEF profiles [13]. Furthermore, the IEF profiles of newt GTH were markedly different from those of bullfrog, suggesting a Accepted July 22, 1986 Received June 18, 1986 possible difference in biological properties be- tween these species [13-15]. However, receptor- binding activity does not always reflect the biologi- cal activity [16-18]. Therefore, it was necessary to elucidate what kinds of biological properties each IEF component of newt pituitary GTH possesses. In the present study, testosterone and cyclic AMP production by minced newt testes were used to examine the biological activity. We also studied the influence of temperature on testosterone pro- duction, because temperature plays an important role in the steroidogenic effect of GTH on amphi- bian testes [for review, 19, 20]. MATERIALS AND METHODS Isoelectric focusing (IEF) and separation of isoelec- tric components Adult male newts Cynops pyrrhogaster pyrrho- gaster were collected at an appointed station in Hayakawa, Niigata Prefecture in the middle of 116 S. TANAKA, M. Hatrori AND K. WAKABAYASHI July, 1983 (Experiment I) and again in July, 1984 (Experiment II). The pituitaries were removed immediately after collection, and then pituitary extracts were prepared by homogenizing approx- imately 175 glands (Experiment I) and 200 glands (Experiment II) with distilled water (DW), fol- lowed by freezing-thawing, and by centrifugation at 15,000rpm for 20min. The IEF fractionation was carried out on a 25 ml-column with Ampholine pH3.5-10 (LKB Produkter) at 1% concentration in a sorbitol density gradient from 5 to 50%, according to the method described in the previous reports [13, 15]. IEF with the anode at the top of the column was run at 2-4°C for 4 days, the voltage applied being increased stepwise from 300 to 900 V. After focusing, 0.45-ml fractions were collected at a flow rate of 0.1ml/min_ under simultaneous measurement of pH with a flow- through type of glass electrode (Fuji Kagaku Keisoku, SE 1600GC). The fractions containing GTH activity were detected by Anolis RRA (see below). Then, each fraction was dialysed against cold DW for 2 days after adding 1.5ml of PBS containing 1% bovine serum albumin (PBS-BSA), and lyophilized. The materials were stored at —20°C until bioassay. In vitro estimation of biological activity The biological activity of isoelectric GTH com- ponents in the newt pituitary was measured by in vitro testosterone and cyclic AMP production in minced mature zone of newt testes containing androgen-secreting tissue [21, 22]. Testicular tissues were obtained from adult male newts collected in early November, 1983 (Experiment I) and late November, 1984 (Experiment II) at the Same station as mentioned above. Newts were decapitated and only the mature zones of their testes were quickly excised. The testicular tissues were minced with scissors as small as possible in ice-cold Medium 199 (GIBCO Laboratories, Grand Island Biochemistry Company, Grand Is- land, NY) modified for amphibians containing 0.1% BSA (incubation medium). After centri- fugation at 1,000 rpm for 15 min, the supernatant, containing spermatozoa, was removed by suction. This procedure was repeated three times to re- move spermatozoa completely. The testicular tissue pellet was resuspended in the incubation medium. Incubations were performed at 8, 20 or 35°C in a 24-well tissue cluture cluster (Costar, Cambridge, MA) under an atmosphere of 95% O,-5% CO; for 3 hr. The minced testicular tissue equivalent to approximately one-third of testis was incubated in triplicate in 0.5 ml of the incuba- tion medium containing 0.5mM_ 3-isobutyl-1- methylxanthine (Sigma) with or without varying doses of the GTH preparation. After incubation, 50 xl of 10% NaN3 was added to each well to stop steroidogenesis. Each incubation medium was transferred to a glass culture tube and heated at 90°C for 10min in the presence of 1mM theophiline, then centrifuged at 3,000rpm for 20min. Testosterone and cyclic AMP in the supernatants were measured by radioimmunoassay using iodinated tracer, as previously described [23, 24]. Anolis radioreceptor assay (RRA) Anolis RRA was performed with Anolis testicu- lar homogenates as receptor fractions and '**I-rat FSH as radioligand according to the described in the previous reports [13, 15]. '”°I-labeled rat FSH was prepared by radioiodination of NIADDK rat I-3 (supplied by the National Pituitary Agency and the Pituitary Hormones and Antisera Center, NIADDK, NIH) with lactoperoxidase according to the method of Miyachi et al. [25] with minor modification. Nonspecific binding was determined by incubation of the receptor fraction with iodi- nated tracer in the presence of 100 I.U. of pregnant mare serum gonadotropin (Sankyo Manufacturing Co.). Results were expressed as micrograms of NIADDK rat FSH I-3. Statistical analysis In Anolis RRA system, the inhibition curves obtained with isoelectric GITH components and standard preparation were linearized by the method of least squares on logarithmic amount of hormone preparations versus logit B/Bo, and their slopes were compared by Student’s t-test or Cochran-Cox test. Steroidogenic Activity of Newt Gonadotropin 117 RESULTS Experiment I Isoelectric GTH components To obtain isoelectric GTH component prepara- tions, pituitary extract from adult male newts collected in mid-July, 1983 was fractionated by IEF using pH 3.5-10 ampholites. When GTH activity was measured by Anolis RRA, seven peaks (three major peaks, two mediun peaks and two minor peaks) appeared in the alkaline to acidic pH region (Fig. 1). These components were desig- (0) 10 20 30 40 50 60 Fraction Number ug X NIAMDDK rat FSH |I-3/Tube Fic. 1. Isoelectric focusing profile of gonadotropin in the pituitary extract of adult male newt captured in the mid-July, 1983. The fractions were assayed for GTH with Anolis RRA. Dots in the figure repre- sent the pH of the fractions. nated for convenience as components A (pI 9.24), B (pI 8.75), C (pI 8.31), D (pI 7.53), E (pI 6.75), F (pI 5.24) and G (pI 4.98), respectively. This IEF profile was almost similar to that of the mid-July in the previous report [13]. Since the components F and G in the acidic pH region were also a very small amount, they were collected under one fraction as component F/G in the following experi- ment. When the six isoelectric GTH components were assayed by Anolis RRA system in triplicates in several different dilutions, their inhibition curves were Statistically parallel to the standard curve obtained with rat FSH, indicating that it is possible to express the relative amount of each component as Anolis testicular receptor-binding activity of NIADDK rat FSH I-3 (Table 1). In the following results, therefore, the relative potency represented the ratio of biological activity to receptor-binding activity (B: R). Biological activity In the present experiment using doses of the hormone ranging from 0.1 to 6.4ng/well, all the components were effective in stimulating testoster- one production (Fig. 2). Testosterone production was dose-dependently increased by each compo- nent. At the maximal dose (6.4ng/well) of each component, testosterone produced by the minced testicular tissues was 5.19 ng/well (component A), 1.63ng/well (B), 1.96ng/well (C), 2.66ng/well (D), 3.53ng/well (E) and 3.14ng/well (F/G). These values were 4-13 times greater than that without stimulation (0.40ng/well). In addition, the component A with the highest pI showed the highest B: R ratio. Experiment II To elucidate the maximal response of testoster- one production, higher doses of GTH preparation were used in the present experiment. Further- TABLE 1. Inhibition curves of isoelectric GITH components Species R Slope S2E: No. of Points rat FSH — 0.986 = il Zit 0.089 7/ Component A 0997) = 11S) 0.061 4 B —0.976 = 1/33) 0.156 ») C 08999 — 1.306 0.024 4 D 08953 = 11107 0.246 4 EB — 0.987 —1.243 0.099 6 F/G — 0.980 = Avo 0.169 4 Slopes are from plots of logit % bound vs log of dilution factors or hormone concentration. All the slopes are not significantly different from the slope of rat FSH. 118 S. Tanaka, M. Hatrori AND K. WAKABAYASHI o = ~ fe) Cc oO Cc (eo) () o (eo) 2) o - 0.1 0.4 1.6 6.4 Isoelectric GTH Component (ng/well) Fic. 2. Stimulation of in vitro testosterone production by the isoelectric GTH components obtained from newt pituitary extract in mid-July, 1983. Testes were from adult newts captured in early November. The minced testicular tissue equivalent to approx- imately one-third of a testis were incubated at 20°C for 3hr in 0.5ml of modified Medium 199 for amphibians with or without the GTH component. The hormonal dose of each component was express- ed as the Anolis testicular receptor-binding activity of NIADDK rat FSH I-3. The points are means of triplicate incubations. The bar and vertical line rep- resent the mean and SE of three controls. more, incubation was carried out at different temperatures to examine the effects of tempera- ture on steroidogenic activity. At the same time, cyclic AMP production was assayed. Isoelectric GTH preparations were obtained by the same method as described in Experiment I. The IEF profile of newt pituitary GTH collected in mid-July, 1984 is shown in Figure 3. The pls of the isoelectric GTH components were quite similar to those of Experiment I, although the relative amount of each component was somewhat differ- ent. o Q a) od lop) ily ac op) ve © SZ Q Q = x Zz x< fo) 0 10 20 30 40 50 = Fraction Number Fic. 3. Isoelectric focusing profile of gonadotropin in the pituitary extract of adult male captured in mid- . July, 1984. Effect of temperature on biological activity a) Testosterone production Since the available amount of each component was limited, the range of doses used in this experiment differed according to the component. When incubated at 35°C, the basal control level was 2.18+0.15ng/well, and component F/G was the most potent of all the components tested 55 59 Testosterone (ng/well) 4 10 40 100 400 Isoelectric GTH Component (ng/well) = cAMP (pmole/well) Isoelectric GTH Component (ng/well) Fic. 4. Stimulation of in vitro testosterone and cyclic AMP production by minced testes in late Novem- ber, 1984 with the isoelectric GTH components at 35°C. Incubation conditions and symbols are the same as those in Fig. 2. (Fig. 4). The maximal steroidogenic activity of component F/G occurred at 100 ng/well (ca. 23 times over control), but at the highest dose (300 ng/well) testosterone production showed a slight decrease. The other five components showed maximal steroidogenic activity at the highest dose tested (ca. 15 to 20 times over control). Thus, there was no difference in potency among each isoelectric GTH component. In an incubation at 20°C, at the lower doses (4 to 10ng/well) which are similar to those used in Experiment I, the component A with the greater Steroidogenic Activity of Newt Gonadotropin 119 Testosterone (ng/well) 4 10 40 100 400 Isoelectric GTH Component (ng/well) 12.5 o1 S ~ 2 [e) £ jo} o. = < Oo 4 10 40 100 400 Isoelectric GTH Component (ng/well) Fic.5. Stimulation of in vitro testosterone and cyclic AMP production by minced testes in late Novem- ber, 1984 with the isoelectric GTH components at 20°C. Incubation conditions and symbols are the same as those in Fig. 2. alkaline pI value showed the highest stimulation in testosterone production (Fig.5). In each of the isoelectric components maximal steroidogenic activity was 7-17 times as great as that without stimulation (1.29+0.20 ng/well). When incubated at 8C, the response was lower than at 20 and 35°C. Maximal steroidogenesis caused by the isoelectric GTH components was 3-9 times as much as that of control stimulation (0.79 +0.02 ng/well) (Fig. 6). b) Cyclic AMP production The effect of all the isoelectric GTH compo- nents on cyclic AMP production in the minced testicular tissue was measured (Figs. 4, 5 and 6). At 8 and 20°C, all the components stimulated cyclic AMP production only at the highest doses tested. The maximal response was only 2-5 times as great as that of the control (2.58+0.31 pmol /well at 8°C and 2.45 +0.69 pmol/well at 20°C). At 35°C, a similar increase in cyclic AMP was noted in the four components except components A and B. o = ™~™ o (= oO = fe) ® n” (e) ” () | ae 4 10 40 100 400 Isoelectric GTH Component (ng/well) 12s 010. = oO iS Uc = Qa ~ 5. a = < 2. () 4 10 40 100 400 Isoelectric GTH Component (ng/well) Fic. 6. Stimulation of in vitro testosterone and cyclic AMP production by minced testes in late Novem- ber, 1984 with the isoelectric GTH components at 8°C. Incubation conditions and symbols are the same as those in Fig. 2. DISCUSSION With respect to amphibian GTH, Licht and his co-workers have reported that pituitaries of both anuran and urodele species have two kinds of GTH which correspond to mammalian and avian LH and FSH, respectively (for review, [3, 26]). In the study on urodele Ambystoma LH and FSH, however, their biochemical and biological prop- erties are not fully confirmed, because of difficulty of collecting their pituitaries in large quantities [27]. On the other hand, to observe all the components of GTH in as natural as possible and small amount of materials, we developed the IEF technique, coupled with RIA or RRA, and eluci- dated the IEF profiles of several amphibian GTH activity [13-15]. When newt pituitary extracts were fractionated by IEF, and GTH activity was measured by two RRA systems; one is Xenopus RRA, which is thought to be LH-specific, and the other is Anolis RRA, five GITH components appeared in the alkaline to neutral pH regoin, and 120 S. TANAKA, M. HatroriI AND K. WAKABAYASHI Xenopus RRA-positive components closely cor- responded to Anolis RRA-positive ones [13]. On the other hand, in the IEF profiles of bullfrog GTH, Xenopus RRA-positive components were found in the alkaline pH region, whereas Anolis RRA-positive components were found in the neutral to acidic pH region [14, 15]. Thus both RRA-positive components were quite independ- ent, indicating distinct separation of LH and FSH similar to that of mammalian and avian species. Therefore, it was estimated that the biological properties of newt GTH may be somewhat differ- ent from those of bullfrog LH and FSH. Fur- thermore, IEF profile of newt GTH varied de- pending on seasonal reproductive state; only in July when spermatogenesis was active, Anolis RRA gave one or two exceptional components in the neutral to acidic pH region [13]. Since the pls of these additional components seemed to agree with those of Ambystoma FSH prepared by Licht et al. [27] when estimated by its chromatographic behavior, it was very interesting to elucidate whether or not all the components might possess steroidogenic activity. First, in the present study, it was confirmed that IEF profiles of newt pituitary GTH in mid-July have two additional components F and G in the neutral to acidic pH region, which were obtained by Anolis RRA [13]. Secondly, it was clearly demonstrated that component F/G in the neutral to acidic pH region stimulated testosterone pro- duction similar to the other five components with alkaline to neutral pIs, when the biological activity of each component was examined on the basis of in vitro testosterone production in minced mature zone of newt testes captured in November. Thus, with respect to steroidogenic activity in the homo- logous testes, the specificity of each of the isoelec- tric components in newt pituitary GTH was not clearly distinct. However, this was in contrast to an earlier report that in secretion of Ambystoma testicular testosterone Ambystoma LH is about 50 times more potent than Ambystoma FSH, although Ambystoma FSH is not highly purified compared with LH [11]. In the range from 0.1 to 6.4ng/well used in Experiment I, the components with the greatest alkaline pI value showed the highest B: R ratio, — indicating that the hormonal actions of this compo- nent are efficiently amplified in the biological response to the final step. When incubated at 20°C in Experiment II, similar results were obtained in the lower dose range, but not at higher doses tested. Since the testis of amphibians living temperate zone shows a discrepancy between spermatogene- sis and steroidogenesis, depending on tempera- ture, the importance of temperature to testicular function has been pointed out for a long time, and many studies have already been performed in vivo on the effect of temperature and gonadotropin upon testicular function (for review, [19]). Our previous study [20] on newt testes with mammalian LH and FSH has shown that FSH is more potent in stimulating spermatogenesis at the higher temper- ature (18°C), whereas LH is potent in testosterone production at the lower temperature (8 C), as well as the results in Rana esculenta [28] and Triturus cristatus carnifex [29]. In the present study, the incubation at 8 and 35°C in addition to 20°C was performed because our previous studies in respect to newt testicular function and environmental temperatures suggested that approximately 8°C is optimal for steroidogenesis, and 20°C for sperma- togenesis [22, 30] and the temperature of 35°C seemed to be lethal for newt. We had expected that incubation at 8°C would result in the highest increase in testosterone production, but the pres- ent results indicated that testosterone production increases with increasing temperatures. Similar results were obtained in bullfrog although a significant decrease in androgen secretion was observed at 36°C [10]. On the other hand, Kubokawa and Ishii [31, 32] reported that gona- dotropin receptor of newt testes has an optimal temperature of affinity for rat FSH between 15 and 20°C, but not around 37°C. These discrepancies might be due to differences in incubation time or seasonal fluctuations in the testicular sensitivity to GTHs. It is well established that in mammals the action of GTHs in their target organ is mediated by cyclic AMP. In the present study, almost all the isoelectric GIH components at the highest dose significantly stimulated cyclic AMP production. Similar stimulation was found at three different Steroidogenic Activity of Newt Gonadotropin 121 temperatures, suggesting that an increase in cyclic AMP production is not dependent on tempera- ture. In the range of doses with no clear stimula- tion of cyclic AMP production, almost all the isoelectric GTH components stimulated testos- trone production. This result may be consistent with the fact that in mammals receptor-occupation of less than 1% of the whole LH receptor is sufficient to elicit a maximal steroidogenic re- sponse in Leydig cells [33]. Recently, Abe [34] reproduced the differentia- tion of newt primary spermatocytes into sperma- tids in vitro, and moreover Nishikawa and Abe [35] demonstrated that the percentage of cell that progressed to an advanced stage is not increased by the addition of fetal bovine serum, ovine FSH, 5a-DHT and testosterone propionate at 22°C. On the other hand, secondary spermatogonia undergo some mitotic divisions in vitro but soon degener- ate, which can be stimulated by neither mamma- lian FSH or LH [36]. Furthermore, Muller and Licht [37] reported that bullfrog LH as well as FSH stimulates the proliferation of spermatogonia in the frog testes. Hanaoka ef al. [6] found that bullfrog FSH (acidic GTH-III, pI 6.2) stimulates only about 3 times as potently as bullfrog LH (basic GTH-IV, pI 9.3) the increase in testicular weight in hypophysectomized young Xenopus. Thus, there was no evidence indicating apparent FSH specificity for spermatogenesis. Therefore, it is possible that all the isoelectric components of newt pituitary GTH may stimulate some stages of the spermatogenic process. To clarify whether or not each isoelectric GITH component of newt pituitary with potency in testosterone production also stimulates spermatogenesis, further studies will be necessary. ACKNOWLEDGMENTS We are grateful to Professor K. Kurosumi of this Institute for his encouragement during this work. We are also grateful to Dr. Y. Hanaoka of the Institute for discussing this paper and Dr. A. F. Parlow of the Pitui- tary Hormones and Antirera Center and National Pituitary Agency, NIADDK for supplying rat FSH. i) Nn 10 11 13 REFERENCES Licht, P. and Papkoff, H. (1974) Separation of two distinct gonadotropins from the pituitary gland of the bullfrog Rana catesbeiana. Endocrinology, 94: 1587-1594. Papkoff, H., Farmer, S. W. and Licht, P. (1976) Isolation and characterization of luteinizing hor- mone from amphibian (Rana catesbeiana) pituita- ries. Life Sci., 18: 245-250. Licht, P., Papkoff, H., Farmer, S. W., Muller, C. H., Tsui, H. W. and Crews, D. (1977) Evolution of gonadotropin structure and function. Recent Prog. Horm. Res., 33: 169-248. Takahashi, H. and Hanaoka, Y. (1981) Isolation and characterization of multiple components of basic gonadotropin from bullfrog (Rana catesbeiana) pituitary gland. J.Biochem., 90: 1333-1340. Takahashi, H. and Hanaoka, Y. (1985) Character- ization of bullfrog gonadotropin molecules in com- parison with mammalian hormones. In “Current Trends in Comparative Endocrinology”. Ed. by B. Lofts and W. N. Holmes, Hong Kong Univ. Press., Hong Kong, pp. 187-188. Hanaoka, Y., Hayashi, H. and Takahashi, H. (1984) Isolation and characterization of bullfrog gonadotropins. Gunma Symposia on Endocrinolo- gy, 21: 63-77. Takada, K. and Ishi, S. (1984) Purification of bull- frog gonadotropins: Presence of new subspecies of luteinizing hormone with high isoelectric points. Zool. Sci., 1: 617-629. Licht, P. and Crews, D. (1976) Gonadotropin stim- ulation of in vitro progesterone production in reptilian and amphibian ovaries. Gen. Comp. En- docrinol., 29: 141-151. Muller, C. H. (1977) Jn vitro stimulation of 5a- dihydrotestosterone and testosterone secretion from bullfrog testis by nonmammalian and mammalian gonadotropins. Gen. Comp. Endocrinol., 33: 109- 1VAR Muller, C.H. (1977) Plasma 5a-dihydrotestosterone and testosterone in the bullfrog, Rana catesbeiana: Stimulation by bullfrog LH. Gen. Comp. Endocri- nol., 33: 122-132. Muller, C.H and Licht, P. (1980) Gonadotropin specificity of androgen secretion by amphibian testes. Gen. Comp. Endocrinol., 42: 365-377. Gavaud, J., Licht, P. and Papkoff, H. (1979) In vitro stimulation of cyclic-AMP production in Rana catesbeiana ovaries by homologous gonadotropins. Gen. Comp. Endocrinol., 38: 83-92. Tanaka, S., Takikawa,H., and Wakabayashi, K. (1981) Seasonal variation in pituitary gonadotropin in the adult male newt, Cynops pyrrhogaster pyrrho- gaster, revealed by isoelectric focusing technique 14 15 16 17 18 20 21 22 23 24 Ds) 26 122 and radioreceptor assay. Endocrinol. Japon., 28: 335-345. Tanaka, S., Hanaoka, Y. and Wakabayashi, K. (1983) A homologous radioimmunoassay for bull- frog basic gonadotropin. Endocrinol. Japon., 30: 71-78. Tanaka, S., Park, M. K., Takikawa, H. and Waka- bayashi, K. (1985) Comparative studies on the elec- tric nature of amphibian gonadotropin. Gen. Comp. Endocrinol., 59: 110-119. Licht, P. and Midgley, A.R., Jr. (1976) Jn vitro binding of radioiodinated human follicle-stimulating hormone to reptilian and avian gonads: Radioligand studies with mammalian hormones. Biol. Reprod., 15: 195-205. Licht, P. and Midgley, A.R., Jr. (1976) Competi- tion for the in vitro binding of radioiodinated human follicle-stimulating hormone in reptilian, avian and mammalian gonads by nonmammalian gonadotro- pins. Gen. Comp. Endocrinol., 30: 364-371. Licht, P. (1980) Relationship between receptor binding and biological activities of gonadotropins. In “Hormones, Adaptation and Evolution”. Ed. by S. Ishu, T. Hirano and M. Wada. Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin, pp. 167-174. Lofts, B. (1974) Reproduction. In “Physiology of the Amphibia II”. Ed. by. B. Lofts, Academic Press, New York and London, pp. 107-218. Tanaka, S. and Takikawa, H. (1984) Amphibian and reptilian gonadotropin: Biological activity. Gunma Symposia on Endocrinology, 21: 37-61. Imai, K. and Tanaka, S. (1978) Histochemical and electron microscopic observation on the steroid hormone-secreting cells in the testis of the Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster. Dev. Growth Differ., 20: 151-167. Tanaka, S. and Iwasawa, H. (1979) Annual change in the testicular structure and sexual character of the Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster. Zool. Mag., 88: 295-305. Hattori, M., Sakamoto, K. and Wakabayashi, K. (1983) The presence of LH components having different ratios of bioactivity to immunoreactivity in the rat pituitary glands. Endocrinol. Japon., 30: 289-296. Hattori, M. and Wakabayashi, K. (1983) Different profiles of isoelectric avian luteinizing hormone components in biological activity and immunoreac- tivity. Endocrinol. Japon., 30: 551-560. Miyachi, Y., Vaitukaitis, J.L., Nieschlag, E. and Lipsett, B. (1972) Enzymatic radioiodination of gonadotropins. J. Clin. Endocrinol., 34: 23-28. Licht, P. (1979) Reproductive endocrinology of 27 28 29 30 Si 3, 33 34 35) 36 37 S. TANAKA, M. Hatrori AND K. WAKABAYASHI reptiles and amphibians: Gonadotropins. Ann. Rev. Physiol., 41: 337-351. Licht, P., Farmer,S.W. and Papkoff, H. (1975) The nature of the pituitary gonadotropins and their role in ovulation in a urodele amphibian (Ambysto- ma tigrinum). Life Sci., 17: 1049-1054. Kort, E. J. M. de (1971) “Het interstitium testis bij de groene kikker, Rana esculenta: Een histometrisch en histochemisch onderzoek.” Grafisch Bedrijf Fa. Lammers en Zn., Terborg. Vellano. C., Sacerdote,M. and Mazzi, V. (1974) Effects of mammalian gonadotropins (FSH and LH) on spermatogenesis in the crested newt under different temperature conditions. Monitore Zool. Ital., 8: 177-188. Tanaka, S., and Takikawa, H. (1983) Seasonal changes in plasma _ testosterone and 5a- dihydroteststerone levels in the adult male newt, Cynops pyrrhogaster pyrrhogaster. Endocrinol. Japon., 30: 1-6. Kubokawa, K. and Ishii, S. (1980) Follicle-stim- ulating hormone (FSH) receptors in the testis of the newt, Cynops pyrrhogaster, and comparison of temperature dependency of the receptors with those of the vertebrates. Gen. Comp. Endocrinol., 40: 425-433. Kubokawa, K. and Ishii, S. (1984) Adaptation of testicular follicle-stimulating hormone receptors to ambient temperatures in vertebrates: Equilibrium analysis. Gen. Comp. Endocrinol., 54: 277-282. Dufau, M. L., Tsuruhara, T., Horner, K. A., Podesta, K. A. and Catt, K. J. (1977) Intermediate role of adenosine 3’: 5’-cyclic monophosphate and protein kinase during gonadotropin-induced ster- oidogenesis in testicular interstitial cells. Proc. Natl. Acad. Sci. USA, 74: 3419-3423. Abe, S. (1981) Meiosis of primary spermatocytes and early spermiogenesis in the resultant spermatids in newt, Cynops pyrrhogaster in vitro. Differentia- tion, 20: 65-70. Nishikawa, A. and Abe,S. (1983) Progression thoughout all stages of meiosis from the early prophase of newt primary spermatocytes in vitro. Dev. Growth Differ., 25: 323-331. Abe, S. and Tanaka, S$. (1980) Behavior of the secondary spermatogonia of the newt, Cynops pyr- rhogaster in in vitro culture. Dev. Growth Differ., 22: 851-857. Muller, C.H. and Licht, P. (1978) Gonadotropin control of testicular function in the anuran am- phibian, Rana pipiens. In “Comparative Endocri- nology”. Ed by P.J.Gaillard and H.H.Boer, Else- vier/North-Holland, New York, pp. 85. ZOOLOGICAL SCIENCE 4: 123-134 (1987) Neuropeptide Y (NPY)-like Immunoreactive Neurons in the Brain and Pituitary of the Amphibian Rana catesbeiana DANIEL CAILLIEz!, JEAN-MICHEL DANGER!, ANN C. ANDERSEN! Juuia M. Potak’, GEORGES PELLETIER®, KOSUKE KAWAMURA‘, SAKAE KIKUYAMA* and Husert Vaupry!”> ‘Groupe de Recherche en Endocrinologie Moléculaire, UA CNRS 650, Unité Alliée a TINSERM, Faculté des Sciences, Université de Rouen, 76130 Mont-Saint-Aignan, France, Department of Histochemistry and Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London WI20HS, United Kingdom, *>MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l Université Laval, Québec GIVG42, Canada, and *Department of Biology, School of Education, Waseda University, Tokyo 160, Japan ABSTRACT—The distribution of neuropeptide tyrosine (NPY) and the C-terminal peptide of the pro-NPY molecule (C-PON) was examined in the brain of the frog using the indirect immunofluores- cence and the peroxidase-immunoperoxidase techniques. Very dense populations of perikarya contain- ing simultaneously NPY- and C-PON-like immunoreactivity were localized in various regions of the brain, in particular in the pallium, the posterocentral nucleus of the thalamus, the mesencephalic tegmentum, the mesencephalic cerebellar nucleus, the dorsal and ventral infundibular nuclei and the preoptic nucleus. The co-existence of NPY- and C-PON-like material within the cell bodies indicates that frog NPY derives from a pro-hormone which exhibits a high degree of sequence homologies with mammalian pro-NPY. A dense network of nerve fibers containing NPY and C-PON immunoreactive material was found in the infundibular nuclei, coursing towards the internal zone of the median eminence and the pituitary stalk and terminating in the pars intermedia of the pituitary. The presence of a neuronal system containing simultaneously NPY and C-PON in the infundibulo-hypophyseal complex supports the view that these peptides may participate in the neuroendocrine regulation of the frog © 1987 Zoological Society of Japan pituitary. INTRODUCTION Neuropeptide tyrosine (NPY) was isolated origi- nally from porcine brain and sequenced by Tate- moto [1, 2]. This 36-amino acid peptide exhibits considerable sequence homologies with various peptides of the pancreatic polypeptide family mainly with peptide tyrosine-tyrosine (PYY) and avian pancreatic polypeptide (APP) [2]. Although the presence of these latter peptides has been initially reported in the central nervous system, there is now clear evidence that APP- and PYY- like immunoreactivities in the brain are actually Accepted September 30, 1986 Received July 31, 1986 > To whom reprints should be requested. due to authentic NPY [3]. Using immunohisto- chemical methods, NPY containing neurons have been localized in the brain of various mammalian species including rat [4-6], monkey [7, 8], and man [9-11]. Combination of radioimmunoassay with high performance liquid chromatography has established the identity of NPY in the brain [3, 12]. All these studies have shown that NPY has a widespread distribution and is present in extremely high concentrations, especially in the cerebral cortex and the hypothalamic area. Thus, it was postulated that NPY may act as both a neu- romediator and a neurohormone [5, 13]. In sub-mammalian vertebrates, very few studies con- cerning the distribution of NPY have been re- ported. NPY has been localized and identified in the retina of the fish Carassius auratus [14] and in 124 D. CAILLIEZ, J.-M. DANGER et al. the central nervous system of the amphibian Rana ridibunda [15]. Recently the nucleotide sequence of the neuronal-specific gene encoding the NPY precursor molecule has been determined from human pheochromocytoma [16]. The deduced pre-pro-NPY amino acid sequence exhibits two potential sites for proteolytic cleavage which would generate three peptides: a 28 residue signal peptide, NPY and a C-terminal 30 amino acid peptide called C-PON [16]. Antisera have been raised against this cryptic peptide and co- localization of C-PON and NPY in the same neurons has been reported [17]. In the present study, we examined the distribu- tion of NPY and C-PON immunoreactive neurons in the brain and pituitary of the amphibian Rana catesbeiana using both the indirect immuno- fluorescence and the peroxidase-antiperoxidase techniques. MATERIALS AND METHODS Tissue preparation Adult bullfrogs (Rana catesbeiana) 300g body weight were caught in April in their natural environment and maintained at constant tempera- ture for one week. The fixation was performed by transcardial perfusion of a 4% paraformaldehyde solution in 0.1 M pH7.3 sodium cacodylate buffer. The brains were carefully dissected and post-fixed in the same fixative for 4hr. Then the tissues were washed overnight in a 5% sucrose solution and transferred to cacodylate buffer containing 10% sucrose. The brains were embedded and sectioned in a cryostat (Frigocut 2700, Reichert Jung). Consecutive sections (8 um thick) were processed for the indirect immunofluorescence or the perox- idase-antiperoxidase immunohistochemical proce- dure. The slices were incubated overnight at 4°C in a humid atmosphere with antiserum to NPY or to C-PON at the respective working dilutions 1:600 and 1:200. Antisera Antibodies to NPY (n°4603) [9] were raised in rabbits by injection of a mixture of synthetic porcine NPY, methylated bovine serum albumine | (BSA) and complete Freund’s adjuvant. Anti- bodies to C-PON (n’JN8) [17] were raised in rabbits by injection of synthetic human C-—PON coupled to BSA with glutaraldehyde. Indirect immunofluorescence The tissues were rinsed and incubated 1 hr with fluorescein-isothiocyanate-conjugated anti-rabbit y-globulins (GAR/FITC; Nordic Labs) at the working dilution 1:60. The sections were rinsed, mounted in glycerol and examined under a Leitz Orthoplan microscope. Peroxidase-Antiperoxidase (PAP) The sections were rinsed and incubated for 1 hr with goat-anti-rabbit y-globulins diluted 1:30 in Tris-HCl pH 7.6 buffer. The sections were rinsed and incubated for an additional hour with rabbit peroxidase-antiperoxidase solution (diluted 1: 250) (Nordic Labs). The enzymatic activity was revealed using a diaminobenzidine (0.03%)- hydrogen peroxide (0.01%) mixture in Tris-HCl buffer. Finally the sections were rinsed, dehy- drated, mounted and examined under a Leitz Orthoplan microscope. Controls No reaction product could be observed on control sections using normal rabbit serum as the primary antibody. Pre-incubation of the diluted NPY-antiserum with 10°-°M NPY resulted in the complete loss of staining. As synthetic C-PON was not available, it was impossible to perform additional control using pre-absorbed C—-PON antibodies. RESULTS NPY and C-PON-immunoreactivities are widely distributed throughout the brain of the frog with remarkable co-distribution. The specificity of the immunoreaction was shown by a complete loss of staining after pre-absorbtion of the NPY antiserum with the homologous synthetic antigen 10° °M and after substitution of the first antiserum for normal rabbit serum. The indirect immunofluorescence and the PAP procedures revealed C-PON and NPY immunoreactive perikarya and nerve fibers in NPY-like Immunoreactive Neurons 125 various brain regions (Fig. 1). terminals are shown with small dots. The number Stereotaxic mapping of the NPY and C-PON of circles approximates the number of cells in a immunoreactivities is shown in Figure2. Cell givensection. Abbreviations according to Wada er bodies are indicated as large filled circles and a. [18] have been listed in legend to Figure 2. Fic.1. (a) PAP technique reveals the dark-stained processes emanating from a C-PON immunoreactive cell body located in the infundibular nucleus (500). (b, c) Im- munofluorescence labelling of NPY containing perikarya and plexus of fibers in the medial (525) and in the ventral pallium close to the lateral ventricle (x 325): 126 D. CAILLIEZ, J.-M. DANGER et al. Owing to the identical distribution of C-PON and NPY-containing fibers, only one schema depicting these fibers is shown on Figure 2. Telencephalon The dorsal (PD) and medial (PM) portion of the pallium showed numerous perikarya and varicose fibers containing NPY and C—PON. Both antisera showed a moderate number of cells in the pars medialis of the amygdala (Am) (Fig. 3) and in the posterior entopeduncular nucleus (NEP). Scarce cells were seen with NPY and C—PON antisera in the nucleus of the diagonal band of Broca (NDB). Axon processes emanating from cell bodies were visible (Fig. 1b, c). Diencephalon Both antisera revealed immunoreactive cells in the posterocentral nucleus of the thalamus (NPC) and in the ventromedial area of the thalamus (AVM). The dorsal (NID) (Fig. 4) and ventral (NIV) infundibular nuclei showed the greatest accumulation of cell bodies immunoreactive for NPY and C—PON. There were also cell bodies in the diencephalic extension of the interpeduncular nucleus (NIP) and in the preoptic nucleus (NPO) along the third ventricle. A dense accumulation of fibers was seen at the periphery of the dorsal habenular nucleus (NHD) and in the ventral infundibular nucleus (NIV). The most prominent NPY and C-PON bundle was found coursing from the infundibulum through the median eminence (ME) towards the pars intermedia (PI) of the pituitary. Mesencephalon The C-—PON and the NPY antisera showed immunoreactive cell bodies and nerve fibers in the optic tectum. The highest density of immunoreac- tive perikarya was observed in the superficial grey layer (SGS). Positive perikarya could also be seen in the central (SGC) and periventricular grey (SGP) subdivisions. Immunoreactive fibers were seen in all the tectum subdivisions especially in the inner superficial white layer. Cells and fibers were also observed in other regions of the mesencepha- lon i.e. the dorsal (NAD) and ventral (NAV) parts of the anterior tegmentum and the part just posterior to the torus semicircularis (TS). Scarce nerve fibers were found in the reticular isthmic nucleus (NRIS). Conversely, no positive elements could be stained in the nucleus profundus Fic. 2. Schematic drawing of frontal sections of the frog brain. Coordinates are taken from the topographic atlas of Wada et al. [18] and given in mm from the reference zero point defined as the anterior margin of the pars impar tecti mesencephali which is the apex of the angle formed by the junction of the dorsal midline of the two optic lobes. For convenience the drawings portray the left part of the brain. First half sections indicate the anatomical regions; the second ones describe the density and distribution of NPY and C-PON fibers; the third and the fourth ones show the location of NPY and C—PON perikarya respectively. Abbreviations—AL: amygdala, pars lateralis, Am: amygdala, pars medialis, AC: anterior commissure, AVA: area ventralis anterior thalami, AVL: area ventrolateralis thalami, AVM: area ventromedialis thalami, CGL: corpus geniculatus laterale, E: epiphysis, GC: griseum centrale rhombencephali, LFB: lateral forebrain bundle, ME: median eminence, MFB: medial forebrain bundle, NAD: nucleus antero- dorsalis tegmenti mesencephali, NAS: nucleus accumbens septi, NAV: nucleus anteroventralis tegmenti mesencephali, NBPC: bed nucleus of the pallial commissure, NCER: nucleus cerebelli, NDB: nucleus diagonal band of broca, NDLA: nucleus dorsolateralis anterior thalami, NDMA: nucleus dorsomedialis anterior thalami, NEP: nucleus entopeduncularis, NHD: nucleus habenularis dorsalis, NHV: nucleus habenularis ventralis, NID: nucleus infundibularis dorsalis, NIV: nucleus infundibularis ventralis, NIP: nucleus interpeduncularis, NLS: nucleus lateralis septi, NMNT: nucleus mesencephalicus nervi trigemini, NMS: nucleus medialis septi, NPC: nucleus posterocentralis thalami, NPL: nucleus posterolateralis thalami, NPO: nucleus preopticus, NR: nucleus rotundus, NRIS: nucleus reticularis isthmi, NRS: nucleus reticularis superior, NT: motor nucleus of the trigeminal nerve, OC: optic chiasma, ON: optic nerve, OT: optic tract, PaC: pallial commissure, PC: posterior commissure, PD: pallium dorsale, Pdis: pars distalis hypophysis, PI: pars intermedia hypophysis, PLd: pallium laterale, pars dorsalis, Plv: pallium laterale, pars ventralis, PM: pallium mediale, PN: pars nervosa hypophysis, SGC: stratum griseum centrale tecti, SGP: stratum griseum periventricularis tecti, SGS: stratum griseum supeficiale tecti, ST: striatum, STd: striatum, pars dorsalis, STv: striatum, pars ventralis, TS: torus semicircularis. NPY-like Immunoreactive Neurons 127 Fic. 2-1 128 D. CaAILLiEz, J.-M. DANGER et al. Fic. 2—2 NPY-like Immunoreactive Neurons 129 130 D. CAILLiez, J.-M. DANGER et al. a b Fic. 3. Serial parasagittal sections of the posterior telencephalon (T) and the anterior diencephalon (D). Using the PAP technique, NPY (a) and C-PON (b) antisera show intense staining of fiber networks in the amygdala (A), the lateral forebrain bundle (LFB) and the anterior thalamus. This latter tract of fibers (arrow head) surrounds the habenular nuclei and courses downwards to the rostro-lateral infundibulum. (X20). ON: optic nerve. Fic. 4. Serial adjacent sections through the dorsal infundibular nucleus. The same cell bodies (arrow heads) are stained by NPY (a) and C-PON (b) antisera (x 200). NPY-like Immunoreactive Neurons 131 mesencephali. In the posterior mesencephalon a moderate number of perikarya and fibers was observed in the interpeduncular nucleus (NIP). A dense accumulation of cells and fibers was seen in the cerebellar nucleus of the mesencephalon (NCER). Metencephalon The cerebellum was totally devoid of im- munoreactivity to both antisera. Rhombencephalon tracts of C-PON and NPY- immunoreactive fibers were seen to run ventrally and dorsally in the medulla oblongata. However, no cell bodies were revealed in this brain region. Two dense Pituitary A dense plexus of C-PON and NPY im- munoreactive fibers was seen in the pars interme- dia (PI) close to the pars nervosa (PN) (Fig. 5). Immunoreactive terminals appeared to innervate most parenchymal cells of the pars intermedia. No immunoreactivities were seen in the pars distalis (Pdis) and only scarce nerve terminals were stained with C-PON and NPY antisera in the pars nervosa. yaa es 4 : = "AR ee we CD hE z y~ : ‘ ; : , SS are ef ee” : aot ae eo) on a Gees 2 = = PL. + ay’ ae ee = = yy : = : Py . eae oe Pee, PN = ee Pdis BSS on. > a i e+, Bee * ce, - z SIs, oo a Fine gfx re, ae. > = tv se e byes <* es te eee D re pS et %. oe - ee = "er See Se a Ly ee oy Fic: 5. DISCUSSION Specificity of the NP Y-immunoreactive neurons, as is the case for all regulatory peptides, is not absolute. It cannot be ruled out that the NPY antiserum cross-reacts with structurally related peptides. By means of radioimmunoassays, we have shown that the cross-reactivities of our NPY antiserum with PY Y and APP, two peptides which exhibit a high degree of sequence homologies with NPY, were 0.4% and 0.1% respectively [10]. However, it is important to stress that determina- tion of the specificity of antisera by radioim- munoassay techniques does not necessarily agree with immunocytochemical results: immunocy- tochemistry represents a non competitive immuno- logical system to detect covalently-linked antigens whereas radioimmunoassays measure the competi- tion of soluble antigens with a radiolabelled tracer. Thus, although our radioimmunoassay results indi- cated that the antigenic determinant recognized by the NPY antiserum was the N-terminal region of the molecule (unpublished data), we cannot ex- clude that the antiserum contains an additional C-terminal-directed subpopulation of antibodies which would be detected only under the conditions of immunocytochemistry. Thus, it is not possible Serial sections through the pituitary. NPY (a) and C-PON (b) nerve terminals are located in the pars intermedia (PI) being most abundant in the vicinity of the pars nervosa (PN). A few fibers are also present in the neural lobe, close to the intermediate lobe (arrows). The pars distalis (Pdis) is devoid of NPY or C—PON innervation ( X 240). 132 D. CAILLIEZ, J.-M. DANGER et al. to exclude that NPY-related molecules exhibit higher cross-reactivity in the conditions of im- munohistochemistry than in those of radioimmu- noassays. In addition, other molecules structurally related to NPY may also account for the NPY-like immunoreactivity detected in the frog brain. In this respect, it is interesting to mention that APY, a regulatory peptide recently characterized in anglerfish pancreatic extracts exhibits 64% se- quence homologies with porcine PYY [19]. The existence of such a molecule, as yet unknown, in the frog brain cannot be ruled out. Therefore a degree of caution is always warranted in interpret- ing immunohistochemical results and despite the fact that preabsorption of NPY antiserum with NPY completely abolished the immuno- histochemical reaction, we refer to NPY im- munoreactive neurons as “NPY-like” or “NPY- containing” cells. This study demonstrates the widespread dis- tribution of NPY-like neurons throughout the brain of the frog Rana catesbeiana with the greatest innervation in the pallium (considered comparable to the hippocampal area in mammals), the lateral amygdala, the entopeduncular nucleus and the diencephalon. These distributions are similar to those reported in another anuran species, Rana ridibunda [15]. In mammals several authors, found comparable distribution of NPY, in particu- lar in the amygdaloid complex, the hippocampus and the medial basal hypothalamus. Taken together, the results of these studies, including the present one, suggest that in general NPY- containing neurons are distributed similarly in the brain of many vertebrate species. No significant differences were found in the present study between the distributions of NPY immunoreactivity and C-PON immunoreactivity. In addition we often observed co-existence of NPY and C-PON in the same cell bodies. The fact that C-PON immunoreactivity occurs within NPY- positive neurons yields two important conclusions. i) NPY-like immunoreactivity detected in the frog brain most probably corresponds to authentic NPY, since structural differences are to be expected in the cryptic region of structurally related peptides which originate from distinct precursor molecules. To illustrate this assertion one may consider the three opioid precursors pro-opiomelanocortin, pro-enkephalin A and pro- dynorphin which generate the chemically related opioid peptides endorphins, enkephalins, dynor- phin and a-neoendorphin, but exhibit no sequence homologies within the non-opioid regions of their molecule. ii) Both the NPY and C-PON domains of the pro-NPY molecule have been sufficiently preserved during evolution to make immunohis- tochemical detection of these neuropeptides possible. The visualization of a dense plexus of NPY- containing fibers coursing from the infundibular region towards the internal zone of the median eminence, the pituitary stalk and the intermediate lobe of the pituitary suggests that NPY (and/or C— PON) might exert a hypophysiotropic function. This hypothesis has received strong support in that synthetic NPY was found capable of inhibiting alpha-melanotropin (a-MSH) release in vitro by neurointermediate lobe of Rana ridibunda [20]. More recently, NPY has been shown to inhibit a-MSH and f-endorphin secretion in another anuran species, Xenopus laevis [21]. Thus, it appears that in amphibians, NPY could play a physiological role in the inhibitory control exerted by the hypothalamus upon the intermediate lobe secretion. Whether C-PON is only a by-product generated during pro-NPY processing or whether it has a specific role of its own (e.g. modulation of the hypophysiotropic effect of NPY) remains to be determined. It is interesting to note that noradrenergic fibers have been shown -to innervate the intermediate lobe of the amphibian pituitary [22, 23] and that f-adrenergic agonists stimulate a-MSH release by isolated neurointermediate lobes [24]. In the brain of mammals, co-occurence of NPY and _ nor- epinephrine has been documented [25, 26]. Thus, possible co-existence of NPY (and C—PON) with noradrenaline in nerve terminals of the frog pars intermedia and possible modulation of [- adrenergic-induced a-MSH secretion deserve further investigation. Quantification of NPY by radioimmunoassay in the brain of Rana ridibunda [15] has shown that, in addition to the diencephalon, the telencephalon and the mesencephalon contain high concentra- NPY-like Immunoreactive Neurons 133 tions of the peptide. Sephadex G—S0 gel chroma- tography indicated that the immunoreactive com- ponent had a molecular weight similar to synthetic porcine NPY [15]. The abundance of NPY and C-— PON containing neurons in the telencephalon and mesencephalon of Rana catesbeiana shown in the present study suggests that one of these peptides or both may have neurotransmitter or neuromodula- tor functions in the frog brain. This hypothesis is given strong support by the recent observation that perfusion of the brain of Rana catesbeiana with a solution containing NPY induced a dose-related reduction of cytochromes [27]. In addition, in- tracerebroventricular administration of NPY was found to elicit marked modification of the sexual [28] and feeding behavior [29-31] in mammals. Therefore, further studies are required to investi- gate the potential actions of NPY- and C-PON- like peptides in the brain of amphibians. ACKNOWLEDGMENTS This research was supported in part by research grants from INSERM (84-6020 and 86-4340), CNRS and France-Québec exchange program. REFERENCES 1 Tatemoto, K., Carlquist,M. and Mutt, V. (1982) Neuropeptide Y a novel brain peptide with struc- tural similarities to peptide YY and pancreatic polypeptide. Nature, 296: 659-660. 2 Tatemoto, K. (1982) Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. USA, 79: 5485-5489. 3. Di Maggio, D. A., Chronwall, B. M., Buchanan, K. and O’Donohue, T. L. (1985) Pancreatic poly- peptide in rat brain is actually neuropeptide Y. Neuroscience, 15: 1149-1157. 4 Allen, Y.S. and Adrian, T. E. (1983) Neuro- peptide Y distribution in the rat brain. Science, 221: 877-879. Sa bellctier, G., Guy, J., Allen, Y. S. and Polak, J. M. (1984) Electron microscope immunocytochemical localization of neuropeptide Y (NPY) in the rat brain. Neuropeptides, 4: 319-324. 6 Chronwall, B.M., Di Maggio, D. A., Massari, V. J., Pikel, V. M., Ruggiero, D. A. and O’Donohue, T. L. (1985) The anatomy of neuropeptide-Y- contaning neurons in the rat brain. Neuroscience, 15: 1159-1181. ietendrysS HC. YJonessE.G? and Emson, P. C. 10 11 12 13 14 15 16 17 (1984) Morphology, distribution and_ synaptic relations of somatostatine and neuropeptide Y-im- munoreactive neurons in rat and monkey neocortex. J. Neurosci., 4: 2497-2517. Smith, Y., Parent, A., Kerkérian, L. and Pelletier, G. (1985) Distribution of neuropeptide Y im- munoreactivity in the basal forebrain and upper brainstem of the squirrel monkey (Saimiri sciureus). J. Comp. Neurol. 236: 71-89. Pelletier, G., Désy, L., Kerkérian, L. and Coté, J. (1984) Immunocytochemical localization of neu- ropeptide Y (NPY) in the human hypothalamus. Cell Tissue Res., 238: 203-205. Adrian, T. E., Allen, J. M., Bloom, S. R., Ghatei, M.A., Rossor, M.N., Roberts, G. W., Crow, T. J., Tatemoto,K. and Polak, J. M. (1983) Neu- ropeptide Y distribution in human brain. Nature, 306: 584-596. Dawbarn, D., Hunt, S. P. and Emson, P. C. (1984) Neuropeptide Y : regional distribution, chromato- graphic characterization and immunohistochemical demonstration in post-mortem human brain. Brain Res., 296: 168 —-173. Lundberg, J. M., Terenius, L,. Hokfelt, T. and Tatemoto, K. (1984) | Comparative immunohis- tochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripher- al neurons, J. Neurosci., 4: 2376-2386. O’Donohue, T.L., Chronwall, B.M., Pruss, R. M., Mezey, E., Kiss, J. Z., Eiden, L. E., Massari, V.J., Tessel, R. E., Pickel, V.M., Di Maggio, D. A., Hotchkiss, A.J. and Zukowska-Grogec, Z. (1985) Neuropeptide Y and peptide YY neuronal and endocrine systems. Peptides, 6: 755-768. Osborne, N.N., Patel, S., Terenghi,G., Allen, J.M., Polak,J.M. and Bloom, S.R. (1985) Neuropeptide Y (NPY)-like immunoreactive ama- crine cells in retinas of frog and goldfish. Cell Tissue Res., 241: 651-656. Danger, J. M., Guy, J., Benyamina, M., Jégou, S., Leboulenger, F., Coté, J., Tonon, M. C., Pelletier, G. and Vaudry, H. (1985) Localization and iden- tification of neuropeptide Y (NPY)-like im- munoreactivity in the frog brain. Peptides, 6: 1225- 1236. Minth, C. D., Bloom, S. R., Polak, J. M. and Dix- on, J. E. (1984) Cloning, characterization and DNA sequence of human cDNA encoding neuropeptide tyrosine. Proc. Natl. Acad. Sci. USA, 81: 4577- 4582. Gulbekian, S., Wharton, J., Hacker, G. W., Varn- dell, I. M., Bloom,S.R. and Polak, J. M. (1985) Co-localization of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON). Peptides, 6: 1237-1243. 134 18 19 20 75) Vip) 23 24 Wada, M., Urano, A. and Gorbman, A. (1980) A stereotaxic atlas for diencephalic nuclei of the frog Rana pipiens. Arch. Histol. Jpn. 43: 157-173. Andrew, P. C., Hawke, D., Shively, D. E. and Dix- on, J. E. (1985) A nominated peptide homologous to porcine peptide YY and neuropeptide Y. Endo- crinology, 116: 2677-2681. Danger, J. M., Leboulenger, F., Guy, J., Tonon, M.C., Benyamina,M., Martel, J.C., Saint- Pierre, S., Pelletier,G. and Vaudry, H. (1986) Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an a-MSH-release inhibiting factor. Life Sci., 39: 1183-1192. Verburg-van Kemenade, L., Jenks, B., Danger, J. M., Saint-Pierre, S., Pelletier, G. and Vaudry, H. (1987) Evidence that an NPY-like peptide is a melanotropin-inhibiting factor in the pars interme- dia of the amphibian Xenopus laevis. Peptides, in press. Enemar, A. and Falck, B. (1965) On the presence of adrenergic nerves in the pars intermedia of the frog Rana temporaria. Gen. Comp. Endocrinol., 5: 577-583. Nakai, Y. and Gorbman, A. (1969) Evidence for doubly innervated secretory unit in the anuran pars intermedia. II. Electron microscopic studies. Gen. Comp. Endocrinol., 13: 108-116. Tonon, M. C., Leroux, P., Stoeckel, M. E., Jégou, S., Pelletier, G. and Vaudry, H. (1983) Catechol- aminergic control of a-melanocyte-stimulating hor- mone (MSH) release by frog neurointermediate lobe in vitro: evidence for direct stimulation of a-MSH release by thyrotropin-releasing hormone. Endocrinology, 112: 133-141. 755) 26 Dai] 28 28 30 31 D. CAILLiEz, J.-M. DANGER et al. Everitt, B. J., Hokfelt, ,T., Terenius, L., Tatemo- to, K., Mutt, V. and Goldstein, M. (1984) Dif- ferential co-existence of neuropeptide Y with catecholamines in the central nervous system of the rat. Neuroscience, 11: 443-462. Hokfelt, T., Lundberg, J.M., Lagercrantz, H., Tatemoto, K., Mutt, V., Lindberg, J., Terenius, L., Everitt, B. J., Fuxe, K., Agnati, L. and Goldstein, M. (1983) Occurence of neuropeptide Y (NPY)-like immunoreactivity in catecholamine neurons in the human medulla oblongata. Neurosci. Lett., 36: 217— 222. Saito, T., Kanno, T., Tatemoto, K. and Mutt, V. (1983) Dose-related effect of neuropeptide Y stimu- lating respiratory chain in the bullfrog brain. Biomed. Res., 4: 173-178. Clark, J.T., Kalra, P.S. and Kalra, S. P. (1985) Neuropeptide Y stimulates feeding but inhibits sexual behavior in rats. Endocrinology, 117: 2435— 2442. Stanley, B.G., Chin, A.S. and Leibowitz, S. F. (1985) Feeding and drinking elicited by central injection of neuropeptide Y: evidence for hypothal- amic site(s) of action. Brain. Res. Bull., 14: 521— 524. Stanley, B.G., Daniel, D.R., Chin, A\S: “and Leibowitz, S. F. (1985) Paraventricular nucleus in- jections of peptide YY and neuropeptide Y pref- erentially enhance carbohydrate ingestion. Peptides, 6: 1205-1211. Levine, A.S. and Morley, J.E. (1984) Neuro- peptide Y: a potent inducer of consummatory behavior in rats. Peptides, 5: 1025-1029. ZOOLOGICAL SCIENCE 4: 135-144 (1987) Comparison of Reproductive Activities between Two Japanese Quail Lines Selected with Regard to Photoperiodic Gonadal Response Hisao HonpA, TADASHI O1sur! and Takao KonisHi’ Kanebo Institute for Cancer Research, Tomobuchicho 1—5—90, Osaka 534, Japan ABSTRACT— KR and KN lines of Japanese quail selected for photoperiodic gonadal response through five generations were maintained ten more generations with relaxed selection. Males of KR line have maintained their responsiveness of cloacal glands to “continuous light treatment (CLT)” by increasing the size of cloacal glands, while those of KN line have maintained irresponsiveness. Females of KR line maintained the trait to lay the first egg earlier in age than those of KN. Other characteristics noted are as follows: 1. Responses of cloacal glands in males (decrease in the size of cloacal glands) and oviposition in females (stop of the oviposition) to the light condition of short days were more distinct in KN than in KR line. 2. Females of KR line tend to lay foggy eggs (translucent milk-white material veils egg surface pattern of dark spots and patches). 3. Females of KR line have breast feathers with black dots all over, while black dots were restricted around the throat in females of KN. 4. The incubated eggs of KR line hatched earlier than those of KN. 5. Serum thyroxine (T,) level was higher in KR than in KN under long photoperiod (LD 16:8) and normal temperature (23°C), but there was no difference in the serum triiodothyronine (T3) level between two lines. 6. The birds of KR line grew faster, and their body weights were larger than those of KN line in generations with selection. The difference of body weight, however, disappeared after selection was relaxed. © 1987 Zoological Society of Japan INTRODUCTION Two lines of Japanese quail (KR and KN lines) were established concerning the potency of repro- duction under photoperiodic conditions [1]. In KR line, males responded to “continuous light treat- ment (CLT)” by increasing their cloacal gland size, and females laid eggs at earlier ages and between 08:00 and 14:00 under LD 16:8 (light on 01: 00- 17:00). In KN line, males were irresponsive to CLT, and females laid eggs at later ages and between 16: 30 and 08:00. That is, the birds of KR line were more sensitive to photoperiodic manip- ulations in regard to their reproductive activities than those of KN line. These two lines were maintained for ten more generations with relaxed Accepted September 18, 1986 Received July 7, 1986 Present address: ‘Department of Biology, Nara Women’s University, Kitauoyanishi-machi, Nara 630, and *Aburabi Laboratories, Shionogi-Seiyaku Co., Ltd., Koka-cho, Koka-gun, Shiga 520-34, Japan. To whom reprints should be requested. i selection. In this report, we examined 1) whether genetical traits previously reported have been retained or not, 2) whether there is any difference in the short day photorefractoriness [2-4], a characteristic response of this species to short days, between these two lines, and 3) several other characteristics specific to each line. MATERIALS AND METHODS Maintenance of lines Breeding colonies (25 females and 12 males) of KR and KN lines of Japanese quail (Coturnix coturnix japonica) have been raised in large cages (178” x91? x96 cm). Eggs were collected and stored within limits of two weeks in a chamber where temperature was kept at 14°C and relative humidity at 80%. Eggs were then transferred to an incubator in which temperature and relative humidity were kept at 37°C and 60%, respectively. Eggs were turned every three hours. Rearing conditions of the birds after hatching were the 136 H. Honpa, T. Otsu AND T. KonrisHI same as described previously [1]. Measurement Incubation period until hatch was recorded. During incubation, eggs were placed separately in cotton wool on a plate to inhibit egg-to-egg contact, because the time of hatching synchronizes within an hour or two when the eggs are incubated in contact [5]. In females, the age at first egg was recorded individually. The number of eggs laid and the percentage of foggy eggs (translucent milk-white material veils egg surface pattern of dark spots and patches) were recorded. Breast feather pattern of black stipples was checked when the birds matured (The stippled feather pattern does not appear in males). In males, the size of cloacal glands (width or volume) was measured before and after CLT (continuous light treatment of 32hr on 42 days after hatching) with a caliper to check the respon- siveness to CLT [1]. Volume of a cloacal gland was calculated by using a formula, volume = WLH/6, where W, L and H are width, length and height of a cloacal gland protrusion in cm, respectively. In both males and females, responsiveness to short day treatments was examined by transferring them to an LD 8:16 photoperiod (light on from 10:00 to 18:00) from an LD 16:8 photoperiod (light on from 06: 00 to 22:00) or by placing them under an LD 8:16 throughout the experimental periods. Serum levels of triiodothyronine (T3) and thy- roxine (T,) were measured by radioimmunoassay in both males and females. Radioimmunoassay kits (Eiken, Tokyo) for T; (I-LK12, cross reacti- vity with T, is less than 0.1%) and for T, (I-LK22, cross reactivity with T3 is less than 0.1%) were used. Birds were reared for three weeks under respective condition (LD 16:8 and 23°C, LD 16:8 and 10°C or LD 8:16 and 23°C), and starved for about four hours before sacrificed. Blood was taken from 13:00 to 15:00 to avoid fluctuation of the data due to diurnal changes of the hormones. Average values were presented with standard errors (SE). For statistical analysis, we used the N88-basic programs [6]. Student’s t-test, y°-test, two-way analysis of variance (ANOVA) and Dun- can’s multiple range test were employed. Logistic analysis of body weight Body weight was measured once every week and the difference in body growth between KR and KN lines was examined by logistic analysis as described elsewhere [7]. The increase of body weight was approximated by a logistic curve which was ex- pressed by the analytical function, w(t)=W,/(1+ exp(—k(t-a))), where w(t) is body weight at ¢ days after hatching, W,is the final body weight of a full grown body, and a is an inflection point of a logistic curve. The least square method by a personal computer was used to fit a logistic curve to average values of body weight at every mea- sured ages (e.g., solid circles in Fig. 1). Then, we can obtain probable values of Wy, a and k. We note W, for a parameter to indicate the final body weight. Oe 100 Fic. 1. An example of fitting of a logistic curve to the increase of body weight. Ordinate: body weight (g). Abscissa: age (days after hatching). A series of average values (population size, N=6—20) of body weight is plotted by solid circles. The logistic curve fitted to the data is characterized by W, (final body weight), a (inflection point) andk. A gradient of the tangent of a curve at the inflection point is k W,/4. RESULTS Characteristics noted in females 1. Age at first egg Age at first egg has been Reproductive Activities of Two Quail Lines 17 TABLE 1. relaxed selection” Average age* at first egg of each generation during maintenance of lines with No. of No. of Generation Birds KR line eras KN line F; 38 42.2 + 0.46 47 AS aa2 0-51" * Fs 31 42.6 + 0.52 24 Ae 0.30" "* Fy 54 42.0 + 0.34 28 45.0 + 0.86* Fio 16 42.6 + 0.74 25 47.2 + 0.76** Fio 31 44.2 + 0.49 32 48.4 + 0.51*** Fi, 30 42.2 + 0.61 Si ASE 0.78" ** Fi 29 43.3 + 0.74 32 ANS) (® SEN pata F,3 DS 41.8 + 0.59 25 43.0 + 0.54 Fy, 32 42.3 + 0.65 31 A evict0.02" Average 287 ae ino 920) 2S AT OE O25" a: Values are mean+SE (days after hatching). b: Data of generations with selection (F,—F,) are presented in Table 3 in the previous paper [1]. Significant difference between KR and KN lines: mb 05ee, POOL =" P<000n recorded in every generation with relaxed selec- tion and results are shown in Table 1. Two-way ANOVA showed that the difference in age at first egg among generations is not significant, but the difference between lines is highly significant (P< 0.002). The average age at first egg of KR line is 10 Bird number O1 0 i I | Tis 40 50 4.3 days earlier than that of KN. Distribution of ages at first egg in F,4 generation is shown in Figure 2 as an example. 2. Effect of short days Figure 3 shows the effect of alternation of long and short days on egg production. When the birds were transferred from 60 Days after hatching Fic. 2. Distributions of ages at first egg. Average ages with SE are 42.3+0.65 (N=32) days after hatching for KR line, and 47.5 +0.62 (N=31) days for KN line. Data from birds of F;4 generation. Open and solid bars indicate KR and KN lines, respectively. 138 H. Honpba, T. OlsHi AND T. KoniIsHI *lo Laying birds cee Zealies) OM, Fic.3. Variation of the number of laying birds under alternate photoperiodic conditions. Ordinate: ratio (per- centage) of laying birds to the total number of females in a day. Abscissa: age (days after hatching). Population sizes of females of KR and KN lines are from 24 to 32 and from 22 to 33, respectively. Solid and broken lines: birds of KR and KN lines, respectively. Photoperiodic conditions are shown at the bottom of the figure. Open areas: long days (LD 16:8) except for 0-14 days under continuous light. Stippled area: short days (LD 8:16). Solid arrow: date of inbreeding (61 days). Arrow head: days of first eggs. Data from F jo generation. individual cages to large cages for inbreeding, they suddenly stop laying for about two weeks (arrow). When the birds were transferred from long days to short days at 168 days, they stopped laying soon. KR line stopped to lay eggs 1-2 days later than KN. When the birds were transferred from short to long days at 215 days, KR started to lay eggs about 6 days earlier than KN. This corresponds to the difference in the age at first egg (Fig. 2, arrow head in Fig. 3). 3. Ratio of the number of foggy eggs to that of normal ones There are two types of eggs, 1.e., foggy eggs (upper three eggs in Fig. 4) and normal ones (lower three). The ratio of foggy eggs 1s 82.3% (the number of eggs examined, N=232) and 14.5% (N=249) for KR and KN, respectively. Chi-square test showed that the difference is highly significant (P<0.01). 4. Incubation periods until hatch Figure 5 shows frequency of hatching in every four hours during incubation. Incubation period for KR line was 5.7 hr shorter than that for KN (403.3+1.0hr for KR vs. 409.0+1.1 hr for KN, P<0.001). 5. Breast feather pattern Females of KR line (left in Fig. 6) show distinct black stipples all over the breast. On the other hand, females of KN — Fic. 4. Comparison between foggy and normal eggs. There are many dark spots and patches on the surface of normal eggs (lower three of the figure), and in foggy ones translucent milk-white material veils egg surface (upper three). The minimum scale of the measure on the right is in mm. line (right) show stipples only around the throat. Quantitative data are shown in Table2. The difference in breast feather pattern between KR and KN lines is highly significant (P<0.01, 77- test). Reproductive Activities of Two Quail Lines 139 20 Hatched egg number oO 390 400 410 420 430 Incubation time (hours ) Fic. 5: Distribution of incubation periods until hatch. Ordinate: the number of hatched eggs in every four hours. Abscissa: incubation time (hours). Data from KR line (open bars) and KN line (solid bars) are shown. Average incubation periods with SE are 403.3+1.0hr for KR line (N=86) and 409.0+1.1hr for KN line (N=104, P<0.001). Data from F, generation. Fic. 6. Comparison of breast feather patterns between KR and KN lines. Distinct black stipples are all over the breast in females of KR line (left), forming a contrast to females of KN line (right) which show stipples only around the throat. Characteristics noted in males 1. Response of cloacal gland size to CLT Growth rate of the cloacal gland in Fy generation was measured. Ratios of the cloacal gland size (width) after CLT to that before CLT were 1.46+ TaBLE2. The number* of females having each breast feather pattern Pattern KR Iine KN line Stipples all over 43 8) Intermediate 3 18 Stipples only 0 24 around the throat Total 46 51 a: Data from females of F,4 and F,; generations. 0.045 (N=20) for KR line, and 1.06+0.012 (N= 18) for KN line (P<0.001). Thus, the genetical trait with selection, which was reported previously [1], has been retained after selection was relaxed. 2. Response of cloacal gland size to short days Cloacal gland size was measured every week under alternate photoperiodic conditions. Indi- vidual records for KR and KN lines are shown in the upper part of Figure 7. Response patterns to short days are classified into three types (a, b and c at the lower part of Fig.7). Type a does not respond to short days at all. Type c birds are 140 H. Honpa, T. Otsu! AND T. KonisHI 14 425 18459232 259 Fic. 7. Change of cloacal gland size under alternate photoperiodic conditions. Ordinate: cloacal gland volume in logarithm (cm*). Abscissa: age (days after hatching). The size was measured every week. KR, KN: representation of volume change of individuals in superposition which belong to KR line (N=19) and KN line (N= 18), respectively. a, b, c: volume change patterns of types a, b and c, respectively. Data from males of Fj generation. The bottom of the figure shows photoperiodic conditions as described in the legend of Fig. 3. The open arrow head indicates the date on which CLT was performed. defined as those which respond to short days and _—in KR and KN lines. There are more birds of type reduce the cloacal gland volume to less than bin KR line than in KN line. In contrast, there are 0.5cm*. Type b responds to the second short day _less birds of type c in KR than in KN line (P<0.05, treatment but not to the third short day treatment.. _y-test). Table 3 indicates the number of birds of each type 3. Maturation under short days When Reproductive Activities of Two Quail Lines 141 TABLE 3. The number* of males showing each type of response to alternate photoperiodic condition Type? KR line KN line Type a 1 Z Type b 16 9 Type c 2 7 Total 19 18 a: Data from males of Fj generation. b> Seer Big: ¢/. Cloacal gland volume (cm?) Fic. 8. Growth of cloacal glands under short days. The short day condition continues from the day of hatch- ing until 133 days. KR, KN: representation of cloa- cal gland growth in superposition (13 and 16 individ- uals, respectively). See the legend of Fig.7 for further explanation. Data from males of F,3 genera- tion. quail were raised under short days, all the birds of KR line matured but 19% KN line did not mature (Fig. 8). Days required to attain half size of the maximum cloacal gland are 64.1+1.33 days and 89.5 + 6.08 days (P<0.01) for KR and KN, respec- tively. 4. Comparison of body growth between the two lines The body weight was recorded to be significantly larger in KR than in KN after selec- tion (F, generaton) [1]. The body weight after relaxed selection (F,. and F,3 generations) were measured under two rearing conditions as shown in Table 4. In either condition, differences of the body weight between KR and KN lines of F,, and F,3 generations (25 and 32 days old) are small in comparison with differences between those of F; generation (28 days old). When the birds of 50 days old, at which the birds had fully grown, were compared, the difference of the body weight in F,> generation is also small. When the logistic analysis is applied to data of generations with selection (F, and F;) and those after selection was relaxed (F)> and F,3), the final body weight of Fi and F;3 generations did not show much differences (1-2 g) between KR and KN lines, while those of F, and F; generations showed greater differences (10- 14g) (Table 5). Although the logistic analysis has been done only on mean body weights, the results well coincide with those in Table 4. Thus, the trait of superiority in body weight in KR over KN has not been retained after selection was relaxed. Serum levels of T3 and T4 The results are shown in Figure9. Two-way ANOVA showed significant differences in T3 levels between rearing conditions (P<0.01) and between lines (P<0.05) in males and between rearing conditions (P< 0.05) in females. As shown in Figure 9a and b, serum level of T3 does not differ between KR and KN lines under long days and 23°C. Under short days (23°C), the level of T; was higher than under long days (23°C) except for females of KR line. In the case of low temperature (10°C), the result was not definite. The level of T, was low and close to the limit of detection by radioimmunoassay, and the level varied in each experiment. However, there was a definite tendency that the T, levels of males and 142 H. Honpa, T. OIsHi AND T. KonlisHI TABLE 4. Comparison of body weight between KR and KN lines Rearing KR line‘ bd toe Generation® Age? 24L-8L? 5 28 1 D5 1 32 8L* 5 28 13 25 16 By 2A 84 4 50 12 50 KN line‘ 76.3 + 1.00 (33) 66.9 + 1.91 (20) 85.1 + 1.60 (20) 77.6 + 1.01 (30) SG. £ i. CS) 77.8 + 1.52 (15) 100.7 + 1.53 (17) 97.8 + 1.38 (20) 65.5 + 0.86 (30)*** 62.6 + 1.11 (18) 82.0 + 1.16 (18) 66.9 + 1.12 (32)*** 58.2 + 1.45 (17) 75.8 + 1.76 (17) 88.1 + 1.07 25)" 96.9 + 1.16 (18) a: All data are from male birds. b: Days after hatching. c: Body weight in grams (mean+SBE). The number of birds is shown in parentheses. d: Birds were reared under LD 24: 0 for the first 2 weeks and the photoperiod was changed to LD 8:16. e: Birds were reared under LD 8: 16 throughout the experimental periods. Statistical difference between KR and KN lines: *** P< 0.001. TABLE5. Final body weights in grams (W,) obtained by logistic analysis* Generation KR line KN line Difference FD 110.4 96.5 13.9 Fs 95.5 84.0 WS F,> 106.8 104.7 DM F,3 101.8 100.9 0.9 a: Data from male birds. See “Materials and Methods” and Fig. 1 for the details of the analysis. b: Analyzed from the observed data by Konishi and Oishi [1]. c: Analyzed from the observed data by Konishi and Oishi (unpublished). females in long days under 23°C were higher in KR than in KN line. Figure 9c and d shows the examples of the data obtained. Two-way ANOVA showed significant differences in T, levels between rearing conditions (P<0.01) and between lines (P <0.05) in males and between lines (P<0.05) in females. The value of males in long days under 23°C is significantly higher than those in short days under 23°C (P<0.001) and long days under 10°C (P<0.01). No remarkable difference was observed between conditions of short days (23°C) and long days (10°C). DISCUSSION KR and KN lines, which were selected for five generations and maintained for ten generations thereafter with relaxed selection, have retained the characteristics for photoperiodic gonadal response noted in our previous paper [1]. The birds of KR line have a high potency of reproduction, that is, males of KR line are easily stimulated to increase the size of cloacal glands by CLT, and females lay the first eggs earlier. In contrast, the cloacal gland size of KN males hardly increases after CLT, and females tend to stop laying eggs sooner when they are transferred from long to short days. Superiority in body weight and growth rate in KR line over KN line [1] has not been maintained after selection was relaxed, i.e., we were unable to find any difference in body weight between KR and KN lines at F,. and F,3 generations, because the body weight of KN line increased and reached the similar value as that of KR line after selection was relaxed. This is interesting in connection with the finding by Kawahara [8, 9] that unconscious selection of wild quail enhanced body growth and sexual maturation. According to the response of cloacal glands to short photoperiods, three types were classified, 1,e., type a: does not respond to short photo- periods, type b: responds to the second short photoperiod but not to the third short photo- period, and type c: responds to the second and the third short photoperiods by showing a decrease of the cloacal gland volume to less than 0.5cm?. These types are comparable to types I, II and III in Reproductive Activities of Two Quail Lines 143 KR KN (ng/100m| ) ry) 300 0 Fic. 9. KR KN (ug/100ml) oO un Serum T; (a, b) and T, (c, d) concentrations. Open columns: long days (LD 16:8) and 23°C. Stippled columns: short days (LD 8:16) and 23°C. Striped columns: long days and 10°C. Vertical lines of each column represent SE. a: T; concentration (ng/100 ml) in males of KR and KN lines. b: T3 concentration in females of KR and KN lines. ¢c: T, concentration ( «g/100 ml) in males of KR and KN lines. d: T, concentration in females of KR and KN lines. Population size: 6-15 for T;, and 4-10 for Ty. *a: P<0.005, *b: P< W020 005 sade <0: 0le “err <0.05; our previous report [4]. That is, KR line includes more type I birds and KN line includes more type III birds, but one to one correspondence could not be established. We found several other characteristics specific to each line, which had not been described in the previous paper [1], 1, e., 1) females of KR line lay more foggy eggs, 2) females of KR line show more distinct dotted breast feathers than those of KN, and 3) incubation period required for hatching is several hours shorter in KR than KN line. It remains unsolved whether these characteristics correlate each other, whether these reflect the characteristics of wild and domesticated quails as suggested in our previous paper (1), or whether these reflect the habitats of different populations which were the ancestors of each line. The results of serum T3 concentration confirmed the previous paper [1], showing no statistical difference between KR and KN lines. Higher T3 level in males under short than long photoperiods also confirmed the previous results [3]. Although serum T, concentration was low and varied among each experiment (some variations in the plasma T, level have been reported in birds [10, 11]), the level was always higher in KR than KN line under long days and 23°C. This is interesting in connec- tion with the recent report by Follett and Nicholls [12]. They showed that the thyroid plays an important role in the gonadal response of Japanese quail to short days when the birds are transferred from long to short days, 1.e., thyroidectomized males do not show the rapid testicular regression and moult which occur in controls and if T, 1s administered at certain period of the photo- periodic treatments normal regression does occur under short days. Therefore, the difference in the photoperiodic gonadal response between KR and KN lines might be attributable to the difference in the thyroid activity. In addition, as reviewed by Sharp and Klandorf [13], intimate interactions between day lengths, gonadal steroids and thyroid gland function in Galliform birds lead us to study the relation between thyroid and gonadal activity of KR and KN lines in future experiments. ACKNOWLEDGMENTS We are grateful to Professor Emeritus Masaru Kato of Kyoto University for his encouragement throughout this inverstigation. Thanks are also due to Messrs. Yoshio 144 H. Honpa, T. OISHI AND T. KONISHI Asai, Keiji Hara and Takaaki Himoto for their assistance in animal care and Ms. Kumiko Tanaka for her technical 8 assistance on radioimmunoassay. Japanese quail. J.Exp. Zool., 220: 311-319. Kawahara, T. (1973) Comparative study of quan- titative traits between wild and domestic Japanese REFERENCES Konishi, T. and Oishi, T. (1985) Characteristics of two lines of Japanese quail selected for response to “Continuous light treatment”. Zool. Sci., 2: 549- quail (Coturnix coturnix japonica). Exp. Animals, 22 (Suppl.): 139-150. Kawahara, T. (1976) History and useful characters of experimental strains of Japanese quail (Coturnix coturnix japonica). Exp. Animals, 25: 351-354. (in Japanese) Ile 10 Astier, H. (1980) Thyroid gland in birds: Structure Oishi, T. (1978) Effect of short days in the photo- and function. In “Avian Endocrinology”. Ed. by A. periodic testicular response of Japanese quail. En- Epple and M.H. Stetson, Acad. Press, New York, viron. Cont. Biol., 16: 35—40. pp. 167-189. Oishi, T. and Konishi, T. (1978) Effects of photo- 11 Herbuté, S., Pintat, R., Parés, N., Astier, H. and period and temperature on testicular and thyroid Baylé, J.D. (1983) Comparison of plasma thyrox- activity of the Japanese quail. Gen. Comp. Endocri- ine levels following short exposure te cold and TRH nol., 36: 250-254. administration in intact and pituitary autografted Oishi, T. and Konishi, T. (1983) Variation in the quail (Coturnix coturnix japonica). Gen. Comp. photoperiodic cloacal response of Japanese quail: Endocrinol., 49: 154-161. Association with testes weight and feather color. 12 Follett, B.K. and Nicholls, T. J. (1985) Influences Gen. Comp. Endocrinol., 50: 1-10. of thyroidectomy and thyroxine replacement on Freeman, B.N. and Vince, M.A. (1974) Develop- photoperiodically controlled reproduction in quail. ment of the Avian Embryo, Chapman and Hall, J. Endocrinol., 107: 211-221. London. 13. Sharp, P. J. and Klandolf, H. (1985) Environmen- Ishii, S. (1983) Programs of Statistical Methods for Biologists by N&88-Basic, Baifukan, Tokyo. (in Japanese) Honda, H., Tanaka, K., Minamino, T. and Konishi, T. (1982) Control of contour feather growth of tal and physiological factors controlling thyroid function in Galliforms. In “The Endocrine System and the Environment.” Ed. by B. K. Follett, S. Ishi and A. Chandola, Japan Sci. Soc. Press, Tokyo/ Spring-Verlag, Berlin, pp. 175-188. ZOOLOGICAL SCIENCE 3: 145-150 (1986) © 1987 Zoological Society of Japan Structure, Innervation, Persistence, and Effects of Juvenile Hormone on the Prothoracic Glands in Adult Blattella germanica (L.) (Dictyoptera, Blattellidae) LAKSHMI K. HAZARIKA and Ayopuya P. Gupta! Department of Entomology and Economic Zoology, Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, NJ 08903, USA ABSTRACT— During a study of the structure, degeneration pattern, and effects of juvenile hormone on the integrity of adult prothoracic glands (PTGs) in Blattella germanica (Dictyoptera, Blattellidae), we found that the nymphal PTGs were characterized by the presence of interglandular bridges in addition to crossovers. The glands are innervated posteriorly by the prothoracic gland nerve, originating from the prothoracic ganglion. In adults, the PTGs, although degenerate, persist throughout the adult life, maintaining themselves as two nonglandular strands, composed of two muscle bands, and thick connective tissue sheath. The breakdown of the PTGs can be partially prevented by applying juvenile hormone (JH), suggesting that JH is necessary for maintaining the structural integrity of the PTGs. INTRODUCTION The prothoracic glands (PTGs) are nymphal structures, but in some dictyopterans they are present in the adults as well. After adult emer- gence, they gradually degenerate and break down, as reported in Leucophaea maderae [1, 2], Peri- planeta americana [3], and Nauphoeta cinerea [4, 5]. It is believed that degeneration commences soon after adult emergence [2], owing to absence of JH [6], and is visible in the external appearance of the gland. It is also believed that the PTGs in the adults are endocrinologically non-functional [1]. The persistence of these so-called non- functional PTGs in adult cockroaches is an in- teresting phenomenon, since they disappear com- pletely in adults of other insects. Therefore, the objectives of this study are to describe the gross anatomy and nervous innervation of the PTGs in the last (6th) instar nymphs, and their progressive degeneration and residual persistence in the adults, and the effects of JH on PTGs of adultoids Accepted September 18, 1986 Received May 23, 1986 " To whom reprints should be requested. of Blattella germanica L. MATERIALS AND METHODS Insects B. germanica colony was maintained at 28+2°C on Purina lab chow and water. Sixth instar nymphs and adults of various ages were used in this study. Newly emerged male and female adults were separated each day from rearing jars and placed in groups of 15-20 individuals in Dixie cups with food and water. In this study, we used the PTGs of 5 adults for each of day 1 (which represented an adult immediately after emerg- ence), day 7 (end of the first reproductive cycle), day 50 (about middle age), and day 120/150 (old age for male and female, respectively) to prove that the PTGs persisted throughout the adult life span. For scanning electron microscopic (SEM) observations, these PTGs were fixed in toto according to the method described below for histology. The fixed PTGs were air-dried, mounted on aluminium stubs, and coated with gold paladium for 2—4min in a Technic sputterer. They were examined in a Hitachi S—510 SEM at 146 L. K. HAZARIKA AND A. P. GUPTA 5kV at different magnifications. Wholemount for gross anatomical study Two-to-four hours prior to dissection, 6th-instar nymphs and adults were injected with 20 to 30 yl of 0.25% methylene blue in double-distilled water with the help of a 100 41 Hamilton syringe fitted with a 30-gauge needle. After cutting open the prothorax and neck ventrally in ice-cold physiolog- ical saline, 5 prothoracic ganglia (PG) attached with the blue-stained PTGs were also prepared after fixing in toto (as described in the following section) and whole-mounted in Permount or emer- sion oil for light microscopic observations. PTG- attached ganglia were also examined under the SEM. Histology PTGs from ten 6th instar nymphs and ten one-day-old adults were fixed in toto with 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2) and postfixed in 1% osmium tetroxide in the same buffer (pH 7.2). They were then dehydrated in ethanol series and dealcoholized with propylene oxide. These tissues were used for preparing resin blocks with epon-araldite mixtures. One-to-two pem-thick sections were cut with Sorvall MT2-B Ultramicrotome. Sections were stained with basic fuchsin and couterstained with methylene blue. Composite diagrams were made through light microscopic observations. Application of juvenile hormone analogue (JHA) and measurement of PTG widths Two four-day-old 6th-instar nymphs were treat- ed with two ul of 100 ppm R—20458 (6, 7-epoxy-1- (p-ethylphenoxy)-3, 7-dimethyl-trans-2-octene) in acetone on the dorsal side of the abdomen with an ISCO microapplicator fitted with a precalibrated 100 ul Hamilton syringe. The average widths of the two glands at their widest points were meas- ured in normal adults (day-1, -7, -50, -120, and -150) and JHA-treated adultoids (day 1-3) accord- ing to [7]. RESULTS The PTGs of B. germanica (L.) are a pair of Fic. 1. Light micrograph of the prothoracic glands (PTG) of a 6th-instar female nymph showing the crossing over (X) and the interglandular bridge (arrow). 400. Fic.2. Scanning electron micrograph showing the prothoracic gland nerve (PGN) (arrow) connected to the prothoracic ganglion (PG). The remaining nerves were cut during preparation. x 100. irregularly shaped tissue bands (Fig.1). They cross over each other anterior to the PG. Unlike the PTGs of other cockroach species so far studied, the PTGs of the 6th-instar nymph of B. germanica have an interglandular bridge (Fig. 1) below the crossing over. The histology of the bridge is similar to that of the gland. Innervation of PTGs At the base of the 4th peripheral nerve [8] of each side of the PG, a single nerve, the prothoracic gland nerve (PGN), arises and innervates the PTG of the respective side (Fig. 2). In examinations of Persistence of Prothoracic Glands in Adults of Cockroach 147 the methylene blue-stained PGN, we traced the nerve throughout the entire length of the PTG. The PGN appears to receive axons from the PG. Histology of a nymphal PTG and interglandular bridge The longitudinal section of gland (Fig. 3) and the bridge revealed the centrally located striated muscle, a nerve, fine branches of connective tissue, and cells with large nuclei. The boundaries of these cells are not well defined and the nuclei are of variable sizes and shapes, such as, long, elliptical, round and irregular. We did not observe any granules in the cytoplasm. As in L. maderae [1], we also observed in B. germanica that cells Fic. 3. Diagram of a longitudinal section through the center of a nymphal prothoracic gland. C: connec- tive tissue sheath, M: muscle. Fic. 4. Part of a cross section of a nymphal prothoracic gland showing the nuclei (N) and cup-shaped infold- ing (arrow). 1,200. containing elliptical nuclei usually occupy the central part of the gland. Generally, at the broadest area of the gland, there are about 6 to 8 cells. Cytoplasm in the peripheral cells is abundant in comparison with that of the centrally located ones. The cup-shaped cavity with round ex- tracellular basic fuchsin-stainable material can be seen in the cross section of the 6th-instar nymphal PTGs (Fig. 4). Trachea can be seen both external- ly and internally. A branch of the cephalic ventral longitudinal trachea (VLT) (ventral branch of the head [9]) tracheates the PTGs on both sides, appearing externally along the anterior 2/3 of their lengths. It enters and traverses internally in the remainder of the PTGs. Degeneration and persistence of PTGs in adults The PTGs persist throughout the lifespan of male and female B. germanica adults (Fig. 5 and Table 1). However, they undergo degeneration, TaBLE 1. Comparision of PTG widths in nymphs and adults of Blattella germanica (L.) Mean PTG widths* Stages N Gane) SD 6th Nym > 138.60 2.40 Adult 1 day 5 79.20° 5.26 7 day 5 32.80° 2.16 50 day 5 15.40° 4.72 120 day 5 7.80° 2.58 * Means with the same letter are not significantly different at 0.05 level (Student-Newman-Keuls Test). losing their glandular tissues within a few hours of adult emergence (<1-day old), which can be visualized from the cross section of a day-1-adult PTG (Fig. 6). During this process, the PTGs form lacunae or intercellular spaces and thickening and protrusion of the connestive tissue sheath. Subse- quently, the lacunae disappear from the gland cavity and are replaced by thick connective tissue sheath. Consequently, the PTGs maintain their physical identity in the form of two nonglandular tissue strands, consisting of the muscle, trachea, and connective tissue. When we compared the widths of the PTGs in 148 L. K. HAZARIKA AND A. P. GuPTA Fic. 5. Scanning electron micrographs of prothoracic glands of adults of various ages to show the reduction in size concommitant with advancement of ages. 1: 1-day-old adult, 135. 7: 7-day-old adult, «270. 50: 50-day-old adult, x 450. 120: 120-day-old adult, «4,500. Though PTGs in micrographs 7 and 50 appear to be of same size, their magnifications are different. Persistence of Prothoracic Glands in Adults of Cockroach 149 Fic. 6. Part of a cross section of a 1-day-old female adult PTG showing the lacunae (V), thick connec- tive tissue sheath (C) and its branching (B) into the gland cavity. 1,200. 6th-instar (day 6-7) nymphs and adults of various ages (Table 1), we found that they are significantly different from one another (P=0.05). The mean width of the PTG (8.2 um) of the day-150-adult female (which is not shown in Table 1) was almost identical with that of the day-120-adult male. The widths of PTGs in the remaining adult females of ages, day-1, -7, and -50, were exactly identical with those of the male. Therefore, we did not consider them separately. Table 1 represents the mean PTG widths for both male and female adults. Effect of JHA on adultoid PTGs We found that JHA-treated adultoids of B. germanica contained almost nymphal-sized PTGs (in terms of widths) for 3 days after emergence (Table 2), which may be true for the remaining period of their short lifespan. TABLE 2. PTG widths in JHA-treated adults of Blattella germanica Age Mean widths (Days) (in pm) SD 1 11 118.74 DT DZ. 9 102.04 47.86 3 V 123.91 60.2 DISCUSSION As decribed previously, the PTG cross over anterior to the PG in B. germanica. The signif- icance of the crossing over is, we believe, to provide mechanical support to the PTGs, since they are floating in the hemocoel between the gut dorsally and the ventral nerve cord ventrally. This is common in all dictyopterans so far studied. The presence of an interglandular bridge only in B. germanica is difficult to interpret; it might provide additional mechanical support. This kind of bridge has not been described in any other species. Considering the similarity in histology of the bridge with those of glands, we believe that the bridge may also be responsible for ecdysone synthesis. The origin of the PGN in B. germanica in the PG has been observed also by scanning electron microscope (Fig. 2), in addition to the methylene blue-tracing technique. That the PG innervates the PTG via the PGN in P. americana [10] has been electrophysiologically documented [11] (for review see [12]). In addition to the large nucleated cells in the gland periphery, the thickness of the connective tissues is indicative of the active nymphal state of the PTGs [1, 2]. A thin connective tissue sheath and its fine branches protruding into the gland cavity are characteristic features of nymphal PTGs, whereas, the thick connective tissue sheath and its branchings (Fig. 6) indicate the degenerat- ing adult PTGs [1, 13]. Pronounced cup-shaped infoldings on the sur- face of the gland (Fig. 4) are essential for increas- ing surface area for exchange of materials [12, 14]. The significant differences in the widths of PTGs of the 6th-instar nymphs and adults of various ages (Table 2) has clearly demonstrated the difference in PTG sizes among those insects, presumably indicating the absence of growth of the PTG in adults. Persistence of such so-called endocrinolo- gically nonfunctional glands in adult dictyopterans and Aeshna (Odonata) [15] may suggest a rela- tionship between the two groups [1]. By applying JH exogenously at the critical stage of the last-instar nymph, one can prevent the degeneration of the PTGs in B. germanica adult- oids and allow them to persists as nymphal-sized PTGs. From this finding, we can designate the day 2-4 of 6th instar nymphs as the critical stage, because those adults, which emerged as a result of 150 L. K. HAZARIKA AND A. P. GuPTA JH application on 6th-instar nymphs before or after this age (day 2—4), showed normal degenara- tion pattern of PTGs (personal observations). The differences in PTG widths between day-1-adults and -adultoids, and no difference in PTG widths between the 6th- instar nymphs and day-1 adult- oids, strongly indicate that JH is essential in preserving the integrity of the PTGs. ACKNOWLEDGMENTS This paper is the New Jersey Agricultural Experiment Station Publication No. D—08112-14-86 supported by state funds, U.S. Hatch funds, and Rutgers Research Council. LKH was on sabbatical from Assam Agricultur- al University, Jorhat, India during this study on India Government Scholarship. REFERENCES 1 Scharrer, B. (1948) The prothoracic glands of Leucophaea maderae (Orthoptera). Biol. Bull., 38: 35-45. 2 Scharrer, B. (1966) Ultrastructural study of the regressing prothoracic glands of Blatterian insects. Z.Zellforsch., 69: 1-21. 3 Bodenstein, D. (1953) Studies on the humoral mechanisms in growth and metamorphosis of the cockroach, Periplaneta americana. IJ. The function of the prothoracic glands and the corpus cardiacum. J. Exp. Zool., 123: 413-433. 4 Radwan, W.A. (1977) The prothoracic glands in the cockroach, Nouphoeta cinerea (Olivier). II. Effect of JHA on histological structures of the gland. Bull. Soc. Entomol. Egypte, 61: 91-98. 5 Radwan, W.A. and Novak, V.J. A. (1977) The prothoracic glands in the cockroach, Nauphoeta cinerea (Olivier). I. Histological studies of the glands in the last instar nymph and their degenera- 10 11 12 13 14 15 tion in the adult insect. Bull. Soc. Entomol. Egypte, 61: 79-89. Lanzerin, B. (1975) Programming, induction, or prevention of the breakdown of the prothoracic gland in the cockroach, Nauphoeta cinerea. J. Insect Physiol., 21: 367-389. Hazarika, L. K. (1986) Hemocyte population changes and neuroendocrine complex in Blattella germanica (L.) (Dictyoptera: Blattellidae). Ph.D. Thesis, Rutgers University, New Brunswick, NJ. Pipa, R. L. amd Cook, E. F. (1959) Studies on the hexapod nervous system. I. The peripheral distribu- tion of the thoracic nerves of the adult cockroach, Periplaneta americana. Ann. Entomol. Soc. Am., 52: 695-710. Haber, V.R. (1926) The tracheal system to the German cockroach, Blattella germanica Linn. Bull. Brooklyn Entomol.Soc., 21: 61-92. Chadwick, L. E. (1956) Removal of prothoracic glands from the nymphal cockroach. J. Exp. Zool., 131: 291-306. Richter, K. and Gersch, M. (1983) Electrophys- iological evidence of nervous involvement of the prothoracic gland in the Periplaneta americana. Experientia, 39: 917-918. Pipa, R.L. (1983) Morphological considerations in the integration of nervous and endocrine systems. In “Endocrinology of Insects”. Ed. by G. H. Downer and H. Laufer, Alan R. Liss, Inc., New York, pp. 39—S53. Scharrer, B. (1964) The fine structure of the Blatte- rian prothoracic glands. Z. Zellforsch., 64: 301-326. Dorn, A. and Romer, F. (1976) Structure and func- tion of prothoracic glands and oenocytes in embryos and last larval instar of Oncopeltus fasciatus Dallas (Insecta, Hemiptera). Cell Tissue Res., 171: 331-350. Cazal, P. (1947) Recherches sur les glandes endo- crines rétrocérébrales des Insects II. Odonates. Arch. Zool. Exp. Gén., 85: 55-82. ZOOLOGICAL SCIENCE 4: 151-158 (1987) Long-lasting Effects of Orchiectomy and Its Preceding Procedures on Open Field Behavior in Male Mice TAKEO Macuipa, SHIHO Iso! and Tetsuo NoUMURA” Zoological Institute, Faculty of Science, Hiroshima University, Naka-ku, Hiroshima 730, ‘Biology Division, Tokyo Metropolitan Institute of Gerontology, Itabashi, Tokyo 173, and *Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Saitama 338, Japan ABSTRACT— During the course of investigations aiming to clarify the role of androgen in the ontogeny of open field behavior in male mice, it was found that several manipuiation stresses from transport to surgical castration exert long-lasting effects on measures of the behavior. Male mice of the ICR/SLC strain were divided into five groups, in stepwise addition of the following four treatments to the untreated controls: transport from animal room to laboratory, anesthetization with ether, operation of abdominal opening (sham-orchiectomy), and orchiectomy at 43 days of age. All animals were subjected to the open field test at 70-74 days of age. Scores of ambulation and rearing were the greatest in untreated controls and the smallest in orchiectomized mice, while those of grooming and defecation were the greatest in orchiectomized animals and the smallest in untreated controls. Scores of ambulation and rearing showed a tendency to decrease with addition of treatments. In contrast scores of grooming increased with addition of treatments. However, there were no significant differences in ambulation, rearing and grooming between orchiectomized and sham-orchiectomized animals. On the other hand, weight of adrenals increased with addition of treatments. Through 67 animals examined, adrenal weight negatively correlated with ambulation and positively interacted with grooming. These results indicate that orchiectomy and its preceding procedures at young adults exert long-term effects on later open field behavior in male mice and that orchiectomy itself exerts no significant influences on the © 1987 Zoological Society of Japan open field behavior. INTRODUCTION Several aspects of sociosexual behavior of male rodents are markedly influenced by gonadal androgens (for review, see [1, 2]). In our prelimi- nary experiments, we found that scores of ambula- tion and rearing in the open field were significantly smaller in orchiectomized mice than in intact animals but not significantly different from scores of sham-orchiectomized mice [3]. Similarly, signi- ficant difference was obtained in scores of defeca- tion between intact male mice and sham- orchiectomized animals, although there were no differences between orchiectomized mice and sham-orchiectomized animals. It seems highly probable, therefore, that not only orchiectomy but Accepted September 25, 1986 Received July 31, 1986 also surgical operation itself and/or manipulation of animals preceding operation might act as stressors and cause some behavioral changes in male mice. Procedures of manipulating animals preceding operation of orchiectomy are divided into three steps: (1) transport of mice in the cage from animal room to laboratory, (2) anesthetiza- tion of animals with ether, and (3) operation of abdominal opening. In the present experiments, effects of these procedural steps on several meas- ures of open field behavior were examined in male mice. MATERIALS AND METHODS Animals Sixty seven male mice of the ICR/ SLC strain were used. All animals were obtained at 21 days of age from the Shizuoka Animal Breeding Laboratory, Hamamatsu, Japan. They 152 T. MacuipA, S. Iso AND T. NouMURA were housed in groups of four mice per cage and maintained on pelleted diet and water ad libitum in a temperature- (22+2°C) and humidity- (50% R.H.) controlled animal room on a 14hr/10hr light-dark cycle (lights on at 0700). Treatments At 43 days of age, all animals were randomly assigned to one of the following five groups: (1) untreated controls. Animals were left undisturbed in the animal room until the day of behavioral test. (2) transport. Animals were carried out of the animal room to laboratory. (3) anesthesia. Animals transported to laboratory were anesthetized with ether. (4) sham- orchiectomy. Animals transported to laboratory and anesthetized with ether were given an opera- tion of sham-orchiectomy. (5) orchiectomy. Animals transported to laboratory and anesthe- tized with ether were orchiectomized. Animals that had been transported to laboratory were then carried back to animal room immediately after receiving above treatments. Open filed test When animals reached 70 days of age, they were given open field test daily for five consecutive days. A detailed description of appa- ratus and procedures of the test has been reported elsewhere [4]. Briefly, a 45 cm square open field surrounded by walls of 20cm high was constructed of acrylic plates, mat black in color. In each test, a sheet of black velvet paper, 45 x 45 cm in size, was placed on the floor of the open field. The floor of the field was divided into nine 15cm square units by 1 mm white lines ruled on the velvet paper. The open field was lighted by a fluorescent light source (20 W x2) mounted 150cm above the field. In the daily test administered between 1030 and 1300 hours, each animal was gently removed from its home cage and placed in the center of the field and the following elements of behavior were measured for five minutes: (1) ambulation; num- ber of squares entered by the animal, (2) rearing; number of standing upright on hind legs, (3) grooming; number of bouts of grooming and time in seconds spent grooming, and (4) defecation; number of fecal boli deposited. After each test, animals were placed in a holding cage until all the occupants of the home cage had been tested. Measurement of organ weight Two days after | the end of open field test, animals were killed by overdose of ether and the adrenals and the preputial glands were dissected out and weighed. Statistics Statistical analyses were performed by Mann-Whitney’s U-test and Kruskal-Wallis test for multiple groups or using correlation coefficient (r). RESULTS Cumulated scores for each measure of the open field behavior obtained over five days of testing are shown in Figures 1-3. Scores of ambulation were significantly in- teracted with treatments (H cor (4) =9.56, P<0.05 by Kruskal-Wallis test, Fig. 1a). Mice of the untreated group exhibited significantly more active ambulation than those of the orchiectomized or the sham-orchiectomized group (U(14, 13)=50, P<0.05 between untreated and orchiectomized mice, and U (14, 17)=67, P<0.05 between untre- ated and sham-orchiectomized animals by Mann- Whitney’s U-test), although there were no signi- ficant differences in ambulation between untreated and transported animals (U (14, 10)=36, P>0.05) or among transported, anesthetized, sham- orchiectomized and orchiectomized groups (H cor (3)=3.25, P>0.3). Through five groups, howevy- er, scores of ambulation showed somewhat de- crease with addition of treatments. On the other hand, scores of rearing failed to show any significant interaction with treatments (H cor (4)=8.50, P>0.05, Fig. 1b), although animals of the orchiectomized group exhibited significantly lower scores of rearing than the untreated controls (U (13, 14)=39, P< 0.05). Nevertheless, scores of rearing decreased with addition of treatments, except for anesthetized animals. Significant interaction with treatments was evi- dent in number of bouts of grooming (H cor (4)= 20.90, P<0.001, Fig. 2a). Orchiectomized animals exhibited significantly greater number of bouts of grooming than either anesthetized (U (13, 13)= 33.5, P<0.05), transported (U (13, 10)=10, P< 0.05), or untreated mice(U (13, 14)=14.5, P< 0.05), although they failed to show significantly different numbers of bouts of grooming from Open Field Behavior in Male Mice 153 oa oO oO a 400 : | ; 1 0 4 LSihehl, | ls D) Pe OqeeAy 7 SsO number of squares entered per 5 tests F 3 200 b 100 0 14} |10] 13] J17] 113 Ue ete RA ae IS 1 O number of rearing per 5 tests 1. Cumulated scores of ambulation (a) and rearing (b) obtained in the open field during 5 daily tests in male mice of different groups. U: ntreated, T:transported, A: nesthetized, S: sham-orchiectomized, O:orchiectomized. Each bar represents the mean +standard error. Number of animals used is shown in each column. See text for statistical evaluation. a 10 5 2 14) |10} |13] |17] 113 CO RERP CONE fou: Moan SH ene @. number of bouts of grooming per 5 tests b 90 § O77 n& 60 Ee C8 "2 C-— —E 30 VO ES 0 VEN AON Tey AFA Als} U I A Ss O Fic. 2. Cumulated scores of number of bouts of grooming (a) and time in seconds spent grooming (b) obtained in the open field during 5 daily tests in male mice of different groups. See legend of Fig. 1 for details. sham-orchiectomized animals (U (13, 17)=73, P> 0.1). Significant difference was similarly found in number of bouts of grooming between sham- orchiectomized and transported (U (17, 10)=40.5, P<0.05) or untreated animals (U (17, 14)=55, P<0.05). The number of bouts of grooming increased step by step with addition of treatments. Duration of grooming likewise exhibited a signi- ficant interaction with treatments (H cor (4)= 15.28, P<0.01, Fig. 2b). Animals of the orchiecto- 154 T. Macuipa, S. Iso AND T. NOUMURA mized group spent significantly longer time for grooming than those of either anesthetized (U (13, 13)=45, P=0.05), transported (U (13, 10)=8, P <0.05) or untreated group (U (13, 14)=32, P< 0.05), but failed to exhibit a significant difference in duration of grooming from sham-orchi- ectomized mice (U (13, 17)=82, P>0.1). Signi- ficant difference was achieved in scores of duration of grooming between sham-orchiectomized and transported animals (U (17, 10)=41.5, P<0.05). There were no significant differences, however, in duration of grooming among _ sham-orchi- ectomized, anesthetized and untreated animals (H cor (2)=3.86, P>0.1), and between transported and anesthetized (U (10, 13)=42, P>0.05) or untreated mice (U (10, 14)=64, P>0.1). Dura- tion of grooming increased with addition of treat- ments, but not for transported. Scores of groom- ing were negatively interacted with ambulatory activity: correlation was significant between the number of bouts of grooming and the number of squares entered (r (67)= —0.39, P<0.005). Statistically significant differences were obtained in scores of defecation among groups of different treatments (H cor (4)=13.34, P<0.01, Fig. 3). Animals of orchiectomized group eliminated signi- ficantly greater number of fecal boli than sham- 24 12 } 5 : 1) es 1S hiss: fl eye Aripace State Cumulated scores of defecation obtained in the number of fecal boli deposited per 5 tests Fic. 3. open field during 5 daily tests in male mice of | different groups. See legend of Fig. 1 for details. orchiectomized, anesthetized or untreated animals (U(13517)=355,U (135 13) =37 SyomUi(1314) = 29, respectively, P<0.05). There were no signi- ficant differences in scores of defecation, however, between orchiectomized and transported groups (U(13, 10)=44, P>0.1) and among mice of untreated, transported anesthetized and sham- orchiectomized groups (H cor (3)=2.85, P>0.3). Daily changes in several measures of the open field behavior during five days of testing were shown in Figure 4. In the five groups of different treatments, daily scores of ambulation invariably exhibited a significant decrease with repeated testings (r (70) = —0.37, r (50) =—0.47, r (65) = —0.44, r (85) =—0.39 and r (65)=—0.46 for untreated, transported, anesthetized, sham- orchiectomized and orchiectomized groups, re- spectively, P<0.005). On the other hand, in scores of rearing, signi- ficantly negative correlation with days of testing was only found in the transported group (r (50)= —0.31, P<0.05), while no consistent correlation was obtained between scores of rearing and days of testing in the other four groups (r (70)= — 0.13, r (65) =—0.17, r (85) =—0.16, and r (65) = —0.23 for untreated, anesthetized, sham-orchiectomized and orchiectomized groups, respectively, P> 0.05). By contrast, daily scores of grooming tended to increase with repeated testing. Positive correlation was significant between duration of grooming and days of testing in the orchiectomized (r (65) = 0.42, P<0.001) and the anesthetized groups (r (65) =0.28, P<0.05). In the other groups, however, positive correlation between scores of grooming and the repeated testing failed to reach statistical significance (r (70) =0.12, P>0.1, r (S50) =0.26, P >0.05, and r (85)=0.20, P>0.05 for untreated, transported and anesthetized groups, respectively. Table 1 shows the body weight and the weights of adrenals and preputial glands in animals of different experimental groups. Significant interac- tion was obtained between the body weight and the different treatment two days after exposing five daily tests of behavior (H cor (4) =12.8, P<0.02). — Orchiectomezed animals had significantly smaller body weight than mice of the untreated and the anesthetized groups (U (13, 14)=32, and (U (13, Open Field Behavior in Male Mice 155 number of squares entered number of rearing Aa time in seconds spent grooming | 2SR ST 2eAE U i (Ie panel ey) ia 1.23415 (13) ill LZ S425 alee S405 day of testing A > O (17) (13) Fic. 4. Changes in scores of ambulation, rearing and grooming following daily testing in the open field. See legend of Fig. 1 for details. 13) =40, respectively, P<0.05). Sham-orchi- ectomized animals were likewise smaller than untreated controls (U (17, 14)=58, P<0.05). Through the five groups, the body weight de- creased with the addition of treatments. Weight of the adrenals was significantly interact- ed with treatments (H cor (4)=33.6, P<0.001). Adrenals of the orchiectomized animals were significantly heavier than those of anesthetized, transported and untreated mice (U (13, 13)=31.5, (U (13, 10)=1, and U (13, 14) =13, respectively, P <0.05), although there were no significant differ- 156 T. MAcHIDA, S. Iso AND T. NOUMURA TABLE 1. experimental groups Group Body weight (no. of mice) (g) Untreated (14) 39772-2056 Transported (10) 38.4+0.6 Anesthetized (13) 38.8+0.7 Sham-orchiectomized (17) 37.4+0.6 Orchiectomized (13) 36.6+0.8 See text for statistical evaluation. ences in the weight of adrenals between orchiecto- mized and sham-orchiectomized groups (U (13, 17)=63, P>0.05). Animals of the sham- orchiectomized group possessed significantly heavier adrenals than mice of the transported and the untreated groups (U (17, 10)=5.5, and U (17, 14)=32.5, P<0.05). Adrenals of the anesthetized animals were significantly heavier than those of the transported and the untreated mice (U (13, 10) = 22.5, and U(13, 14)=46, P<0.05). Adrenal weights increased with the addition of treatments. On the other hand, weight of the preputial glands was significantly reduced by orchiectomy (orchiectomized vs. sham-orchiectomized mice, U (35 17)=1., P< 0205). “Among athe) other tour groups with intact testes, however, no significant difference was found in the weight of preputial glands (H cor (3) =4.63, P>0.2). Through 67 animals examined, significant cor- relations were obtained between measures of the open fleld behavior and the body weight or the weight of adrenals. Scores of ambulation exhibited a positive correlation with body weight and a negative correlation with adrenal weight (r (67) = 0.39, P<0.005, and r (67)=0.27, P<0.05, respec- tively). Significantly positive correlation was evi- dent between the number of bouts of grooming and the weight of adrenals (r (67)=0.54, P< 0.001). DISCUSSION The present experiments clearly demonstrated that castration caused significant changes in meas- ures of the open fleld behavior in male mice of the Body weight and weights of adrenals and preputial glands in male mice of different Adrenals Preputial gland (mg/10g B.W.) 13s Ol 20.2+1.4 eae Il 20.8+0.9 Date Oe DUE Z-S 2.4+0.1 19.4+1.3 2.5+0.2 Dal Or7 ICR/SLC strain. This is in good agreement with the reports of Broida amd Svare [5, 6] showing that locomotion in the home cage is significantly reduced by castration in males of the CS7BL/6]J, DBA/2J and Rockland-Swiss strains. However, in the present experiments, orchiectomized animals exhibited slightly smaller scores of ambulation than sham-orchiectomized mice. It seems there- fore likely that removal of testes could exert only very small but no significant effect on male mice in lowering ambulatory activity in the open field. Similarly, the finding that orchiectomized animals exhibited slightly larger scores of grooming and defecation than sham-orchiectomized mice like- wise indicates a minor involvement of testis in these measures of the open field behavior in male mice. Further experiments are required to clarify these differences. It is well known that stimulation of animals in infancy results in significant modifications of be- havioral responses in adulthood (for review, see [7, 8]). Although the literature unequivocally indicates that stimulation by handling for the first 20 days of life is most effective in inducing permanent changes in later behavior in rats and mice, Machida et al. [9] successfully restricted the effective period of infantile handling to the second 10 days after birth in mice: handling from 11 to 20 days of age, but not from 1 to 10 days, significantly elevated later activities of ambulation and rearing in male amd female mice. In the present experi- ments, a single exposure of male mice to orchiec- tomy and its preceding procedures at 43 days of age exerted significant influences on behavioral reactivities at 70 days of age. In this connection, Goodrcik [10] previously reported that gentling for Open Field Behavior in Male Mice Lou) 42-51 days of age significantly affected scores of grooming at 73 days of age in male rats. It is interesting that not only stimulation in infancy but also exposure to certain stimuli in later life produce long-term behavioral changes in animals. In the present experiments, significant correla- tions were evident between measures of the open field behavior and the addition of treatments: scores of ambulation and rearing were significantly decreased with addition of treatments, while those of grooming and defecation were increased. On the other hand, it was found that addition of treatments at 43 days of age significantly decreased the body weight and increased the weight of adrenals at 76 days of age. It has been well known that exposure of immature rodents to stressful stimule, i.e., handling or electric shock, affects the later behaviors and the function of the hypothala- mo-hypophyseal-adrenocortical (HHA) system [11, 12]. This manipulation also alters plasma levels of corticosterone in rats [11, 13]. Therefore, in the present experiments, there is every probabil- ity that four kinds of treatments acted as stressors, incited the function of HHA system and caused elevated secretions of adrenocorticotropic hor- mone (ACTH) and adrenocortical hormones which provoked long-term alterations of behavior- al responses in later life. Determination of plasma titers of these hormones are required to confirm the probability. It has been known that repeated exposure to the open field causes a decrease in ambulation and an increase in defecation and grooming in rats and mice (for review, see [14]). We have also reported a similar decrease in scores of ambulation and rearing and an increase in grooming following successive tesing in mice [4, 9, 15]. In the present experiments, five daily exposures to the open field caused a decrease in ambulation and rearing and an increase in grooming. The results are in good agreement with our and others’ previous findings. In this connection, Nagy and Holm [16] found a significant increase in defecation following re- peated testing in mice of the C3H strain. In our experiments, however, mice of the ICR/SLC strain eliminated a very small number of fecal boli in the open field and therefore we were unable to detect daily changes in defecation. It is apparent that defecation is not a valid measure for the ICR/SLC strain. A significant negative correlation has well documented between defecation and ambulation in various strains of mice (for review, see [14]). Throughout 67 mice examined in this experiment, however, inverse correlation was evident between scores of grooming and ambulation. Correlation of ambulation with variables other than defecation has seldom been reported (for review, see [17]). This is the first to demonstrate significant inverse correlation of ambulation with grooming scores. ACKNOWLEDGMENTS This work was supported in part by a Grant for Life Science Research Project “Discovery of Factors Regula- ting Aging” from the Institute of Physical and Chemical Research to T.N. REFERENCES 1 Hart, B. L. (1974) Gonadal androgen and socio- sexual behavior of male mammals: A comparative analysis. Psychol. Bull., 81: 383-400. 2 Lloyd, J. A. (1975) Social behavior and hormones. In “Hormonal Correlates of Behavior. Vol. 1. A Lifespan View”. Ed. by B. E. Eleftheriou and R. L. Sprott, Plenum Press, New York, pp. 185-204. 3. Machida, T. (1985) Effects of androgen on spon- taenous activities of male mice. Proc. Chugoku- Shikoku Branch, Zool. Soc. Japan, 37: 24 (Ab- stract). 4 Machida, T., Matsumoto, K., Kobayashi, S. and Noumura, T. (1982) Age-associated changes in the open field behavior of male mice of the ICR/SLC strain. Zool. Mag., 91: 263-271. 5 Broida,J. and Svare, B. (1983) Genotype mod- ulates tetosterone-dependent activity and reactivity in male mice. Horm. Behav., 17: 76-83. 6 Brioda, J. and Svare, B. (1984) Sex differences in the activity of mice: Modulation by postnatal gonad- al hormones. Horm. Behav., 18: 65=78. 7 Denenberg, V. H. (1964) Critical periods, stimulus input, and emotional reactivity: A theory of infantile stimulation. Psychol. Rev., 71: 335-351. 8 Ader, R. (1975) Early experience: Emotinal be- havior and adrenocortical function. In “Hormonal Correlates of Behavior. Vol. 1. A Lifespan View”. Ed. by B.E. Eleftheriou and R. L. Sprott, Pleum Press, New York, pp. 7-33. 9 Machida, T., Kurando, T. and Tosaki, M. (1982) Effects of different periods of handling during 10 11 12 13 158 infancy on later open field behavior in mice. J. Sci. Hiroshima Univ., Ser. B, Div. 1, 30: 259-270. Goodrick, C. L. (1971) Variables affecting free ex- ploration responses of male and female Wistar rats as a function of age. Dev. Psychol., 4: 440—446. Levine, S. (1962) Plasma free corticosteroid re- sponse to electric shock in rats stimulated in infancy. Science, 135: 795-796. Haltmeyer, G. C., Denenberg, V. H. and Zarrow, M. X. (1967) Modification of the plasma cortico- sterone response as a function of infantile stimula- tion and electric shock parameters. Physiol. Behav., 2: 61-63. Denenberg, V.H., Brumaghim, J. T., Haltmeyer, G.C. and Zarrow, M. X. (1967) Increased adreno- 14 15 16 LZ T. Macuipa, S. Iso AND T. NouMuURA cortical activity in the neonatal rat following hand- ling. Endocrinology, 81: 1047-1052. Archer, J. (1973) Tests for emotionality in rats and mice: A review. Anim. Behav., 21: 205-235. Machida, T., Kobayashi,S. and Noumura, T. (1983) Effects of different sizes of postweaning grouping on open field behavior in male mice. J.Sci. Hiroshima Univ., Ser. B, Div. 1, 31: 155-162. Nagy, Z.M. and Holm, M. (1970) Open field be- havior of C3H mice: Effect of early handling, field illumination and age at testing. Psychon. Sci., 19: 273-274. Walsh, R.N. and Cummins, R. A. (1976) The open-field test: A critical review. Psychol. Bull., 83: 482-504. ZOOLOGICAL SCIENCE 4: 159-166 (1987) © 1987 Zoological Society of Japan Geographic Variation in the Tail of the Japanese Salamander, Hynobius lichenatus, with Special Reference to Taxonomic Bearing MasAto HASuUMI and HISAAKI IWASAWA Biological Institute, Faculty of Science, Niigata University, Niigata 950-21, Japan ABSTRACT—Tail vertebrae were observed radiographically in 283 males and 49 females of adult Hynobius lichenatus collected during the breeding season from 19 localities in northeastern Japan. The frequency of specimens with broken and regenerated tails was much higher when the sites of oviposition were in swifter flowing streams. The relative tail length of specimens with normal tails tended to be greater at higher latitude. It is difficult to distinguish externally most regenerated tails from normal tails. The dispersions of relative tail length, however, were remarkably greater in most localities when data from specimens with normal, questionable, and regenerated tails were used. It is, therefore, necessary to take notice of tail regeneration in the measurement of tail length for taxonomic purposes. INTRODUCTION Relative tail length, that is tail length/snout-vent length, is one of the diagnostic characters for study of intra- and interspecific variation in urodeles. Since tail-autotomization among salamanders is restricted to most plethodontids and some sala- mandrids [1], tail length of specimens with com- plete and unautotomized tails is used taxonomical- ly in these urodelen species [2, 3]. As far as we know, however, there are no taxonomic papers that take into consideration regeneration following tail breakage in other urodeles. Hynobius is a genus of the family Hynobiidae that contains the most primitive living salamanders [4]. Hynobius lichenatus is widely distributed in northeastern Honshu, the mainland of Japan. Sato [5] reported that this species had considerable geographic variation in morphology, but he did not provide enough data. We reinvestigated the taxonomic characters of this species in the minutest detail, taking into account the various localities, and drew some new conclusions. Varia- tion in the length of the tail is dealt with in this paper. Accepted August 1, 1986 Received March 11, 1986 MATERIALS AND METHODS During the breeding seasons of 1983-1985, 283 adult males and 49 adult females of Hynobius lichenatus Boulenger were collected from sites of Oviposition in nineteen localities in northeastern Honshu. Sample sites are shown in Table 1. Sample sites 16-19 nearly correspond to the southern limit of distribution for this species. The stream index for sites of oviposition, divided into five types, shows that the stream flows more rapidly at higher type numbers. Additionally, in sample site 8, salamanders were trapped in road- side ditches on their way to the natural site of Oviposition. As soon as possible after collection, animals were anesthetized with 0.01% p-aminobenzoic acid ethyl ester aq. and measured. All measure- ments were made to the nearest 0.1 mm with slide calipers. The specimens were then fixed in 10% formalin. The following morphological data were recorded: snout-vent length, measured from the tip of the snout to the posterior angle of the vent; tail length, from the posterior angle of the vent to the tip of the tail; and axilla-groin length, from the posterior angle of the forelimb to the anterior angle of the hindlimb. Proportions were repre- sented as relative to snout-vent length. Radiographs of all specimens were taken using 160 TABLE 1. Sample site (Altitude) Maedanome, Goshogawara-shi 1) Aomori Pref. (320m) 2) Kudoji, Hirosaki-shi Aomori Pref. (240m) 3) Mt. Ajara-yama, Ohwani-machi Aomori Pref. (340m) 4) Mase-keikoku, Hachimori-machi Akita Pref. (50m) 5) Natsuzaka, Takko-machi Aomori Pref. (300m) 6) Kawamata, Tamayama-mura Iwate Pref. (180m) 7) Hirukawa, Ohmagari-shi Akita Pref. (180m) 8) Idosawa, Ichinoseki-shi Iwate Pref. (570m) 9) Yamanome, Ichinoseki-shi Iwate Pref. (120m) 10) Iragawa, Atsumi-machi Yamagata Pref. (100m) 11) Hataya, Yamanobe-machi Yamagata Pref. (610m) 12) Mt. Ninohji-dake, Shibata-shi Niigata Pref. (720m) 13) Hibara, Kitashiobara-mura Fukushima Pref. (820m) 14) Yutagami, Tagami-machi Niigata Pref. (80m) 15) Kamijoh, Kamo-shi Niigata Pref. (40m) 16) Tanne, Kashiwazaki-shi Niigata Pref. (160m) 17) Mt. Atema-yama, Tohkamachi-shi Niigata Pref. (800m) 18) Okushiobara, Shiobara-machi Tochigi Pref. (930m) 19) Fujiwara, Minakami-machi Gunma Pref. (740m) * See text. SOFRON equipment (TYPE SRO-MS0, SOKEN CO., LTD., Tokyo). The tail vertebrae observed were divided into four types as follows: a. exter- nally broken tails, including obviously regenerated tails, with tail vertebrae half-cut the same as the broken parts; b. regenerated tails not distinguish- able from normal tails externally but with partial or incomplete tail vertebrae (Fig.1B, C); c. M. HAsumMI AND H. IwASAWA Sample site, site of oviposition, stream index, and number of specimens used in this study oe : No. of specimens Oviposition site P and stream index Male Female fountain, 2 yD 0 fountain, 2 HLL 0 fountain, 2 3 0 roadside pool, 1 2 0 roadside fountain, 2 15 0 roadside stream, 3 18 0 fountain-flowing pond, 2 34 4 - 13 12 pool, 1 5) 0 torrent, 5 7 0 fountain, 2 5 0 swamp stream, 4 12 3 fountain-flowing ditch, 3 9 4 torrent, 5 44 10 fountain-flowing pool, 2 17 i torrent, 5 10 0 brook, 4 9 0 brook, 4 9 3 fountain-flowing ditch, 2 48 12 questionable tails with tail vertebrae somewhat abnormal but not partial; and d. normal tails with tail vertebrae normal and complete (Fig. 1A). The presence of correlation between two vari- ables was tested with Spearman’s rank correlation coefficient. For statistical analysis of relative tail length, data from the specimens having normal tails (type d) were used. The significance between Salamander Tail 161 Fic. 1. Radiographs of tails: A. normal vertebrae; B. fragmentary vertebvrae; C. no vertebrae. Scale: Smm. two sample means was tested with the median test. For histological observations, regenerated tails were embedded in paraplast, sectioned serially at 8 ym, and stained with Delafield’s hematoxylin and eosin. RESULTS Interpopulation variation was found in the ratios of the four types of tail vertebrae, and sexual difference in the ratios was also recognized in the specimens collected at sample sites 7, 12, and 14 (Fig. 2). The frequency of specimens with exter- nally broken and regenerated tails was much higher where the sites of oviposition were the swifter flowing streams (Table1 and Fig. 2). Animals with newly cut tails were not found among specimens having broken tails. No signi- ficant inverse correlation was found between the frequency of male specimens with broken and regenerated tails and the altitude (r,= —0.0725, a=(0.05; Table 1 and Fig. 2). The mean relative tail length did not differ significantly (a2 =0.05) between males and females in sample site 7, but was significantly greater in males than in females in sample sites 8 (P<0.05), 12 (P<0.05), 13 (P<0.05), 14 (P<0.02), and 19 (P<0.005) (Table 2). The relative tail length of this species tended to be greater at higher latitude (Tables 2 and 3). The mean relative tail length of the males in sample site 15 was significantly less than the means in other sites, except for sample sites 9 and 10 (Table 3). The absolute value of the relative tail length of the specimens collected in sample site 5 was the greatest of all (Tables 2 and 3). When data from specimens with normal, questionable, and regenerated tails were com- bined, the maximum and minimum relative tail lengths were 0.911 and 0.463, respectively, and these values were both found in males with regenerated tails (compare with the values shown in Table 2). The relative axilla-groin length was significantly (P< 0.001) greater in females (0.495 + 0.014, mean+SD) than in males (0.466+0.014). In the cross sections of the regenerated tails having no vertebrae, cartilage was observed to surround the spinal cord (Fig. 3A), while in the cross sections of the regenerated tails with frag- mentary vertebrae, chondral cartilage or fragmen- tary bone tissue was seen (Fig. 3B). DISCUSSION 1. The tail as a taxonomic character Most salamanders seem to regenerate complete tails after suffering tail breakage, so it may be difficult to distinguish externally regenerated tails from normal tails. However, only herpetologists studying tail-autotomizable salamanders have pointed out the above-mentioned phenomenon [1, 6, 7]. In amputation-regeneration experiments on the tails of Ambystoma larvae, Holtzer et al. [8] showed that larvae regenerated all parts of the tail, but the lengths of the regenerated tails ranged from 20% to 35% below those of the controls. It seems, however, that tail regeneration has not been taken into consideration in tail-unau- totomizable salamanders, so that the lengths of tails including regenerated ones have been used for taxonomic comparison. Therefore, in most papers [9-14] in this field the relative tail length in each urodelen species shows a remarkable variation in comparison with other taxonomic characters, such as relative head length, trunk length, and axilla- groin length. In fact, when data from specimens 162 M. HASUMI AND H. IwAsawa PACIFIC OCEAN Fic. 2. The ratios of the four types of tail vertebrae in each sample site. The size of circles reflects sample size (See Table 1). with normal, questionable, and regenerated tails are combined, the dispersions of relative tail length are greater and the means of relative tail length are less in most localities (Fig. 4). The main reasons why relative tail length is significantly greater in males than in females may be that relative axilla-groin length is significantly greater in females than in males, and that breeding males have knife-edged tails so that the tails are longer. The relative lengths of many regenerated tails were less than those of normal tails, but the relative lengths of a few were greater. In addition, TABLE 2. Salamander Tail Geographic variation of relative tail length of specimens with normal tails SS Sample Mean snout-vent length Mean relative tail length 163 HE site We: +2SE (range) (mm) +2SE (range) 1 0 Z 6 67.4+2.4 (63.9—71.6) .804 + .020 (.770—.844) 3 2 59.6 (58.2—61.0) .781 (.780— .782) 4 10 61.9+0.7 (60.0—63.6) .782 + .018 (.742 — .827) 5 9 60.6+1.8 (56.7—66.1) .827 + .029 (.765 — .890) 6 8 67.9+2.7 (63.7—74.8) .786 + .037 (.700— .844) 7 21 68.7+ 1.6 (63.0—76.1) .795 + .021 (.715 — .877) 8 6 58.8+2.4 (54.5—63.6) .763 + .014 (.750—.785) 9 3 59.4+2.9 (56.8—61.7) .724+ .107 (.618—.793) Male 10 1 67.8 701 11 3 63.5+5.5 (60.0—69.0) .764 + .032 (.732—.781) 12 5 68.1+4.3 (63.0—73.8) 802 + .013 (.790— .828) 13 5 57.4+1.9 (54.4—60.2) .751+.017 (.728—.771) 14 13 68.0+2.3 (61.8—75.2) .766 + .028 (.680— .846) 15 4 67.6+2.2 (64.3—69.1) .633 + .034 (.607— .682) 16 2 73.8 (73.0—74.6) .786 (.764— .808) 7/ 0 18 1 59.4 795 19 29 66.0+1.3 (58.1—74.1) .760 + .012 (.678— .820) ih 4 67.0+3.2 (65.2—71.7) .747 + .050 (.715— .822) 8 6 60.1+1.8 (57.6—62.8) .724+ .019 (.688—.759) 12 3 68.9+5.4 (64.1—73.5) .663 + .032 (.637—.693) Bomle 13 3 60.4+3.3 (58.7—63.7) .672 + .060 (.612—.705) 14 6 69.6+3.7 (65.0—77.0) .717 + .022 (.678— .748) 15 0 18 1 63a 658 19 7 69.4+2.2 (65.9—73.9) .695 + .024 (.655 — .738) TABLE 3. Median test for the mean relative tail length (x) of specimens with normal tails X min. X max. Sample site LSoveGhO)\RS (‘SemlseetOws 11 1455) 44> (16). 16" (48), 7 12.2715 of males Sample site Cs ouleaiats: 19Mnine 8 7 of females Sas Each category is arranged in linear as less value comes left. Each bar indicates the groups that the difference is not statistically significant (a=0.05). However, the mean values of males in sample sites 19 and 4 are significantly less than the means in sample sites 4 and 2, respectively. Sample sites with no adequate number of specimens having normal tails are shown in parentheses. the relative tail length showed a definite latitudinal variation, so it cannot always be determined from external observations that shorter tails must be regenerated ones. In conclusion, it is necessary to take notice of tail regeneration, sexual difference, and latitudinal variation when cosidering tail length for taxonomic purposes. 164 M. HAsuMI AND H. Iwasawa Fic. 3. Cross sections of regenerated tails: A. no vertebrae; B. fragmentary vertebrae. s: spinal cord, c: cartilage, cc: chondral cartilage, b: bone tissue, m: muscle, d: dermal gland, v: blood vessel. Scale: 0.5mm. 2. Causes of tail breakage The breakage of tails in tail-autotomizable salamanders was the result of traumatic events, such as attacks by predators, rock falls, entrap- 0.90 «) O B 8 ) a 8 5 0.80 § oc a fe) 4 © = z WwW 2 e S =] Qo.70+ @ c) ow re 7p) @ @ O \ & © = e e 2 e ey e a e = 0.60 e é - e | 0.50 e [Soto tif) Lica Sayin N 10> 6 13> 6 12+ 5 43 — 13 10 — 6 EEE: 22 8 12 14 14 ment, and so on [1]. Predators Previous investigators thought that tail autotomy in tail-autotomizable salaman- ders reflects predation pressure from any predator so long as the tail is attacked at least at some time [7, 15, 16]. The tails of tail-unautotomizable salamanders, too, may be injured by potential predators. So differential predation pressure ought to contribute to the differences in the ratios of the four types of tail vertebrae in Hynobius lichenatus. Shaffer [7] reported that snake densi- ties (potential predators) were found to decrease with elevation, and a significant inverse correlation was found betweerr tail loss and elevation; that is, tail loss and the regenerated tails were equivalent expressions. However, this correlation was not found in the present study. Biting Kusano [17] suggested based on his observations on Hynobius nebulosus tokyoensis during the breeding season that tail breakage was Fic. 4. Comparison of relative tail length. Left vertical lines indicate mean + 2SE when data from specimens with normal, questionable, and regenerated tails are combined. Whereas right vertical lines indicate mean+2SE when only data from specimens having normal tails are used. Open circle: normal tail, semisolid circle: questionable tail, solid circle: re- generated tail. Salamander Tail 165 the result of biting between breeding males, because the percentage of animals with cuts in the tail was higher in males than in females, and some breeding males which appeared in the pond were seen to lose their tail tips during their stay in the pond. It seems, however, that only tail tips or small parts of caudal fins would be lost in such cases. Larval cannibalism Cannibalism during the larval period easily happens when larval density is high. It seems unlikely, however, that differential tail breakage resulted from cannibalism in the sites of oviposition, because the ratios of the four types of tail vertebrae differed according to sex. Rapid stream The present results suggest that tail breakage in Hynobius lichenatus is due to rapid stream flow in most cases. During early spring thaw streams often overflow so rapidly that breeding adults may be carried away and injured because they are unable to resist the current. Moreover, the salamanders with just broken tails are even less able to resist and are carried away anew. Breeding males have knife-edged thin tails and stay longer in the stream, so they may be injured more frequently than females. It seems, however, that larvae are not so strongly influenced by the streams even in these sites, because the streams flow more slowly after the breeding season, except when there is occasional heavy rain. If tail breakage happens in the larval period, no difference according to sex would be found in the ratios of the four types of tail vertebrae. It is conceivable, therefore, that the breakage of tails usually happens to adults rather than to larvae. At any rate, the causes of tail breakage in this species seem to be very complex. 3. Supplementary comments on the causes of tail breakage in three sample sites Sample site & Roadside ditches in a forest were dug a year before the present collection, so tail breakage is not due falling into these ditches. A torrent was found near this site. Since the incidence of regenerated tails in the specimens collected at this site was quite high, the torrent may be the natural site of oviposition. However, because little difference according to sex was found in the ratios of the four types of tail vertebrae, it is also possible that the tails were injured during the construction of the road. Sample site 15 This site is on either side of a railroad line and is almost a static body of water into which water from fountains flows. On both sides of the railroad line are vertical precipices 5m high faced with blocks of stone. Thawing water cascades down from parts of the precipices during the breeding season, so it is conceivable that some salamaders fall down the precipices when they come to the site of oviposition, and some may fall down with the cascading water. These may be the causes of a higher frequency of specimens with regenerated tails in this site. Sample site 19 This site is a roadside ditch 20 cm wide, and a body of water about 30m long remains throughout the year because fountains flow into the ditch. More than 100 pairs of egg sacs were found in this ditch, and larval density was remarkably high. There was little difference according to sex in the ratios of the four types of tail vertebrae, and few specimens had severely broken tail vertebrae. It seems, therefore, that most tail breakage at this site is due to cannibalism of the tail in the larval period. ACKNOWLEDGMENTS We are grateful to Professor N. Nara of Hirosaki University for giving us information on the sites of oviposition. We are also grateful to Professor K. Kobayashi and Dr. I. Sasagawa of Nippon Dental Uni- versity for the use of SOFRON equipment. We wish to express our gratitude to Professor K. Takata of Niigata University for his advice on statistical analysis. REFERENCES 1 Wake, D.B. and Dresner, I. G. (1967) Functional morphology and evolution of tail autotomy in salamanders. J. Morphol., 122: 265-306. 2 Brodie, E.D., Jr. (1970) Western salamanders of the genus Plethodon: systematics and geographic variation. Herpetologica, 26: 468-516. 3 Crump, M.L. (1977) Intrapopulation and _ inter- specific variation of “standard” morphological char- acters of four closely related South American salamanders (Bolitoglossa), with description of habi- tat preferences. Herpetologica, 33: 415—426. 4 Dowling, H.G. and Duellman, W. E. (1973) Sys- tematic herpetology: a synopsis of families and 10 11 166 higher categories. Hiss Publications, New York, p. 6. Sato, I. (1943) Monogragh of Japanese tailed ba- trachians. Nippon Shuppan-sha, Osaka, pp. 106-119. (In Japanese) Muchmore, W. B. (1955)Brassy flecking in the sala- mander Plethodon c. cinereus, and the validity of Plethodon huldae. Copeia, 1955: 170-172. Shaffer, H. B. (1978) Relative predation pressure on salamanders (Caudata: Plethodontidae) along an altitudinal transect in Guatemala. Copeia, 1978: 268-272. Holtzer, H., Holtzer, S. and Avery, G. (1955) An experimental analysis of the development of the spinal column. IV. Morphogenesis of tail vertebrae during regeneration. J. Morphol., 96: 145-171. Nambu, H. (1983) A preliminary note on the sala- mander (Hynobius sp.) found in Ooyama-machi, Toyama Pref., and Oumi-machi, Niigata Pref., Central Japan. Bull. Toyama Sci. Mus., 5: 75-83. (In Japanese with English abstract) Krebs, S.L. and Brandon, B. A. (1984) A new species of salamander (family Ambystomatidae) from Michoacan, Mexico. Herpetologica, 40: 238-245. Matsui, M. and Miyazaki, K. (1984) Hynobius take- 2 13 14 15) 16 Uy) M. HAsumI AND H. IwAsSAWwA dai (Amphibia, Urodela), a new species of salaman- der from Japan. Zool. Sci., 1: 665-671. Morris, M. A. and Brandon, R. A. (1984) Gynogenesis and hybridization between Ambystoma platineum and Ambystoma texanum in Illinois. Copeia, 1984: 324-337. Nambu, H. (1984) Notes on Japanese salamander Hynobius lichenatus collected from a mixed egg- doposited pool in Minakami, Gunma Pref., Japan. Bull. Toyama Sci. Mus., 6: 73-78. (In Japanese with English abstract) Kraus, F. (1985) Unisexual salamander lineages in northwestern Ohio and southeastern Michigan: a study of the consequences of hybridization. Copeia, 1985: 309-324. Brodie, E. D., Jr., Johnson, J. A. and Dodd, C. K.., Jr. (1974) Immobility as a defensive behavior in salamanders. Herpetologica, 30: 79-85. Dodd, C. K., Jr. and Brodie, E. D., Jr. (1976) De- fensive mechanisms of neotropical salamanders with an experimental analysis of immobility and the effect of temperature on immobility. Herpetologica, 32: 269-290. Kusano, T. (1980) Breeding and egg survival of a population of a salamander, Hynobius nebulosus tokyoensis Tago. Res. Popul. Ecol., 21: 181-196. ZOOLOGICAL SCIENCE 4: 167-174 (1987) The Sperm Length and the Internal Reproductive Organs of Drosophila with Special References to Phylogenetic Relationships Fuyuo Hrmara and Haruo Kurokawa! Biological Institute, Ehime University, Matsuyama, Ehime 790, and ‘Institute of Biological Sciences, The University of Tsukuba, Ibaraki 305, Japan ABSTRACT—Forty species of Drosophila, including 2 subfamilies, 7 genera, 10 subgenera, were examined for the sperm length in relation to the evolutionary trend. The sperm length varied broadly from 0.2mm to about 10mm among species of the family Drosophilidae. In the subfamily Drosophili- nae to which many species belong, the more advanced genera and subgenera were clearly characterized by having longer sperm than the primitive ones. The lengths of the testis and the ventral receptacle (the sperm storage organ of the female) were highly correlated with the sperm length. This suggests that the non-homologous reproductive organs between sexes became to have a structural correlation along with the evolutionary processes due to a selection for the size of the sperm. All species examined were sorted into six classes to see whether or not the sperm length correlates with the number of spermatogonial divisions. Results showed that the species with smaller number of the spermatocytes per cyst generally produce the longer sperm. The sperm length, however, was very variable among species within the same class, showing that the number of spermatogonial divisions is not a definitive prerequisite to decide the sperm length. In contrast with this, the volume of the cytoplasm of the first spermatocytes just prior to the meiosis showed a high correlation with the sperm length in nine species, each belonging to either of the four different classes. The relation between the dose of the Y chromosome and the sperm length was © 1987 Zoological Society of Japan discussed based on our results obtained here. INTRODUCTION The insects have sperm storage organs in the female to make her possible to fertilize numerous eggs without further copulations for a certain period of time. This type of the internal fertiliza- tion can avoid waste of the sperm. This may serve the purposes of the most insect species employing the strategy that the continuity of the species is chiefly dependent on high fecundity. The sperm storage organs of female, thus, are required to possess the structure and function favorable for the survival of the sperm without losing the ability of fertilization. There have been several studies concerning the sperm length in Drosophila [1-3]. Hess and Meyer [4] showed the species difference of the sperm length among the five species of the hydei sub- group and D. repleta of the repleta subgroup. Accepted September 9, 1986 Received July 24, 1986 Members of the obscura species group have been known to produce the sperm with two or three different sizes in each individual [5-8]. Beatty and Sidhu [6] suggested that the variation in the sperm length among Drosophila might be related to their taxonomic positions. Thus, we can regard the sperm length as species-specific and considerably different among species. In the present article, we report the lengths of the sperm, the testis and the ventral receptacle of 40 species of the family Drosophilidae with special references to the phylogenetic relationships. We also examined the relation between the sperm length and the number of spermatogonial divi- sions. The latter has a clear evolutionary trend that the more advanced species of Drosophila tend to show a smaller number of divisions [9]. MATERIALS AND METHODS Two suofamilies, 7 genera, 10 subgenera includ- ing 40 species in total were examined for the 168 F. HIHARA AND H. KUROKAWA lengths of the sperm, the testis and the ventral receptacle (Table 1). Flies of the laboratory stocks were imaged on the usual food adding sufficient dry yeast, although the sperm length was proved not to be affected by the nutritional conditions [8]. Wild-caught flies were also used when the species were difficult to culture in the laboratory. Flies were dissected in Ephurussi-Beadle’s Ringer solu- tion. After removing the wall of the seminal vesicle, cover slip was put on the sperm which were free from the organ. A phase contrast microscope was used for the observation of the unstained sperm. Individual sperm was drawn by using camera lucida apparatus and measured by tracing with curvimeter on the whole length of the drawing. The lengths of the testis and the ventral receptacle were measured by the same methods. The volume of the cytoplasm of the spermato- cytes was calculated from the diameters of the cells and the nuclei. The mature spermatocytes which were clearly distinguished by the network structure of mitochondria surrounding the nucleus were chosen for the measure. The both spermatocytes of species to be measured and D. melanogaster were put together in a field under the microscope because the diameter changed gradually by the increasing pressure of the cover slip with the evaporation of the medium. The data from 20 spermatocytes in each species were used to obtain the means of the cell volume. RESULTS The sperm length The means and the ranges of the sperm lengths of 40 species examined are listed in Table 1. The sperm length was highly variable among species of the family; two species of the subgenus Scaptodro- sophila produce the shortest sperm, 0.21 and 0.32 mm, respectively, and D. hydei of the subgenus Drosophila produces the longest one reaching over 10mm, being the other species range between these values. Because the sperm of D. hydei has a long end piece in its tail which is consisted only of the axial filaments and is frequently apt to be cut off under manipulations, the entire sperm length could hardly be measured. In the subfamily Steganinae, number of species examined was so little that we could not speculate the phylogenetic relationships among them. Gen- erally, species of this subfamily tend to produce the shorter sperm than the subfamily Drosophili- nae; the sperm lengths of two species of the genus Amiota were both about 0.6mm and those of three species of the genus Leucophenga were from 1.1 to 1.4mm. The body sizes of the adults of five species in this subfamily were larger or nearly equal as compared with species of the subfamily Drosophi- linae. Thus, the body size does not have a correlation with the sperm length. This fact is also applicable to species of the subfamily Drosophili- nae; D. immigrans, for example, has nearly the same body size as D. hydei but its sperm length is much shorter than the latter (Table 1). Among the subfamily Drosophilinae except for the genus Drosophila, two species of the genus Scaptomyza produce shorter sperm, a species of the genus Mycodrosophila, on the contrary, produces much longer one, although only one species, M. shikokuana, was concerned here. In the genus Drosophila, three species of the sub- genus Hirtodrosophila were different in the sperm length; 0.36mm in D. qguadrivittata, 2.78 mm in D. alboralis and 3.04 mm in D. nokogiri. The most members of this subgenus were not compiled in Table 1 because the data have not sufficiently been provided. However, it can be conjectured from our unpublished data that there is wide inter- specific variation concerning the sperm length, reflecting that the Hirtodrosophila is composed of several species groups and subgroups which are widely diverged morphologically [10]. Eight species of the subgenus Sophophora being composed of five different species subgroups [11] show rather small difference among the species. Among these, three species, D. ananassae of the ananassae subgroup and D. auraria and D. rufa of the montium subgroup produce longer sperm than the others. In the subgenus Drosophila, the sperm length widely varies from 1.3mm of D. sterno- pleuralis to about 10mm of D. hydei. Most species of the immigrans species group consistently produce the shorter sperm. In con- trast with this, members of robusta (D. sordidula, D. lacertosa), funebris (D. funebris), repleta (D. Sperm length of Drosophila TABLE 1. Summary information for the length (mm) of the sperm, the testis and the ventral receptacle in species of the family Drosophilidae Species Genus Amiota Subgenus Amiota dispina Subgenus Phortica variegata Genus Leucophenga Subgenus Trichiaspiphenga argentosa Subgenus Leucophenga maculata magnipalpis Genus Microdrosophila Subgenus Microdrosophila purpulata Genus Mycodrosophila shikokuana Genus Liodrosophila aerea Genus Scaptomyza pallida graminum Genus Drosophila Subgenus Scaptodrosophila coracina throckmortoni bryani Subgenus Hirtodrosophila quadrivittata alboralis nokogiri Subgenus Dorsilopha busckii Subgenus Sophophora simulans lutescens melanogaster ficusphila suzuki auraria ananassae rufa Subgenus Drosophila sternopleuralis immuigrans sulfrigaster subtilis curviceps bizonata brachynephros histrio pengi nigromaculata sordidula lacertosa virilis funebris hydei 169 Sperm Testis Ventral Mean Range receptacle 0.64 0.58—0.76 = = 0.63 O52 Oe == 0.92 1.38 el = iSO) 3.60 = 1.08 O17) = 1222 1.16 0.97—1.33 3.85 15S) 1.06 O86 bale = 2.29 ssW 5.08—5.46 == 9.41 2.47 DY DAY! = 6.78 0.31 0.28—0.33 DD 1.50 0.58 0.52—0.65 = 1.68 0.21 020-023 0.76 —= 0.32 0.29—0.34 0.77 0.70 0.90 0.84—0.95 —= = 0.36 0350587) — 0.53 2.78 2.62—2.89 4.24 6.30 3.04 DSB) —= = 1.10 EO 1.80 1.88 lS 1AOS S125 1.62 1.74 iL.Sy 1.19—1.66 2.42 4.43 1.81 15631897 2.41 3.76 1.84 1.64—1.92 = S211 2D? 1.96—2.48 3.82 4.27 Depo 2AO—2239 3.05 3.47 2.30 225—238 3.96 6.26 S.0// 3.399 6.24 e22 1.29 NAS 1.39 2.65 2.65 1.76 S18 2.87 3.98 1.98 Leip 24S == 6.01 2.02 1.96—2.06 2.97 2.60 2PSi— SO 6.25 6.31 DAT Dalim Sle 3555 4.14 Desi 2 i—-3.09 3.86 5.50 3923 3: 16—3:28 5.14 Theol Sail 3.59—3.86 6.23 6.64 3.85 31 JAS EL 08 5.40 7.29 5.24 SV S857 7.62 6.67 5.44 5.26—5.66 8.44 8.93 6.47 Nei 7/ Sul 8.39 8.18 8.29 7.06—9.92 10.50 12297 A030 === 28.77 47.20 170 F. HIHARA AND H. KUROKAWA repleta ~10 funebris 8.3-84 robusta 52-54 melanica quingsig 3.7 histrio virilis a 65 28 Hirtodrosophila (hirticornis) 3.0 immigrans Liodrosophila 25 1.8-26 Scaptomyza 03-06 Dorsilopha 14 Hirtodrosophila 04-28 Leucophenga eS melanogaster Amiota 18-54 oe obscura Micro drosophila 0.05-03 11 Scaptodrosophila 0.2-0.8 Fic. 1. Phylogenetic representation concerning the sperm length among the genera or the subgnera within the family Drosophilidae (the first letter capitalized) and the species groups of the genus Drosophila. For detailed explanations on phy- logenetic treatments of the taxa, see Throckmorton (2 hydei) and virilis (D. virilis) species groups pro- duce the longer ones. The subgenus Drosophila, thus, consists of more various members concerning the sperm length than the subgenus Sophophora. Figure 1 shows phylogenetic relationships rel- evant to the sperm length of the major taxa and groups within the family Drosophilidae. The phylogenetic tree adopted is mostly based on that presented by Throckmorton [12]. Evidently, the sperm length tends to become longer in advanced genera, subgenera or species groups as compared to primitive ones. It remained unknown, however, whether or not the sperm length can reflect a closer relationships among relatives within a certain species subgroup. The lengths of the testis and the ventral receptacle In the fourth column of Table 1, the testis lengths of 28 species are presented. There was an — obvious correlation between the sperm and the testis length among 27 species examined (r=0.948, D.F.=25, t=14.818, P<0.001). As shown in Figure 2, the relative length of testis to the sperm length is about 1.5 when the sperm length exceeds 3mm. However, it becomes larger when the sperm length is less than 3mm. This implies that the spacial proportion of the elongating spermatid bundles to the whole length of the testis decreases as species have the shorter testis and vice versa. The lengths of the ventral receptacle of female are shown in the last column of Table 1. The ventral receptacle consists of structurally two different parts, the basal and the folded or coiled ones. Most of the sperm stored is found in the latter part and, accordingly, the basal part is regarded to have a role of a passage of the sperm. Thus, the length of the folded or coiled part is indicative of structural correlation between the sperm and the ventral receptacle. But, the figures in this column were obtained by measuring the whole length of both parts, because the boarder was not exactly distinct. In the most species, the length of the ventral receptacle was 1.5—3.0 times as much as that of the sperm. A high correlation was found between the sperm length and the total length of the ventral receptacle ((i—0!9325) Der: — 3 hat — IS s00nela 0.001). The length of the ventral receptacle is also highly correlated with that of the testis (r=0.978, D.F.=23, t=22.607, P<0.001). These high cor- relations well suggest that the sperm which accom- plish their mission from male to female might be a predominant factor of the selection to determine the structure of the non-homologous organs be- tween sexes. Correlation between the sperm length and num- ber of spermatocytes per cyst Kurokawa and Hihara [9] reported that the more advanced forms of Drosophila tend to show a smaller number of spermatogonial divisions and, consequently, a smaller number of spermatocytes per cyst. Species listed in Table 1 were sorted into 6 classes by number of spermatocytes per cyst regardless of their taxonomic positions (Table 2). The 64 cell class comprises two species of the Sperm length of Drosophila 171 ratio testis/sperm 1 Z 3 sperm 5 6 7 8 length (mm) Fic. 2. Correlation between the sperm length (mm) and the ratio of the testis length to the sperm length. Each point corresponds to the species listed in Table 1. TABLE 2. The mean sperm length among the classes sorted by the number of spermatocytes per cyst Number of Number of Sperm length (mm) spermatocytes species per cyst* observed Mean Range 64 2 0.26 Ot Or2 32 6 0.51 0.20—0.89 24 2 2.69 2.60 —2.78 16 22 be 7) ODS. (16)** (13) (2:25) (1.29—5.37) 12—14 2 4.48 S/N =D Taster 9 5.47 2.3) (ee) * Data from Kurokawa and Hihara [9]. ** Figures in parentheses of the 16 cell class are obtained from 13 species limited to Genus Drosophila. *** D. hydei was excluded from this class because precise sperm length could not be measured. subgenus Scaptodrosophila and the 32 cell class includes Microdrosophila purpurata, D(H). quad- rivittata, and four species of the obscura group, D. obscura, D. bifasciata, D. imaii and D. pseudoob- scura all of which are not listed in Table 1 because they were reported before [8]. The 24 cell class comprises D(H). alboralis and D. curviceps. The 16 cell class contains 22 species of many genera. Thirteen species of the genus Drosophila are also shown for comparison because the most species of the other classes are members of the genus Drosophila. The 12-14 cell class comprises D. pengi and D. sordidula and the 7-8 cell class contains eight species of the genus Drosophila. 172 F. HIHARA AND H. KuROKAWA Results presented in Table2 clearly show a tendency that the mean sperm lengths of these classes increase as numbers of spermatocytes per cyst decrease. However, the length of the longest sperm of a given class frequently exceeds that of the shortest sperm of the next class as shown in the ranges. The sperm of D. rufa of the 16 cell class, for example, is much longer than that of D. pengi or D. brachynephros which belongs to the 12-14 and 7-8 cell class, respectively. Further, as shown in parenthesis in Table 2, the mean sperm length within the 16 cell class shifted remarkably when species of the genus Drosophila were chosen. Accordingly, number of spermatocytes per cyst might not be a definite prerequisite for decision of the sperm length, admitting that an indication that the sperm length increases in species with a smaller number of spermatocytes per cyst is in evidence. Correlation between the sperm length and the cell volume of the mature spermatocyte It is proved by more detailed observations that the volume of the mature spermatocytes is con- siderably different among species even if they have the same number of spermatocytes per cyst. A correlation between the sperm length and the cell volume of the mature spermatocytes just prior to relative cell volume 1 2 3 4 5 6 7 relative sperm length Fic. 3. Correlation between the relative sperm length and the relative cell volume with range in nine species of the genus Drosophila. bif; D. bifasciata, bus; D. busckii, mela; D. melanogaster, biz; D. bizonata, pen; D. pengi, nig; D. nigromaculata, vir; D. virilis, lac; D. lacertosa, fun; D. funebris. Fig- ures after the species marks are the number of the first spermatocytes per cyst. the meiosis of nine species is shown in Figure 3. Both the values of the sperm length and the cell volume are relatively expressed on the basis of assigning a 1.0 to those of D. melanogaster. More strictly, the cell volume means the volume of the cytoplasm; the nuclear volume was deduced from the whole cell volume. When the whole cell volume was adopted, the correlation became somewhat indistinct. The relative cell volume of D. busckii, D. melanogaster and D. bizonata all belonging to the 16 cell class is 0.75, 1.00 and 1.30, respectively and obviously correlated with the relative sperm length of each species, 0.6, 1.0 and 1.6. In four species, D. nigromaculata, D. lacertosa, D. virilis and D. funebris, all belonging to the 7-8 cell class, a positive correlation between the relative sperm length and the relative cell volume is demonstrated as well. Although D. pengi is one of the species of the 12-14 cell class, both the sperm length and the cell volume are nearly equivalent to D. nigromacu- lata of the 7-8 cell class. These facts indicate that the sperm length would correlate more intimately to the cell volume than to the number of spermato- cytes per cyst. DISCUSSION We examined for the sperm length of 40 species of Drosophila with special references to their phylogenetic relationships. Results obtained clear- ly indicate that the more advanced species tend to produce the longer sperm. This is in accordance with the study where four species of Drosophila were compared as concerned with the sperm length [3]. Our results also indicate that the cytoplasmic volume of the mature spermatocyte would have a primary cause in determination of the sperm length. This can be conjectured that large spermatocyte makes possible to increase the doses both of the ribosomes relating translation of sperm proteins and of the mitochondria forming mitochondrial derivatives of the sperm tail. Since elongating spermatids do not concern de novo synthesis of proteins [13], gene expression neces- sary for spermiogenesis might be accomplished in stages of spermatocyte. Although the sperm head becomes longer as the whole length of the sperm Sperm length of Drosophila 173 increases [7, 14], the major part of the mature sperm is occupied by the tail which is consisted of the axoneme and the mitochondrial derivatives both being furnished by the materials from the cytoplasm of spermatocyte. Our results obtained here well agreed with evidences presented by Virkki [15] for the beetle, the family Alticidae, where the number of sperma- tozoa had been stepwise reduced from the primi- tive to the modern forms, sometimes accompanied by an extra large size of spermatocytes. In species of the hydei subgroup, sperm length have thought to be dependent on the Y chromo- some dose; the sperm length varies from 1.2mm for D.nigrohydei which has the smallest Y chromosome in this group, up to 6.6mm for D. hydei which possesses the largest Y chromosome [4]. In D. melanogaster as well, males with a complete or a partial deficient Y chromosome, namely, XO, XY°, XY", and with duplications of the Y chromosome, XYY, produce the sperm on the average 1.2, 1.3, 1.5 and 3.5mm long, respect- ively [16]. These facts have led to regard the Y chromosome as one of the causative factors of the sperm elongation. However, Gould-Somero et al. [17] reexamined the sperm length by means of duplicated Y chromosomes but were unable to repeat the observations by Hess and Meyer; clearly the presence of the extra Y chromosome per se was insufficient to elongate the length of the normal sperm of D. melanogaster. On the bases of the facts mentioned above, we must regard that the role of the Y chromosome on the sperm elongation is remained obscure as yet. In the present study, we postulate a determinant role of the cytoplasmic volume of spermatocyte than the Y chromosome dose for the sperm elongation in Drosophila. The volume of mature spermatocyte might be partially dependent to the number of multiplication divisions of spermatogo- nia which is thought to be strictly regulated by a certain gene(s) in each species [9]. The final determinant of the volume of spermatocyte, however, remains uncertain. In the present study, it can be affirmed that the length of the sperm is highly correlated with those of the internal reproductive organs of both sexes. Yanders and Perras [3] and Beatty and Sidhu [6] have already pointed out a close relation of the length between the testis and the ventral recepta- cle and they have a positive association with the sperm length in a limited species of Drosophila. Okada [10] gave a phylogenetic considerations on the subgenus Hirtodrosophila in which he assumed each set of character states to have been differenti- ated from a primitive to an advanced one. Steps of character differentiations of the testis and the ventral receptacle are: (1) banana-shaped, (2) with coils and with only a few small tite folds, (3) loosely and irregularly folded, (4) with proximal coils and distal folds, and finally, (5) with coil alone, respectively. We confirmed evolutionary changes in structures of these organs as proposed by him. Physiological and behavioral changes of the inseminated female are affected by several factors transmitted from the male accessory gland but not by the sperm themselves [18]. This fact shows that the female reproductive tracts have two distinct functions; the storage role of the sperm accepted and the receptor role of the informative molecules related to the physiological changes of the repro- ductive system. Structural association of the inner reproductive organs between sexes might be a result of natural selection for the former function. REFERENCES 1 Dobzhansky, Th. (1934) Studies of hybrid sterility. I. Spermatogenesis in pure and hybrid Drosophila pseudoobscura. Z. Zellforsch. Mikrosk. Anat., 21: 169-223. 2 Cooper, K. W. (1950) Normal spermatogenesis in Drosophila. In “Biology of Drosophila”. Ed. by M. Demerec, Wiley, New York, pp. 1-16. 3. Yanders, A] E= jand> Bernas, J P.(1960) » Sperm length in four Drosophila species. Dros. Inf. Serv., 34: 112. 4 Hess,O. and Meyer, G.F. (1963) Chromosome differentiations of the lampbrush type formed by the Y chromosome in Drosophila hydei and Drosophila neohydei. J. Cell Biol., 16: 527-539. 5 Polikansky, D. (1970) Three sperm sizes in D. pseudoobscura and D. persimilis. Dros. Inf. Serv., 45: 119-120. 6 Beatty, R.A. and Sidhu, N.S. (1970) Polymegaly of spermatozoan length and its genetic control. Proc. Roy. Soc. Edin., B71: 14-28. a 10 11 2 174 Beatty, R. A. and Burgoyne, P.S. (1971) Size classes of the head and flagellum, of Drosophila spermatozoa. Cytogenetics, 10: 177-189. Kurokawa, H., Matsuo, Y. and Hihara, F. (1974) A study on sperm length and body size of Drosophi- la bifasciata. Annot. Zool. Japon., 47: 140-146. Kurokawa, H. and Hihara, F. (1976) Number of first spermatocytes in relation to phylogeny of Drosophila (Diptera: Drosophilidae). Int. J. Insect Morphol. Embryol., 5: 51-63. Okada, T. (1967) A revision of the subgenus Hir- todrosophila of the old world, with descriptions of some new species and subspecies (Diptera, Dro- sophilidae, Drosophila). Mushi., 41: 1-36. Bock, I.R. and Wheeler, M.R. (1972) The Dro- sophila melanogaster species group. Studies in Genetics VII. Univ. Texas. Publ., 7213: 1-102. Throckmorton, L. H. (1975) The phylogeny, ecolo- gy, and geography of Drosophila. In “Handbook of Genetics”. Ed. by R.C. King, Plenum Press, New 13 14 MS) 16 17 18 F. HIHARA AND H. KUROKAWA York and London, pp. 421-469. Lindsley, D. L. and Grell, E. H. (1969) Sper- miogenesis without chromosomes in Drosophila melanogaster. Genetics, 61: Suppl. 1, 69-78. Hauschteck-Jungen, E. and Mauer, B. (1976) Sperm dysfunction in sex ratio males of Drosophila subobscura. Genetica, 46: 459-477. Virkki, N. (1969) Sperm bundles and phylogenesis. Z. Zellforsch. Mikrosk. Anat., 101: 13-27. Hess, O. and Meyer, G. F. (1968) Genetic activities of the Y chromosome in Drosophila during sperma- togenesis. Adv. Genet., 14: 171-189. Gould-Somero, M., Hardy, R. and Holland, L. (1974) The Y chromosome and sperm length in D. melanogaster. Exp. Cell Res., 87: 397-398. Hihara, F. (1981) Effects of the male accessory gland secretion on oviposition and remating in females of Drosophila melanogaster. Zool. Mag., 90: 307-316. ZOOLOGICAL SCIENCE 4: 175-181 (1987) © 1987 Zoological Society of Japan Two Peculiar Species of Corophiid Amphipods (Crustacea) from the Seto Inland Sea, Japan AKIRA HIRAYAMA Laboratory of Biology, Department of the Liberal Arts, Asia University, 5-24-10 Sakai, Musashino-shi, Tokyo 180, Japan ABSTRACT—Two species of the genus Corophium (Corophium lobatum n. sp. and C. sinense Zhang, 1974) taken from the shallow water of the Seto Inland Sea are described and figured. C. Jobatum n. sp. has the peculiar, fused urosome, and its uropod 2 arise from the base of the urosome instead of the distal part. This new species is clearly distinguished from the known corophiid amphipods by these characteristics. C. sinense Zhang, 1974, originally described from the Shangtung Peninsula, North China, is new to Japan. The specific characteristics of this species are observed in the mandibular palp that the terminal segment is reduced and directly attaches to the apex of the proximal one. INTRODUCTION During an assessement survey of the marine environment in the Seto Inland Sea, Japan, Mr. N. Sawada collected some specimens of peculiar amphipods from a sandy-mud bed of In-no-shima Island, Hiroshima Prefecture, and sent me for identification. Through a close exmination it became apparent that they were composed of two species of the genus Corophium. One species possesses a fused, laterally rounded urosome. This characteristic suggests that the species belongs to Section C of Crawford’s system [2, 5, 6], but the uropod 2 of this species attaches to the laterobasal part of its urosome and is well developed. Such an uropod is not found among the corophiid amphi- pods previously known. On the basis of these characteristics, I will describe it in this paper as a new species of Corophium. The other species collected in the present survey is Corophium sinense Zhang, 1974 [8], originally described from the Shantung Peninsula, North China. This spe- cies, that is new to Japan, is redescribed also in this paper. All the specimens here described are deposited in the collection of the Biological Laboratory, Department of the Liberal Arts, Asia University, Tokyo. Corophium lobatum n. sp. (Figs. 1-2) Description of the holotype ( female) Body: Semicylindrical. Rostrum and_ eyes small. Pereonites subequal in size; coxae discon- tinuous in series. Pleonal epimeron 1 rounded, ventrally armed with 3 plumose long and short setae; pleonal epimeron 2 similar to epimeron 1 in form, twice in largeness, ventrally with 3 plumose setae; pleonal epimeron 3 1.5 times as broad as Fic. 1. Accepted July 3, 1986 Received May 12, 1986 Corophium lobatum n. sp. Holotype: female, 2.16mm. 176 A. HIRAYAMA 0.5mm :A,B,.01,G2 0.2mm;C.D.G1.1,J.K.L.MN.OP.R,S.T O.1mm:E.F.G2,H,1,J1,S1 0.05 mm: P1 Fic. 2. Corophium lobatum n. sp. Holotype: female, 2.16mm. A: Head and antennal. B: Antenna 2. C: Upper lip. D: Lower lip. E: Maxilla 1. F: Maxilla 2. Gl: Right mandible. G2: Left mandible. H: Maxilliped. I and I1: Gnathopod 1. J and J1: Gnathopod 2. K: Coxae 5-7. L: Pereopod 1. M: Coxa 3 and gill. N: Pereoped 2. O: Pleonal epimera 1-3. P and P1: Pleopod 1. Q: Urosome in dorsal view. Q2: Urosome in ventral view. R: Uropod 1. S and $1: Uropod 2 and outer ramus (S1). T: Uropod 3 and telson. Corophium from the Seto Inland Sea 177 epimeron 2, rounded and setaceous posteriorly, without plumose setae. Fused urosome even or slightly concave dorsally, provided with 2 spines on both laterodistal ends, anteroventrally with uro- pod 1 and anterolaterally with uropod 2. Antennae: Antenna 1 about 1/3 as long as body length; segment 1 of peduncle armed with 2 inner-proximal and 1 outer-distal spines; flagellum 6-articulate, each segment excluding proximal and terminal ones provided with 1 aesthetasc. Both antennae 2 broken and only first two segments remained for each; segments 1-2 of penduncle equal in size, inner-distal process of segment 1 almost reaching apex of segment 2, armed with 2 spines, segment 2 inner-distally armed with 2 opposite spines. Mouthparts: Mandibular process of lower lip medium. Inner plate of maxilla 1 rudimentary, lacking setae; outer plate provided with 7 tooth- like, simple spines; proximal segment of palp developed as corophiids, terminal segment provid- ed with 8 apical small setae. Both plates of maxilla 2 equal in size, outer plate provided with 1 plumose seta on outer side of apex. Both mandi- bles similar to each other, provided with 3 acces- sory blades of which the proximal one is bifid; palp biarticulate, terminal segment attached to a slight- ly upper level against middle of the proximal one. Inner plate of maxilliped slender, provided with 4 pinnate setae on inner 1/3 part from apex; outer plate rectangular, reaching 1/3 point from palpal article 2, dispersively setaceous; palp 4-articulate, terminal segment half the length of segment 3, provided with 1 apical slender spine. Gnathopod 1: Coxa 1 extending forward, slen- der, ventrally armed with 1 long pinnate seta. Propod shorter than carpus, palm oblique, armed with 2 spines, posterior margin medially armed with 1 pronounced and 1 smaller spines. Dactyl falcate, extending far beyond palm when closed; grasping margin smooth. Gnathopod 2: Corophi- id-fashion. Carpus about 2/3 as long as merus. Propod almost as long as merus and carpus combined, produced posterodistally. Dactyl half the length of propod, grasping margin armed with 1 pronounced tooth. Pereopods 1-2: Similar to each other. Merus prominently expanded and a little produced ante- rodistally. Carpus shorter than half the posterior length of merus. Dactyl falcate, slender, longer than merus and carpus combined. Pereopods 3-5: Coxae 5-7 similar to each other, subequal in size, armed with 1 posterodistal small seta. Other parts broken. Pleopod 1: Peduncle subsquare, prominetly expanded backward, armed with 1 anterior plu- mose seta; rami unclearly segmented, inner ramus shorter than outer ramus; terminal swimming setae longer than rami. Uropods: Uropod 1 slender, not extending beyond uropod 2; peduncle a little produced outer-distally, provided with 6 outer and 1 inner- distal spines; rami similar to each other, equal in size, 1/3 as long as peduncle, on outer margin armed with 2 spines, bifid apically, inner tooth more produced than the outer one, apical concavi- ties armed with 1 pronounced spine. Uropod 2 lobate, broad, extending beyond peduncle of uropod 3, biramous; peduncle armed with margin- al fringe; rami short, subequal in length, inner ramus slender, provided with 2 apical small setae, outer ramus semi-oval, broad, provided with 10 small, dispersively pinnate setae on outer and apical margins. Uropod 3 uniramous, lobate, shorter than half the length of uropod 2; peduncle broader than long, provided with 3 small pinnate setae on outer margin; rami leaf-like, provided with 14 small setae of which the greater part are pinnate, armed with 2 apical spines. Telson: Very small, semi-oval, without setae. Remarks In the known corophiid amphipods, those spe- cies (Sections B and C [2, 5, 6] with fused urosome as that of the present new species) have longest and best developed uropod 1, and their uropod 2 is rather reduced and attaches to the upper part of the urosome. In contrast to them, the uropod 1 of the present new species is slender and not so developed, and its uropod 2 is well developed and attaches to the base of the urosome. Hence, the new species is clearly distinguishable from the other corophiids with fused urosome by these differences. On the other hand, the present new species has peculiar mandibular palp in which the terminal segment attaches to the middle of its 178 A. HIRAYAMA proximal one; this type has been known in only one species, Corophium crassicorne Bruzelius [1, 3, 5], that belongs to Section C of Crawford’s system. The present new species would be closest to C. crassicorne. Material examined Holotype: Female, 2.16mm. Type locality: Sandy bed in the shallow water of In-no-shima Island, Hiroshima Prefecture, Japan. Date: Aug. 1, 1984. Collector: Nobuo Sawada. Collection No.: Asia Univ. 3. Corophium sinense Zhang, 1974 (Figs. 3-5) Corophium sinense Zhang, 1974 [8], pp. 139-146. Description of Female Body: Depressed. Rostrum small, eyes invisi- ble. Coxae discontinuous in series; pleonal epimera 1-3 gradually broadened at twice in turn, epimeron 3 provided with 1 posteroventral tooth; urosome segmented. Antennae: In antenna 1, segment 1 of peduncle armed with 1 outer-distal spine, subequal to segment 2 in length; each segment of flagellum except for the terminal one provided with 1 aesthetasc. In antenna 2, segment 1 of peduncle provided with 1 bifid process, segments 2-3 lacking spines, segment 5 half the length of segment 4; flagellum biarticulate, terminal segment rudimentary. Mouthparts: Mandibular process of lower lip rudimentary, inner lobe broad. Molar process rugose, provided with 1 short plumose seta; palp biarticulate, terminal segment reduced, directly jointed to the proximal one. Inner plate of maxilla 1 rudimentary, unarmed; outer plate with 6 bifid spines; palp biarticulate, proximal segment very small, apex of terminal segment with 2 opposite rows of 8 spines and 7 stiff setae. Plap of maxilliped 4-articulate, terminal segment much reduced, apically provided with 1 stout, long spine. Gnathopod 1: Coxal1 prominently produced forward, not attenuate, ventrally provided with 3 long plumose setae. Propod subequal to carpus in length; transverse palm a little produced forward, armed with many bifid spines. Dactyl extending beyond palm when closed, grasping margin finely pectinate on middle part. Gnathopod 2: Corophi- id-fashion. Dactyl falcate, nearly reaching the base of propod when closed, grasping margin unarmed with spines and teeth. Pereopods 1-2: Similar to each other. Merus, propod and dactyl subequal in length. Pereopods 3—4: Similar to each other in form, pereopod 4 prominently longer than pereopod 3. Carpus about half the length of merus, extending postero- distally, armed with 1 pair of spines on outer side, 7 or 8 spines on outer-distal end gradually growing in length. Propod of peropod 3 provided with 1 locking spine. Pereopod 5: Broken. Pleopod 1: Peduncle square, setaceous on outer margin; both rami equal in length, proximal segment of inner ramus dilated medially; terminal swimming setae 2/3 as long as rami. Uropods: Uropod 1 extending beyond uro- pods 2-3; peduncle twice as long as inner ramus, 3 stout spines on inner margin gradually making larger, outer margin spinose; both rami spinose, especially inner ramus provided with 3 apical pronounced spines, outer ramus a little longer than inner ramus. Peduncle of uropod 2 a little longer than rami, provided with 1 inner-distal enormous spine; rami equal in length, marginally with many spines (6 or 7). Uropod 3 uniramous, half as long as uropod 2, peduncle as long as wide, dilated distally, both lateral margins armed with 5 spines respectively, outer spines stouter than inner ones; Fic. 3. Corophium sinense Zhang, 1974. Female, 6.83 mm. Corophium from the Seto Inland Sea 179 SS SESE ~ 8 Ai) (7 oS | Ve AG Sara = y V 1.0mm:A 0.5mm :BC.C1LKL.M 0.2mm:DEF.G.HALL1 05mm:J.N 0.2mm:H2,J1 Fig. 4. Corophium sinense Zhang, 1974. Female, 6.83mm. A: Head. B: Antennal. Cand Cl: Antenna 2. D: Upper lip. E: Lower lip. F: Maxilla 1. G: Maxilla 2. H and H1: Right mandible. H2: Left mandi- ble. Iand 11: Maxilliped. JandJ1: Gnathopod 1. K: Gnathopod 2. L: Pereoped 1. M: Pereopod 2. N: Pereopod 3. 180 A. HIRAYAMA 1.0mm:S, 0.5mm:PQR 05mm:0.T,U,V Onin RU Fic. 5. Corophium sinense Zhang, 1974. Female, 6.83mm. O: Pereopod 4. P: Coxae 5-7. Q: Pleonal epimera 1-3. Rand R1: Pleopod 1. S: Urosome. T: Uropod 1. U: Uropod 2. V: Uropod 3 and telson. ramus long elliptical, marginally armed with 10 stiff setae and 1 apical spine. Remarks The present specimens well agree with the female of C. sinense described by Zhang [8] from the Shantung Peninsula, North China, though several differences are observed. The Japanese specimens lack the prominent process that was found on the mid-medial face of the peduncular segment 1 of antenna 1 in Zhang’s material, and the basal spine of the peduncular segment 1 on the antenna 1 found in the present specimens is not found in Zhang’s; moreover, the peduncular seg- ment 4 of antenna 2 is armed with one middle and one inner-distal spines in his specimens, but the present specimens lack them. It seems to me that these differences in the antennae 1-2 are mere local variation. As far as Zhang’s figure and description are seen, the maxilliped palp of his material lacks one apical stout spine. He may have failed in observing the spine because it is masked by many setae. One more difference, though minor, is found in the pereopods: The carpus of peropods 1-2 of his material is longer than the propod, but the two segments are subequal in length in the present specimens. Although I have not yet examined any specimen of the male of this species, Zhang [8] clearly demonstrated the presence of sexual dimorphism in the antenna 2 for his material from China. The combination of this sexual dimorphism and the segmented urosome is a characteristic of the member of Section A-1 of Crawford’s system [2, 5, 6]. In addition to this character combination, this species has noticeable characteristics that the terminal segment of its mandibular palp is reduced instead of equal or subequal to the proximal one in length and attaches to the apex of the proximal one. The former character is known only in this species up to date, but the latter is known in C. arenarium Crawford, 1937 [2] and C. orientale Schellenberg, 1928 [4, 7], both of which belong to the same Section A-1 of Crawford’s. C. sinense | would be related to C. arenarium and C. orientale. Corophium from the Seto Inland Sea 181 Material examined Two females: One described and figured, 6.83 mm. Locality: Sandy-mud bed in the shallow water of In-no-shima Island, Hiroshima Prefec- ture, Japan. Date: Aug. 1 to 2, 1984. Collector: Nobuo Sawada. Collection No.: Asia Univ. 2. ACKNOWLEDGMENTS I wish to thank Mr. Nobuo Sawada of the Marine Ecological Research Co., Ltd., Osaka, for giving me the opportunity to examine the present materials and his personal communication with the figures. Thanks are also due to Dr. E. L. Bousfield of the National Museum of Canada for his critical reading of the manuscript and his comments. Further, I sincerely thank Dr. Yoshihide Suzuki of the Biological Laboratory, Asia University, for providing me with working facilities and for his critical comments on the manuscript. REFERENCES 1 Bousfield, E.L. (1973) Shallow-water gammaridean 2 Amphipoda of New England, Cornel Univ. Press, Ithaca, New. York. p. 312. Crawford, G.I. (1937) A review of the amphipod genus Corophium, with notes on the British species. J. Mar. Biol. Assoc. U. K., 21: 589-630. Hirayama, A. (1984) Taxonomic studies on the shal- low water gammaridean Amphipoda of West Kyushu, Japan. II. Corophiidae. Publ. Seto Mar. Biol. Lab., 29: 1-29. Schellenberg, A. (1928) Amphipoda in Cambridge Expedition to Suez Canal. Trans. Zool. Soc. London, 22: 638-692. Shoemaker, C.R. (1947) Further notes on the amphipod genus Corophium from the east coast of America. J. Wash. Acad. Sci., 37: 47-63. Shoemaker, C.R. (1949) The amphipod genus Corophium on the west coast of America. J. Wash. Acad. Sci., 39: 66-82. Stock, J.H. (1960) Corophium volutator forma orientalis Schellenberg, raised to specific rank. Crus- taceana, 3: 188-192. Zhang, W. (1974) A new species of the genus Corophium (Crustacea, Amphipoda, Gammaridea) from the southern coast of Shantung Peninsula, North China. Stud. Mar. Scinica, 9: 139-146. 4s 2 wuld joe > bela woth to apoquicane SIE ee, PMY sees}, beatin ent trageives Hh CORRE) BAD ‘Khatwitd ate vist Absent fo rotor Bio -ORe itis py. jist ld aC) eanwtH” FET rae a Haw U5 cial Rpts ties pas tees bts seh Ise, hS adie? , ree eee See et - -snrmevant wise wol seal sii? shactona> PPCSAER TS toe, aap om sonibaqrst ney ale othe xxi mayer hea ‘agp Pee ai trad rycen a biociacqinth Pie Ee evade W A ese i pee BEE np EOL) a. ee ‘FG teat Jeow —— ne Ane SEND ey ie 0085 ys ie ae > cgcigmgast® te tenieo tens-- ened i a gristEyyVO.9 af bseisn {OUR THUY aithyace arto eS .. $e war uit (eUeL) We gered a uber j } grasione 4% moaitieeos sf} anal but? .sathl ah a3 2 aay Pao e eels ithe yh (ealizerrETs y tr) REEVE od ae be 1S He Tei : path LL IEEERS f r =f} for hee we oe elie x | *" , q 2 ¥) i ee | e a ly Fe re e : +4! i nT vn 4 be OTE Par =fk, ral Veer peer} saa by e Bae. tas 0 arraialiidy 3? .wietrteh Br LEM bee I SERN ES e- | ' ce 4 AGA - MOIAERTIGRISO ets % Ue Ob) to age MIGTS Fo Bee aa rg es ix ant’ bie HS wie ce Sirs 0 Saray, eh eer a Sa ao eT aT Oo aermcreny, LER i tn ; be vei ak Ps a 3% ibe ate als eesa ae Mes .¥ i A $585 tw y = , ae ee ha » senisg repre. stoned Sete ‘avaainialt iiereren. 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Eooet apart Se eer ti: rte ZOOLOGICAL SCIENCE 4: 201-204 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Prolactin Cells of Clarias batrachus in Response to Corpuscles of Stannius Extract Administration SHYAM P. SRIVASTAV, KRISHNA Swarup! and Asal K. SRIVASTAV Department of Zoology, University of Gorakhpur, Gorakhpur 273 009, India ABSTRACT—Corpuscles of Stannius extract adminis- tration increased the activity of prolactin cells. INTRODUCTION Removal of corpuscles of Stannius (CS) causes a rise in serum calcium level [1—4] which is corrected by administration of CS extract [5-8]. This suggests that CS secrete hypocalcemic principle (s). So far, two hypocalcemic principles—‘hypocal- cin’ [9] and ‘teleocalcin’ [10] have been reported from the CS. Recently, a PTH-like substance (parathyrin) has been localized immunocytochemi- cally in the eel CS [11]. Extracts of CS have been found to be effective in inducing hypocalcemia in mammal [12], bird [13], amphibians [14] and intact fishes [2, 6, 9, 15, 16]. Induced hypocalcemia is met with an increased activity of parathyroid gland in tetrapods [17-19 ]. But in fishes, parathyroid gland is absent and in this group prolactin has specific hypercalcemic capacities [20-23]. There exists no earlier report regarding the response of administration of CS extract on prolactin cells in fish. In the present study, therefore, we have attempted to study such a response in a freshwater catfish, Clarias bat- rachus. MATERIALS AND METHODS Sixty adult male specimens of Clarias batrachus Accepted September 22, 1986 Received May 19, 1986 " To whom correspondence should be sent. (23-27 cm/80—120 g) were collected locally during the first week of September and acclimatized to the laboratory conditions (temp. 23-25°C) for two weeks prior to use. They were then divided into two numerically equal groups—A and B. Group A: Control fish received intraperitoneally 1ml/100g body weight of saline (0.6% NaCl solution; physiological saline) and were kept in tap water. Group B: Experimental fish received intraperi- toneally CS extract in a dosage of 5mg/ml/100¢g body weight and were maintained in tap water. The CS used in this study were surgically removed from both sexes of adult Clarias bat- rachus (September—October is post-spawning period; in females during September the ovary displays large number of immature oocytes and unovulated mature oocytes in the process of resorption whereas in males the lobules show compactness, the interlobular septa become thick with numerous blood cells and the germ cells which are inactive during the period of functional maturity begin to divide). The glands were stored in ice and used almost immediately. Prior to use, the glands were weighed (weight of CS of average fish, 0.6mg) and homogenized in ice-cold saline (0.6% NaCl solution). The homogenate was centrifuged for 10 min at 5,000 rpm and the super- natant was collected. Final volume of the super- natant was made up so that every 1ml solution contained extract from 5 mg of CS. Six specimens from each group were anaesthe- tized with MS 222 at 1/2, 1, 2, 4 and 6hr following the injection and the blood samples were collected 202 S. P. SRIVASTAV, K. SWARUP AND A. K. SRIVASTAV by the sectioning of caudal peduncle. An initial sampling of blood (from 6 fish kept separately under similar conditions) was also taken before the start of the experiment (Zero hour). After clotting of the blood the sera were separated by centrifuga- tion at 3,500 rpm and analysed for serum calcium level according to Trinder’s [24] method. Pituita- ries were fixed in aqueous Bouin’s fluid and Bouin-Hollande fixative. After routine processing in graded series of alcohol and clearing in xylene, tissues were embedded in paraffin wax. Serial sections were cut at 4-6 wm and stained according to Herlant tetrachrome and Heidenhain’s azan techniques. The nuclear diameter was measured with the aid of ocular micrometer. Each nucleus was measured along its long and short axes and mean value was calculated. From each group 300 nuclei were measured at every interval (Fifty nuclei were measured from each fish). The data are presented as mean+standard deviation of the mean. Differences in the serum calcium level and nuclear diameter among different groups were analysed by Student’s t-test. RESULTS The serum calcium level, before the start of the experiment (Zero hour), was recorded as 9.77+ 0.42 mg/100 ml. In C. batrachus, CS extract administration evoked hypocalcemia after 1/2 hr which continued up to 1 hr. Thereafter, the serum calcium level increased progressively reaching the normal value after 4 and 6 hr of the initiation of the experiment (Fig. 1). The prolactin cells of fish from group A (saline injected) showed no change in response to the treatment. The prolactin cells possessed indistinct boundaries, the nuclei, however, were distinct. The cytoplasm was noticed granular and azocarmi- nophilic and erythrosinophilic in nature (Fig. 2). The first perceivable change in the prolactin cells of specimens of group B (CS extract-treated) was observed at 2hr as the gland exhibited degranula- tion and increase in nuclear size (Fig. 1). These changes were pronounced and the nuclei became hyperchromatic after 4 hr of the treatment (Fig. 3). O----0 Saline injected Taw ES Graces Ihioetad 12.0 IL, sh Se tn ae. b c £ =) iia ‘ Ges (o>) SQ Im RE D0 og wa Yo vw ou sé Q = OG 2o reer} >) Zane HOURS Fic. 1. Serum calcium level and nuclear diameter of prolactin cells after CS extract administration. a, b, c and d indicate significant differences compared with matching controls, P<0.05, P<0.02, P<0.01 and P<0.001, respectively. Six hours after the initiation of the experiment, the prolactin cells showed recuperation and the nuclei had a tendency towards reduction in their size (Figs. 1 and 4). DISCUSSION In tetrapods, where parathyroids are the hyper- calcemic glands, there is an increase in nuclear size to meet the induced hypocalcemia [17-19]. In C. batrachus, CS extract induced hypocalcemia ren- ders prolactin cells to undergo similar changes (degranulation, increase in nuclear size and chro- maticity i. e. increased synthesis and release of the hypercalcemic factor) at hour 2 which is maximum at hour 4. This is evidently to meet the hypocalce- mic challenge and to restore the serum calcium Effects of CS Extract on Fish Prolactin Cells 203 Fic. 2. Prolactin cells of saline-injected C. batrachus showing granular cytoplasm and well marked nuclei. Herlant tetrachrome. 900. Fic. 3. Prolactin cells of C. batrachus showing hyper- chromatic nuclei and degranulation after 4hr of CS extract administration. Herlant tetrachrome. X 900. Fic. 4. Prolactin cells of C. batrachus showing recu- peration of secretory granules after 6hr of CS ex- tract administration. Herlant tetrachrome. 900. level. Obviously, when the serum calcium level reaches normocalcemia at hour 6, the activity of these cells retards. These observations lend sup- port to the suggestion of Pang et al. [7]‘---the hypocalcemia caused by treatment with Stannius corpuscle homogenate might elicit the release of prolactin”. The present data suggest that calcium has a role in the regulation of prolactin secretion. Several reports agree with the present results showing that calcium levels in the blood and/or the environment are the main factors regulating the activity of prolactin cells [25-27]. Contrary to it, Olivereau and Olivereau [28] have suggested that sodium, and not calcium, plays a major role in the regulation of prolactin secretion. The reduced role of calcium in the regulation of prolactin cells has also been advocated by Ball et al. [29] and Olivereau et al. [30]. It has also been shown that the amount of prolactin released in vitro, by theTilapia pituitary is not affected by the calcium concentration [31] but it is controlled by the osmotic pressure of the incubation medium [32]. ACKNOWLEDGMENT The authors are thankful to M/S Sandoz Ltd., Basel for generous gift of MS 222 and UGC, New Delhi for financial assistance. REFERENCES 1 Fontaine, M. (1964) C. R.Acad. Sci., 259: 875-878. Pang, P. K. T. (1971) J. Exp. Zool., 178: 1-8. 3.0 Pane eo Keb Pang ROK: and . Sawyer, WH: (1973) Endocrinology, 93: 705—710. 4 Fenwick, J.C. (1976) Gen. Comp. Endocrinol., 29: 383-387. 5 Chan, D. K. O., Rankin, J. C. and Chester Jones, I. (1969) Gen. Comp. Endocrinol., Suppl., 2: 342-353. 6 Lopez, E. (1970). Z. Zellforsch. Mikrosk. Anat., 109: 566-672. 7 ‘Pang. Pa ket banee ROK -and) ‘(Gnihith R- W- (1975) Gen. Comp. Endocrinol., 26: 179-185. 8 Kenyon, C.J., Chester Jones, I. and Dixon, R.N. B. (1980) Gen. Comp. Endocrinol., 41: 531-538. 9 Pang, P.K.T.,. Pang,R.K. and Sawyer, W. H. (1974) Endocrinology, 94: 548-555. 10 Ma, S.W. Y. and Copp, D. H. (1978) In “Compa- tative Endocrinology”. Ed. by P. J. . Gaillard and H.H. Boer, Elsevier/North Holland Biomedical Press, Amsterdam, pp. 283-286. 11 Lopez, E., Tisserand-Jochem, E.M., Eyquem, A., Milet, C., Hillyard, C., Lallier, F., Vidal, B. and 12 13 14 15 16 17 18 19 20 21 Vip 204 MacLotyre, I. (1984) Gen. Comp. Endocrinol., 53: 28-36. Leung, E. and Fenwick, J. C. (1978) Can. J. Zool., 56: 2333-2335. Srivastav, A. K. and Swarup, K. (1982) Experi- entia, 38: 869-870. Pandey, A.K., Krishna, L., Srivastav, A. K. and Swarup, K. (1982)Experientia, 38: 1314-1315. Swarup, K. and _ Srivastav, S. P. (1982) J. Adv. Zool., 3: 62-66. Ogawa, H. and Sokabe, H. (1982) Gen. Comp. Endocrinol., 47: 36—41. Swarup, K. and_ Srivastav, A. K. (1981) Molec. Biol., 27: 287-290. Swarup, K. and Srivastav, A. K. and Tewari, N. P. (1979) Gen. Comp. Endocrinol., 37: 541-545. Krishna, L. and Swarup, K. (1985) Herpetologica, 41: 65-70. Pang, P.K.T., Schreibman, M.P., Balbontin, F. and Pang, R. K. (1978) Gen. Comp. Endocrinol., 36: 306-316. Olivereau, M. and Olivereau, J. (1978) Cell Tissue Res., 186: 81-96. Wendelaar Bonga, S. E., Grevan, J. A. A. Cell. and 23 24 25 26 27 28 29 30 31 32 S. P. SRIVASTAV, K. SWARUP AND A. K. SrIVASTAV Mein, C. (1978) Gen. .Comp. Endocrinol., 34: 91. Wendelaar Bonga, S. E. and Van der Meij, J. G. A. (1980) Gen. Comp. Endocrinol., 40: 342. Trinder, P. (1960) Analyst, 85: 889-894. Wendelaar Bonga, S. E. (1978) Gen. Comp. En- docrinol., 34: 265-275. Wendelaar Bonga, S.E. (1978) In “Comparative Endocrinology”. Ed. by P. J. Gaillard and H. H. Boer, Elsevier/North Holland, Amsterdam, pp. 259-262. Wendelaar Bonga,S.E. and Van der Meij, J. C. A. (1980) Gen. Comp. Endocrinol., 40: 391-401. Olivereau, M. and Olivereau, J. (1983) Cell Tissue Res., 229: 243-252. Ball, J.N., Uchiyama,M. and _ Pang, P.K.T. (1982) Gen. Comp. Endocrinol., 46: 480—485. Olivereau, M., Olivereau, J. M., Aimar, C., Cham- bolle, P. and Dubourg, P. (1983) Gen. Comp. En- docrinol., 52: 51—5S. Grau, E.G., Nishioka, R.S. (1979) Am. Zool., 19: 875. Grau, E.G., Nishioka, R.S. and Bern, H. A. (1981) Gen. Comp. Endocrinol., 45: 406—408. and Bern, H.A. 205 INSTRUCTIONS TO AUTHORS ZOOLOCIGAL SCIENCE publishes contri- butions, written in English, in the form of (1) Reviews, (2) Articles, and (3) Communications of material requiring prompt publication. A Review is usually invited by the Editors. Those who submit reviews should consult with the Editor-in- Chief or the Managing Editor in advance. Articles of less than 6 printed pages and communications less than 3 printed pages will be published free of charge. Charges will be made for extra pages (7,000 yen/page). A Communication cannot ex- ceed 4 printed pages. 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Bak bes, ‘ WK A a ays Ee PW a : ee tees Ot ee. iy La ' : ‘ a” ’ tal - - 7 tat i 74 3 Ls j eee saa 7 ‘ * oo i Late he lt ewes reo nash ey te ee Cert , ; Dtel y ' - * : t< {7 oi 7 mi Fah LR Tr Be Ue ye Os | he eo ; et FEES Faq 5 fT: = ‘ ‘ f . 7 rf 4 ty 14 fone yous py ai a a ‘ey See piy iy bys roRS «ee praki* Ee 5 ‘ . : 5 ie pee oe ai ae re : 3 ‘ fib $e: Are ays i ' i dues ised ss el i payee hE Le I TRA ee r » #3 Pd fs ii wes ; : ox % t 4 ‘ J » RD | “ad 4 j 1 ) i % ve }f% oy 1 s ’ ” ths a A tf me ¢ v Th ie ai a en i i ae i RG aT eet cae 22 We Ove E uf eon i PY Aye ee aa ie heat aiv4 4 ' ¥ ive) | k f y ae. ; ey 2 tee: + ‘ A LA at { i c = iJ ee : 2a) ? : rey 4 a7 ti - : or ebiw On lores ae, scotia ¥ ra% yeh } ‘ Aree , * a. + p J Rep} . ipl c i he ‘ Published by the Japanese Society of Developmental Biologists The journal is devoted to the publication of original papers dealing with any aspects of developmental phenomena in all kinds of organisms, including plants and micro-organisms. Papers in any of the following fields will be considered: developmental genetics, growth, morphogenesis, cellular kinetics, fertilization, cell division, dormancy, germination, metamorphosis, regeneration and pathogenesis, at the biochemical, biophysical and analytically morphological levels; reports on techniques applicable to the above fields. At times reviews on subjects selected by the editors will be published. Brief complete papers will be accepted, but not preliminary reports. Members of the Society receive the Journal free of charge. Subscription by institutions is also welcome. Forthcoming Papers in DGD, Vol. 29, No. 1. REVIEW: Y. NAGAHAMA: 17a, 208-dihydroxy-4-pregnen-3-one: 6 K. MrrsunaGa, Y. Fustno and I. Yasumasu: Probable role of A teleost maturation-inducing hormone allylisothiocyanate-sensitive H*, K*-ATPase in spicule calcifica- A. Fustwara, K. Asami and I. YaAsumasu: Induction of fertiliza- tion in embryos of the sea urchin, Hemicentrotus pulcherrimus tion membrane formation and cyanide-insensitive respiration in| 7. 4 Kamar and T. Ontaki: Pattern regulation of the leg disc of sea urchin eggs by the treatment with dimethylsulfoxide followed the fleshfly, Sarcophaga peregrina 2) Eo Geioaulon on gin wee [pele 8. S. YasuGi, K. Hayasui, K. TakiGucui, T. Mizuno, M. Mocun, J. H. Quertier, E. Battus and J. Bracuer: Cytological effects R. Kopama, K. Acata and G.Ecucui: Immunological rela- of heat-shocks on Xenopus oocytes and eggs tionships among embryonic and adult chicken pepsinogens: A D. I. pe Pomerat and A. Carr: Heat shock may relieve a post- study with monoclonal and polyclonal antibodies transcriptional block on 6 crystallin synthesis in cultures of chick 9 A. Pycarp, J.-C. LaBBE, G. PEAUCELLIER, F. LE BOUFFANT, C. LE embryo neuroretinal cells K. IsHipA, M. Oxuno, S. Morisawa, T. Monri, H. Mouri, M. Waku and M. Morisawa: Initiation of sperm motility induced by cyclic AMP in hamster and boar PeucH and M. Doree: Changes in the activity of the matura- tion-promoting factor are correlated with those of a major cyclic AMP and calcium-independent protein kinase during the first mitotic cell cycles in the early starfish embryo Development, Growth and Differentiation (ISSN 0012-1592) is published bimonthly by The Japanese Society of Developmental Biologists, Department of Biology, School of Education, Waseda University, Tokyo 160, Japan. 1987: Volume 29. Annual subscription U. S. $ 110.00 including air speed delivery except Japan. Application to mail at second class postage rate is pending at Jamaica, IN 1431 U.S.A. Outside Japan: Send subscription orders and notices of change of address to Academic Press, Inc., Journal Subscription Fulfillment Department, 6277 Sea Harbor Drive, Orlando, FL 32887, U. S. A. Send notices of change of address at least 6-8 weeks in advance. 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There is no need for a bulky stand. * Hydraulic remote control ensures totally vibration-free operation. * 3-D movements achieved with a single joystick. Micromanipulators Microelectrode pullers Stereotaxic instruments NARISHIGE SCIENTIFIC INSTRUMENT \ ees / LABORATORY CO., LTD. 4-9-28, Kasuya, Setagaya-ku, Tokyo 157 JAPAN Telephone: 03-308-8233 Telex: NARISHG J27781 (Contents continued from back cover) innervation, persistence and effects of juve- nile hormone on the prothoracic glands in adult Blattella germanica (L.) (Dictyoptera, Blattellidae) Fukuda, M., Y. Nakano, K. Yamanouchi, Y. Arai and H. Furuya: Suppressive effect of right-side anterior hypothalamic lesion on Ovarian compensatory hypertrophy in rats (COMMUNICATION) Srivastav, S.P., K. Swarup and A. K. Srivas- tav: Prolactin cells of Clarias batrachus in response to corpuscles of Stannius extract administration (COMMUNICATION) Behavior Biology Machida, T., S. Iso and T. Noumura: Long- lasting effects of orchiectomy and its preced- ing procedures on open field behavior in male mice Taxonomy Hasumi, M. and H. Iwasawa: Geographic variation in the tail of the Japanese salaman- der, Hynobius lichenatus, with special refer- ence to taxonomic bearing Hihara, F. and H. Kurokawa: The sperm length and the internal reproductive organs of Drosophila with special references to phy- logenetic relationships Hirayama, A.: Two peculiar species of corophiid amphipods (Crustacea) from the Seto Inland Sea, Japan Sawada, I. and G. Kugi: Three new species of avian dilepidid cestodes from Oita Prefec- ture, Japan Instructions to Authors ZOOLOGICAL SCIENCE VOLUME 4 NUMBER 1 FEBRUARY 1987 CONTENTS REVIEWS Katagiri, Ch.: Role of oviducal secretions in mediating gamete fusion in anuran amphibians. ....0.2. a8 Wee: + Vee eee Matsuno, A.: Ultrastructural classification of smooth muscle cells in invertebrates and vertebrates oe ORIGINAL PAPERS Physiology Yamashita, S. and R. Tuji: Phototactic be- havior of the orb weaving spiders, Argiope amoena and Nephila clavata............. Yamashita, S.: Dimming reaction of the orb weaving spider, Argiope amoena ......... Ando, M.: Regulation by intracellular ala- nine of water transport across the seawater Celintes iinet set a. Sees oie seo etree ae Ando, M., Y. Furukawa and M. Kobayashi: Comparison of seawater and fresh-water eels for the effects of L-alanine on water transport across the intestine Ble G10) 10: (e/e1e, 6: (eel 10. @| 1@)0)¢\.0: vis 18) elle le Cell Biology Matsuno, A.: Ultrastructural studies on de- veloping oblique-striated muscle cells in the cuttlefish, Sepiella japonica Sasaki ....... Iwasa, F., Y. Hasegawa, S. Ishijima, M. Oku- no, T. Mohri and H. Mohri: Effects of cal- modulin antagonists on motility and acro- some reaction of sea urchin sperm ....... Takagi, Y., T. Suzuki and C. Shimada: _Isola- tion of a Paramecium tetraurelia mutant with short clonal life-span and with novel life- cycle features eee eer ece ee eee see eee ee ee eee ee Genetics Kohno, S., M.Kuro-o, C. Ikebe, R. Kata- kura, Y. Izumisawa, T. Yamamoto, H. Y. Lee and S. Y. Yang: Banding karyotype of Korean salamander: Hynobius Boulenger leechii Obara, Y.: Karyological differentiation be- tween two species of mustelids, Mustela 15 Vas, 31 3 45 53 61 Ws 81 erminea nippon and Meles meles anakuma wo elila sae fva@@ougowe ness eee ao 87 Immunology Ahmad, R. A., B. L. James and A. B. Kamis: Acquired resistance against Microphallus pygmaeus in the laboratory mouse ....... 95 Biochemistry Okai, Y., S. Ishizaka and U. Yamashita: A DNA synthesis inhibitory peptide from hu- man embryo fibroblasts—characterization of biological properties” .. 32... sep eeeeee 99 Kobayashi, K. and S. Horiuchi: Myofibril de- gradation by tail lysosomes from metamor- phosing bullfrog tadpoles. 2.eesyaseeeer 107 Developmental Biology Kato, S. and K. Kurihara: The intracellular supporting network in the Leydig cells of larval salamander skin (COMMUNICA- TION) Yoshizaki, N.: Isolation of spermatozoa, their ultrastructure, and their fertilizing capacity in two frogs, Rana japonica and Xenopus laevis (COMMUNICATION) Endocrinology Tanaka, S., M. Hattori and K. Wakabayashi: Steroidogenic activity of isoelectric gonado- tropin components in the pituitary of adult male newt, Cynops_ pyrrhogaster pyr- rhogaster Cailliez,D., J.-M. Danger, A.C. Andersen, J. M. Polak, G. Pelletier, K. Kawamura, S. Kikuyama and H. Vaudry: Neuropeptide Y (NPY)-like immunoreactive neurons in the brain and pituitary of the amphibian Rana catesbeiana Honda, H., T. Oishi and T. Konishi: Com- parison of reproductive activities between two Japanese quail lines selected with regard to photoperiodic gonadal response Hazarika, L.K. and A. P. Gupta: Structure, (Contents continued on inside back cover) Issued on February 15 Printed by Daigaku Printing Co., Ltd., Hiroshima, Japan / OLOGICAL EIS: “An International Journal _ ZOOLOGICAL SCIENCE The official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Hideshi Kobayashi (Tokyo) Toshin-building, Hongo 2-27-2, Bunkyo-ku, Managing Editor: — eases Tokyo 113, Japan. Tel. (03) 814-5675 Seiichiro Kawashima (Hiroshima) @ificers Assistant IOUS: ibe: President: Nobuo Egami (Tsukuba) Teco Machida (Hiroshima) Secretary: Yasuto Tonegawa (Urawa) Sumio Takahashi (Hiroshima) Treasurer: Tadakazu Ohoka (Tokyo) Librarian: Shun-Ichi Véno (Tokyo) Editorial Board: Howard A. Bern (Berkeley) Walter Bock (New York) Aubrey Gorbman (Seattle) Horst Grunz (Essen) Robert B. 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New subscriptions and renewals begin with the first issue of the current volume. All rights reserved. No part of this publication may be reproduced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1987, The Zoological Society of Japan Publication of Zoological Science has been supported in part by a Grant-in-Aid for Scientific Publication from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 4: 209-222 (1987) © 1987 Zoological Society of Japan REVIEW Gonadotropin Action on Gametogenesis and Steroidogenesis in Teleost Gonads YOSHITAKA NAGAHAMA Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444, Japan INTRODUCTION The primary role of pituitary gonadotropin in the physiological regulation of gonadal function is well established. In most cases, however, gonado- tropin action on gonadal development is not direct, but through the biosynthesis of gonadal steroid hormones which in turn mediate various stages of gametogenesis including oocyte growth (vitellogenesis), oocyte maturation, spermatogen- esis and spermiation. Surprisingly little is known about the endocrine details of gonadal ster- oidogenesis in lower vertebrates. Over the past several years, a series of studies in our laboratory using several species of teleosts as experimental animals has provided important new information about the endocrine control of oocyte maturation in females and spermiation in males, and the interactions of gonadotropin with ovarian and testicular cell types and the mechanisms of its action. This article briefly reviews the endocrine control of vitellogenesis, oocyte maturation and spermiation in teleosts, and describes our recent data, mainly based on the amago salmon, Oncorhynchus rhodurus, on the mechanism by which gonadotropin exerts its action on the pro- duction of two major naturally occurring steroid hormones in salmonid reproduction, estradiol-178 and 17a, 208-dihydroxy-4-pregnen-3-one (17a, 208-diOHprog) (Fig. 1). Received December 23, 1986 Cholesterol ' Pregnenolone > Progesterone | CH, CH, 9 ee) HO-C-H 2 208 -HSD 3 ny = O NZ oO 5 17a—Hydroxyprogesterone — 17a,20B-Dihydroxy- =. CG anlivase mis= | 4-pregnen-3-one 5 Androstenedione 17B-HSD C> | me OH S ® ah Aromatase ash > Q V ® S HO = Testosterone —— Estradiol-17B 2. Fic. 1. Pathway of steroid biosynthesis in the ovary of salmonids. 178-HSD, 17-hydroxysteroid dehy- drogenase; 208-HSD, 20-hydroxysteroid dehy- drogenase. GONADOTROPIN It is well established that in teleosts, as in other vertebrates, pituitary gonadotropins are the major hormones which stimulate various ovarian activi- ties. Although a number of biochemical studies have been conducted to purify piscine gonadotro- pins, the question of whether teleosts possess one or two types of gonadotropins in the pituitary gland is controversial at present. A glycoprotein- rich gonadotropin with a molecular weight of 25,000-40,000 (glycoprotein-rich “maturational” gonadotropin) has been purified in several teleosts [1,2]. This type of gonadotropin has been reported to stimulate almost all gonadal activities including gametogenesis and steroidogenesis. We 210 Y. NAGAHAMA purified a similar gonadotropin from the chum salmon, Oncorhynchus keta, pituitaries [3]. The molecular weight of this gonadotropin was esti- mated to be 38,000-—40,000 by gel filtration, and that of the two distinct subunits, designated a and B, to be about 15,000 and 20,000 by SDS- polyacrylamide disc gel electrophoresis. The biological nature and peptide structure of this gonadotropin are very similar to those of other piscine glycoprotein gonadotropins. There is now biochemical evidence that teleosts, similar to other higher vertebrates, probably pos- sess two gonadotropins. Idler and his colleagues [2], who used affinity chromatography on Con A-Sepharose as one of the purification procedures, have isolated two gonadotropins, carbohydrate- rich “maturational” gonadotropin and carbohy- drate-poor “vitellogenic” gonadotropin in several teleosts including chum salmon. This second gonadotropin has been reported to be responsible for the uptake of vitellogenin into the oocyte, although glycoprotein-rich gonadotropin itself has also been shown to mediate the uptake of the vitellogenin. More recently, two distinct carbohy- drate gonadotropins have been identified, for the first time in any teleost, in chum salmon [4]. Physicochemical characterization of these two gonadotropins revealed that each consisted of two subunits. It is unknown, however, whether these two carbohydrate gonadotropins have different roles in the regulation of gonadal function in this Hypothalamus Gonadotropin ‘ Gonadotropin v Follicle layer ‘ Estradiol-17 B A QS ( (?) : 0 Liver ) | | a HEE ’ 4 Vitellogenin et s OS ®eoae Fic. 2. Hormonal regulation of vitellogenesis in tele- osts. (See the text for details). species. Further data on the possibility of more than one gonadotropins in teleosts come from a number of morphological and histochemical stu- dies which have shown that in some species at least, there are two distinct gonadotropin-secreting cells in the pituitary gland [5]. In this paper, the term gonadotropin is taken to refer to the gly- coprotein-rich “maturational” gonadotropin. HORMONAL REGULATION OF GAMETOGENESIS A. Oocyte growth After oogonia undergo proliferation by mitotic divisions, they become oocytes and enter a period of growth. During growth, the increase in oocyte size is very considerable. For example, in salmo- nids a young oocyte may be about 50m in diameter, and the fully developed egg is between 3.0mm and 5.0mm in diameter. Simultaneously with the growth the oocyte nucleus goes through the early stages of prophase but is arrested in diplotene stage of the first meiotic division. The increase in oocyte size is due mainly to the accumulation of yolk proteins [6]. In teleosts, as in most other vertebrates, the site of synthesis of the precursor protein of yolk, vitellogenin, is the liver. A model of hormonal regulation of vitellogenesis in teleosts is provided in Figure 2. Estrogen, estradiol-17 in most cases, produced by the ovary under the influence of gonadotropin, is introduced o——o Estradiol-178 (ng/ml) 17a ,20B-Dihydroxy—4—pregnen-3-one (ng/ml) June 20 July 25 Aug 27 Sept. 26 Oct.9 Oct. 24 Fic. 3. Changes in plasma estradiol-172 and 17a, 208- dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon. Gonadotropin Action on Steroidogenesis 211 into the vascular system and stimulates the hepatic synthesis and secretion of vitellogenin. Vitel- logenin is then selectively taken up from the bloodstream by developing oocytes. In all of the species studied so far, plasma concentrations of estradiol-17 have been reported to be high during the main vitellogenic growth period of the ovarian cycle [7,8] (Fig. 3). It has been suggested that gonadotropin is involved in the process of incor- poration of vitellogenin into the oocyte [9-11]. However, the endocrine details of this process are unknown and need to be investigated at the molecular level. B. Oocyte maturation Postvitellogenic oocytes are still physiologically immature in that they cannot be fertilized. For the oocytes to be fertilized they must undergo final oocyte maturation. This process is a prerequisite for successful fertilization and consists of the breakdown of the germinal vesicle (GVBD), chromosome condensation and extrusion of the first polar body (Fig.4A,B,C). It is generally accepted that in lower vertebrates three major Fic. 4. Scanning electron micrographs of the fractured surface of rainbow trout oocytes during salmon gonadotro- pin-induced germinal vesicle breakdown in vitro. A, A fully grown immature oocyte with the germinal vesicle (GV) located halfway between the center and the oocyte periphery. F, ovarian follicle. B, An oocyte 24 hr after the onset of incubation with salmon gonadotropin. The germinal vesicle (GV) is located at the oocyte periphery. C, An oocyte 72 hr after the onset of incubation with salmon gonadotropin. Bar represents 100 ~m. D, Scanning electron micrograph of the fractured surface of a rainbow trout ovarian follicle consisting of thecal layers (T), granulosa layers (G) and the zona radiata (Z). Bar represents 54m. E, Scanning electron micrograph of a granulosa layer preparation, consisting purely of granulosa cells. Z, zona radiata. 212 mediators, gonadotropin, maturation-inducing hormone and maturation-promoting factor, are involved in the induction of oocyte maturation [12-16]. These mediators function sequentially at the levels of the follicle layer, the oocyte surface and the oocyte cytoplasm (Fig.5). The one exception to the intraovarian control of oocyte maturation in lower vertebrates appears to occur in the catfish, Heteropneustes fossilis. In this case, gonadotropin seems to stimulate the interrenal tissue to produce corticosteroids which in turn act on the oocytes to induce maturation [17]. It is now well established that in teleosts, the preovulatory surge of gonadotropin triggers a cascading series of biochemical events which lead to final oocyte maturation. An increase in gona- dotropin levels during oocyte maturation and ovulation has been reported in several teleosts [2]. Follicle-enclosed full-grown postvitellogenic oocytes of some teleosts undergo GVBD in vitro when they are incubated with gonadotropin. However, denuded oocytes are incapable of responding to gonadotropin [16]. Cyanoketone, a specific inhibitor of 3-hydroxy-A°-steroid dehy- drogenase, completely abolishes the maturational effects of gonadotropin and pregnenolone, but not of steroids such as progesterone or 17a, 20/- diOHprog [18]. Thus, the action of gonadotropin in inducing oocyte maturation appears to be Pituitary Gonadotropin y Maturation-inducing hormone Follicle layer v Oocyte Maturation—promoting factor Ovary Oocyte maturation Fic.5. Hormonal regulation of oocyte maturation in lower vertebrates. (See the text for details). Y. NAGAHAMA dependent on the synthesis of a secondary steroid- al effector, maturation-inducing hormone. In several teleosts, a variety of C21 steroids such as progesterone, 17a-hydroxyprogesterone, 17a, 206-diOHprog, cortisol and deoxycorticosterone have been demonstrated to be effective in inducing meiotic maturation in vitro [16, 19-21]; among them, 17a, 208-diOHprog has been reported to be the most effective steroid. C19 steroids, especially testosterone, have been shown to induce GVBD only at high concentrations. C18 steroids including estradiol-17, are generally not effective in induc- ing oocyte maturation in teleost oocytes. There have been no studies on the purification and characterization of the naturally occurring maturation-inducing hormone in any vertebrate. In vitro data described above strongly suggest that C21 steroids are responsible for the induction of oocyte maturation in teleosts. Recently, we purified and characterized the maturation-inducing hormone of amago salmon from media in which immature but fully grown folliculated oocytes of amago salmon had been incubated for 18-24 hr with partially purified chum salmon gonadotropin (SGA, Syndel Lab., Canada) [22]. Twenty separate fractions were obtained by the use of reversed-phase high performance liquid chromatography and their maturation-inducing activity was assessed by a homologous in vitro GVBD assay (Fig.6A). Maturation-inducing activity was found only in one fraction which had a retention time coinciding exactly with 17a, 20/- diOHprog. The purity and final characterization of this active fraction were further confirmed by thin layer chromatography (Fig. 6B) and mass spec- trometry with authentic 17a, 20¢-diOHprog. Thus, the maturation-inducing hormone of amago salmon was identified as 17a, 208-diOHprog (Fig. i), In collaboration with Dr. A. Kambegawa (Teikyo University), a specific radioimmunoassay for 17a,208-diOHprog was developed in our laboratory and has been applied to measure blood concentrations of this steroid during the sexual maturation of several species of salmonids [23- 25]. In all of the species studied so far, 17a, 208-diOHprog levels were low in vitellogenic females but were strikingly elevated in mature and Gonadotropin Action on Steroidogenesis 213 > —_ 00 MI activity (% GVBD) a oO 5 10 15 20 Fraction number Fic. 6. # Identification of maturation-inducing hormone of amago salmon. A, Maturation-inducing activity (MI) of various fractions of the 50% methanol phase of incubation media separated by high performance liquid chromatography. B, Thin layer chromatography of fraction 10 of the 50% methanol phase (right lane) and various steroid standards (left lane; from top to bottom, progesterone, androstenedione, 17a -hydroxyprogesterone, testosterone, 17a , 208 -dihydroxy-4- pregnen-3-one. The solvent system of benzene: acetone (4:1) was used). See [22] for details. ovulated females (Fig. 3). The increase in plasma 17a, 206-diOHprog levels correlated well with a dramatic rise in plasma gonadotropin levels [25]. In the masu salmon, Oncorhynchus masou, the peak of plasma 17a, 20@-diOHprog levels was observed 2-4 days prior to ovulation, coincident with the occurrence of GVBD in oocytes [24]. These data, together with those from other labo- ratories, indicate that 17a, 208-diOHprog is the major naturally occurring maturation-inducing hormone common to several species of salmonids [26-32]. It is also possible that 17a, 20@-diOH- prog acts as an important steroidal mediator of oocyte maturation in several nonsalmonid teleosts [19, 20, 33-38]. C. Spermiation In male salmonids, a marked increase in the relative amount of mature spermatozoa to total germ cells occurs during the later stages of spermatogenesis. Nonetheless, milt can not nor- mally be collected from these fish. In addition, these spermatozoa do not seem to have the capacity to initiate motility. Spermiation generally occurs immediately before or during the spawning period when the majority of germ cells have completed spermatogenesis. Although the term spermiation is widely used by investigators in fish reproduction, the detailed morphological and physiological bases of this process are not known. In this review, spermiation is defined as the release of motile sperm from the genital pore by gentle pressure on the abdomen. Spermiation has been considered to be under hormonal control. Injection of pituitary extracts or gonadotropins stimulates spermiation in several teleosts [39, 40]. It is generally assumed, however, that in teleosts exogenous gonadotropin does not act directly to induce spermiation but works in concert with testicular somatic elements to stimu- late the production of steroidal mediator(s). In this connection, it was of great interest for us to measure plasma levels of maturation-inducing hormone, 17a, 208-diOHprog. 17a, 208-DiOH- prog levels were found to be low during the period of spermatogenesis and dramatically elevated at the time of spermiation in amago salmon [41]. The close association between high blood levels of 17a, 208-diOHprog and spermiation has also been found in other salmonids [23, 28, 32, 42]. These 214 Y. NAGAHAMA results suggest that 17a, 208-diOHprog is involved in the process of spermiation in salmonids. This suggestion was strongly supported by our studies demonstrating that injections of 17a, 208-diOH- prog were effective in inducing in vivo spermiation in amago salmon about one month prior to the normal spawning period [43]. It was of further interest that gonadotropin action on spermiation was accompanied by a dramatic increase in blood levels of 17a, 208-diOHprog [43]. Considered together, these results provide evidence to suggest that 17a, 208-diOHprog is a testicular steroidal mediator of gonadotropin-induced spermiation in salmonids. Recently a similar suggestion has been made for goldfish [44]. MECHANISMS OF GONADAL STEROIDOGENESIS The preceding findings led to the conclusion that in salmonids at least, two biologically important ovarian steroid hormones are estradiol-178 and 17a, 208-diOHprog (Fig. 1). These findings per- mitted a study of the role of the follicle layer in the production of these two steroids. The ovarian follicle layer of teleosts, as in that of other vertebrates, consists of two major layers: the thecal layer, containing fibroblasts, capillaries, collagen fibers and large cells designated as special thecal cells, and the granulosa layer, composed of a single population of granulosa cells [45] (Fig. 4D). One of the major features of salmonid ovaries is that the follicles they contain are large, about 3-5mm in diameter, and develop synchro- nously, an enormous advantage for biochemical studies, since a large number of follicles at the same stage of development can be obtained easily. The large size of the follicles has also facilitated the development of a simple dissection technique to separate the ovarian follicle of salmonids into two layers (thecal cell layer and granulosa cell layer) [46] (Fig. 4A), making it possible to elucidate the relative contributions of these layers and gonado- tropin in the production of estradiol-17@ and 17a, 208-diOHprog in amago salmon and rainbow trout. A. Ovarian estradiol-17f biosynthesis 1. Two-cell type model In amago salmon, estradiol-17P levels in the plasma increase during vitellogenesis and rapidly decline prior to oocyte maturation. This seasonal pattern is reflected in the ability of the ovarian follicle to produce estradiol-178 in response to gonadotropin [8]. Using various follicular prepara- tions obtained from vitellogenic amago salmon, we examined the effects of partially purified chinook salmon gonadotropin (SG—G100) or chum salmon gonadotropin (SGA) on estradiol-17£ production. Both gonadotropins stimulated estradiol-17 pro- duction by intact follicles and thecal and granulosa layer co-culture preparations, but not by the isolated thecal or granulosa layers. These results indicated that both layers are necessary for gona- dotropin-stimulated estradiol-178 production. In contrast, gonadotropins greatly stimulated testos- terone production by thecal layers but only slightly stimulated testosterone production by the other follicular preparations. Incubation of granulosa layers with exogenous testosterone resulted in elevated estradiol levels, whereas isolated thecal layers incubated with testosterone produced rel- atively small amounts of estradiol-178 which should be attributed to contamination of thecal layer preparations with granulosa cells [8]. We further investigated the role of the thecal layer and the granulosa layer in estradiol-172 production using cyanoketone [47]. Thecal layers incubated with SG—G100 secreted large amounts of testosterone but not estradiol-17 into incuba- tion medium. After incubation of granulosa layers in this medium estradiol-178 levels increased. Cyanoketone inhibited SG—G100-induced testos- terone production of thecal layers and also inhi- bited estradiol-178 production by granulosa layers when they are incubated in this medium. Howev- er, addition of cyanoketone only to the granulosa layer incubations did not inhibit estradiol-178 production. Furthermore, in vitro incubation experiments with various steroids showed that granulosa layers have a limited capacity to metab- olize exogenous pregnenolone, progesterone and 17a-hydroxyprogesterone. Considering all of these data, a two-cell type Gonadotropin Action on Steroidogenesis 215 Gonadotropin GTH-receptor a 7 a Cholesterol cAMP 5 = Pregnenolone S BR Testosterone eS > = : | Aromatase | eile m am Estradiol-17 ra} B Fic. 7. Two-cell type model for the production of estra- diol-17 in the ovarian follicle of salmonids. (See the text for details). model in the production of follicular estradiol-17f has been proposed in amago salmon [8, 45, 48] (Fig. 7). In this model, the thecal layer, under the influence of gonadotropin, secretes aromatizable androgen, mainly testosterone, which is converted to estradiol-17 by granulosa layers. In a recent study we have found that the thecal layer from amago salmon and the granulosa layer from the rainbow trout, Salmo gairdneri, could produce the WQ_ Ringer [eamaeSes Estradiol-178 (ng/ml) same effect as has been reported using combina- tions of thecal and granulosa layers from the same species. The reciprocal use of amago salmon granulosa and rainbow trout thecal layers is also effective (Nagahama, unpublished) (Fig. 8). This finding implies that there may be little species specificity of each of these cell layers among salmonids. This two-cell type model is the first report in lower vertebrates and of evolutionary interest considering the situation in mammals and birds. Since first proposed by Falck [49], numer- ous studies of isolated ovarian cell types from several mammalian species have attempted to elucidate the relative contributions of the various cells and gonadotropins in the overall process of estrogen biosynthesis. A two-cell type model similar to that in teleosts has been demonstrated in mammals [50], but in this case two kinds of gonadotropins, _follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are re- quired, each of which acts upon separate follicular cell types. The theca interna cells, under the influence of LH, produce androgens that are transfered to granulosa cells, where, in the pres- ence of FSH, they are converted to estrogens. In some larger mammals such as monkeys, the thecal Amago-1 Rainbow-1 Amago-G Rainbow-G Amago-T Rainbow-T Amago-T Rainbow-T + 4: + + Amago-G Rainbow-G Rainbow-G Amago-G Fic. 8. The effect of various combinations of the thecal (T) and granulosa (G) layers from vitellogenic amago salmon and rainbow trout in response to 1 g/ml chum salmon gonadotropin (SGA). 216 Y. NAGAHAMA interna tissue is considered to be an additional source of estrogens. In the domestic hen, under gonadotropin stimulation progesterone is pro- duced by the granulosa cells, most of this diffusing to the thecal cells where it is converted to estradiol-178 [51]. This is in sharp contrast to salmonids and mammals and thus be of evolution- ary interest. 2. Thecal layers Using biochemical methods, we have identified the aromatizable androgens produced by thecal layers in response to SG-G100. Thecal layers were incubated with SG—G100 and ether extracts of media from these incubates were fractionized (4.5 ml/fraction) by reversed-phase high perform- ance liquid chromatography. Aromatase activity of each fraction was assessed by the capacity of isolated granulosa layers to produce estradiol-17£ from exogenous testosterone. Among 27 fractions separated, one fraction produced large amounts of estradiol-17@ when added to the granulosa layer Estradiol-178 (ng/ml) 5 10 15 A) 2S) Fraction number Fic. 9. incubation (Fig. 9A). This fraction had a retention time that coincided with authentic testosterone. Another fraction with a retention time coinciding with authentic androstenedione also produced estradiol-17£, but its production was much lower compared with the other fraction. The purity and final characterization of these fractions were con- firmed by thin layer chromatography (Fig. 9B) and mass spectroscopy. Thus, we conclude that the major aromatizable androgen to be produced in the amago salmon thecal layer in response to gonadotropin is testosterone (Adachi and Naga- hama, unpublished). In vitro production of testosterone by the isolated thecal layer preparations obtained each month during vitellogenesis and oocyte maturation has revealed that the capacity of the thecal layer to produce testosterone in response to salmon gona- dotropin gradually increased during the course of vitellogenic growth and peaked during the post- vitellogenic period; this capacity of thecal layers was maintained by the period of oocyte maturation B Identification of aromatizable androgen produced by thecal layers of vitellogenic amago salmon. A, Aromatase activity of various fractions of the 50% methanol phase of incubation media separated by high performance liquid chromatography. Aromatase activity was assessed by the capacity of 10 isolated granulosa layers to produce estradiol-17£ from 100 ng/ml exogenous testosterone. B, Thin layer chroma- tography of fractions 18 and 19 of the 50% methanol phase (right lane) and various steroid standards (left lane, from top to bottom, progesterone, androstenedione, 17a-hydroxyprogesterone and testosterone). The solvent system of benzene: acetone (4: 1) was used. (See the text for details). Gonadotropin Action on Steroidogenesis 217 and ovulation. An identical seasonal pattern of stimulation of testosterone production was observed when thecal layers were incubated with forskolin and agents known to raise intracellular cyclic AMP (cAMP). These results are consistent with the general hypothesis that gonadotropin- induced testosterone production is mediated by the adenylate cyclase-cAMP effector system (Kanamori, unpublished). The site of gonadotro- pin action on the thecal layer remains unclear, but our recent in vitro data using steroid synthesis inhibitors suggest that its major site is on the pathway from cholesterol to pregnenolone. 3. Granulosa layers The foregoing findings indicate the presence of aromatase enzymes in granulosa cells of amago salmon vitellogenic follicles. Aromatase activity increased during the period of vitellogenesis to reach a peak in late vitellogenesis, and then declined rapidly in association with the ability of the oocyte to mature in response to gonadotropin [52]. These findings, combined with our observa- tions on the enhanced capacity of the thecal layer to produce testosterone in the postvitellogenic period, provide strong evidence that the decrease in the capacity of postvitellogenic follicles to produce estradiol-178 is due, in part, to the decrease in aromatase activity during the course of oocyte development. This is in contrast to the situation reported in the rat where the decreased secretion of estradiol-17 immediately after ovula- tion has been explained by a diminished supply of aromatizable androgens [53]. The mechanism of the induction or activation of the amago salmon granulosa cell aromatase system is unknown at this point. It is known that in certain mammals, one of the major roles of gonadotropin action on follicular estradiol-178 secretion is to stimulate aromatase activity; the most fun- damental regulator is FSH [50]. However, very few studies have been conducted in non- - mammalian species. As discussed above, isolated amago salmon granulosa cells have no capacity to produce testosterone and estradiol-176 from en- dogenous substrates in response to gonadotropin. Furthermore, there was no significant difference in estradiol-17@ production by granulosa layers incu- bated with testosterone in the presence or absence of SG-—G100 at any stage of follicle development [52]. In addition, pituitary homogenates from vitellogenic amago salmon were unable to induce aromatase activity (Kagawa, unpublished). Never- theless, aromatase activity in granulosa cells in- creases during vitellogenesis to reach a peak in late vitellogenesis and thereafter rapidly declines in the postvitellogenic period. In goldfish, human chorionic gonadotropin (HCG) slightly but significantly enhanced the conversion of exogenous testosterone to estradiol- 17 by ovarian follicles at the primary yolk stage. Furthermore, forskolin, an adenylate cyclase acti- vator, stimulated conversion of exogenous testos- terone to estradiol-17£ by vitellogenic follicles of goldfish even when cyanoketone was present in the medium [54]. Since cyanoketone completely blocked forskolin-induced endogenous production of estradiol-17/, the observed stimulated effects of forskolin can apparently be accounted for by an enhancement of endogenous aromatase activity in response to forskolin. Taken together, these results suggest that gonadotropin stimulates the induction of aromatase activation in goldfish vitel- logenic follicles via an adenylate-cyclase-cAMP- dependent step. A similar situation seems to occur in the medaka, Oryzias latipes (Sakai and Nagaha- ma, unpublished). In contrast, Sire and Depeche [55] reported that highly purified salmon glycopro- tein maturational gonadotropin inhibited aroma- tase activity in pre- and early vitellogenic follicles of rainbow trout. Thus, the mechanisms by which aromatase activity in teleost ovarian follicles is regulated merit further investigation and are of evolutionary interest. B. Ovarian 17a, 208-diOHprog biosynthesis 1. Two-cell type model The identification of the maturation-inducing hormone in salmonids as 17a, 208-diOHprog per- mitted a study of the role of the follicle layer in the production of this hormone. 17a,208-DiOHprog production by intact follicles at different stages of development clearly showed that the capacity of the follicles to respond to gonadotropin by syn- thesizing and secreting this steroid is acquired immediately prior to the natural maturation period 218 Y. NAGAHAMA [25]. Using isolation techniques similar to those used for the studies on follicular estradiol-17£ production, three follicular preparations from postvitellogenic amago salmon and rainbow trout were incubated with or without chum salmon gonadotropin (SGA). 17a-Hydroxyprogesterone and 17a, 208-diOHprog were measured by specific radioimmunoassay [48, 56]. In vitro production of 17a, 208-diOHprog by co-culture preparations was remarkably enhanced by gonadotropin, but neither isolated thecal layers nor isolated granulosa layers were capable of producing substantial amounts of 17a, 208-diOH- prog in response to gonadotropin. These in vitro data clearly indicate that the interaction of both thecal and granulosa layers is necessary for the production of 17a, 208-diOHprog in response to gonadotropin. Measurement of 17a-hydroxypro- gesterone concentrations of media from the same experiment revealed that isolated thecal layers produced large quantities of 17a-hydroxyproges- terone, but granulosa layers did not respond to gonadotropin. In contrast, levels of 17a-hydroxy- progesterone in media from co-culture incubations peaked at 12 hr and rapidly decreased concom- itantly with a rapid rise in 17a, 208-diOHprog levels. The presence of 208-hydroxysteroid dehy- drogenase (208-HSD), the key enzyme involved in the conversion of 17a-hydroxyprogesterone to 17a, 206-diOHprog (Fig. 1), has been demon- strated in the granulosa layers, since this layer produced 17a, 208-diOHprog when incubated with exogenous 17a-hydroxyprogesterone. When all of these in vitro data are combined, a two-cell type model has been proposed for the first time in any vertebrate for the follicular production of maturation-inducing hormone, which is summa- rized in Figure 10. [48,56]. In this model, the thecal layer produces 17a-hydroxyprogesterone that traverses the basal lamina and is converted to 17a, 208-diOHprog by the granulosa layer where gonadotropin acts to enhance the activity of 208-HSD. However, the extent to which thecal layers contribute to the production of 17a, 20f- diOHprog is still not clear, since in some cases thecal layer preparations produced this steroid in response to gonadotropin. Although our granulo- sa layer preparations are completely free of Gonadotropin [er-receptor Le 7 cAMP 17a,208 —Dihydroxy—4-pregnen-3-one Cholesterol Pregnenolone THECA 17a —-Hydroxyprogesterone GTH-receptor 17a—-Hydroxyprogesterone cAMP 319171104 NVIYVAO 20B-HSD |< GRANULOSA Fic. 10. Two-cell type model for the production of 17a, 20-dihydroxy-4-pregnen-3-one in the ovarian folli- cle of salmonids. (See the text for details). attached thecal layers, it is extremely difficult to obtain pure thecal layer preparations from post- vitellogenic follicles. 2. Thecal layers Studies of in vitro 17a-hydroxyprogesterone producton by thecal layers at different stages of development showed that the thecal layer only begins secreting 17a-hydroxyprogesterone im- mediately prior to the maturation of oocytes in vitro. Thus, in addition to 20@-HSD activation in the granulosa cells, the availability of 17a-hy- droxyprogesterone may also play a major role in the enhancing action of gonadotropin on follicular biosynthesis of 17a, 208-diOHprog (Kanamori, unpublished). The first step of the stimulating effect of gonadotropin in the thecal layer appears to be receptor-mediated activation of adenylate cyclase and formation of cAMP, the major site of action probably occurring at the steroidogenic step be- tween cholesterol and pregnenolone. Further- more, gonadotropin-induced 17a-hydroxyproges- terone production by the thecal layer is abolished by cyclohexamide and puromycin, but not by actinomycin D and cordycepin, suggesting that stimulating effects of gonadotropin in the thecal layer require the synthesis of a new protein (Nagahama and Adachi, unpublished). 3. Granulosa layers The granulosa cells of salmonids provide a Gonadotropin Action on Steroidogenesis 219 unique system in which to study gonadotropin regulation of a steroidogenic enzyme, in this case 208-HSD, since precursor steroids cannot be produced by these cells; granulosa cells of amago salmon have little cholesterol side-chain cleavage activity. This allows the indirect quantification of 208-HSD activity by the measurement of 17a, 208-diOHprog production when these cells are incubated with exogenous 17a-hydroxyprogester- one [57,58]. Granulosa cells obtained from full- grown follicles responded to gonadotropin by enhancing 208-HSD activity when exogenous 17a- hydroxyprogesterone was added to the incubation medium. The action of gonadotropin on 208-HSD enhancement was mimicked by forskolin, by dibutyryl cAMP (dbcAMP), but not dbcGMP, and by two phosphodiesterase inhibitors, theophylline and 3-isobutyl-1-methylxanthine [57]. Further- more, gonadotropin and forskolin caused a rapid accumulation of cAMP with maximal levels at 30-60min (Kanamori, unpublished). These findings are consistent with the view that cAMP is the second messenger of gonadotropin action. In addition to cAMP, calcium appears to play an important role in the gonadotropin regulation of ovarian steroidogenesis in mammals. Recent evi- dence for the widespread role of calmodulin in the regulation of intracellular calcium led us to deter- mine whether calmodulin was involved in the responses to gonadotropin and cAMP. For this purpose, three calmodulin inhibitors (trifluoro- perazine, TFP, N-(6-aminohexy]l)-1-naphthalene- sulfonamide hydrochloride, W5 and N-(6- aminohexyl)-5-chloro-1-naphthalenesulfonamide, W7) were used, and the effects of these inhibitors on the gonadotropin-, cAMP- and _forskolin- induced enhancement of 20@-HSD in the amago salmon granulosa cells have been investigated (Nagahama, unpublished). Both TFP and W7 strongly prevented SGA-, dbcAMP-, and fors- kolin-stimulated conversion of exogenous 17a-hy- -droxyprogesterone to 17a, 20$-diOHprog in a dose-dependent manner. W5 also inhibited 17a, 208-diOHprog production, but was less effective than the other two inhibitors. These results suggest that calmodulin plays some role in the gonadotropin regulation of 206-HSD activation in the amago salmon granulosa cell. The precise role and pathways through which calmodulin affects gonadotropin-induced 208-HSD activation remain to be determined. A very important question concerns the molecu- lar mechanisms of granulosa cell 208-HSD activa- tion. We have examined the effects of cyclohex- amide, puromycin, actinomycin D and cordycepin on the production of 17a, 208-diOHprog by chum salmon gonadotropin and dbcAMP in the amago salmon granulosa layer. When varying concentra- tions of one of the inhibitors were added to the incubation medium containing 17a-hydroxypro- gesterone, stimulatory effects of SGA and dbcAMP on the production of 17a, 208-diOHprog were significantly inhibited. Thus, effects of gonadotropin and dbcAMP upon both transcrip- tional and translational processes appear to be essential intermediate steps in the activation of 208-HSD. Our time course studies further suggest that de novo synthesis of 208-HSD in vitro in response to gonadotropin and dbcAMP occurs, and consists of gene transcriptional events within the first 6 hr of exposure to gonadotropin and dbcAMP and translational events 6-9 hr after exposure to gonadotropin and cAMP [58]. C. Testicular 17a, 208-diOHprog biosynthesis Incubation of testicular fragments from sper- miating rainbow trout and amago salmon with gonadotropin (SGA) or 17a-hydroxyprogesterone resulted in a highly significant increase in 17a, 208-diOHprog levels in the incubation medium [41]. These results indicate that the testis is the principal source of 17a, 206-diOHprog biosynthe- sis. However, the testicular cell types responsible, as well as the intratesticular mechanisms involved in controlling testicular steroidogenesis in teleosts, remain unresolved. Therefore we investigated whether various testicular preparations of spermiating salmonids could produce two major testicular steroids, 11-ketotestosterone and 17a, 208-diOHprog, in response to gonadotropin and 17a-hydroxyprogesterone [59]. Three different testicular preparations, intact testicular fragments, sperm-free testicular fragments, and isolated sperm from spermiating rainbow trout, were incu- bated in Ringer for 18 hr in the continuous presence or absence of SGA and 17a-hydroxypro- 220 Y. NAGAHAMA gesterone. Both SGA and 17a-hydroxyprogester- one significantly stimulated 11-ketotestosterone by intact and sperm-free testicular fragments. Sperm preparations did not produce 11-ketotestosterone in any incubation condition. These results indicate that the site of 11-ketotestosterone production in the testis is its somatic cell elements. Whether 11-ketotestosterone production is limited only to Leydig cells or whether it also occurs in Sertoli cells is open to question, since our sperm-free testicular fragments included both Leydig cells and Sertoli cells. Chum salmon gonadotropin also stimulated 17a, 208-diOHprog production by intact testicular fragments, but not by sperm-free testicular frag- ments and sperm preparations (Fig. 11). 17a-Hy- droxyprogesterone markedly stimulated 17a, 20/- diOHprog production by intact testicular frag- ments and sperm preparations, but not by sperm- free testicular preparations. A preliminary study with thin-layer chromatography has also shown that after incubation of sperm preparations with 17a-hydroxyprogesterone, 17a, 206-diOHprog was identified as one of the major metabolites (Ueda and Nagahama, unpublished). Taken together, these results seem to indicate that the (ng/mg tissue) 17a,208-Dihydroxy—4—-pregnen-3-one Treatment R G17 (ig/ml) 1 01 Intact Sperm-free Sperm testicular fragment Fic. 11. Jn vitro production of 17a , 208 -dihydroxy-4- pregnen-3-one in response to chum salmon gonado- tropin (SGA, G) and 17a -hydroxyprogesterone (17) by testicular fragments and isolated sperm. R, Rin- ger solution. distribution of 20G-HSD in the amago salmon testis is limited only to sperm. It is a subject for further studies to define the cellular source of precursor steroids such as 17a-hydroxyprogester- one. CONCLUSION Inasmuch as 17a, 208-diOHprog was identified as the major mediator of oocyte maturation, we now have two known biologically important mediators of oocyte growth and maturation in salmonids, estradiol-178 and 17a, 208-diOHprog. It is now established a clear-cut synergism of ovarian thecal and granulosa layers for the produc- tion of these two steroid hormones. In both cases, the thecal layer is the site of precursor synthesis and the granulosa layer is the site of conversion of the precursors to the final products. Thus, normal growth and maturation of the oocyte depend upon a high degree of coordination of these layers. More complete understanding of the interactions of gonadotropin with ovarian somatic cells will provide new and important information about the endocrine activities of ovarian follicle cells in relation to oocyte growth and maturation. Our recent binding studies with ['*°I]-chum salmon gonadotropin demonstrated specific binding to both thecal and granulosa layers from vitellogenic and postvitellogenic amago salmon follicles (Kana- mori, unpublished). Certainly, studies on the temporal and functional nature of these bindings provide further insight into the mechanisms of interactions of gonadotropin with thecal and gran- ulosa layers. Endocrine details of vitellogenin uptake into the oocyte from the vascular system are not known. It is necessary to endeavor to investigate whether gonadotropin or other pituitary hormones have a mediator effect of this vitellogenin uptake by oocytes. As yet, we know very little about the mode of action of 17a, 208-diOHprog to induce oocyte maturation. Since the site of 17a, 20- diOHprog action is considered to be at or near the oocyte surface, further work must be performed to determine the identity and functional significance of the surface receptor of 17a, 208-diOHprog. 17a, 208-DiOHprog has also been shown to be Gonadotropin Action on Steroidogenesis a major mediator of gonadotropin-induced sper- miation in amago salmon and goldfish. A shift in the steroidogenic pattern from androgens to 17a, 208-diOHprog seems to occur in the testis im- mediately prior to or during the spermiation period. Although the question of which cell type is the principal source of testicular production of 17a, 208-diOHprog still remains unresolved, it is likely that the interaction of somatic cells and sperm is necessary. It is noteworthy that sperm contain 208-HSD. These findings may be one of the best documented examples of the relationship between testicular steroidogenesis and male germ cell maturation in nonmammalian vertebrates, and will certainly provide the basis for a study at the molecular levels of hormonal regulation of male germ cell development. ACKNOWLEDGMENTS The studies from our laboratory described in this article have been aided in part by Grant-in-Aid for Special Project Research (Project Nos. 58119008 and 61134041) and scientific research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1 Burzawa-Gérard, E. (1982) Can. J. Fish. Aquat. Sci., 39: 80-91. 2 Idler, D.R. and Ng, T. B. (1983) In “Fish Physiolo- gy”. Ed. by W.S. Hoar, D.J. Randall and E.M. Donaldson, Academic Press, New York, Vol. IXA, pp. 187-221. 3 Ueda, H., Nagahama, Y. and Takahashi, H. (1984) Gunma Symp. Endocrinol., 21: 21-35. 4 Kawauchi, H., Suzuki, K., Nagahama, Y., Adachi, S., Naito, N. and Nakai, Y. (1986) In “Pars Distalis of the Pituitary Gland—Structure, Function and Regulation”. Ed. by F. Yoshimura and A. Gorb- man, Elsevier Sci. Publ. B. V., pp. 383-390. 5 van Oordt, P.G. W.J. and Peute, J. (1983) In “Fish Physiology”. Ed. by W.S. Hoar, D.J. Randall and E. M. Donaldson, Academic Press, New York, Vol. IXA, pp. 137-186. 6 Wallace, R. A. (1985) In “Developmental Biology”. Ed. by L. W. Browder, Plenum Press, New York, Vol. 1, pp. 127-177. 7 Fostier, A., Jalabert,B., Billard, R., Breton, B. and Zohar, Y. (1983) In “Fish Physiology”. Ed. by W.S. Hoar, D.J. Randall and E.M. Donaldson, Academic Press, New York, Vol. IXA, pp. 277- 10 IL 13 13) 24 25 26 74] 28 29 Pp S72: Kagawa, H., Young, G. and Nagahama, Y. (1985) In “Salmonid Reproduction”. Ed. by R. N. Iwamoto and S. Sower, Washington Sea Grant Program, University of Washington, Seattle, pp. 20-25. Sundararaj, B.I., Nath, P. and Burzawa-Gérard, E. (1982) Gen. Comp. Endocrinol., 46: 93-98. Le Menn, F. and Burzawa-Gérard, E. (1985) Gen. Comp. Endocrinol., 57: 23-36. Ng, T. B. and Idler, D. R. (1983) In “Fish Physiolo- gy”. Ed. by W.S. Hoar, D.J. Randall and E.M. Donaldson, Academic Press, New York, Vol. IXA, pp. 373-404. Wasserman, M.J. and Smith, L. D. (1978) In “The Vertebrate Ovary”. Ed. by R.E. Jones, Plenum Press, New York, pp. 443-468. Masui, Y. and Clarke, H. J. (1979) Int. Rev. Cytol., 57: 185-282. Schuetz, A. W. (1979) J. Steroid Biochem., 11: 695-699. Kanatani, H. and Nagahama, Y. (1980) Biomed. Res., 1: 273-291. Goetz, F. W. (1983) In “Fish Physiology”. Ed. by W.S. Hoar, D.J. Randall and E.M. Donaldson, Academic Press, New York, Vol. IXB, pp. 117- 170. Sundararaj, B.I., Goswami, S.V. and Lamba, V. (1979) J. Steroid Biochem., 11: 701-707. Young, G., Kagawa, H. and Nagahama, Y. (1982) J. Exp. Zool., 224: 265-275. Jalabert, B. (1976) J. Fish. Res. Board Can., 33: 974-988. Iwamatsu, T. (1980) J. Exp. Zool., 211: 231-239. Nagahama, Y., Hirose, K., Young, G., Adachi, S., Suzuki, K. and Tamaoki, B. (1983) Gen. Comp. Endocrinol., 51: 96-105. Nagahama, Y. and Adachi, S. (1985) Dev. Biol., 109: 428-435. Ueda, H., Hiroi, O., Hara, A., Yamauchi, K. and Nagahama, Y. (1984) Gen. Comp. Endocrinol., 53: 203-211. Yamauchi, K., Kagawa, H., Ban, M., Kasahara, N. and Nagahama, Y. (1984) Bull. Jpn. Soc. Sci. Fish., 50: 2137. Young, G., Crim, L. W., Kagawa, H., Kambegawa, A. and Nagahama, Y. (1983) Gen. Comp. Endocri- nol., 51: 96-105. Idler, D. R., Fagerland, U. H. M. and Ronald, A. P. (1960) Biochem. Biophys. Res. Commun., 2: 133- 1377 Campbell, C. M., Fostier, A., Jalabert, B. and Trus- cott, B. (1980) J. Endocrinol., 85: 371-378. Fitzpatrick, M.S., Van Der Kraak, G. and Schreck, C. B. (1986) Gen. Comp. Endocrinol., 62: 437-451. Fostier, A., Breton, B., Jalabert, B. and Marcussi, 30 31 32 3)3) 34 35 36 37 38 39 40 4] 42 43 222 O. (1981) C.R. Acad. Paris, Ser. III, 293: 817-820. Hirose, K., Adachi,S. and Nagahama, Y. (1985) Bull. Jpn. Soc. Sci. Fish., 51: 399-403. Scott, A.P., Sheldrick, E.L. and Flint, A.P.E. (1982) Gen. Comp. Endocrinol., 46: 444-451. Truscott, B., Idler,D.R., So, Y.P. and Walsh, J.M. (1986) Gen. Comp. Endocrinol., 62: 99-110. Scott, A.P., MacKenzie, D.S. and Stacey, N.E. (1984) Gen. Comp. Endocrinol., 56: 349-359. Kagawa, H., Young, G. and Nagahama, Y. (1983) Bull. Jpn. Soc. Sci. Fish., 49: 1783-1787. Stacey, N.E., Peter, R.E., Cook, A.F., Truscott, B., Walsh, J.M. and Idler, D. R. (1983) Can. J. Zool., 61: 2646-2652. Kobayashi, M., Aida, K. and Hanyu, I. (1985) Bull. Jpn. Soc. Sci. Fish., 51: 1085-1091. Levavi-Zermonsky, B. and Yaron, Z. (1986) Gen. Comp. Endocrinol., 62: 89-98. Greeley, M.S., Jr., Calder, D.R., Taylor, M.H., Hols, H. and Wallace, R. A. (1986) Gen. Comp. Endocrinol., 62: 281-289. Clemens, H. P. and Grant, F. B. (1965) Copeia, 2: 174-177. Billard, R., Fostier, A., Weil, C. and Breton, B. (1982) Can. J. Fish. Aquat. Sci., 39: 65-79. Ueda, H., Young, G., Crim, L.W., Kambegawa, A. and Nagahama, Y. (1983) Gen. Comp. Endocri- nol., 51: 106-112. Scott, A. P. and Baynes, S.M. (1982) In “Proceed- ings of the International Symposium on Reproduc- tive Physiology of Fish”. Ed. by C. T. T. Richter and H.J. Th. Goos, Pudoc. Wageningen, The Nether- lands, pp. 103-106. Ueda, H., Kambegawa,A. and Nagahama, Y. (1985) Gen. Comp. Endocrinol., 59: 24-30. 44 45 46 47 48 49 50 51 52 53 54 3) 56 57 58 SY Y. NAGAHAMA Kobayashi, M., Aida, K. and Hanyu, I. (1986) Bull. Joe SOEs SCw JS. Ss V5. Nagahama, Y. (1983) In “Fish Physiology”. Ed. by W. S. Hoar, D.J. Randall and E.M. Donaldson, Academic Press, New York, Vol. IXA, pp. 223- DSS. Kagawa, H., Young, G., Adachi, S. and Nagahama, Y. (1982) Gen. Comp. Endocrinol., 47: 440-448. Young, G., Kagawa, H. and Nagahama, Y. (1982) Biomed. Res., 3: 659-667. Nagahama, Y. (1984) Gunma Symp. Endocrinol., 21: 167-182. Falck, B. (1959) Acta Physiol. Scand., 47, Suppl. 163: 1-101. Dorrington, J.H. and Armstrong, D. T. (1979) Re- cent Prog. Horm. Res., 35: 301-342. Huang, E.S., Rao,K.J. and Nalbandov, A. V. (1979) Biol. Reprod., 29: 310-315. Young, G., Kagawa, H. and Nagahama, Y. (1983) Biol. Reprod., 29: 310-315. Suzuki, K. and Tamaoki, B. (1980) Endocrinology, 107: 2115-2116. Tan, J.D., Adachi,S. and Nagahama, Y. (1986) Gen. Comp. Endocrinol., 63: 110-116. Sire,O. and Depeche, J. (1981) Reprod. Nutr. Dev., 21: 715-726: Young, G., Adachi,S. and Nagahama, Y. (1986) Dev. Biol., 118: 1-8. Nagahama, Y., Kagawa, H., Adachi, S. and Young, G. (1985) J. Exp. Zool., 236: 371-375. Nagahama, Y., Young, G. and Adachi, S. (1985) Dev. Growth Differ., 27: 1213-1221. Ueda, H., Kambegawa,A. and Nagahama, Y. (1984) J. Exp. Zool., 231: 435-439. ZOOLOGICAL SCIENCE 4: 223-232 (1987) © 1987 Zoological Society of Japan Effect of Host myo-Inositol Deficiency on Hymenolepis diminuta (Cestoda) MUHAMMAD M. KHAN and YUEN K. IP Parasitology Laboratory, Department of Zoology, National University of Singapore, Kent Ridge, Singapore, 0511, Republic of Singapore ABSTRACT—The absorption of myo-inositol and its incorporation into phosphatidylinositol by Hymenolepis diminuta in rats fed with normal pellets (Purina Laboratory Chow), were concentration and time dependent. Glucose uptake was similar in both groups of worms recovered from control and myo-inositol deficient rats. Deletion of myo-inositol from host diet caused no effect on the establish- ment of H. diminuta in its host. The worms obtained from rats fed with myo-inositol deficient diet were heavier in terms of wet weight than those from rats fed with control diet. No apparent difference in the length of the worms was noted. Compared to worms from rats fed with control diet, egg production was significantly higher (P<0.01) in those recovered from the rats fed with myo-inositol deficient diet. The patent period was reduced by one day in the worms parasitized in myo-inositol deficient rats. The infectivity of H. diminuta eggs was similar in both groups of worms. INTRODUCTION myo-Inositol appears to be an important car- bohydrate for Hymenolepis diminuta, as the organ- ism is synthesizing [1] and actively absorbing it [2]. It has been identified and shown to be unevenly distributed along the length of the worm [3]. Phosphatidylinositol is an essential constituent of biomembranes which is involved in transport phenomena. The cell membrane does not function satisfactorily if inositol lipids are not present in adequate concentration [4]. Formation of phos- phatidylinositol from labelled ‘C-glucose, 'C- palmitate and **P-orthophosphate has been estab- lished [1, 5]. However no published information is available for the formation of phosphatidylinositol from labelled *H-myo-inositol in H. diminuta. Thus one of the aims for the present investigation was to elucidate the pattern of absorption of _myo-inositol and its incorporation into phospha- tidylinositol in H. diminuta. myo-Inositol is essential for the survival and growth of a number of microorganisms and mammalian cells in culture [6-9]. It has been Accepted October 3, 1986 Received August 16, 1986 reported that growth, development and reproduc- tion of parasite may be markedly sensitive to the feeding activity, nutrient intake and digestive physiology of its host [10]. Several studies have been carried out to investigate the influence of host dietary carbohydrates, proteins and vitamins on the different aspects of growth and fecundity of H. diminuta [11-19]. However, no such information is available for myo-inositol. The present inves- tigation was therefore undertaken to elucidate the effect of host diet, with or without myo-inositol, on the establishment, growth and reproduction of the parasite. In order to compare the physiological activities of the worms from control and myo- inositol deficient rats, glucose uptake studies were also performed. MATERIALS AND METHODS Male rats of the Sprague-Dawley strain weighing 100 to 130g at the time of infection were used as definitive hosts in all experiments. Adult Tenebrio sp. served as intermediate host. myo-Inositol uptake and its incorporation into phospholipids Six rats were infected with 10 cysticercoids each. 224 M. M. KHAN AnD Y.K. Ip Before and after infection, the rats were fed with water and food (Purina Laboratory Chow) ad libitum. Ten-day old worms were flushed from the excised gut with saline solution [20]. myo-Inositol uptake studies, with 10 worms in each incubation medium, were undertaken as described by Ip and Fisher [2]. Tritiated myo-inositol was used with specific activity adjusted to 2.5 ~Ci/umol. Wet weight of the worms was recorded before the experimental incubation. Samples of parasites were homogenized and extracted according to Folch et al. [21]. The water washes from the Folch procedure as well as the water extract of the residue were combined. This fraction (water soluble fraction) contained free carbohydrates which were separated by paper chromatography. In order to study the incorporation of absorbed 3H-myo-inositol into phospholipids, the washed total lipid fraction, obtained from the organic phase, was evaporated to dryness under nitrogen. Phospholipids were separated by thin layer chro- matography. Glucose uptake Six rats were divided into two groups of three. Twenty cysticercoids were force-fed to each rat. Ten-day old worms for glucose uptake studies were prepared as described for the growth experi- ment. They were randomized, incubated and extracted according to Ip and Fisher [2]. Standard incubation conditions were two minutes. ‘4C- Glucose (1mM, 3mM, 5mM) was used with specific activity adjusted to 0.07 #Ci/umol. Up- take velocities were expressed in terms of pmol glucose absorbed/g ethanol extracted dry weight per 2 min. Establishment, growth and reproduction of H. diminuta Twenty or thirty cysticercoids, depending on the nature of the experiment, were force-fed to each rat host. The experimental rats were caged individually. Before and after infection, the rats were provided with water and food (Purina Laboratory Chow or Synthetic diets) ad libitum. Parasites were flushed from the excised guts with saline solution [20] at the end of the experimental period. Flushed parasites were washed in several changes of the same medium before any weight and length measurements were taken. All weighings were done to the nearest 0.1 mg. Worm lengths were determined after relaxing them in cold tap water for 45 min. The synthetic diet was prepared according to Read et al. [22]. However, myo-inositol was deleted from it to prepare myo-inositol deficient diet. Water was added to the requisite amount of the dry material to make a thick paste. Freshly prepared diet was fed to the rats daily in clean and dry bowls ad libitum. In order to study the effect on establishment of the parasite in the host, each of the two groups of three rats was fed with either the control or myo-inositol deficient diet. After two days, 30 cysticercoids were fed to each rat of both groups. The synthetic diets were continued for another five days followed by five days of normal pellets (Purina Laboratory Chow) to eliminate any possi- ble effect of the diets on the growth of the parasite. The rats of both groups were then sacrificed. The worms, flushed from the excised gut with saline solution, were counted to determine the degree of infection. To examine the effect of deleting myo-inositol from the diet on the growth of H. diminuta rats infected with 30 cysticercoids were fed with Purina Laboratory Chow during the first five days of infection to eliminate any possible diet effect on the establishment of the worms. After five days one group was fed myo-inositol deficient diet, whereas the other group received the control diet for another five days. Wet weight and length of these ten-day old worms were then determined. To examine the effect of deleting myo-inositol from the host diet on reproduction of H. diminuta, six rats were infected with 20 cysticercoids each. These rats were maintained on their respective diets as for the growth experiment. To investigate the patent period, three faecal samples from each host of both groups were regularly examined every day beginning 13 days post infection. Egg counts were made for every 24 hr as described by Holmes [23]. At the end of the experiment (25 days postinfection) the worms were flushed from the excised gut with saline solution. Worms collected from each host were counted to establish rela- myo-Inositol Deficiency Effect on H. diminuta 225 tionship with egg production. Infectivity of H. diminuta eggs In order to determine the infectivity of the eggs obtained from the worms recovered from the control and myo-inositol deficient rats, beetles were starved for 24 hr at room temperature. Infection experiments were carried out in six small containers measuring 9cm in diameter and 6cm in height. Ten beetles were introduced into each container. To three containers, eggs from the worms recovered from control rats were adminis- tered, whereas the other three received eggs from the worms recovered from myo-inositol deficient rats. The eggs were offered according to the method of Voge [24]. The infectivity of H. diminuta eggs in the host was examined by beetle dissection after a period of two weeks as described by Voge [24]. Chromatographic Analyses Water soluble fraction was reduced to dryness on a rotary evaporator in vacuo and the residue dissolved in a small volume of deionized water before passing through tandem columns of Dowex 50(H*) and Dowex 2(OH_). The resultant sam- ple was dried and redissolved in small volume of deionized water. This sample was examined by paper chromatography on Whatman 3 MM paper as described by Gray and Frankel [25]. Carbohy- drates were visualized with silver nitrate method of Trevelyan et al. [26]. Neutral lipids were separated from phospho- lipids by ice cold acetone. Phospholipids were separated into classes on Silica Gel G plates using two dimensional system of Rouser et al. [27]: chloroform/methanol/25% ammonia (65 :35:5 v/ v) followed by chloroform/acetone/methanol/ acetic acid/water (10:4:2:2:1 v/v) in the second dimension. In the present work Silica Gel slurry was prepared by mixing 50g of Silica Gel G (Merck) in 100 ml of deionized water. This slurry was applied to the glass plates (20x 20cm) at a thickness of 0.25mm. Lipid spots were detected by exposing the developed plates to iodine vapours. The spots were identified by comparing with phospholipid standards obtained from Sigma Chemical Co. Determination of radioactivity All samples were counted for 10 min in an BETAmatic BASIC Liquid Scintillation Counter (KONTRON Analytical). Aliquots of water solu- ble fraction, containing *H-myo-inositol, were dried and mixed with Biofluor (NEN) for count- ing. Radioactive paper chromatograms were cut into 1cm strips parallel to the origin. Each strip was then counted in a scintillation vial containing 10 ml of Liquifluor (NEN). Identified phospholi- pids were scraped from the plates into scintillation vials containing 1ml of water and 10ml of Biofluor. Radioactivity was determined as stated above. Statistical analysis Data presented graphically were plotted as least Square regression lines wherever applicable. The data were analyzed statistically by Student’s t-test for significant differences. RESULTS myo-Inositol uptake and its incorporation into phospholipids These studies were carried out by incubating 10-day old worms with 7H-myo-inositol at different concentrations and over different incubation periods to investigate the absorption and incor- poration of myo-inositol into water soluble and lipid fractions. The observations were the result of single set of experiment. It is apparent from Figure 1 that the absorption of myo-inositol in water soluble fraction and the free myo-inositol, isolated from water soluble fraction by paper chromatography, was linear and constant with respect to time up to 4 hr of incubation. Its uptake also occurred in a concentration dependent man- ner (Table 1). The incorporation of absorbed 7H-myo-inositol into total phospholipid and phosphatidylinositol fractions was linear and constant as a function of time (Fig. 2). An early labelling of phosphatidyl- inositol was also observed. The incorporation of absorbed myo-inositol into total phospholipid and phosphatidylinositol fractions was non-linear with 226 M.M. KHAN AND Y.K. Ip 0.5 0.005 0.4 0.004 0.3 0.003 V V OMe 0.002 0.1 0.00] ‘ae, | 2 3 4 | 2 3 4 Time (hr) Time (hr) Fic. 1. Absorption of 0.1 mM 7H-myo-inositol by H. Fic. 2. Incorporation of 0.1 mM *H-myo-inositol into diminuta as a function of time (hr). V= umol/g wet the total phospholipid and phosphatidylinositol frac- weight. &=radioactivity in water soluble fractions; tions by H. diminuta as a function of time (hr). V= i = radioactive myo-inositol isolated by paper chro- pmol/g wet weight. [—™=total phospholipid; matography from water soluble fractions. A = phosphatidylinositol. TABLE 1. Rates of myo-inositol uptake; total phospholipid and phosphatidylinositol syntheses as a function of myo-inositol concentrations te 8 Phosphatidylinositol He ae S, Total mde Total myo-inositol : synthesis ne Myo-inositol synthesis faa phospholipid t uptake % myo-inositol concentration % total : % total pmol/g wet wt ee 3 incorporated into as (mM) myo-inositol a myo-inositol per 4hr the phospholipid uptake 3 uptake fraction 0.05 0.081 2s0 97.45 2.187 0.10 0.257 1.210 91.60 15323 0.50 0.806 0.570 66.20 0.868 1.00 0.996 0.328 55.10 0.596 2.00 1.474 0.372 71.80 ODI, respect to myo-inositol concentration (Fig.3). “mol/g wet weight per 4 hr respectively. The Lineweaver Burk transformation of the data (Fig. | Tespective K,, values were 0.186 and 0.086 mM. 4) revealed V,,.x values for total phospholipid and It was expected that all radioactivity would be phosphatidylinositol fractions as 0.0086 and 0.005 recovered in the phosphatidylinositol fraction. myo-Inositol Deficiency Effect on H. diminuta 227 0.008 0.002 0.50 1.00 [Ss] 1.50 2.00 Fic. 3. Incorporation of *H-myo-inositol into the total phospholipid and phosphatidylinositol fractions by H. diminuta as a function of myo-inositol concentrations. V= umol/g wet weight per 4hr; S=myo-inositol concentration in mM; [=total phospholipid; a= phosphatidylinositol. 17S Fic. 4. Lineweaver-Burk plot of *H-myo-inositol incorporation into the total phospholipid and phosphatidylinositol by H. diminuta as a function of myo-inositol concentrations. V= ymol/g wet weight per 4 hr; S=myo-inositol concentration in mM; {j= total phospholipid; 4 = phosphatidylinositol. However, when lipid fractions were subjected to two-dimensional thin layer chromatography, 97.45% and 91.6% of the total phospholipid radioactivities were attributed to phosphatidylino- sitol at 0.05 mM and 0.1 mM of substrate concen- trations respectively. Whereas only 66.2%, 55.1% and 71.8% of the total phospholipid radioactivities were attributed to phosphatidylinositol at respec- tive 0.5mM, 1.0mM and 2.0mM of external myo-inositol concentrations (Table 1). It is evident from Table 2 that increasing incuba- tion time also increased total phospholipid and phosphatidylinositol syntheses from absorbed radioactive myo-inositol. However no change in phosphatidylinositol synthesis, expressed as per- cent myo-inositol incorporated into the phospholi- pid fraction, was noted. It was further observed that with increasing external myo-inositol concen- 228 TABLE 2. M.M. KHAN AND Y.K. Ip Rates of myo-inositol uptake; total phospholipid and phosphatidylinositol syntheses as a function of time at 0.1mM °*H-myo-inositol Phosphatidylinositol Phosphatidylinositol Total - synthesis synthesis ipi Incubation noted ene a total % ase is gee iinone (lat) pmol/g wet wt myo-inositol incorporated into the myo-inositol uptake phospholipid fraction uptake 1/4 0.049 0.279 37.60 0.742 1/2 0.073 0.366 61.70 0.592 0.112 0.615 39.10 ES OZ 2 0.221 0.616 37.50 1.640 4 0.447 0). 5) 48.10 1.144 trations the amount of radioactivity incorporated into the total phospholipid and phosphatidylinosi- tol fractions relative to the quantity of absorbed radio-myo-inositol decreased (Table 1). The total myo-inositol absorbed by the worm was measured as the sum of water-soluble and total phospholipid radioactivities. The total myo-inositol uptake appeared to increase with respect to concentration and incubation time (Tables 1 and 2). The recovery of radioactivities in free myo-inositol, during 1/4, 1/2, 1.0, 2.0 and 4.0 hr of incubation, were 12.3%, 15.59%, 14.3%, 14.4% and 10.96% of the absorbed myo-inositol in the water soluble fraction respectively at 0.1mM concentration. No attempt was made to identify the other radioactive metabolites. Glucose uptake In order to study biomembrane absorption activity in both groups of worms, glucose uptake studies were undertaken. It is evident from Table 3 that there was no apparent difference (P >0.05) in the absorption of '*C-glucose at 1mM, 2mM and 3mM of external glucose concentrations. Establishment, growth and reproduction of H. diminuta Deletion of myo-inositol from the diet appeared to have no effect on the establishment of the parasite in its host (Table 4). The results of the experiment on growth (Table 5) demonstrated that the worms from the hosts fed with myo-inositol deficient diet were comparative- TaBLeE3. Absorption of C-glucose by Hymenolepis diminuta Glucose Uptake velocity [ ~mol/g ethanol extracted dry wt/2min + S.E. (n)] concentration = 5 (mM) Control group myo-Inositol deficient group 1 WNW ae 13) (8) 1720027) 3 15.40 + 2.13 (3) 7/05) ae O79) 5) ISAs ae 0.97 () 1588) ae 2.32 (3) TABLE 4. Effect of myo-inositol on the establishment of Hymenolepis diminuta Average number Recovery Diet group Cysticercoids/rat of worms/rat % +S.E. (n) SEN), Lea Control 30 26.00 + 1.00 (3) COLOOni=Es ono myo-Inositol deficient 30 24.67+3.05 (3) 82.23+ 10.16 myo-[nositol Deficiency Effect on H. diminuta TABLES. Effect of myo-inositol on the growth of Hymenolepis diminuta Number of worms Total wet Average wet Average length Be eceroep per host weight (g) weight(mg) per worm (cm)+S.E. Control 34 0.996 29.29 18.56 + 3.38 Control 29 0.638 22.00 15241 =: 1-92 Control 30 0.872 29.06 171 a 2.355 myo-Inositol deficient 34 ES P45) 38.97 19.32 +186 myo-Inositol deficient 21 1.298 48.07 16.41 + 4.15 myo-Inositol deficient 30 dS 59.16 Pips Vis Ven 48 15 fo) ° ° x< = tO <= + n SS E — ° = 5 ~ om > LJ 17 18 19 20 21 ee 23 24 Days on experimental diets Fic. 5. Egg production of H. diminuta in control and myo-inositol deficient rats. &=control rats; [J myo-inositol deficient rats. Vertical bars around the points show standard errors. Each point represents mean of three determinations. ly heavier than those recovered from the hosts fed with control diet. This indicates that the growth of the worms was enhanced when myo-inositol was deleted from the host diet. No significant differ- ence (P >0.05) in the length of worms was found when the hosts were presented diets with or without myo-inositol. A comparison of rates of egg production from H. diminuta in rats fed with control or myo-inositol deficient diet is shown in Figure 5. Egg production in H. diminuta was remarkably higher when the rats were fed myo-inositol deficient diet. A considerable variation in daily egg output was observed. In the early period, a low level of egg output was noted in both groups of rats. A rapid rise in egg output was observed in both groups from the 18-21 day postinfection and stabilized with minor fluctuations thereafter. In the myo- inositol deficient rats, the H. diminuta egg output raised more rapidly than in the rats fed with control diet. The worms from the myo-inositol deficient rats gained patency 15 days postinfection. However, the patency was delayed by one day for worms parasitizing rats fed with control diet. Infectivity of H. diminuta eggs Beetles examination after a period of two weeks revealed no significant difference in the infectivity of H. diminuta eggs, obtained from both groups of rats. DISCUSSION The function of myo-inositol in higher verte- brates has been suggested as a vitamin, a phos- 230 M.M. KHAN AND Y.K. Ip phate storage agent, a phosphagen and the most important of all, as a component of phospholipids. Best et al. [28] reported the lipotropic function of myo-inositol in rats regardless of the type of fats used. It is therefore possible for myo-inositol to affect the physiology of H. diminuta through its functions discussed above. The present study confirms the observations of Ip and Fisher [2] that myo-inositol uptake in H. diminuta involves, at least in part, a mediated process (Table 1). The absorption of myo-inositol was observed to be time (Fig. 1) and concentration dependent (Table 1). In order to study the fate of absorbed myo-inositol, water soluble fractions obtained as a function of time were subjected to paper chromatography. Only 10.96% to 15.59% myo-inositol was recovered from these fractions indicating that it was being rapidly metabolised. Other radioactive metabolites were not identified. The incorporation of absorbed myo-inositol into the total phospholipid and phosphatidylinositol fractions were also linear with respect to time (Fig. 2). Total phospholipid and phosphatidylinositol syntheses, when expressed as percent synthesis of total myo-inositol uptake, revealed similar phe- nomenon (Table 2). However, there was no change in the amount of radioactivity of phospha- tidylinositol relative to the quantity of myo-inositol incorporated into the phospholipid fraction (Table 2). The incorporation of absorbed myo-inositol into the lipid fractions was also found to be concentration dependent (Fig.3). In order to study the incorporation of *H-myo-inositol into different classes of phospholipids, the lipid frac- tions were analyzed by thin layer chromatography. The recovery of phosphatidylinositol, when ex- pressed as percent synthesis of total myo-inositol uptake or of myo-inositol incorporated into phos- pholipid fraction, was highest at lowest myo- inositol concentration. It, however, decreased with the increase in external myo-inositol concen- tration (Table 1). At higher concentrations the lower recovery of phosphatidylinositol could be due to the metabolism of myo-inositol to some other compounds. H. diminuta can absorb and metabolize glucose [29-33]. Since myo-inositol is an important com- ponent of biomembrane, glucose uptake studies were therefore undertaken to examine the biomembrane activity in the worms. The results indicated that there was no difference in glucose uptake in worms under both conditions. Hence there may not be significant changes in the lipid components of the membrane that would affect the membrane transport processes. Ginger and Fair- bairn [1] reported synthesis of myo-inositol and its incorporation into phospholipids from glucose in H. diminuta. It is possible that the worm can maintain its membrane syntheses for growth and reproduction with its limited capability of forming inositol from glucose. Similarly, deletion of myo- inositol from the host diet appeared to have no effect on the establishment of the worms in its host. It is evident from Table 5 and Figure 5 that the growth and egg production of H. diminuta were enhanced with myo-inositol deficient diet. Such condition also resulted in the reduced time to their patency. Esotcott [6], Beadle [7] and Eagle et al. [9] indicated that myo-inositol was essential for the survival and growth of microorganisms. Stimula- tion of growth by myo-inositol has been observed in higher animals [8]. Hayashi et al. [34] reported that the growth rate of the rats maintained on the myo-inositol deficient diet did not differ from that of the control rats. Addis and Chandler [13] observed that the addition of myo-inositol in the basic diet, deficient in B; and Bz complex, did not significantly alleviate the stunting effect caused by such vitamin deficiency. However, such conclu- sions were drawn basing only on the length of the worms. The present study indicated that the growth and egg production were, in fact, markedly increased when myo-inositol was omitted from the host diet. This was in contrast to the results of earlier workers [6-9] who considered myo-inositol as a growth factor in microorganisms and higher animals. The stimulating effect of male dog bile on the egg output of H. diminuta in both male and female rats had been reported by Beck [15]. Goodchild [35] observed that bile was necessary for normal growth and development of adult H. diminuta in the rat host. Robert [36] reported that H. diminuta required bile salts for its normal development. It is now well established that triacylglycerol and myo-Inositol Deficiency Effect on H. diminuta 231 cholesterol levels in liver were markedly raised in the rats maintained on the basal diet without myo-inositol [34]. It is therefore possible that during the syntheses of bile acids and salts from the elevated cholesterol level in the liver, an alteration in the intestinal physiology favourable to H. diminuta might have occurred, which increased the growth and egg production of the worms. In the present study we found that both growth and egg production in H. diminuta were retarded in the presence of myo-inositol. However, it does not imply that H. diminuta has no requirement for myo-inositol. It is definitely an important compo- nent of biomembrane. Bly [37] reported increase of peristaltic activity in the stomach and small intestine of dogs in the presence of myo-inositol which may produce a transitry cathartic effect. Such effect may interfere with the growth and development of H. diminuta as a gut parasite. There are other vitamins which either have no effect on the establishment and growth of H. diminuta or retard its growth. Choline or pan- tothenate deficient diets have no effect on the establishment or growth of this worm [19]. Addi- tion of riboflavin in the host diet, however, causes retardation in its growth [19]. Although these vitamins do not favour the growth of this parasite, it still needs them as nutritional requirement [19]. Figure 5 displayed the pattern of egg production of H. diminuta in the presence or absence of myo-inositol from the host diet. The retardation of growth and in egg production in worms from rats fed with control diet as compared to those from myo-inositol deficient rats indicates the existence of a less favourable environment in the former hosts. The day to day variations in egg counts in both groups of rats were due to the shedding of gravid proglottids at irregular intervals. In the early period, 17 and 18 days postinfection, a low level of egg output occurred. It happened only when the worms were just beginning to shed gravid proglottids. The higher rate of egg production by worms from myo-inositol deficient rats was very obvious from the 18 days postinfection. However, the infectivities of H. diminuta eggs from both groups of rats were similar, indicating that myo- inositol deficiency in the diet neither affects its hatchability nor normal development. ACKNOWLEDGMENTS This project is supported by a grant RP 145/82 from the National University of Singapore. REFERENCES 1 Ginger, C.D. and Fairbairn, D. (1966) Lipid me- tabolism in the helminth parasites. Il. The major origins of the lipids of Hymenolepis diminuta (Cesto- da). J. Parasitol., 52: 1097-1107. 2 Ip, Y.K. and Fisher, F.M., Jr. (1982) Membrane transport of inositol by Hymenolepis diminuta (Ces- toda). J. Parasitol., 68: 53-60. 3 Ip, Y.K. and Fisher, F. M., Jr. (1982) Quantitative determination of inositol by Hymenolepis diminuta (Cestoda). J. Parasitol., 68: 593-598. 4 Michell, R.H. (1979) Inositol phospholipids in membrane function. Trans. Biochem. Sci., 4: 128- it 5 Webb, R.A. and Mettrick, D.F. (1971) Pattern of incorporation of **P into the phospholipids of the rat tapeworm Hymenolepis diminuta. Can. J. Biochem., 49: 1209-1212. 6 Esotcott, E. V. (1928) Wilder’s Bios, the isolation and identification of “Bios I”. J. Phys. Chem., 32: 1094-1111. 7 Beadle, G. W. (1944) An inositolless mutant strain of Neurospora and its use in bioassays. J. Biol. Chem., 156: 683-689. 8 McIntire, J.M., Schevergerl, B.S. and Eluejhem, C.A. (1944) The nutrition of the cotton rat (Sigmodon hispidus hispidus). J. Nutr., 27: 1-9. 9 Eagle, H., Oyama, V.I., Levy, M. and Freeman, A.E. (1956) myo-Inositol as an essential growth factor for normal and malignant human cells in tissue culture. Nature, 128: 845-847. 10 Mettrick, D.F. and Podesta, R. B. (1974) Ecologi- cal and physiological aspects of helminth-host in- teractions in the mammalian gastrointestinal canal. In “Advances in Parasitology”. Ed. by B. Dawes, Academic Press, London, 12: 183-278. 11 Hager, A. (1941) Effects of dietary modifications of host rats on the tapeworm Hymenolepis diminuta. Iowa State Coll. J. Sci., 15: 127-153. 12 Chandler, A.C. (1943) Studies on the nutrition of tapeworms. Am. J. Hyg., 37: 121-130. 13. Addis, C. J., Jr. and Chandler, A. C. (1944) Studies on the vitamin requirements of tapeworms. J. Parasitol., 30: 229-236. 14 Addis, C. J., Jr. and Chandler, A. C. (1946) Further studies on the vitamin requirements of tapeworms. J. Parasitol., 32: 581-584. 15 Beck, J.W. (1952) Effect of diet upon singly established Hymenolepis diminuta in rats. Exp. 16 7 18 19 20 21 22 23 24 Mi) 26 i 232 Parasitol., 1: 46-59. Roberts, L.S. and Mong, F,N. (1973) Develop- mental physiology of cestodes. XIII. Vitamin Be requirement of Hymenolepis diminuta during in vitro cultivation. J. Parasitol., 59: 101-104. Hall, A. (1983) Dietary protein and the growth of rats infected with the tapeworm Hymenolepis dimi- nuta. Br. J. Nutr., 49: 59-65. Keymer, A., Crompton, D.W.T. and Singhvi, A. (1983) Mannose and the “crowding effect” of Hymenolepis in rats. Int. J. Parasitol., 13: 561-570. Platzer, E.G. and Robert, L.S. (1970) Develop- mental physiology of cestodes. VI. Effect of host riboflavin deficiency on Hymenolepis diminuta. Exp. Parasitol., 28: 393-398. Read, C.P., Rothman, A.H. and Simmons, J.E., Jr. (1963) Studies on the membrane transport, with special reference to parasite-host integration. Ann. N.Y. Acad. Sci., 113: 154-205. Folch, J., Lees,M. and Sloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Read, C.P., Schiller, E.L. and Phifer, K. (1958) The role of carbohydrates in the biology of cestodes. V. Comparative studies on the effects of host dietary carbohydrates on Hymenolepis spp. Exp. Parasitol., 7: 198-216. Holmes, J.C. (1970) The effect of crowding on fecundity of Hymenolepis diminuta . In “Experi- ments and Techniques in Parasitology”. Ed. by A. J. Macinnis and M. Voge, D. H. Freeman and Co., San Francisco, pp. 49-50. Voge, M. (1970) Laboratory maintenance of para- sites. In “Experiments and Techniques in Parasitolo- gy”. Ed. by A.J. Macinnis and M. Voge, D.H. Freeman and Co., San Francisco, pp. 130-131. Gray, H.E. and Fraenkel, G. (1953) Fructomal- tose, a recently discovered trisaccharide isolated from honeydew. Science, 118: 304-305. Trevelyan, W.E., Procter, D. P. and Harrison, J.S. (1950) Determination of sugars on paper chromato- grams. Nature, 166: 444-445. Rouser, G., Kritchevsky, G. and Yamamoto, A. 28 2) 30 Sit 32 33 34 35 36 37 M. M. KHAN AND Y.K. Ip (1967) Column chromatographic and associated procedures for separation and determination of phosphatides and glycolipids. In “Lipid Chromatog- raphic Analysis”. Vol. I. Ed. by G. Y. Marinetti, Marcel Dekker Inc., New York, pp. 99-162. Best,C.H., Ridout,J.H., Patterson,J.M. and Lucas, C.C. (1951) A statistical evaluation of the lipotropic action of inositol. Biochem. J., 48: 448- 452. Laurie, J.S. (1957) The in vitro fermentation of carbohydrates by two species of cestodes and one acanthocephala. Exp. Parasitol., 6: 245-260. Read, C. P. and Rothman, A. H. (1958) The role of carbohydrates in the biology of cestodes. VI. The carbohydrates metabolized in vitro by some cy- clophyllidean species. Exp. Parasitol., 7: 217-223. Phifer, K. (1960) Permeation and membrane trans- port in animal parasites : The absorption of glucose by Hymenolepis diminuta. J. Parasitol., 46: 51-62. Phifer, K. (1960) Permeation and membrane trans- port in animal parasites : Further observations on the uptake of glucose by Hymenolepis diminuta. J. Parasitol., 46: 137-144. Overturf, M. (1966) Jn vivo and in vitro uptake and distribution of C'*-glucose by Hymenolepis diminu- ta. Comp. Biochem. Physiol., 17: 705-713. Hayashi, E., Maeda, T. and Tomita, T. (1974) The effect of myo-inositol deficiency on the lipid meta- bolism in rats. I. The alteration of lipid metabolism in myo-inositol deficient rats. Biochim. Biophys. Acta, 360: 134-145. Goodchild, C. G. (1958) Growth and maturation of the cestode Hymenolepis diminuta in bileless hosts. J. Parasitol., 44: 352-362. Roberts, L. S. (1980) Development of Hymenolepis diminuta in its definitive host. In “Biology of the Tapeworm Hymenolepis diminuta”. Ed. by H.P. Arai, Academic Press, New York, pp. 357-423. Bly,C.H., Heggeness,E.W. and Naset, E.S. (1943) The effect of pantothenic acid and inositol added to whole wheat bread on evaculation time, digestion and absorption in the upper gastrointesti- nal tract of dogs. J. Nutr., 26: 161. ZOOLOGICAL SCIENCE 4: 233-242 (1987) Morphometrical Features of Rod Outer Segments in Relation to Visual Acuity and Sensitivity in the Retina of Rana catesbeiana Y OSHIHIKO TSUKAMOTO Department of Anatomy, Hyogo College of Medicine, Mukogawa, Nishinomiya, Hyogo 663, Japan ABSTRACT— Morphometry of the rod outer segment in the bullfrog retina was performed to clarify the structural bases of functional local specialization along the vertical meridian. The length, cross-sectional area, and density of the outer segments showed distinct differences between the red and green rods, between the central and peripheral parts, and between the dorso-central and ventro-central portions. The outer segments of red rods are always longer than those of neighboring green rods in all locations, while the cross-sectional areas are nearly equal. The red and green rod outer segments are smaller in length and in cross-sectional area, and higher in density at the periphery than at the center, in both the dorsal and ventral retinas. On the other hand, when measured at about 2 mm from the optic disc, they are larger in length, smaller in cross-sectional area, and higher in density at the dorso-central retina than at the ventro-central retina. At the dorso-central “area centralis”, although the diameter of the rod outer segment decreases (being accompanied with the increase in density), the increase in length complements the decrease in diameter. Since the volume of the outer segment decreases little, visual acuity is thought to increase with almost no decrease in sensitivity. The above findings are substantiated by observations of the thickness and angular spread of the retina using whole eyes embedded in © 1987 Zoological Society of Japan celloidin. INTRODUCTION The bullfrog retina has been noted to have local specialization. The content of visual pigments is two the three times greater in the dorsal than in the ventral retina [1,3]. Vitamin A,-based pigments are much denser in the dorsal retina (porphyropsin zone) [1-4]. The consequent changes in the spectral sensitivity are considered to adapt to the environmental light [1]. In addition, the environ- mental influences on the bullfrog retina are thought to differ in light intensity between the dorsal and ventral halves. This is suggested by the fact that macrophages in the subretinal space are more abundant in the ventral than in the dorsal retina [5]. The area centralis is shaped like a crescent and Accepted October 29, 1986 Received September 19, 1986 lies above the optic disc in Rana [6,7]. The topographic map of ganglion cells indicates that the area centralis extends horizontally like a streak in the dorso-central part of the bullfrog retina. The peak densities of photoreceptors and ganglion cells are located about 2 mm dorsal to the horizon- tal meridian [8]. This may largely contribute to the functional differences between the dorsal and ventral retina of the bullfrog. Although the density of photoreceptors is re- lated to visual acuity, it does not offer any information about sensitivity. The morphological parameter related to sensitivity is the volume of the photoreceptor outer segment which contains the visual pigments. It has been recently shown that the area with the highest concentration of visual pigments is located 3-4mm just dorsal to the optic disc and is shaped like a semicircular band [3]. Since visual acuity and sensitivity are both important functions but generally contradic- 234 Y. TSUKAMOTO tory, the relationship of them in the bullfrog area centralis is interesting. In this report, first, the retinal thickness and angular spread are surveyed using bullfrogs with different sizes. Second, the length, cross-sectional area, cell density and other parameters of the red and green rod outer segments (ROS) are examined along the vertical meridian of the retina. It will be shown that in the area centralis the larger length of the outer segment compensates its smaller cross- sectional area, thus keeping its volume constant. MATERIALS AND METHODS Preparations and preliminary fixation Adult bullfrogs (Rana catesbeiana), 12-14cm in length, were used under room light after more than 5 hr of illumination. Two syringe needles, passing through the anterior and posterior poles of the cornea, were inserted as far as the vitreous body, while the eyeball was held by the upper jaw, which had been removed by decapitation. The intraocular perfusion of the fixative was carried out with a syringe, using one of the two holes as an inlet, the other as an outlet. After 10 min of this preliminary fixation, the eye was gently enucle- ated. One of two different fixative solutions was used from this step, as described below. Fic. 1a. Photomicrograph of vertical section of a bull- frog left eye, embedded in celloidin, cut into a 20 ~m thick section, and stained with Cason’s Mallory Heidenhain. Upper side is dorsal. Whole eyes embedded in celloidin For embedding the whole eye in celloidin resin, a solution of 3% glutaraldehyde, 1% paraformal- dehyde, 2% CaCl), and 2% MgCl, in 0.1M cacodylate (pH 7.4) was used as the fixative. The eye was immersed in the fixative overnight follow- ing the above-mentioned preparation. It was dehydrated in ethanol with graded percentages, for one day at each concentration, and substituted by a dehydrated ethyl ether and ethanol mixture. After immersion in 2%, 4%, 8% celloidin for 3-4 weeks each, the sample was transferred to 10% celloidin which was gradually hardened for 3 months. The eye was vertically sectioned with a steel knife, and 20-30 um thick sections passing through the optic disc along the meridional line were obtained. The sections were stained accord- ing to Cason, a modification of Azan staining (Mallory-Heidenhain). 10.4mm on |\ =_ $$ __________ 9.5mm Fic. 1b. Diagram of vertical section of the bullfrog eye shown in Fig. la. R, retina; Ch, choroid; S, Sclera; SC, scleral cartilage; B, blood vessel; OD, optic disc; ON, optic nerve; Co, cornea; I, iris; CP, ciliary pappilla; OS, ora serrata. Most part of the cornea, the anterior and posterior lens surfaces, and the retina are regarded as arcs of the circles having their centers on the optic axis (horizontal line). Their radii (r) are shown in units of mm. The intersection of the retina and the optic axis is regarded as the retinal center. Optic disc is located 0.3mm ven- trally. Morphometry of Bullfrog Retinal Rods 235 After the eyeballs were embedded in celloidin, the horizontal and vertical diameters of the sclera showed only 1-3% of shrinkage. The retina, which is stiffly attached to the choroid and sclera complex, is thought to hardly change in length in the tangential direction. However, it was possible that a considerable shrinkage might occur in a radial direction. Pieces of retina embedded in Araldite For embedding pieces of retina in epoxy-resin, 2% OsQO, in 0.1 M phosphate buffer (pH 7.4) was used as the fixative. After the above-mentioned preparation, the anterior parts of the eye were removed. The posterior hemisphere was divided into quadrants by horizontal and then vertical sectionings. Tissues were furthermore immersed in the fixative for 1.5 hr. Strips of retina (1.5- 2mm in width) along the meridional line were cut out from the dorsal and ventral halves. Each strip was cut into 5—7 rectangular pieces. One angle of each piece was cut off for reference of direction. These pieces were numbered, separately dehy- drated and embedded in Araldite. Three retinas from different frogs, denoted as retina 1 (right eye), 2 (right eye), and 3 (left eye) in the follow- ing, were used for morphometry of the ROS along the vertical meridian. The sections of 0.5 um in thickness were cut in radial (retina 1 and 2) and tangential (retina 1 and 3) directions, and stained TaBLE 1. Comparison in parameters of occular apparatus between two bullfrogs A and B at different growth stages body length and weight meridional length of the retina angle encompassing the retina radius of curvature of the retina eye ball: equatorial dia. ——+_—— =ratio axial dia. lens: equatorial dia. ; ——+_—__——— =ratio axial dia. mma ot la STR RR REN IRAP A = Frog A Frog B 12cm and 200g 14cm and 300g 16.7 mm 19.2 mm 173° 188° 5.4mm 5.7 mm 10.4 mm 11.5 mm 9.5mm Se 10.5 mm ck 6.3 mm 5.6mm 41mm a 3.7 mm HLS =e Fic. 2. Parts of the retina shown in Fig. 1a, dorsal (a) and ventral (b) to the optic disc. Abbreviations are the same as in Fig. 1b. The thickness of the retina, indicated by bars, is about 2 times greater in the dorsal than in the ventral. 236 Y. TSUKAMOTO Fic. 3. Light micrographs of ROS in longitudinal sec- tion taken from three different areas in a retina; (a) about 2.5 mm ventral to the center, (b) about 2 mm dorsal to the center, and (c) close to the dorsal ora serrata, at the same magnification. R, red rod; G, green rod. with toluidine-blue for light microscopy. In par- ticular, for the retina 1, radial sectioning was followed by reembedding and tangential section- ing. Ultrathin sections were also obtained for electron microscopy with the cutting plane trans- verse to the ROS. These sections were stained with uranyl acetate and lead citrate. During processing of Araldite embedding, the retina was estimated to shrink 7-12% in a tangen- tial direction. Microscopy and measurements Zeiss Ultraphot and Olympus Vanox light microscopes were used with plan-apochromat objective lenses of 20 x, 40 x, 100 x (oil). Sec- tions of the whole eye were photographed with a Nikon Multiphoto camera. An electron micro- scope (JEM-100CX) was used at 80kV. Morphological measurements were carried out on enlarged photographs. The length of tortuous lines, and the sectional area of cells, were calcu- lated with a microcomputer (Sord M243) linked to a digitizer (Summagraphics, Bit Pad One). All the data are presented in this paper with no correction for shrinkage. RESULTS Morphology of the whole eye The vertical section of a whole eye gives general morphology of the ocular apparatus of the bullfrog eyeball (Fig. 1). Morphometric parameters of the left eyes of two male bullfrogs with body lengths of 12 and 14cm are compared (Table 1). The inner aspect of the sclera is made of hyaline cartilage. This cartilage is thickest at the posterior pole and extends anteriorly close to the equatorial plane. The eye and its lens are greater in the frog with the greater body weight [9]. The meridional length of the retina is also greater in the larger eye. This is based on two factors. The radius of curvature of the retina and the angle encompassing the retina are both greater in the larger eye. On the other hand, the ratios of the equatorial versus the axial diameter in the eyeball and in the lens are almost constant. Their shapes are regarded to be homolo- gous. Morphometry of Bullfrog Retinal Rods 237 70 ; @ 4 Red rods ti aN a \ = Rd > a b oN < 40 te eset . = i : : / : 60130 ‘ © 4 Green rods Ps 20 ° E o—~ n es 200 pee a Spam a 2) eee be tee aes eee ——— cs 100 od- A @-@A ra =a fe) 7 5 3 1 1 3 5 7 9 (Dorsal) (Ventral) Distance from the center (mm) Fic. 4. Upper) Vertical variations in the length of red (closed) and green (open) ROS in retinas 1 (circle) and 2 (triangle) from two bullfrogs. The maximal length is given at about 2 mm dorsal to the center, such as retina 1: red=62+4, green=49+3, and retina 2: red=66+3, green=52+2. The length is prominently short at 1-3 mm ventral to the center, such as retina 1: red=37+2, green=23+3, and retina 2: red=46+4, green =36+3. It becomes shorter toward the periphery, e.g. reaching 21 +3 (retina 1, red) at 7.4 mm ventral to the center, and 24+3 (retina 1, green) at 7.7 mm dorsal to the center. (Mean +standard deviation in units of ym. The number of counts is 20 for red rods and 10 for green rods at each position). The margins are located 8.4 mm dorsal and ventral to the center in both retinas 1 and2. Lower) The thickness of the retina is greatest at about 2 mm dorsal to the center, such as retina 1: 220 wm, retina 2: 240 um. It has the smallest value of about 100 um at the margin. The data are obtained from the retinas embedded in Araldite. Retinal thickness and the length of ROS The thickness of the retina is given by the distance from the inner limiting membrane to the basement of the pigment epithelium. The retina has the greatest thickness at about 2mm dorsal to the center, which is one and a half to two times larger than that at about 2mm ventral to the center (Figs. 2 and 4, below; the retinal center is defined as the intersection of the retina and the optic axis as shown in Fig. 1b). The retina becomes thinner toward the periphery. The discrimination of the red and green ROS in radial section is based on the differences in their lengths and the position of their basal ends; the green ROS is shorter and located to the scleral side as compared with the red ROS (Fig. 3). Since it is often difficult to find the green ROS near the ora serrata, no data are displayed for them in this location. The lengths of the red and green ROS are also greatest at about 2mm dorsal to the center, and prominently short at 1-3mm ventral to the center. Their lengths become shorter toward the periphery, reaching the minimal values at the margin (Fig. 4, upper). The cross-sectional area and the density of ROS Cross sections of the red and green ROS are observed by electron microscopy at the height where they both appear. The sectional planes have been adjusted so as to be perpendicular to the long axis of the ROS. The red and green ROS in cross section can be discriminated on electron micro- graphs by cues of the number and depth of disc 238 Y. TSUKAMOTO Fic. 5. Electron micrographs of ROS in cross section taken from three different areas in a retina; (a) about 3 mm ventral to the center, (b) about 2mm dorsal to the center, and (c) near the dorsal ora serrata, at the same magnification. R, red rod; G, green rod. incisures [10]; the disc incisures of the red rod are greater in number and longer in depth than those of the green rod (Fig. 5). The sectional areas of the red and green ROS are prominently small at 2-3mm dorsal to the center, showing valley-like curves in the vertical variation. In contrast, these values are greatest at 2-4mm ventral to the center. Their sectional areas tend to become smaller in more peripheral portions, reaching the minimal values at the margin (Fig. 6). The densities of the red and green ROS have peaks at 2-3mm dorsal to the center. On the other hand, their densities are almost constant in the ventral retina. Extremely close to the margin, it is often difficult to discriminate the green rods from the red ones. The density totaling both types of rods tends to increase sharply near the ora serrata in both the dorsal and ventral retinas (Fig. 7, upper). No prominent differences are found in the density ratio of red rods to green rods (R/G ratio) between the dorsal and ventral retinas. While the R/G ratio varies moderately from 5.4 to 7.3 in the central retina, it has a tendency to increase to some extent close to the margin (Fig. 7, below). DISCUSSION Features in relation to the bullfrog growth The body length and the eye-lens weight of bullfrogs are parameters showing the degree of their growth [9,12]. Clear correlations are observed by Makino-Tasaka et al. [3] between the body length and the contents of protein, visual pigment, and dopamine in the bullfrog retina. In the present study, also, both the eyeball and the retina are greater in size in the larger bullfrog. The growth of the retina seems to be related to the increases in both the radius of curvature and the angle encompassing the retina. An increase in the angle encompassing the retina means that the ora serrata moves anteriorly. This suggests that the retinal field in the vicinity of the ora serrata is capable of growing [9]. The ROS small in both length and cross-sectional area are present in the extreme peripheral retina. These ROS are thought to be in the process of growing. Adult bullfrogs Morphometry of Bullfrog Retinal Rods 239 range from 12 to 18cm in body length [12]. Since relatively juvenile adult bullfrogs (12 and 14cm) are used for the present study, it is reasonable that their organs have some growing activities. According to Duke-Elder [6], the lens in the tadpole, like that of fish, is spherical and close to the cornea; in the adult frog it moves posteriorly and becomes somewhat flattened in an antero- posterior direction. The value of 1.3 has been quoted as the ratio of the equatorial versus the axial diameter of the lens in a certain kind of frog (Rabl, 1898 in [6]). The value of 1.5 is obtained as this ratio for the bullfrog lens in the present study. 50 Sectional area ( pm?) Ww Oo 20 10 50 ro £ i 40 5 = oe 30 G 5 ae 20 VU 7) é 10 sf The bullfrog lens is distinctly far from a sphere; its anterior surface is prominently flat. The volume of ROS and the content of visual pigment The data represented by filled circles in Figures 4 and 6 of both the length and cross-sectional area of the ROS were derived from the same retina (retina 1). The product of the mean length and the mean cross-sectional area of the red ROS at each position on the meridional line of the retina gives the mean volume. In the central half of the retina, the mean volumes of the red ROS approximately wane ied is ®@ #® Red rod oo Green rod 9 7 5 3 (Dorsal) 1 1 3 5 7 9 (Ventral) Distance from the center (mm) Fic.6. Vertical variation in the sectional area of red (closed, upper) and green (open, lower) ROS in retinas 1 (circle) and 3 (rectangular) from different bullfrogs. The maximal area is given at 2-5 mm ventral to the center, such as retina 1: red=36+4, green=38+4, and retina 3: red=47+7, green= 47+4. The area is prominently small at 2~3 mm dorsal to the center, such as retina 1: red=24+3, green=25+3, and retina 3: red=22+3, green=18+4. It becomes smaller toward the periphery, e.g. reaching 9+2 (retina 1, red) at 7.4 mm ventral to the center, and 13+3 (retina 3, green) at 8.4 mm dorsal to the center. The margins are located 8.9 mm dorsal and ventral to the center in the case of retina 3. (Mean+standard deviation in units of ym?*. The number of counts is 20 for red rods and 10 for green rods at each position). 240 Y. TSUKAMOTO x10 60 e ew Red rods 50 (=| ) i \ = SS SO (Tp) O ag 10F oo Green rods cc) Metter ees ae a— 8 0 0 gg —05,—0n Se) 7 5 3 (Dorsal) © 11 reat iG a an Oe) fa Py 5 % ——=- o> ad __—— © —_ ea — et? ee | { 1 3 5 7 9 (Ventral) Distance from the center (mm) Fic. 7. Upper) Vertical variation in the density of red (closed, upper) and green (open, lower) ROS in retinas 1 (circle) and 3 (rectangular). The maximal density is given at 2-3 mm dorsal to the center, such as retina 1: red=28, green=S, and retina 3: red=29, green=4.5. The density is rather small at 2-5 mm ventral to the center, such as retina 1: red=17, green=2.5, and retina 3: red=13, green=2. It sharply increases at the extreme margin, where discrimination of the green rod from the red rod is difficult. Therefore the values totaling both types of rods are displayed with half-closed symbols, e.g. reaching 45 (retina 3) at 8.4mm dorsal to the center, and 65 (retina 1) at 7.4 mm ventral to the center. (units: 10°/mm7). Lower) The ratio of red to green rods ranges from 5.4 to 7.3 between the dorsal and ventral locations about 5 mm distant from the center. It increases near the margin, e.g. reaching 12 (retina 3) at 6.9 mm ventral to the center. range from 1,500 to 1,800 um* (Fig. 8, upper). Their differences, less than 20%, are rather small compared with those of the mean lengths and sectional areas. When the mean volume of the red ROS is multiplied by the mean density at each position given in Figure 7, the product indicates the red ROS volume per unit area (mm?) (Fig. 8, lower). The pattern illustrated by these values is quite similar to the meridional profile in the topographic map of visual pigment in the bullfrog retina [3]. The red ROS volume per unit area is about two times as great in the dorsal as in the ventral areas. This coincides with the observation of Reuter et al. [1] that the number of visual pigment molecules per unit area is two to three times as great in the dorsal as in the ventral areas, assuming the relation that the content of visual pigment is proportional to the ROS volume. The area centralis The ROS volume can be considered to have a close relationship to visual sensitivity. On the other hand, the density of the ROS is one of the determining factors of visual acuity. The ROS are “slender” and high in density in the dorsal areas but are “stocky” and low in density in the ventral areas of the bullfrog retina. In the area centralis a few millimeters dorsal to the optic disc, the ROS show a peak in density and are reciprocally small in sectional area. Since the increase in length com- plements the decrease in cross-sectional area, the volume of the ROS decreases little. It is suggested that visual acuity increases with almost no decrease Morphometry of Bullfrog Retinal Rods 241 20 s @ x 10 ° 400 Z50 500 550 600 Wavelength, nm Fic. 5. Typical recordings showing spectral reflectance changes with the progress of coloring response of iridophores elicited by electrical nervous stimulation (biphasic square pulses, 10 V in strength, 1.0 msec in duration, 1 Hz). Spectral curve A was recorded in physiological saline, while those from B to H were obtained at 12, 29, 35, 41, 46, 50 and 60 sec after the initiation of stimuli, respectively. The wave peak moved from 372 to 388, 404, 420, 440, 458, 482 and finally to 510 nm, progressively. Arrows indicate positions of the peaks of those spectral reflectance curves. 100 bis 7) c ° Q @ 50 '2)) & ie fe) O 0 350 400 450 500 550 Wavelength, nm Fic. 6. Relationship between the spectral peak wavelength and the magnitude of coloring response of damselfish iridophores. Each plot was obtained on different split-fin preparation. The coloring re- sponses at varying levels were induced by nor- epinephrine, tolazoline or increased K* at various strengths. Abscissa, wavelength of the spectral peak in nm. Ordinate, magnitude of response as a per- centage of the level of full coloring response attained during a 5-min application of 5x 10-°M_nor- epinephrine at the end of each series of measure- ment. to the position of the spectral peak. The measure- ments were made by the monochromator system, and the results were shown in Figure 6, where the magnitude of the coloring response was plotted against the wavelength of the peak. The linear regression was calculated as follows: y=0.655x—251 where y is the grade of the coloring response in percentage, and x is the wavelength of the peak in nm. It was found that, while y proceeds from 0 to 100, x changes from 383 to 536. Then, we studied the effect of changing the angle of incidence of light hitting the plane of the body surface on the spectrum of the reflected light. The skin preparations in which iridophores assumed the maximally and sustained colored state by treating with 5x10-°M norepinephrine were exclusively employed, since the strongest and stable reflectivity could be obtained. In the actual measurements using the MCPD-100 system, the inclination of the skin piece was appropriately varied in reference to the direction of light-rays emitted from the rigidly fixed light-guide setup. When the incident rays were perpendicular to the 250 H. KasuKkKawa, N. OSHIMA AND R. FuJII plane of the skin preparation, the angle of inci- dence was justly defined as 0°. In this particular series of measurements, the spectral peak wavelength in this geometrical condition was found to be 530nm, as exhibited in Figure 7. The incident angle was then increased up to 40°. Between each group, one way analysis of variance showed no significant difference. That is, the spectral peak of the reflected light was found to stand practically unmoved disregarding the angle of incidence. 950 R ro) Wavelength, nm on WwW ro) ae a a ) 10 20 30 40 Incident angle, degree Fic. 7. Relationship of the angle of incident light-beam to the spectral peak wavelength of the light reflected from iridophores in a damselfish skin piece. A full coloring response of the cells had previously been induced by 5X10-°M norepinephrine. Angle of incidence varied from 0 to 40°. Each point is the mean of 5 measurements on different animals. Ver- tical lines indicate SE. Hyposmotic effect on iridophore reflectivity As expected, a decrease in the osmolarity of the solution bathing a skin preparation induced color- ing of the iridophores. For example, the iri- dophores exhibited a moderate coloring response, when the normal saline (282.4mOsm) was re- placed with a little hyposmotic solution of 191.8 mOsm, as displayed in Figure 8. The rate of the response to the hypotonicity, however, was signif- icantly lower than that elicited by sympathomimet- ic substances of supramaximal strengths in the normal saline. In the left part of this particular recording, the rapid and pronounced response to norepinephrine at 5X10 °M is exhibited as a control. The hypotonicity (191.8 mOsm)-induced response shown around the middle was about half as large as the maximal level caused by the amine of the mentioned strength. With a further lower- ing of the tonicity of the media, the magnitude of the response progressively increased, and the response became even larger than that attainable by the amine alone (control response), when the tonicity was lowered to 151.8 mOsm (cf. Table 1). Using the MCPD-100 system, then, the effects of the hyposmolarity on the spectrum of light reflected from the iridophores were investigated more quantitatively. The data obtained were summarized in Table 1. Evidently, the spectral peak shifted towards the longer wavelengths as the tonicity was decreased. Within the region of the tonicity decrease exhibited in Table 1, the phe- nomenon was quite reversible and reproducible, although a further decrease in it infallibly caused the rupture of the cells, resulting in the irreversibil- ity of the phenomenon. The lowest tonicity within the limit of reversibility was as low as 71.8mOsm. At that time, the peak was rising at about 600 nm, and the skin piece assumed yellow with a dash of orange color. As referred to in Tablel, the spectral peak in the maximally colored state induced by norepinephrine was about 530nm (green). By lowering the tonicity of the bathing fluid, therefore, the spectral peak could be brought into the region much longer in wavelength than when norepinephrine alone was added to the normal saline. So far, alpha adrenoceptors have been known to be solely responsible for mediating physiologically the coloring response of the cells [9]. In the next trial, therefore, influences of adrenergic blocking agents of alpha type, i.e. phentolamine and phe- noxybenzamine, on the coloring response-inducing effect of hypotonicity were examined. The results were that neither of them had any influence. These observations apparently exclude the possi- bility that the hypotonic effect is mediated by adrenoceptors or by the liberation of adrenergic neurotransmitter from the neural elements around the effector cells. Light Reflection in Damselfish Iridophore TABLE 1. 251 Effects on the wavelength of the spectral peak reflected from motile iridophores of the blue damselfish, Chrysiptera cyanea, of the hypotonicity of the medium, and the influence of norepinephrine (NE) and adenosine (AS) on the former’s action [Na‘] in test in te Tonicity Wavelength of spectral peak** (nm) solution Gan) (mM) (mOsm In simple saline +10-*MAS +5X10-°MNE 1253 (Caen 282.4 376.0+5.48 372.0+ 4.47 534.44 4.39 100.0 231.8 434.8+ 10.38 422.4+11.35 546.8+ 2.77 80.0 191.8 492.2+16.77 463.8+ 13.65 5962225) 3:03 60.0 ones 562.4+ 6.35 542.0+ 13.65 574.44 3.91 40.0 111.8 581.8+ 5.40 588.0+ 3.39 SPA tae 7 Pre, 20.0 71.8 596.4+ 7.92 586.8+ 8.79 Sw )s).4ae IMO co * Other components (mM): KCI 2.7, CaCl, 1.8, MgCl, 1.8, p-glucose 5.6, Tris-HCl buffer 5.0 (pH 7.2). ** Each value is the mean of 5 measurements followed by SE. F aly ° © 100b | Qa 1) = | =) EO) 2min, 4 5 (eo) teen : -6 -6 NE .5X10_|M : 5X10, M 5X10 _M AS om Tomicityme. 262-6mOsm. 2 SB mOSMm eT 28.214 mOsm, Fic. 8. Typical recording showing the coloring response-inducing action of the hypotonicity (191.8 mOsm) on damelfish motile iridophores, and the influences of 10 * M adenosine (AS) and 5 x 10° M norepinepherine (NE) added to the same medium. Abscissa: time. Ordinate: magnitude of coloring response in percentage, taking maximal coloring response to 5 X 10°-°M NE in the normal saline (282.4 mOsm) as 100%. Norepinephrine and adenosine effects in hypoto- nicity We have already shown that either the coloring response or the reverse process, i.e. the clearing response, of the motile iridophores was induced by the true neurotransmitter, norepinephrine, or the co-transmitter, adenosine, from the same fibers, respectively [9]. In solutions, whose osmolarity was lowered variously, therefore, the effects of those neural substances were examined. The photoelectric recording displayed in Figure 8 in- cludes parts showing their effects under a hypoto- nic condition. Again, the maximal coloring re- sponse to 5X10-°M norepinephrine seen in the left part could be employed as the control, which was followed by a moderate coloring response induced by changing the medium to a hyposmotic saline (191.8 mOsm). Then, 10~*M adenosine was added to the same medium. A large clearing response developed quickly. The nucleoside’s effect was quite reversible. Then, 5x10°°M norepinephrine was added to the hypotonic saline. The moderately colored state was soon augmented to the level apparently beyond that attained by the amine of the same strength in the isotonic saline. The results of a number of such measurements were Statistically treated and summarized in Table 1. Evidently, the spectral curves shifted towards longer wavelengths as the tonicity of the medium was lowered. It is also clear that both adenosine and norepinephrine retained their actions even in the hypotonic media. The effects of adenosine was the most remarkable in 191.8-mOsm saline, and Da H. Kasukawa, N. OSHIMA AND R. Fuym fell to some degree upon further decrease in the tonicity. On the other hand, the shift of the spectral peak by norepinephrine decreased in proportion to the decrease of the tonicity. At osmolarities below 111.8 mOsm, neither adenosine nor norepinephrine showed influences. DISCUSSION Reviewing past morphological and functional observations as well as theoretical treatments of the relevant optics, Huxley [16] and then Land [7] elaborately discussed the light reflection from multilayered structures often found in the surface specializations of many animal species. The reader may refer to these articles especially when a certain phenomenon in which he or she is in- terested must be dealt with theoretically. In the case of the multilayered thin-film system, the highest reflectivity at a given wavelength (A) is known to be actualized when the alternately arranged, optically “less dense” (with suffix “a’’) and “denser” (“b”) layers have the same and definite optical thickness (i.e. the refractive index (n) multiplied by the actual thickness (d)), which equals a quarter of wavelength of the reflective ray. That is, the so-called “ideal” multilayer interference occurs when both n,d, and n,d, are equal to A/4. The interference under such condi- tions should truly be constructive, yielding an extremely effective reflective surfaces. For in- stance, the architecture of the reflecting stack in the iridophores of a clupeid fish has been shown to fit this “ideal” system [17]. Applying the interfer- ence microscopy to the iridophore inclusions from a sprat scale, Denton [18] actually showed that the platelets had an exceedingly uniform thickness of ca. 100nm, and that the cytoplasmic sheets be- tween the crystals were about 135nm thick. The optical thickness of either layer was shown to be practically identical, being about 180 nm. The above authors have also treated of the “non-ideal” system, where n,d, is not identical with n,d, [7,16]. In addition, Bone and Denton [19] and Denton and Land [20] have presented observations on teleostean iridophores which may be explainable in terms of the “non-ideal” interfer- ence phenomenon. For example, the latter investi- gators reported that the spectral peak of light reflected from the iridophores shifted from ca. 650nm to ca. 515nm, when the bathing normal saline for marine teleosteans was replaced with the solution in which the salt concentrations were doubled. The tonicity change should not have influenced the thickness of the crystalline platelets. Thus, the optical change may safely be ascribable to the dehydration-induced thinning of the cyto- plasmic layer sandwitched between the platelets. They further claimed that, when the cells were equilibrated in the isotonic saline, the situation was actually “ideal”. Upon exposure to hyperto- nicity, the system proceeded to a non-ideal state, where the decreased n,d, naturally became un- equal to n,d,. These changes, however, are by no means physiological, but are experimentally in- duced processes. Based mainly on the fine structural observations on the iridophores of the present material, the blue damselfish, Chrysiptera cyanea, we have reported lately that the reflecting platelets were too thin as to produce the ideal-type interference, and further suggested that the characteristic hue displayed by the fish should be due to the interference of the non-ideal nature, being far from the “constructive” interference [8]. In the present study, we could further corroborate our former assumption: By making use of modern optical means, it was definitely proven that the iridophores are motile, and that the essence of motility is the simul- tanenous change in the distance of platelets con- tiguous with each other in all the piles of them within the cells. All our observations to date on the present material indicate evidently that the reflection in the iridophores is explainable in terms of the multilayer interference of the “non-ideal” type. Quite recently, Iga and Matsuno [21], while working on the freshwater goby, Odontobutis obscura, reported that iridophores on their scales are motile. Like many other chromatophore types dealt with by many researchers, these cells are dendritic, and a number of minute reflecting platelets in the cytoplasm disperse from or aggre- gate into the perikarya in response to neural or hormonal principle. There, the multilayer in- terference can not be applicable to the mass of Light Reflection in Damselfish Iridophore 253 randomly oriented platelets. The incident rays are reflected by the simple scattering like in a leucophore. According to the current terminology of the chromatophores [22], the cells studied by those workers may more properly be classified into the leucophores. Lythgoe and Shand [23] also reported that iridophores which were responsible for the beauti- ful blue-green tint of their characteristic longitu- dinal stripe in the neon tetra, Paracheirodon innesi, might be motile. The stripe shows circadian color changes, varying to deep violet at night. According to them, the cells change their con- figuration at the transition of color. A rather narrow peak of the spectrum from these iri- dophores may be due to the interference of the “non-ideal” type. However, the structural organ- ization of the cell as well as the regulatory mechanisms for the cellular motility are quite different from those reported by us on the blue damselfish [8, 9]. Later, the workers on the neon tetra have come to the conclusion that the spacing of the platelets may change daily, being triggered by the action of light on the rhodopsin-like molecules contained in the light-reflecting cells themselves [24]. Huxley [16] and Land [7] soundly described that, in the non-ideal interference, the mean wavelength of the reflectance is given as follows: A— 27,4 2-1), d py) As mentioned earlier in the Introduction, 1.37 and 1.93 may be adopted for n, and n,, respectively. Reflecting platelets of this material have been estimated on transmission electron micrographs to be not more than 5 nm thick [8]. Substituting these values into the above equation, the following equation may be offered for estimating the cyto- plasmic spacing in nm: OM When the body surface assumes the cobalt-blue color (the spectral peak: ca. 465 nm), the cytoplas- mic layer between platelets is calculated to be 163nm thick. We have learned in the study that the spectral peak of light reflected from the iridophores could be moved experimentally be- tween 380 and 530nm. For producing those changes, the thickness of the cytoplasmic sheet should vary within the region between 132 and 186 nm, theoretically (Table 2). That is, when the coloring response in the iridophores proceeds from 0 to 100%, the net increase in the spacing should be about 54nm, while the rate of increase is 42%. When the skin coloration changes from the charac- teristic cobalt-blue to dark violet hue, and to green, the estimated rates of decrease and increase in the spacing thickness may be approximately 19 and 14%, respectively. It has also been shown that when the optical thicknesses of two alternate layers are different, both the maximal reflectance and the bandwidth of the principal spectral peak decrease depending upon the grade of the difference [7, 16]. Actually, the reflectance of the blue damselfish skin is much lower than that of the silvery trunk or belly skins of TaBLE2. Summary of the relation of skin colors to motile activities of the iridophores and melanophores in the dermis of the blue damselfish, Chrysiptera cyanea EE Chromatic state Excitement Excitement of animal Noa darkening pallor Skin hue Cobalt-blue Dark violet Light green Peak wavelength (nm) 465 380 530 Calculated distance between guanine 163 132 186 platelets Gan) Increase in distance he _19 14 from normal (%) ag Coloring response 0 100 of iridophore (%) aa Aggregation response 4 0 100 of melanophore (%) 254 H. Kasukawa, N. OSHIMA AND R. FudII a number of teleosteans, including, for instance, the sprats, sardines, cutlasses, silvery smolts of salmonids, where the “ideal” interference by large paralleled platelets has been confirmed to occur for the high reflectivity. One may call to mind that the colorations assumed by the blue damselfish are “fluorescence- like”. This is especially specious when the fish are characteristic cobalt-blue. Of course, any colors of the tegument do not belong to the fluorescence. In the latter, the radiating rays are commonly re- stricted within a narrow range of spectrum. The present measurements disclosed clearly that, alike the fluorescence, the spectral peaks of the reflected light were always sharp, if compared with those from the colored surfaces of many other organisms from which the light-rays are absorbed and scat- tered. In other words, the chroma (saturation) of the blue damselfish coloration is very high, accord- ing to the terminology of the Munsell color system, which is being widely used to describe characteris- tics of colors. That is, the hues assumed by this species of fish are much “purer” than those displayed by many other common fishes. Those optical characteristics may afford additional strong supports for the conclusion that the coloration of the iridophores is based on the “non-ideal” multi- layer thin-film interference phenomenon, and that the motility of the cell is due to the alteration of the spacing between the thin platelets. In the present study, it was clearly shown that the hyposmolarity of the bathing medium shifted the spectral peak of the reflection from the iridophores towards longer wavelengths. It was further shown that norepinephrine or adenosine could bring about additive chromatic effects on the cells which had previously been under the in- fluence of the hypotonicity. That is, the spectral shifts induced by the hypotonicity may be a passive and simply osmotic process, being entirely differ- ent from the sequences initiated by the interaction between pigment-motor ligands and membrane- bound receptors. The latter events are of course physiological ones, normally operating in the control of the chromatophore motility in vivo. Preliminary studies have led us to conclude that the tubulin-dynein system is involved in this process (Oshima and Fujii, to be published), although the detailed explanations for the cellular motility still remain to be the subject of future research. Another point worthy to be noted here is that, at a certain degree of the response, the spectral peak did not shift, even when the angle of incidence was varied (cf. Fig.7). We conclude that this is certainly due to the characteristic arrangement of the piles of reflecting platelets in the motile iridophores; the axes of many piles dispose radially from the nucleus which locates around the apical part of the non-dendritic cell [8]. If the parallel light-rays project on the surface of the skin from a certain incident angle, the piles whose axes are almost parallel with the rays are dominantly responsible for the reflective interference. We have already known through morphological studies that such piles are always present in every iridophore in the skin [8,9]. The piles with the axes forming a sufficiently small angles with the incident rays may be concerned with the reflection to a lesser degree by a small number of platelets within each stack. Those having axes crossing with a larger angle with the direction of the rays are practically irrelevant to the reflection, since the number of platelets becomes too small for produc- ing an effective interference. Even under such an artificial condition of being irradiated by parallel rays unidirectionally any part of skin can reflect light macroscopically, since, as mentioned above, every iridophore contains at least one or few piles among many of them ready for reflecting incident rays, i.e. only a small part within the area occupied by an iridophore is reflective [8]. Consequently, the fractional area of light reflection becomes small, resulting in the weak net reflectance of the skin. In their habitat, i.e. under the coral sea water, on the other hand, the fish receive scattered blue light-rays from every quarter. Thus, the incident rays from near the observer are reflected rather effectively and definitely towards the latter by the mechanism we are now concerned. Against the blue background deep in the sea water, therefore, the fish are rather faintly visible with the characteristic bluish hues with the higher chroma. An important problem to be discussed next is that the black-pigment containing melanophores underlying the iridophores must have a sup- Light Reflection in Damselfish Iridophore 255 plemental but very important role in producing the characteristic hues, especially in increasing chroma value of the coloration. The dark sheet of light-absorbing melanin may certainly be service- able in decreasing the intensity of stray light coming from inside the body of the small fish. If there were no dark layers, the light coming out through the skin is inevitably filtered by the iridophore layer, tending to assume a hue com- plementary to the intrinsic reflective one, 1.e. reddish, yellowish or greenish tone, when the iridophores reflect blue green, cobalt-blue or violet rays, respectively. The same dark sheet should also be useful in preventing rays to invade the body, thus leading to a reduction of stray light-rays under the tegument. When melanosomes are so well dispersed as to fill dendritic processes of the melanophore, fur- thermore, each iridophore becomes light-insulated from the adjacent ones, because, as already described by us elsewhere [8, 9], peripheral halves of the processes are bent and inserted into the space among the iridophores to form screens partitioning each of them off. Stray light from the adjacent cells may thus be shut off by the dark wall. Such architectural features may certainiy enhance the chroma or the purity of the colora- tions displayed by the integument. When the melanosomes aggregate into the melanophoral perikarya, such as when the fish is in the excitement pallor or when the in vitro skin piece is adrenergically stimulated, the iridophores become rather easily accessible by the stray light- rays both from their bottom and sides. Under such a condition, a considerable amount of diffused and scattered rays from both the dermal side and the surrounding iridophores may actually penetrate the iridophore, resulting in the lightening of the skin. Furthermore, the hues complementary to the reflective peaks of the iridophores are added as the stray light-rays pass through the iridophore layer. Consequently, the broadening of the spectral peak of the reflectance occurs, and the skin assumes greenish white hue. Above explanations on the reflective characteristics of the blue damselfish skin are comprehensively summarized in Table 2. We have just shown that the characteristic colorations and their changes of a blue damselfish species, Chrysiptera cyanea, are predominantly due to the motile activities of the iridophores present in the uppermost part of the dermis in a single layer. It should also be stressed here that the changes are entirely graded or continuous in term of the wavelength within the region between the near UV and the green. Rather incredibly, those are resulted entirely from a physical phe- nomenon, i.e. the thin-film interference, being totally independent of the changes in the spectral absorption characteristics of certain pigmentary substances. In such a way, the blue damselfish iridophores possess an extremely elaborate machinery within them to realize the remarkable but subtle changes of skin coloration. As one of almost terminally evolved motile cell types, we may be able to compare this optical effector cell even to the striated muscle cell, which has univer- sally been believed to be the most sophisticated mechanism for fulfilling its part, i.e. very fast and effective production of tension. Such an exquisite optical mechanism has never been reported before in any sort of biological structures. Thus, we dare to categorize the motility of the iridophores of the material to be of an entirely new type. The presence of such motile iridophores as described here is not restricted to this particular species of fish. Up to the present time, for instance, we have actually found very similar ones in a closely related species belonging to the same genus, the yellowtail blue damselfish, C. hemicy- anea (Weber), and in the heavenly damselfish, Pomacentrus coelestis Jordan et Starks (Kasukawa et al., unpublished observations). At least within the genera Chrysiptera and Pomacentrus, both belonging to the family Pomacentridae (damsel- fishes), therefore, one may safely suppose that some other unexamined species possess the iri- dophores of the same category. On the other hand, our preliminary survey also indicates that such unique iridophores are not existing in many fishes even if they were taxonomi- cally included into the so-called damselfish group (the family Pomacentridae). Instead, those fishes possess immotile iridophores which are quite identical with those found and described almost ubiquitously among many teleostean families [7, 18, 22]. The role of such iridophores among the 256 H. Kasukawa, N. OSHIMA AND R. Fuiil assemblage of chromatophores in the color man- ifestations of some damselfish species will be given in a separate article (Kasukawa and Oshima, to be published). What a signification or an advantage do the characteristic hues and their changes have in the life of the blue damselfish? We like lastly to consider this interesting ethological problem, which should at the same time be an extremely important one for the fish themselves in fulfilling their strategy for survival. Using the herring, Clupea harengus, for in- stance, Denton and Nicol [17] have shown that iridophores existing in the skin must take a decisive role in camouflaging the fish against the marine blue background. Their morphological studies indicated that iridophores were present widely over the trunk, and that all the reflecting platelets within them were arranged very parallel with the median plane of the body, i.e. in the sagittal planes. When the fish is swimming normal- ly, therefore, the platelets were very perpendicular to the plane of the sea surface. Thus, the body surface can be likened to a vertically held mirror. In the sea, the light intensity is of course higher from above and lower from below, although not so much like in the air. On the other hand, the horizontal components of the intensity are much more even in the water, since light-rays are scattered at the rough surface and inside the sea water. That is, the fish receives rays from both sides nearly equally. It is easily understandable, therefore, that, when an observer sees the fish swimming on the same level, the mirror equivalent reflects the light of the same intensity and color, resulting in the invisibleness of the organism. If the observer looks at the fish at a certain angle, either of depression or of elevation, both the rays reflected at the body surface and those directly reaching the eyes by passing close to the fish have practically the same intensity and the spectrum, since both groups of rays have practical- ly the same absolute value of the angle in reference to the vertical reflective plane. Thus, the image of the fish becomes obscure against the oceanic blue, as if it were not there. This type of optics explaining the cryptic coloration may be applicable to oceanic migratory fishes very commonly [7, 18]. According to the results of our morphological as well as optical investigations, the participation taken by the blue damselfish iridophores must be quite different from the above explanation. Every- where on the skin, the iridophores reflect light-rays but only those incident from the directions close to the position of the observer, as we have just discussed here. The reflectivity is low and much restricted to a narrow range within the spectrum depending upon the periodicity of the iridophore platelets assumed at that time. Thus, the mirror effect as explained above in the migratory fishes can not be applied. So far aS we are aware, no observations have been recorded up to the present time on the ethological significance of characteristic colora- tions of the species or of those closely related. We have made only few preliminary investigations into these subjects, too. Therefore, it may be too early to speculate the problem. But, we dare to presume that the rather faint but highly saturated coloration assumed by them must be a signal coloration or more properly the recognition one for other individuals of the same species. Among the corals, the cobalt-blue color should be very conspicuous. Conversely, the same color might be useful as the crytic means when they are apart from the coral reef to escape from their predators. There, the blue tint may be useful in concealing themselves into the marine blue water. Further works are justly needed to establish the roles of their characteristic colorations as well as of their ex- traordinary rapid changes amid their beautiful habitat. ACKNOWLEDGMENTS We thank Professor T. Kajiwara, Department of Chemistry, Professor M. Shimazu and some other mem- bers of the Department of Physics, for their helpful discussion on the optical problems. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The monochromator employed was procured by the amount supplementary to the Makoto Seiji Memorial Prize to R. F., 1983. 10 11 12 Light Reflection in Damselfish Iridophore REFERENCES Kawaguti, S. (1965) Electron microscopy on iri- dophores in the scale of the blue wrasse. Proc. Japan Acad., 41: 610-613. Kawaguti, S. and Kamishima, Y. (1966) Electron microscopy on the blue back of a clupeid fish, Harengula zunasi. Proc. Japan Acad., 42: 389-393. Kawaguti,S. and Kamishima, Y. (1966) A _ sup- plementary note on the iridophore of the Japanese porgy. Biol. J. Okayama Univ., 12: 57-60. Hachisu, S. (1975) Nacreous pigments. Prog. Orga- nic Coatings, 3: 191-220. Hiramoto, Y., Shimoda, S. and Shoji, Y. (1979) Refractive index of the protoplasm in sea urchin eggs. Dev. Growth Differ., 21: 141-153. Denton, E.J. and Land, M. F. (1967) Optical prop- erties of the lamellae causing interference colours in animal reflectors. J. Physiol. (London), 191: 23- 24P. Land, M.F. (1972) The physics and biology of animal reflectors. Prog. Biophys. Mol. Biol., 24: 75-106. Oshima, N., Sato, M., Kumazawa, T., Okeda, N., Kasukawa, H. and Fujii, R. (1985) Motile ir- dophores play the leading role in damselfish colora- tion. In “Pigment Cell 1985: Biological, Molecular and Clinical Aspects of Pigmentation”. Ed. by J. Bagnara, S.N. Klaus, E. Paul, and M. Schartl, Univ. Tokyo Press, Tokyo, pp. 241-246. Kasukawa, H., Oshima, N. and Fujii, R. (1986) Control of chromatophore movements in dermal chromatic units of blue damselfish—II. The motile iridophore. Comp. Biochem. Physiol., 83C: 1-7. Kasukawa, H., Oshima,N. and Fujii, R. (1985) Optical analyses of reflection in the motile iri- dophores of blue damselfish. Zool. Sci., 2: 885. Kasukawa, H., Sugimoto, M., Oshima, N. and Fu- jul, R. (1985) Control of chromatophore movements in dermal chromatic units of blue damselfish—I. The melanophore. Comp. Biochem. Physiol., 81C: 253-257. Oshima, N. and Fujii, R. (1984) A precision photo- 13 14 15 16 V7 18 19 20 72) 22 23 24 257 electric method for recording chromatophore re- sponses in vitro. Zool. Sci., 1: 545-552. Fujii, R. and Novales, R. R. (1969) The nervous mechanism controlling pigment aggregation in Fun- dulus melanophores. Comp. Biochem. Physiol., 29: 109-124. Oshima, N., Kasukawa,H. and Fujii, R. (1985) Effects of potassium ions on motile iridophores of blue damselfish. Zool. Sci., 2: 463-467. Fujii, R. and Miyashita, Y. (1975) Receptor mechanisms in fish chromatophores—I. Alpha nature of adrenoceptors mediating melanosome aggregation in guppy melanophores. Comp. Biochem. Physiol., 51C: 171-178. Huxley, A. F. (1968) A theoretical treatment of the reflexion of light by multi-layer structures. J. Exp. Biol., 48: 227-245. Denton, E.J. and Nicol, J. A.C. (1965) Reflexion of light by external surfaces of the herring, Clupea harengus . J. Mar. Biol. Assoc. U. K., 45: 711-738. Denton, E. J. (1970) On the organization of reflect- ing surfaces in some marine animals. Philos. Trans. R. Soc. London, Ser. B, 258: 285-313. Bone, Q. and Denton, E.J. (1971) The osmotic effects of electron microscope fixative. J. Cell Biol., 49: 571-581. Denton, E. J. and Land, M. F. (1971) Mechanism of reflexion in silvery layers of fish and cephalopods. Proc. R. Soc. London, Ser. B, 178: 43-61. Iga, T. and Matsuno, A. (1986) Motile iridophores of a freshwater goby, Odontobutis obscura. Cell Tissue Res., 244: 165-171. Fujii, R. (1969) Chromatophores and pigments. In “Fish Physiology”. Ed. by W.S. Hoar and D.J. Randall, Vol. 3, Academic Press, New York, pp. 307-353. Lythgoe, J.N. and Shand, J. (1982) Changes in spectral reflexions from the iridophores of the neon tetra. J. Physiol. (London), 325: 23-34. Lythgoe, J. N. and Shand, J. (1984) Action spectra for the iridophore light response in the neon tetra. Photochem. Photobiol., 40: 551-553. “i ven Mp Qi ie er, ‘ .* 1. auaiaatoneias’ ORES Wb DOREY, a saleveys a) ae heme | "5 tA i puree ee Wet) Ne hes Paha thy 1G 2) ake FY aay Serr guaaltesety 743 i pre esery Pi RT Ee UD a NTN Ear eine ELE SW tae a ae ae ~, ; how ie wie) ah A akeres ees Cle EEE VEEN ce eo bk ri 9 a ‘ Ea ee lind AGS LD ta BITE a writ “ re we 7 MICRO eS ad cAi Lin i ak Ky Lb t Yee 14 a4 ' DS | A \ av PIP SLAt MP thd pel) ify eerie MEDS ESTE rq rm ‘u PMI RS EG AT Dik S Oye tev eat att rh ae Does Gee Leb regis fe rmal 5 ub ot polryt) i ; winkve) rt Gail aon pits: Ethie Nanas MBs: Anes Vy tare aay Pe Ee Sry vk ay hay © ee As a ee hd Tee iyi hal 4 ae eye Sy es Vries tay Bey CUP EP, OP ipreteny it ce Sd Gat ad eine VeTICL UTD aR ee ae ye ae! KA ej o4 Reo: AVE take, Met ae Ua " PETES, ARS od TE c ign, Se pe Herter 1 Aloe ty Sh A | } i r a oe) pti ayy ti ae fecibtivcn 7 - ? ys yyht. § y >; Ka Seanad Er 4 4 j j ” vied oe nos epi T . : ] “4 , LitNOK (etki. Gr hoger ao ri i y rif } i af 1 ie t er ‘ Tree Tr yor 2 j ‘ é ¥ i : . pope tite yah a rm ' 4 ‘4 ~ ie ‘ k . : OS 2 5 ; ie y iy ‘ i af % ubeweceginn ey volt secsebaebio pb. ANTS ay She Mpa #7120 att we men : a i Wy Hl dat cs THE aE ee Ns a mt wen: & 3 aa aie be WN ABET, 8 4) Y iasietiie - eh a any 16 RRA HE ng a Seite ire sidan Hier aod: aiironntyiy coloas N WP Kalil tae Bh TLE. BS? ai fe ¥ it] pO NII a? 4 es poet ti a - pend LeSHO bac Y. ie! tri Vereruriche “hs A itch ef pass gee ye Sg Pvch@ts orn Om 3 sora ji | weg lf i CeOpoart Lshe pe mS Py A he ee r } when dy Ray} °F ra » deticommnts wiiabs + dete pal 7 Haale Sf rn pug ei Pee habits ; , oe ee “Maeite het punslth he ; yt cae Onan Theta ey ovis oo ey it pea gp ae) CR tae Lavemgh aes aTeyranns Sytem lat neesaiel tu ikon C., f ' "iy ’ j bean. , Dats yh ) FB IeF - Dea it eagle yi pC rel pul ‘aN we ; This NG PY 7? Wel as i oe? ‘ «Pisa aelpla ian aye oiogegtaanr disc ids dati i, ¥ . ~Husthor cal gapth te enti. oe RLM Mee 3) , e ( es on AONE dy a Reeth A sigh vf bes TH para nt A xl ey oe oe Pi iyir Ti Wh Sin Die DCL eet t pocruomkchet aca, LRA DTA pri te i Ne" golieenahe Hy orate Ae ee ; , { ae : my I; inc he “0 = ot Sarg ae ‘lac . oe ZOOLOGICAL SCIENCE 4: 259-264 (1987) Meiosis in the Japanese Gryllacridid Anoplophilus acuticercus Karny 1931 (Orthoptera Saltatoria, Rhaphidophoridae): Amphitelic Orientation of the X and Supernumerary Chromosome(s) ALEJO MESA AND BEatTriz GONt! ‘Departamento de Biologia, Instituto de Biociéncias, Universidade Estadual Paulista “Julio de Mesquita Filho” UNESP, 13500 Rio Claro, Sao Paulo, Brasil, and *Department of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158, Japan ABSTRACT—The karyotype of the Japanese rhaphidophorid Anoplophilus acuticercus exhibits a male chromosome number of 33, comprising six pairs of metacentric or submetacentric autosomes, ten pairs of acrocentric autosomes and a submetacentric X chromosome. The sex determining mechanism is of the XO(male)-type. One of the two individuals studied had one, the other two supernumerary chromosomes. Both the X and the S’s orientate amphitelically in metaphase I and anaphase I, and are reintegrated into one of the daughter nuclei as a single undivided mass of chromatin at telophase I. This meiotic behavior was observed in all the nuclei of both the studied specimens. A tentative reconstruction of the history of Rhaphidophoridae taking into account the karyological data compatible with a recent systematic classification is proposed. The chromosome number of A. acuticercus suggests no clear relationship with Rhaphidophorini, where it has been assigned. As an alternative, the morphology and arboreal habits of A. acuticercus suggest a close relationship to Gammarotettix in the © 1987 Zoological Society of Japan Gammarotettigini. INTRODUCTION The Rhaphidophoridae is a worldwide family of wingless gryllacridoid saltatorial Orthoptera. In recent classifications it is divided into five subfami- lies: Macropathinae, Ceuthophilinae, Dolichopo- dinae, Tropidschiinae and Rhaphidophorinae [1]. All Japanese rhaphidophorids belong to the last- named subfamily, which includes three tribes: Gammarotettigini, with one genus in western North America; Rhaphidophorini, with a number of Asiatic genera including Diestrammena and Tachycines, each having species in Japan; and Troglophilini, comprising the circum-Mediterrane- an Troglophilus with seven described species and the Japanese Anoplophilus with three. Doubts have been expressed by Hubbell (per- sonal communication) as to whether Anoplophilus belongs in the Troglophilini; in his opinion it is Accepted October 6, 1986 Received May 23, 1986 probably a member of the Gammarotettigini. With the intent of investigating this problem using a karyological approach, the senior author visited Japan in the summer of 1985 in a successful attempt to obtain living specimens of Anoplophi- lus. In this paper the karyotype of A. acuticercus is analyzed and its significance in relation to the other known rhaphidophorid karyotypes is con- sidered. Certain unusual features in it relating to the amphitelic orientation of the X and supernu- merary chromosome(s) in the first meiotic division are described and discussed. MATERIALS AND METHODS Five females and two males of Anoplophilus acuticercus Karny 1931 (determined by T.H. Hubbell) were collected in Honshu, Saitama Pre- fecture, Chichibu District, in the vicinity of the Mitsuminejinja Shrine near Ropeway Station at an elevation of 1100 m, on 9 August, 1985. They were all taken at night along trails in the forest, resting 260 A. MESA AND B. GOoNI on tree trunks. Two male specimens (J2 and J3) were dissected in the field and the testes were fixed in a mixture of pure ethanol and glacial acetic acid (3:1 in volume ratio). Semipermanent slides were prepared by squashing follicular testes in a drop of lactoacetic orcein 0.5% after approximately one minute of softening in acetic acid 45%. RESULTS Both the studied individuals of A. acuticercus exhibit a basic karyotype of 2n=33, comprising 16 pairs of autosomes and an X chromosome. The sex-determining mechanism is of the XO(male)- Lo type. Specimen J2 has two relatively large but sligthly unequal supernumerary (S) chromosomes (Fig. la), while J3 has only one (Fig. lc). The autosomes comprise six pairs of metacentric or submetacentric chromosomes and ten pairs of acrocentrics (Fig. 1a). The X and the S’s seem to be submetacentric, although the morphology of each chromosome could not be ascertained with certainty. The S chromosomes are univalent, and their arrangement at metaphase I (MI) is amphitelic (Fig. 2a). The same arrangement is shown by the X chromosomes. As anaphase I (AI) proceeds the S’s and the X remain in the equatorial plate until the beginning of telophase (Fig. 2b). At last moment, S and X join together into a single Fic. 1. Metaphase and diplotene chromosomes of A. acuticercus. a) First metaphase of specimen J2 with bivalents (six of them formed by metacentric or submetacentric autosomes plus 10 acrocentric pairs) arranged in decreasing order of size. b) Diplotene of specimen J2 with two S chromosomes; note the nonhomologous association involving the X and S’s chromosomes. c) Metaphase I of specimen J3 with a single S chromosome. Bar=10pm. Melosis in A. acuticercus 261 EEE Fic.2. Anaphase and telophase chromosomes of A. acuticercus. a) First anaphase of specimen J2 with amphitelic orientation of both X and S’s. b) The X and S’s chromosomes clustered in a single mass of chromatin during early telophase I. c) Late teloph- ase I showing X+S’s mass of chromatin approaching one of the daughter cell nuclei. Bar=10um. mass of chromatin and start a fast movement toward one of the daughter cells (Fig. 2c), being apparently incorporated normally into the nucleus instead of being eliminated in the cytoplasm. At pachytene and diplotene (Fig. 1b) the X remains strongly heteropycnotic, while S; and S; are also heterochromatic but stain less deeply. X, S; and S5 remain connected throughout the prophase stage in the majority of nuclei (Fig. 1b). DISCUSSION First anaphase division is subject to several variables. It depends to some extent on the previous coorientation of the unsplit centromeres of the associated chromosomes in the metaphase configurations. The simultaneous orientation of two centromere halves of a chromosomes to the same pole is called syntelic while the orientation to different poles is called amphitelic. The first event is normally restricted to AI while the second occurs at AII [2]. When amphitelic orientation of univalents (including the X) occurs in MI and AIT, either of the two kinds of events may follow. In the first case the two centromeres start to move toward opposite poles, pulling the respective chromatids which remain connected by a bridge of chromatin [3,4]. In the alternative event the univalents lag behind in the equational plate without any poleward movement of their centro- meres during anaphase stage. When the nuclei enter telophase the lagging chromosomes may finally move rapidly toward one of the poles to be integrated into one of the nuclei, or else may be eliminated into the cytoplasm in the form of micronuclei [5]. In species with localized centromeres, normal reductional division of the chromosomes takes place in AI. An exception to this rule occurs in species of the coleopterous family Lampyridae, where the X always divides equationally in AI and reductionally in all AII [6, 7]. Amphitelic orienta- tion of univalents at MI and even equational division at AI, however rare, have been occa- sionally reported in some species [8-11]. Univa- lent autosomes, originated in accidental asynapsis or in meiosis of hybrids or polysomic cells, as well as the X chromosome in XO males, are obvious 262 A. MESA AND B. GONI candidates for misdivision. In some cases the delay of amphitellically oriented univalents to divide equationally may lead to a failure of the cell to divide, and as a result to the production of unreduced second division products [8, 9]. This is not what happens in A. acuticercus, however, where the first meiotic division proceeds normally. In three instances of misdivision of the X chromosome observed in our laboratories the individuals also carried S chromosomes. This coincidence opens into a question whether the presence of supernumerary chromosome may cause irregularities in the orientation of univalent chromosomes at meiosis. In Passalus perplexus, a passalid beetle, Martins [3] observed that S and X oriented amphitelically in some AI, and that after a period of lagging in the equatorial plate the sister chromatids went to opposite poles though re- mained connected by a thin thread of chromatin. Mesa [4] observed a similar behavior of the X and S chromosomes in some AI nuclei of two speci- mens of the grasshopper Daguerreacris tandiliae. In A. acuticercus the situation is different. The X and S’s remain amphitelically oriented in the equatorial plate, forming at the end of MI a single mass of chromatin which at the last moment moves rapidly to be integrated into one of the daughter nuclei. This belated incorporation prevents the formation of gametes that all lack an X, which would result in an all-male progeny. Karyological information about species of Rhaphidophoridae is not extensive except for the circum-Antarctic Macropathinae, in which group the majority of the species have been studied and the ancestral chromosome number determined to be 2n¢=45 [12-16]. Among the Dolichopodinae (comprising the genera Dolichopoda, Hadenoecus and Euhadenoecus), some species of the first- named genus possess chromosome numbers rang- ing from 2n¢=28 to 35 [17-20]. In Hadenoecus cumberlandicus and Euhadenoecus insolitus, Lamb [21] found the number in bisexual populations to be 2n¢=34+4 XY and 2n# =34+ XX; in parthe- nogenetic populations of both these species the females have the same chromosome number as those of the bisexual ones. Chromosome data have been published on only two Ceuthophilinae: Ceutophilus sp. with 2n ¢ =37 [22] and C. macula- tus (as latebricola) with 2ni=37—39 [23]. Chromosome data on Rhaphidophorinae are also few. Only two members of the tribe Rhaphi- dophorini have been examined: Diestrammena Japanica and Tachycines asynamorus, both with 2né=57, the X metacentric, and the autosomes “rod-shaped”. The first was studied by Makino [24] from specimens collected near Lake Shikotsu in Hokkaido, Japan, and the latter by Mohr and Ecker [25] from specimens taken in a greenhouse in Oslo, Norway, where it has recently become established. In the Troglophilini only two species have been reported upon: Troglophilus cavicola with 2nt=21 [26] and T. neglectus with 2nt =17 [19]. Nothing is known of the chromosomes of half dozen species of the western North American genus Gammarotettix, at present the only member of the tribe Gammarotettigini, or of Tropidischia xanthostoma, restricted to the western edge of North America and the only species of the only genus of the subfamily Tropidischiinae. If high chromosome number is indicative of primitiveness in the Orthoptera, then the Rhaphi- dophorini have the most primitive ancestral karyotype not only among the _ gryllacridoid Orthoptera but indeed among all sexually repro- ducing Saltatoria for which data are available. However, Hubbell and Norton [1] concluded that the Macropathinae are in most respects the most primitive living rhaphidophorids. They pointed out that in addition to shared apomorphic charac- ters and sole occupancy of the Gondwanaland fragments that attest to the monophyletic origin of the subfamily, its taxa exhibit the largest assem- blage of primitive (pleisomorphic) features to be seen in any of the subfamilies. A tentative reconstruction of the history of the Rhaphidophoridae, compatible with that proposed by Hubbell and Norton [1] but taking into account the karyological evidence, is as follows: An ances- tral pre-rhaphidophorid stock already existed in the Triassic (approximately 200 million years ago), prior to the division of Pangaea. In that stock the chromosome number 57-58 was probably com- mon. When Pangaea split up, a species or a group of closely related species with 2n/=45 remained in Gondwanaland, most of the descendants of which have preserved that basic karyotype down to Meiosis in A. acuticercus 263 the present. The same conservatism is manifest in their morphology. In Laurasia most of the lineages evolved toward a reduction in the chromosome number, but a few of them may have maintained the old 57-58 karyotype and given rise to the modern Rhaphidophorini. It is, however, also possible that the chromosome number 57 observed in Rhaphidophorini has been derived from lower numbers by pericentric inversion and centric fission cycles. A search among the more than one hundred unstudied species of this tribe could help to settle this question. The chromosome number of Anoplophilus acu- ticercus is too low to suggest direct relationship with the Rhaphidophorini and too high to make probable any close relationship to Troglophilus. Whether or not Anoplophilus belongs with Gam- marotettix in the Gammarotettigini, as suggested by its morphology and arboreal habits, cannot now be determined. The validity of such an assignment must await the cytological analysis of the latter genus (Gammarotettix). ACKNOWLEDGMENTS The authors are indebted to Dr. T. H. Hubbell of the University of Michigan for identifying the species stu- died, reviewing published information on the genus for us and critical reading on the manuscript, and Drs. T. Yamasaki and Y.N. Tobari of the Tokyo Metropolitan University for constructive comments on the manuscript (T. Y.) and for providing us with field collecting mate- rials. This work was sponsored by the CNP, PIG (Conselho Nacional de Pesquisa, Programa Integrado de Genetica) and the CNP, Research Fellowship (Processo 30 1315/80) grants to the senior author and by a grant from the Ministry of Education, Science and Culture, Japan to B. Goni. REFERENCES 1 Hubbell, T.H. and Norton, R.M. (1978) The sys- tematics and biology of the cave-crickets of the North American tribe Hadenoecini (Orthoptera Saltatoria, Ensifera, Rhaphidophoridae, Dolichopo- dinae). Misc. Publ. Mus. Zool. Michigan Univ., 156: 1-156. 2 Sybenga, J. (1975) Meiotic Configurations. Sprin- ger-Verlag, Berlin, Heidelberg & New York. 3 Martins, V.G. (1982) Citogenética de dezenove especies de Scarabaeoidea (Treze Passalidae e seis Scarabaeidae) (Coleoptera). M. Sc. Thesis, Univer- 10 11 12 13 14 15 16 07, 18 19 sidade Estadual Paulista “Julio de Mesquita Filho” UNESP, Rio Claro, Sao Paulo, Brasil, 62 pp. Mesa, A. (1984) The chromosomes of a relict species of eumastacid: Daguerreacris tandiliae Decamp and Lieberman 1970 (Orthoptera, Eumas- tacoidea, Morseinae). Rev. Brasil. Genet., 7: 219- 229. Morrison, J. W. (1953) Chromosome behavior in wheat monosomes. Heredity, 7: 203-217. Stevens, N.M. (1909) Further studies on the chromosomes of the Coleoptera. J. Exp. Zool., 6: 101-113. Smith,S.G. and Maxwell, D.E. (1953) Post- reduction of the X-chromosome and complete chias- ma interference in the Lampyridae (Coleoptera). Can. J. Zool., 31: 179-192. John, B. and Henderson, S.A. (1962) Asynapsis and polyploid in Schistocerca paranensis . Chromo- soma, 13: 111-147. John, B. and Lewis, R. (1965) Genetic speciation in the grasshopper Eyprepocnemis plorans . Chromo- soma, 16: 308-344. John, B. and Lewis, R. (1965) The meiotic system. Protoplasmatologia, 16: 1-335. Lewis, R. and John,B (1959) Breakdown and restoration of chromosome stability following in- breeding in a locust. Chromosoma, 10: 589-618. Mesa, A. (1965) Karyology of four Chilean species of gryllacridids of the genus Heteromallus (Ortho- ptera, Gryllacridoidea, Rhaphidophoridae). Occas. Papers Mus. Zool., Michigan Univ., 640: 1-13. Mesa, A. (1970) The chromosomes of two species of the genus Australotettix Richards (Gryllacridoidea, Macropathinae). J. Aust. Entomol. Soc., 9: 7-10. Mesa, A. and Mesa, R.S. de (1971) Citologia y evolucién en Macropathinae (Orthoptera, Gryllacri- doidea, Rhaphidophoridae). Rev. Peru. Entomol., 14: 220-224. Mesa, A., Ferreira, A. and Mesa, R.S. de (1968) The karyotype of some Australian species of Macro- pathinae (Gryllacridoidea, Rhaphidophoridae). Chromosoma, 24: 456-466. Mesa, A., Ferreira, A. and Mesa, R.S. de (1969) The chromosomes of three Australian species of gryllacridids (Gryllacridoidea, Rhaphidophoridae). Rev. Peru. Entomol., 14: 220-224. Saltet, P. (1959) La formule chromosomique de Dolichopoda linderi Duf. (Orthoptera, Rhaphi- dophoridae). C.R. Acad. Sci. Paris, 248: 851-853. Saltet, P. (1960) La formule chromosomique de Dolichopoda palpata e D. bolivari (Orthoptera, Rhaphidophoridae). C.R. Acad. Sci. Paris, 250: 2612-2614. Saltet, P. (1967) La formule chromosomique de l’Orthoptere Troglophilus neglectus (Rhaphidophor- idae). C.R. Acad. Sci. Paris, 265: 1313-1316. 20 21 22 23 264 Baccetti, B. (1958) Osservazioni caryologiche sulle Dolichopa italiane. Redia, 43: 315-327. Lamb, Y.R. (1975) Cytogenetic studies of parthe- nogenetic and bisexual populations in two species of cave cricket. Proc. North. Cent. Branch Entomol. Soc. Am., 30: 103. Stevens, N.M. (1912) Supernumerary chromo- somes and synapsis in Ceutophilus (sp.?). Biol. Bull., 22: 4-6. Thompson, C. (1911) The spermatogenesis of an orthopteran Ceutophilus latebricola Scudder with A. MESA AND B. 24 25 26 GONI special reference to the accessory chromosome. 30th Rep. Michigan Acad. Sci., 97-104. Makino, S. (1931) The chromosomes of Diestram- mena japonica Karny, an orthopteran. Zool. Mag., 43: 635-645. Mohr, O.L. and Ecker, R. (1934) The grasshopper Tachycines asynamorus, a new animal for cytolo- gical purposes. Cytologia, 5: 384-390. Baccetti, B. (1961) Cariologia di populazioni parte- nogenetiche e bisesuate de Troglophilus cavicola Koll. IX Int. Kongr. Entomol. Wein. ZOOLOGICAL SCIENCE 4: 265-276 (1987) Examinations of Spindle Structures Modified by T-1, the Mitotic Arrester Tomouiko J. Iron, HipEm1 Sato and Axkio KospayasHt' Sugashima Marine Biological Laboratory, School of Science, Nagoya University, Sugashima-cho, Toba-shi, Mie 517, and ‘Department of Agricultural Chemistry, Faculty of Agriculture, Okayama University, Tsushima naka 1-1-1, Okayama 700, Japan ABSTRACT—The process of inducing barrel-shaped spindle (B-spindle) in fertilized eggs of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, was examined. By elecron mi- croscopy, the microtubule organizing centers (MTOCs) of the spindle poles were seen to be dispersed into many MTOCs and appeared spread out both in sections of whole dividing eggs or iso- lated mitotic apparatus(MA), supporting our previous conclusion that T-1 alters the distribution pat- tern of MTOCs. Spindle assembly in eggs, which were first pulse-treated with T-1 and then insemi- nated after various time intervals, was examined with the polarizing microscope. We confirmed that a 5 min exposure to T-1 was sufficient to produce typical B-spindles when the drug concentration was above 4.2x10~’M in H. pulcherrimus and 1.3X10~°M in P. depressus. Although the birefrin- gence(BR) of spindle decreased with higher concentrations of T-1, the decrease was inversely pro- portional to the length of the interval between brief T-1 application and fertilization. However, the spindle never reverted to the normal spindle form. Suppression of pronuclear migration, which was also induced by T-1 treatment, could be reduced by increasing the time interval. Application of 30% D,O to B-spindle rapidly induced an increase in spindle BR and in spindle dimensions but did not restore the spindle to the original shape of untreated spindles. These results indicate that the T-1 de- pendent alteration of spindle morphology is very difficult to reverse and this effect is independent of the inhibitory effect of the drug on microtubule assembly. We believe that T-1 is a novel molecule © 1987 Zoological Society of Japan which alters the spindle configuration by affecting the centrosome. INTRODUCTION Mitotic poisons have played an important role in the analyses of the functional and structural basis of spindle assembly [1]. For instance, colchicine and its derivatives, Vinca alkaloids, griseofulvin and nocodazole have been applied as inhibitors of tubulin polymerization, whereas taxol has been used to prevent microtubule disassembly. In- formation accumulated by using such mitotic poisons has mainly related to the behavior of microtubule assembly because the major effect of these poisons is to disturb the in vivo tubulin polymerization/depolymerization equilibrium. Additional information has been obtained by shifting various physical parameters such as Accepted October 8, 1986 Received September 4, 1986 temperature and hydrostatic pressure [2-4], or by the partial substitution of D,O for enviornmental H,O [5-7]. In previous works [8-10], Takahashi and Sato showed that D,O converts unpolymeri- zable tubulin molecules to a polymerizable state in dividing cells. Although the use of D,O is a powerful tool in the examination of spindle assem- bly in living cells, the results of in vitro tubulin polymerization experiments with D,O [11] did not coincide with those from the in vivo experiments. This discrepancy may be due to the presence in Abbreviations Used EGTA, ethyleneglycol-bis-(2-aminoethyl ether)-N, N, N’, N--tetraacetic acid; PIPES, piperazine-N, N -bis(2- ethanesulfonic acid; TAME, _ p-tosyl-L-arginine methylester HCI; B-spindle, barrel-shaped spindle; BR, birefringence; ASW, artificial sea water; FSW, filtered sea water; MA, mitotic apparatus; MTOCs, micro- tubule organizing centers. 266 T. J. IroH, H. SATO AND A. KOBAYASHI vivo of unidentified molecule(s) not found in vivo. One may be able to identify and characterize these molecules by using certain molecular probes. T-1 (2-hexyl-5-propyl-1-3-benzene diol), which can be isolated from the culture medium of Pseudomonas sp., is known to alter the spindle morphology of sea urchin eggs causing, (i) a shortening of pole to pole distance, (11) a straight- ening of the spindle microtubules, (iii) a spreading of the polar regions and (iv) a shrinking of the aster size. This modified spindle has been pre- viously termed a “barrel-shaped spindle (B- spindle)” [12,13]. T-1 has also been reported to suppress microtubule assembly both in vivo and in vitro, although spindle morphology is altered without a concurrent decrease in spindle birefrin- gence [14]. Thus, we thought that T-1 affected, in particular, the spatial arrangement of MTOCs in the spindle poles and the centrosome, independent of its inhibitory effect on microtubule assembly. The centrosome is considered to be a kinetic organelle which governs the shape of the mitotic apparatus during cell division [15]. The drug T-1 may play a key role in uncovering additional clues useful in determining the molecular basis of the system regulating spindle morphology. In this paper, we follow the induction of B-spindle by T-1 in sea urchin egg. We describe our electron microscopic observations of the polar region of B-spindle which verify our idea that the centrosome is modified by T-1. We also follow the formation of B-spindle in eggs which were pulse- treated by T-1 before and after fertilization and observe the effects of D,O on the B-spindle. The latter experiments were done in order to deter- mine whether B-spindle induction is independent of the modulation of microtubule assembly. MATERIALS AND METHODS Materials and chemicals All chemicals were purchased from commercial sources and were of certified reagent grade. Mature gametes of Japanese autumn sea urchin, Pseudocentrotus depressus and winter sea urchin, Hemicentrotus pulcherrimus, were used and han- dled as described before [14]. Electron Microscopy Dividing sea urchin eggs were fixed according to the method of Hirano et al. [16]. This fixation medium consisted of 0.1 M phosphate buffer (pH 6.9), 0.1M sucrose, 10mM EGTA, 5mM MgSO,, 2.5% glutaraldehyde, 1% paraformaldehyde and 300 xg/ml saponin. Mitotic apparatus (MA) was isolated as described before [14] and fixed as follows. The isolation medium for MA consisted of 10mM PIPES (pH 6.9), 5mM EGTA, 1mM MgSO,, 1mM TAME, 20% glycerol and 1% Nonidet P- 40. Immediately after isolation, MA was fixed with 2.5% glutaraldehyde in glycerol free MA isolation medium for 60 min. The fixed MA was washed with glycerol free MA isolation medium three times then treated with 0.2% tannic acid in 0.1M phosphate buffer (pH 6.9) containing 0.5 mM MgSO, for 120 min. They were washed three more times with this mixture and postfixed with OsQO, on ice for 60 min. Both eggs and MA were dehydrated through a graded series of ethanol, en bloc stained with lead acetate [17] and embedded in Quetol 651 resin [18]. En bloc staining was used as a routine procedure to increase contrast. Thin sections 80-100 nm in thickness were cut with a diamond knife on Porter-Blum MT2B ultramicrotome. After being placing on a collo- dion coated 200 mesh grid, the sections were stained with uranyl acetate and lead citrate. In some cases, serial sections were made and mounted on collodion- and carbon-coated single slot grids. HITACHI H-300 electron microscope operated at 60 kV was routinely used. The mitotic stages were identified from the distribution pattern of the chromsomes. T-1 Pulse treatment Eggs were treated with T-1 before or after fertilization. In the former case, unfertilized eggs were collected in conical tubes by hand centrifuga- tion and gently suspended for 5 min in artificial sea water (ASW) containing various concentrations of T-1. After washing with filtered sea water (FSW) three times, the pulse-treated eggs were transferr- ed to a watch glass and allowed to settle until T-1 Altered Spindle Structure 267 fertilization. Eggs were fertilized by adding a drop of diluted sperm. When eggs were treated with T-1 after fertiliza- tion, they were first fertilized in a watch glass and allowed to develop for 30 to 40min. After the pronuclei fused, eggs were collected and treated with T-1 in ASW in the way as the T-1 pretreated eggs. In both cases, spindles were stabilized at the metaphase stage as described before [14], and examined under polarizing microscope. In some cases, stabilized eggs were treated with 10 ug/ml of ethidium bromide for 10 min in order to observe the chromosomes. After washing the eggs with stabilizing medium, eggs were examined under an Olympus BH-RFL epifluorescence microscope (Olympus Optical Co., Tokyo) equip- ped with a dichroic mirror (BH-DMV) and a 570nm cut-off barrier filter. A high pressure mercury arc lamp (Osrum HBO 100 W, Berlin- Miinchen, West Germany) was used as the illumi- nating source. Fluorescence was induced by exciting ethidium bromide with 300-350 nm light. Application of DO to T-I treated eggs Because DO is known to increase the assembly of spindle microtubule [6,7], we examined the effect of D,O on microtubule assembly in the B-spindles of T-1 treated eggs using the following procedure. T-1 was applied to fertilized eggs between the pronuclear fusion stage and the break down of the nuclear envelop, and the eggs were allowed to develop until metaphase. In meta- phase, eggs were transferred to 30%(v/v) DO ASW and suspended for 2 min. Then, the spindles were stabilized. In some cases, fertilized eggs at prometaphase were treated with T-1 containing 30% D0 ASW for 5-8 min and then the spindles were stabilized. Fic. 1. Electron eats of a B- ae maneed by 8 8x 10- ™M T. 1 in an egg af H. Paes Gy, K half spindle in early anaphase at low magnification. Note the siightly electron dense materials dispersed at the polar region perpendicular to the axis of the B-spindle (indicated by an arrow). Bar=1ym. (b) High magnification of the polar region in Fig. la. Arrowheads indicate MTOCs. bar=0.5um. 268 T. J. Irou, H. SATo AND A. KosBayAsHI } er es * ‘4 * te bod ?*- * * se, Sas 5 2g ee iy bs s 5 st of . * AJ Re ae ee & — ‘ ~ 4 — -_ os se s 1 3 * Fic. 2. Electron micrographs of isolated MA from H. pulcherrimus eggs. (a) The center of the polar region of a normal spindle. Arrows indicate short microtubules which do not extend into adjacent sections and were commonly seen in the polar region. Arrowheads indicate minute filamentous bridge structures. bar=0.5 um. (b) A half B-spindle in metaphase. The “bridge-rich region” in the spindle pole is observed as a slightly darker area at low magnification. A centriole (c) exists at the center of this region. bar=0.5 um. RESULTS Ultrastructure of the polar region of the B-spindle The improved fixation method developed in this laboratory [16] allowed us to obtain the excellent preservation of spindle structures as well as associ- ated membranous components in dividing eggs of the sea urchin, H. pulcherrimus. Electron dense amorphous material was always seen in the polar regions of the spindle as reported previously [16]. This amorphous material was composed of aggre- gated clusters of tiny granules. Many microtubules terminated in it and appeared to radiate out from it to various directions. As shown in Figure la, B-spindle excluded yolk granules and mitochon- dria just as normal spindles do. Though the distribution pattern of the microtubules was rather difficult to detect at low magnifications, we could identify the spindle axis from the position of the chromosomes. The amorphous material turned out to be distributed in a cloud perpendicular to the axis of the B-spindle. This material did not form a compact aggregate as it does in normal spindles, but rather was in a dispersed state. As shown in Figure 1b, however, these dispersed granules could also act as MTOCs. In a series of cross sections of B-spindle, we confirmed that the MTOCs in the polar region were dispersed and formed a disk. To analyze the distribution pattern of micro- tubules within a B-spindle, isolated MA from dividing eggs of sea urchin, H. pulcherrimus, were examined. In the polar region of isolated MA, the majority of spindle microtubules were radially oriented. As shown in Figure 2a, many short and fragmented microtubules were seen in this region (indicated by arrows). Instead of granules, many filamentous bridges were commonly seen in the polar region which appeared to connect neigh- bouring microtubules each other. At low mag- nification (Fig. 2b), this “bridge-rich region” could be easily distinguished from other areas because of its slightly darker contrast. We considered the possibility that this region could contain an altered centrosome from the isolated MA. The centro- some of the B-spindle in metaphase appeared to be in a spread or flattened form and perpendicularly T-1 Altered Spindle Structure 269 oriented to the spindle axis. The centriolar pair was always located at the middle of the centroso- mal area. The components of centrosome from isolated MA such as the filamentous bridges did not have the same appearance as in the sections of preserved eggs. However, the dispersed state of the MTOCs observed in both specimens is consist- ent with the results obtained from light microscopy as reported before [14]. The electron dense spots whose diameter are around 0.1 to 0.2 4m are artifacts induced during the en bloc staining and probably correspond to secondary deposits of lead carbonate. A three dimensional reconstruction made from serial sections of an isolated B-spindle clearly showed that the centrosome of this B-spindle has a flat or spread appearance (Fig. 3). We also found that the microtubules radiating out from this region were gathered together as loose bundles containing both kinetochore and non-kinetochore 4 c=). SON | SS Re nets — ~ £ v NS Fic. 3. Three-dimensional reconstruction of a half B- spindle at the onset of anaphase. Microtubules, chromosomes and bridge-rich regions were traced through four adjacent sections, bar=1 um. microtubules. T-1 pulse treatment on fertilized eggs The minimum dose of T-1 inducing B-spindle formation was slightly different for the two species of sea urchins. Eggs of P. depressus were less sensitive to T-1 than those of H. pulcherrimus; the minumum dose required to induce B-spindle formation in P. depressus eggs was 1.3x10-°M and 4.2x10~’M for H. pulcherrimus. B-spindle could be induced by pulsing eggs of both species with T-1 if the fertilized eggs were treated with T-1 ASW for 5 min after the stage of syngamy formation. The BR of the spindle decreased as the T-1 concentration increased in the same manner as in the immersion experiments described before [14]. When T-1 pulsed eggs were allowed to develop, mitotic spindles formed sequentially in the first, second, third and fourth division with a slight delay as compared to control eggs. The spindles were still barrel-shaped during these cell cycles. These observations indicated that T-1 did not inhibit the replication cycle of centrosomal components dur- ing the cell cycles despite the morphological modification of the centrosome. However, cyto- kinesis was frequently inhibited and cleavage did not occur until the fourth division. It has been suggested that the position of the cleavage furrow is controlled by the cortical ends of the astral microtubules [19]. The asters of B-spindle are tiny, containing short microtubules which do not extend into the cortex. Therefore, the suppression of cleavage by T-1 might be attributed to the lack of microtubules ending in the cortex. In the fourth division, cleavage furrows formed all at once but were randomly oriented, yielding irregular blasto- meres. T-1 pulse treatment on unfertilized eggs To examine the reversibility of T-1 effect, we followed the assembly of spindles in eggs which were treated with T-1 for 5 min before fertilization and then, after standing for 5 to 240 min, were inseminated. B-spindles could be induced using a concentration of T-1 above 4.2x10°-’M for H. pulcherrimus (Fig. 4). The BR of the B-spindle in eggs fertilized following a pulse of T-1 decreased in 270 T.J. Iron, H. Sato AND A. KoBaAyASHI 323 Soe Fic. 4. _B-spindle induced in eggs of H. pulcherrimus pulsed with T-1 (2.1 x 10~° M) before fertilization. Eggs are fertilized (a) 5 min, (b) 60min and (c) 240 min after application of T-1. When the interval between pretreatment and fertilization is short (5 to 60 min), the B-spindle is situated in the periphery of the egg and a cytoaster frequently existed beside the B-spindle. 1 div.=10 um. Time interval (hr) A B +4 4 f tn ae Povo Ele) 5 : : jofae e 2 i 0 1 Fic. 5. BR recovery in B-spindles induced by pulse-treating eggs of H. pulcherrimus with T-1. The point below arrow A represents the BR of control spindle and those below arrow B, the BR of B-spindle induced by T-1 applied after the syngamy formation. Different symbol shapes correspond to the diferent batches of eggs. The x-axis indicates the length of the interval between T-1 treatment and fertilization. The y-axis indicates the amount of BR in the center of the half spindle. The T-1 concentration used was 4.2107’ M (open symbols) or 2.1x10~° M (closed sym- bols). The sample number for each point ranges from 18 to 20 eggs. inverse proportion to the increase of T-1 concen- trations (Fig.6). When pretreated eggs were fertilized 60 to 240 min after a highly concentrated T-1 pulse, the T-1 induced decrease in BR was less and less apparent as the time of the chase increased. With the 240 min chase, the BR attained normal levels. However, the modified B-spindle morphology persisted and did not revert to the normal spindle shape. With lower concen- trations of T-1, the BR was not altered while the change in spindle shape continued to occur. These results showed that T-1 is more effective in T-1 Altered Spindle Structure 271 Fic.6. Observation of chromosomes in T-1 pulse treated eggs of H. pulcherrimus by fluorescent microscopy. Photographs of (a) and (b) were taken from the same egg at different depth of focus. This egg was fertilized 5 min after the pulse treatment. The chromosomes in photograph (a) are located on a metaphase plate and those in (b) are dispersed, suggesting that the clusters in (a) come from the male pronuclei and the cluster in (b) come from the female pronuclei. In almost all the eggs observed, the two clusters of chromosomes were not in the same focal plane. Egg (c) was fertilized 180 min after pulse treatment. Under these conditions, one still sees two separate chromosomal clusters but the distance between them is less and the clusters are usually observed in the same focal plane. One is in the metaphase state and the other in the dispersed state. The T-1 concentration during the pulse treatment for this egg was 2.1 10~°M. 1 div.=10 um. modifying the morphology of the centrosome and inducing B-spindle formation than in reducing spindle BR. Suppression of syngamy formation by T-I1 pulse treatment When eggs were treated with T-1 before ferti- lization, B-spindles were not formed in the center of eggs but in the peripheral region. A cytoaster was frequently seen beside the B-spindle (Fig. 4). This raises a question. Is pronuclear migration also disturbed by T-1 pulse treatment and is this effect, again, independent of B-spindle assembly? To answer this question, the location of chromo- somes in prometaphase or metaphase was followed by staining the DNA with ethidium bromide. When eggs were washed briefly from 5 to 60 min before fertilization, two chromosomal clusters were seen in each egg in the majority of cases as shown in Figure 6a, b. In many cases, one cluster appeared to be located on a metaphase plate whereas the other one consisted of randomly situated chromosomes. The presence of two chromosomal clusters suggests that T-1 pulse treatment disturbs pronuclear migration. Accord- ing to Sluder and Rieder [20], when the migration and fusion of pronuclei are inhibited in sea urchin eggs, only centrosomes associated with the male pronucleus organize a bipolar spindle. Thus, it is probable that spindles assembled in eggs treated by T-1 before fertilization were organized by centrosomes derived from the male pronucleus. The percentage of eggs forming two chromo- somal clusters decreased with an increase in the 1.0 DCC index 0 1 2 3 4 Time interval (hr) Fic. 7. The proportion of eggs which contain double chromosomal clusters (DCC) vs. the length of time interval between T-1 pulse and fertilization. X-axis indicates time interval length and y-axis indicates the proportion of eggs which contain DCC. Eggs were observed under the fluorescent microscope after staining chromosomes with ethidium bromide. The sample number at each point ranges from 160 to 200 eggs. DD T. J. Irou, H. Sato AND A. KOBAYASHI time between the T-1 pulse treatment and fertiliza- tion (Fig. 7). As shown in Figure 6c, the cluster to cluster distance also appears to decrease with increasing time between the T-1 pulse and fertiliza- tion. When eggs were fertilized 240 min after T-1 pulse-treatment, the separation of the two chro- mosomal clusters was hardly detectable, if it occurred at all during first spindle assembly. Instead, the usual metaphase chromosome plate as seen in untreated cells formed in the center of the egg. These results suggest that T-1 pulse treatment suppresses pronuclear migration. Because this T-1 inhibitory effect was reversible as is the reduction of spindle BR in T-1 treated cells, we presume that the suppression of normal pronuclear migration may be related to the disturbance of microtubule assembly in the sperm aster. Effects of D2O on B-spindle Because D,O is an agent which directly pro- motes microtubule polymerization both in vivo and in vitro [6,7,11], we can rapidly increase microtubule assembly with the proper concentra- tions of D,O. Therefore, eggs pretreated with T-1 were immersed in 30% D,O to determine if there is any relationship between the extent of microtu- bule assembly and B-spindle induction. Because spindle shape often becomes abnormal at higher concentrations of D,O, we chose a rather in- termediate concentration. According to Taka- hashi and Sato [8], 33% DO was enough to show the reversibility of the isotope effects in H. pulcherrimus eggs. At this concentration, the effects of the D,O was reversible and could be repeated. This concentration was high enough to increase the extent of microtubule assembly both in normal spindle and in B-spindle. When eggs were kept in D,O, further development was retarded compared to the control. As shown in Table 1, when eggs were treated with 30% D,O, the B-spindle responded with an increase in BR. The whole shape of the B-spindle became more spherical. Both the length and the width of spindle increased but the length to width ratio hardly changed and its value stayed around 1.0. Astral rays also grew but the dispersed aster did not change (Fig. 8). The increase in the BR and in the dimensions of the B-spindle and the astral rays is clearly due to the isotope effects of D2O on spindle microtubule assembly. The rounding of spindle shape could be due to the increased number of microtubules within the restricted regions of the B-spindle. These results indicate that D,O can affect microtubule assembly in T-1 treated spin- dies. However, the normal shape of the spindle could not be restored merely by increasing micro- tubule polymerization. Fic. 8. Effect of D,O on B-spindle in eggs of H. pulcherrimus. (a) A B-spindle induced with 2.1 x 10-°M T-1 applied after pronuclear fusion. (b) A B-spindle treated with 30% D,O for 2 min in metaphase. Note that the length and width of the B-spindle are increased but that the structure of spindle is not at all restored to the original form of untreated spindle. 1 div.=10 um. DISCUSSION In an earlier paper, we considered the possibility TABLE 1. Change in BR of B-spindle with D,O No. Control T-1 T-1+D,0* Species 1 3.66 + 0.14 (16) DEAD il DOKLO) = 4.18 + 0.49 (20)** H. pulcherrimus D S12 a2 0.27 (20) eds) a2 0, A0 (AD) 3.54 + 0.23 (20)** H. pulcherrimus 3) 3.71 + 0.16 (14) 83,03 ae O13) (U2) Alar OZ (ayer P. depressus * D.O concentration is 30%. **T_1 concentration is 2.1X10 °M. *** T-1 concentration is 1.3 x10°-°M. Numbers in parentheses show the number of examined spindles. T-1 Altered Spindle Structure 273 that the induction of B-spindle formation in sea urchin eggs by T-1 is not due to the inhibitory effect of T-1 on microtubule assembly [14]. The morphological modification of the spindle may be dependent on the redistribution of the MTOCs which normally form the spindle poles. In this article, we intend to determine the cause of the induction of B-spindles. We employed an electron microscopic technique which gave improved ultrastructural preservation to observe the MTOCs of the B-spindle. We saw that the centrosomes in the B-spindle were spread out and a granular material was dispersed perpen- dicular to the spindle axis in sections of preserved eggs. In sections of isolated MA, a number of minute filamentous bridges which appeared to connect neighbouring microtubules were common- ly observed in the polar regions in place of granules. These might be a residual skeletal components of the centrosome which remained throughout the MA isolation procedure. The granules commonly observed in sections of whole eggs were probably lost during the process of spindle isolation. Endo et al. [21] saw clusters of granular material in the polar regions of MA of H. pulcherrimus eggs and called them microtubule organizing granules (MTOGs). The cluster of granules seen in this article did not appear similar to the MTOGs observed by Endo et al. because the MTOGs are denser than and their size is three to four times larger than the clusters in the polar region of the B-spindle. However, the conditions under which the eggs were fixed and sectioned were different in the two cases. Furthermore, T-1 might induce the clusters of granules to disperse and change in shape and size, and therefore, the altered appear- ance of the granules might be ascribed to the effect of T-1 on the centrosome. The results of the electron microscopic examina- tion of B-spindles were in good agreement with those from light microscopy [14]. However, it should be noted that distinctly different spindle pole morphologies were seen in these two different microscopic techniques. The dispersed polar cen- trosome of the B-spindle appeared as a strand of beads in light microcopy [14], but these aggregates were not the same size when seen with electron microscopy. This discrepancy may be due to an artifact caused by specimen modifications which occur during its preparation for light or electron microscopy. The distribution of microtubules appeared to be unaltered by fixation and dehydra- tion since the microtubules were observed to be gathered in loose bundles similar to that seen by light microscopy (compare Figure 5 in ref. [14] to Figure 3 in present article). Recently, Schatten et al. [22] found that the poles of acentriolar spindles in mouse oocytes and eggs were spread out and that the spindle were typically barrel-shaped. In contrast, the centro- somes of normal metaphase spindles in sea urchin eggs are more compact. We believe that one purpose of centrioles may be to gather pericentrio- lar materials into one location, thus producing a centrosomes with a compact shape during meta- phase. Although the centrioles are always located in the center of centrosomes in B-spindle as they are in normal spindle, the morphology of the B-spindles and its dispersed, flattened ellipsoid centrosomes resembles to that of mouse oocytes or mouse eggs. We suggest that the centrioles in B-spindle cannot behave as proper kinetic centers for spindle assembly and cannot properly gather the MTOCs together during metaphase due to the influence of T-1. Next, we attempted to separate the T-1 depend- ent modification of spindle shape from its inhib- itory effect on microtubule assembly. Because spindle BR directly reflects the number of oriented microtubules within the spindle [23], the results obtained from the T-1 pulse experiments are interpreted as following. The reduction in the population density of microtubules following a highly concentrated pulse of T-1 was accompanied by an alteration of the centrosomal configuration. However, the reduction in the number of microtu- bules induced by the T-1 pulse was abolished when the interval between the pulse and fertilization was increased, whereas there was no change in micro- tubular length or in the dispersal of the kinetic centers from which microtubules were assembled. Because the microtubule population density could be restored by increasing the length of time between the T-1 pulse and fertilization, we consid- er the T-1 dependent disturbance of microtubule 274 T. J. Iron, H. SaATo AND A. KOBAYASHI assembly to be reversible. The migration of male and female pronuclei and the formation of pronuclei were also suppressed when eggs were treated with high concentrations of T-1. Again, this suppression was abolished by leaving the eggs unfertilized for more than 180 min after the T-1 pulse. Because the migration of pronuclei is considered to be tightly coupled to sperm aster formation and microtubule assembly [24, 25], these results are a further indication that the inhibition of microtubule assembly within the sperm aster by T-1 can be readily reversed. The results of our work is comparable with that obtained by Sluder and Rieder [20]. They have shown that pulsing sea urchin eggs with Colcemid before fertilization is enough to suppress microtu- bule assembly, while the UV irradiation reverses the effects of the Colcemid. They believe that the long term suppression of microtubule assembly by the pulse of Colcemid was due to the high affinity between tubulin molecules and Colcemid. We suggest that the T-1 dependent suppression of microtubule assembly may also be the result of a similarly tight T-1-tubulin interaction. Despite the similarity between the effect of T-1 and Colcemid, the mechanism of T-1 dependent suppression of pronuclear migration may not be identical to that of Colcemid, especially when considering the different way the eggs recover from the effects of each drug. The T-1 dependent induction of B-spindle is not reversible. This suggests that the morphological modification of spindle by T-1 could be mediated by a different mechanism than that which produces the inhibitory effect on microtubule assembly. The effects of D,O on B-spindle clearly indicate that the inhibition of microtubule assembly does not cause modification of the spindle structure by T-1. D,O is a useful reagent for producing an increase in the amount of polymerized tubulin [6, 7, 11], and the effect of D2O on tubulin polymerization is considered to be due to an increase in proportion of polymerizable tubulin subunits in the living cells [8-10]. In this case, the employment of D,O enhances microtubule assembly. If we use other chemical reagents such as taxol, which bind to tubulin and thereby increase microtubule assem- bly, the binding of that reagent may competitively interfere with the binding of T-1. This competition may produce side effects which make the results difficult to understand. If the reduction in microtubule assembly caused by T-1 is necessary for the formation of B-spindle, then these modified B-spindle configuration should be restored by adding D,O. If the spreading of the centrosome was a result of the partial inhibition of microtubule assembly, then DO might induce the MTOCs in the centrosomal regions to retract into the compact morphology of the control centro- some. However, the addition of D,O to the B-spindle did not alter the dispersed state of its polar centrosomes or its reduced length to width ratio. The BR and dimensions of the B-spindle were increased without a reversion to the normal structure of the spindle, instead changing to an enlarged barrel-shape. Astral fiber length also increased by the application of D,O. These results indicated that the isotope effects of D,O on B-spindle microtubules were similar to those on normal spindles. The modified spindle configura- tion was never converted to the normal shape although D,O promoted the assembly of microtu- bules. We believe, therefore, that the T-1 depend- ent alteration of spindle structure is not due to its effect on microtubule assembly. Sato and Sato [26] have reported the effect of T-1 on dividing endosperm cells of Haemanthus katharinae. In this case, high concentrations of the drug mainly affected the spindle BR, but no alteration of the poles occurred, even when using higher concentrations of T-1. These effects can be explained as due to the absence of a centrosome in the endosperm of H. katharinae. T-1 could not modify the spindle configuration by affecting the centrosome because of the absence of centrosome. Only the secondary effect of the drug, that of modulating microtubule assembly, could be de- tected. Indeed, the T-1 induced change in the plant spindle can be reversed by washing, suggest- ing that T-1 affects only microtubule assembly. Based on these data, we suggest that T-1 affects in particular the MTOCs of the centrosome. In conclusion, we believe that the main effect of T-1 on the induction of B-spindle is to directly cause the dispersion of the MTOCs within the centrosome. Although the inhibitory effect of T-1 T-1 Altered Spindle Structure on microtubule assembly was reversible, the mod- ified spindle could not be converted to a normal morphology, suggesting that T-1 may irreversibly modulate the distribution pattern of the kinetic centers on which microtubules assemble. For this reason, we believe that T-1 will be uniquely useful in the examination of the molecular basis of the physiology of the centrosome and of what deter- mines spindle morphology. We suggest that T-1 binds to some cellular component(s) which partici- pates in the control of the structure of the centrosome, and thereby alters the overall mor- phology of the spindle. Our next goal is to identify and characterize these regulatory molecules using both biochemical and physiological procedures. ACKNOWLEDGMENTS The authors are most grateful to Drs. Makoto Fuku- moto and Koichi H. Kato of the College of General Education, Nagoya City University for the use of their electron microscope and for their various helpful sugges- tions on electron microscopy technique. We also thank Dr. Hiroshi Hayashi of the Institute of Molecular Biology, Nagoya University for critically reading this manuscipt, Dr. Elena McBeath for her help for editing of this manuscript, and Mr. Yoshiharu Murata and Kuzaki Fishermen’s Cooperative Association for supplying us with sea urchins. TJI appreciates the general support from fellowships of the Japan Society for the Promotion of Science for Japanese Junior Scientists. This work was supported by several Grant- in- Aids for Scientific Research provided from the Ministry of Educa- tion, Science and Culture, Japan, whose numbers are 57380016, 58340042, 58380026, 59390006 and 61790232. REFERENCES 1 Dustin, P. (1984) Microtubules, Springer-Verlag, Berlin, Heidelberg, New York, Tokyo, 2nd ed., pp. 482. 2 Inoué, S. (1959) Motility of cilia and the mechanism of mitosis. Rev. Mod. Phys., 31: 402-408. 3 Stephens, R.E. (1973) A thermodynamic analysis of mitotic spindle equilibrium at active metaphase. J. Cell Biol., 57: 133-147. 4 Salmon,E.D. (1975) Pressure-induced de- polymerization of spindle microtubules. II. Thermo- dynamics of in vivo spindle assembly. J. Cell Biol., 66: 114-127. 5 Inouwé, S. and Sato, H. (1967) Cell motility by labile 10 11 12 13 14 15 16 17 18 19 ZS association of molecules. The nature of mitotic spindle fibers and their role in chromosome move- ment. J. Gen. Physiol., 50: 259-292. Sato, H. (1975) The mitotic spindle. In “Aging Gamates”. Ed. by J. Blandau, S. Karger AG, Basel, pp. 19-49. Sato, H., Kato, T., Takahashi, T.C. and Ito, T. (1982) Analysis of D,O effect on in vivo and in vitro tubulin polymerization and depolymerization. In “Biological Functions of Microtubules and Related Structures”. Ed. by H. Sakai, H. Mohri and G. G. Borisy, Academic Press, New York, pp. 211-226. Takahashi, T.C. and Sato,H. (1982) Thermo- dynamic analysis of the effect of D,O on mitotic spindles in developing sea urchin eggs. Cell Struct. Funct., 7: 349-357. Takahashi, T.C. and Sato, H. (1984) Effects of heavy water (D,O) on the length of the mitotic period in developing sea urchin eggs. Cell Struct. Funct., 8: 357-365. Takahashi, T.C. and Sato, H. (1984) Yields of tubulin paracrystals, vinblastine-crystals, induced in unfertilized and fertilized sea urchin eggs in the presence of D,O. Cell Struct. Funct., 9: 45-52. Itoh, T. J. and Sato, H. (1984) The effect of deute- rium oxide (7H,O)) on the polymerization of tubulin in vitro. Biochim. Biophys. Acta, 800: 21-27. Kobayashi, A., Hino, T., Umeyama, K. and Kawazu, K. (1985) Chemical studies on microtubule assembly regulators of microbial origin. 27th Sym- posium on the Chemistry of Natural Products, Hiroshima. Symposium Papers, pp. 343-350. Sato, H., Kobayashi, A. and Itoh, T. J. (1985) The mitotic arresters; Molecular probes to examine the spindle assembly. In “Cell Motility. II. Mechanism and Regulation”. Ed. by H. Ishikawa, S. Hatano and H. Sato, Univ. Tokyo Press, Tokyo, pp. 357- 370. Itoh, T.J., Sato, H. and Kobayashi, A. (1986) T-1 dependent barrel-shaped spindle induction in the sea urchin eggs and the role of T-1 in microtubule assembly. Zool. Sci., 3: 255-264. Mazia, D. (1984) Centrosomes and mitotic poles. Exp. Cell’ Res? 15392 1-15: Hirano, K-I., Masuda,M. and Sato,H. (1984) Ultrastructural study of asters induced by micro- injection with sperm centriolar fraction in sea urchin eggs. Dev. Growth Differ., 26: 435-444. Kushida, H. (1966) Block staining with lead acetate. J. Electorn Microscopy, 15: 90-92. Kushida, H. (1975) Hardness control of the Quetol 651 cured block. J. Electron Microscopy, 24: 299. Conrad, G. W. and Rappaport, R. (1981) Mecha- nism of cytokinesis in animal cells. In “Mitosis/ Cytokinesis”. Ed. by A.M. Zimmerman and A. Forer, Academic Press, New York, pp. 365-396. 20 7a 22 28 276 Sluder, G. and Reider, C. L. (1985) Experimental separation of pronuclei in fertilized sea urchin eggs: Chromosomes do not organize a spindle in the absence of centrosomes. J. Cell Biol., 100: 897-903. Endo, S., Toriyama, M. and Sakai, H. (1985) Mi- crotubule organizing granules (MTOGs) in mitotic sea urchin egg. In “Cell Motility. II. Mechanism and Regulation”. Ed. by H. Ishikawa, S. Hatano and H. Sato, Univ. Tokyo Press, Tokyo, pp. 403-414. Schatten, H., Schatten, G., Mazia, D., Balczon, R. and Simerly, C. (1986) Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proc. Natl. Acad. Sci. USA, 83: 105-109. Sato, H., Ellis, G. W. and Inoué, S. (1975) Micro- 24 75) 26 T. J. Iron, H. Sato AND A. KOBAYASHI tubular origin of mitotic spindle form birefringence. Demonstration of the applicability of Wiener’s equation. J. Cell Biol., 67: 501-517. Schatten, G. and Schatten, H. (1981) Effects of motility inhibitors during sea urchin fertilization. Exp. Cell Res., 135: 311-330. Hiramoto, Y., Hamaguchi, M.S., Nakano, Y. and Shoji, Y. (1984) Colcemid UV-microirradiation method for analyzing the role of microtubules in pronuclear migration and chromosome movement in sand-dollar eggs. Zool. Sci., 1: 29-34. Sato, Y. and Sato, H. (1985) Effect of the mitotic arrester on the spindle assembly in dividing endo- sperm cells of Haemanthus katharinae. Cell Struct. Funct., 10: 540. ZOOLOGICAL SCIENCE 4: 277-283 (1987) © 1987 Zoological Society of Japan Novel Cytotoxic Factors from Tumor Virus-transformed Human Embryo Fibroblasts YASUJI OKAL! Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Machida, Tokyo 194, Japan ABSTRACT— The author found novel growth-inhibiting factors in the culture medium conditioned by SV40-transformed human embryo fibroblasts. They were separated by gel filtration, ion exchange and lectin-affinity column chromatographies from the culture medium and characterized to be a protein or glycoprotein which exhibiting the drastic cytotoxic effect on certain untransformed and transformed cell lines. They were inactivated by the addition of endogenous growth-promoting factors from the same transformed cells. The properties of the factors are compared with those of the other factors reported previously and the biological significance of the factors is discussed from the aspect of the cell growth regulation. INTRODUCTION The cell proliferation is regulated by various growth factors [1]. Amongst these factors, trans- forming growth factor a (TGFa) is produced from various tumor virus-transformed cells which con- fers the transformed phenotype on normal cells [2] and TGFf is produced from normal or trans- formed cells which synergistically potentiates the effect of TGFa and epidermis growth factor (EGF). In this paper, the author will describe the partial purification and characterization of novel growth regulating factors from tumor virus-transformed human cell line. They exhibited a remarkable inhibition of DNA synthesis and a drastic cytotoxic activity in various cultured cell lines. They are compared with the other factors reported pre- viously and the biological significance of the factors is discussed. MATERIALS AND METHODS Cell culture Normal human embryo fibroblast cell line (YH- Accepted October 3, 1986 Received May 8, 1986 1 Present Address: Lab. of Molecular Oncology, Tsukuba Life Science Center, Yatabe-Machi, Tsuku- ba-Gun, Ibaragi 305, Japan. 1) was obtained from a 2-month-old embryo and they are transformed by SV40 at generation 19 as described previously [4]. The cells were grown in Eagle’s minimum essential medium (MEM) sup- plemented with 10% fetal calf serum (FCS, GIB- CO, Grand Island Biological Lab., New York) in 5% CO, and 95% air at 37'C. The medium was changed every other day and the culture was split by a 1:4 ratio every fourth day. Partial purification of the cytotoxic factor from the conditioned medium The recovered medium conditioned by SV40- transformed cells was concentrated with 60% saturation with ammonium sulphate and the pre- cipitate was spun down by a Hitachi RP-20 rotor at 16,000 rpm for 15min. The sample was applied on a Sephadex G-100 column (Pharmacia, 1.8 x 48 cm) and eluted with 10mM phosphate-buffered saline (PBS, pH 7.2) at 5ml per fraction. Then, the active fractions of Sephadex G-—100 chroma- tography were dialyzed against 10mM Tris-HCl buffer (pH 7.5) and applied on a DEAE Sephadex A-25 column (Pharmacia, 0.8x5cm). After washing the column with 10 mM Tris-HCl buffer, it was eluted with a Tris-HCl buffer-containing different NaCl concentrations (0-500mM). The active fraction of DEAE Sephadex A-25 chroma- tography was applied on a Concanavalin A (Con A) -Sepharose column (Pharmacia, 0.8 x 3cm) and 278 Y. OKAI eluted with PBS and PBS supplemented with 0.3 M a-methyl-D-mannoside (a-mM, Sigma). Assay for DNA synthesis and proliferation Mammalian cultured cells (1 x 10*) (Balb/c 3T3, YH-1, SV40-transformed YH-1, PC 10, SK 28, KB, Flow 7,000, GM 258, K 562, IMR 90, L 132, L929, T3M-1, Namarva and Wish) were sus- pended in 0.1ml of RPMI 1640 medium sup- plemented with 10% FCS and 0.1ml of test solution was added. After the cells were cultured for 15 hr at 37°C in 5% CO, and 95% air, they were incubated for further 5 hr in the presence of 1 yCi of [SH] TdR (5 Ci/mmol, Amersham, Eng- land). The culturing was stopped by the addition of trichloroacetic acid (TCA) in 10% and TCA- insoluble fraction was collected on a GF/C mem- brane filter (Whatman Co.) by an aid of a cell harvester. The membrane was washed with 5% TCA and ethanol, dried and its radioactivity was counted in a Beckman scintillation counter. For the assay of cell proliferation activity, the number of viable cells in 10 randomly selected microscopic fields was counted by a dye exclusion test with 0.1% trypan blue. The average cell number per microscopic field was expressed as the cell prolif- eration activity. Analysis of physicochemical and biochemical prop- erties of the cytotoxic factor Heat treatment was carried out at 56°C for 30 min and pH stability was analyzed in 50mM acetate buffer (pH 4.0) and 50 mM Tris-HCl buffer (pH 10.0) for 5 hr at 4°C. After the treatment, the sample was dialyzed against PBS and its activity was assayed. Freezing at —70°C and thawing were repeated twice. Enzyme digestion was performed by bovine pancreas trypsin (Sigma) or proteinase K (Merck) at 50 «g/ml for 30 min. To remove the effects of the exogenously added enzymes on the assay system, soy bean trypsin inhibitor (Sigma) was added to the digest at a final concentration of 50 ug/ml after the trypsin treatment. Proteinase K was separated from the factor’s fractions by a Sephadex G-100 column chromatography, be- cause the proteinase K was positioned at more than 20-30 KDa. RESULTS As a preliminary experiment, the medium con- ditioned by SV40-transformed human embryo fibroblasts was added to the culture of Balb/c 3T3 fibroblasts, the cell growth was considerably en- hanced as compared with that in the fresh medium supplemented with 10% FCS (data not shown). When the medium was concentrated and applied on a Sephadex G-—100 column, the several stimu- lating activities for DNA synthesis of Balb/c 3T3 ee oe =f ) H]TdR INCORPORATION (CPM x 10 i (6.9) 0 10 20 30 FRACTION NUMBER Fic. 1. DNA synthetic activities of Balb/c 3T3 fibro- blasts in the culture medium conditioned by SV40- transformed human embryo fibroblasts in Sephadex G-100 column chromatography. The fresh medium supplemented with 10% FCS or the conditioned medium from SV40-transformed cells was recovered, concentrated, applied on a Sephadex G-100 column and eluted with PBS at 2.5 ml per each fraction. Open and closed circles show the DNA synthetic activities of Balb/c 3T3 fibro- blasts in the fractions from the fresh medium sup- plemented with 10% FCS and the conditioned medium from SV40-transformed cells. Upper arrows in the figure represent the elution positions of molecular weight markers: blue dextran 2,000 (void volume), bovine serum albumin (67 KDa), cytochrome C (13 KDa) and bacitracin (1.5 KDa) from the left to the right. Novel Cytotoxic Factors from Transformed Fibroblasts 279 fibroblasts were observed at void volume, 30-70 KDa and about 10 KDa (closed circles in Fig. 1). Amongst these activities, the major 30-70 KDa activities were also observed in the fresh medium supplemented with 10% FCS (open circles in Fig. 1) and the activity at void volume was considerably shifted to the position at about 10 KDa by a high salt treatment (1 M NaCl) (data not shown). Thus, the author focused here the attention to the dominant activity at about 10 KDa in Figure QO mM 50 mM | | 1. The 10 KDa stimulating activity from the Sephadex G-100 chromatography was dialyzed, applied on a DEAE Sephadex A-25 column, eluted with the buffer containing NaCl at various concentrations and the stimulating activity in the fractions was examined. As shown in Figure 2, three major stimulating activities for DNA synthe- sis were found in the fractions at 0-50, 50-150 and 300-500 mM NaCl. Unexpectedly, a strong inhibi- tory activity for DNA synthesis was separated in 150 mM 300 mM 500 mM [it dbo 5 oa) | >) a x s 4 Ay Vv Q 5 d H z ~ 3 e@ \/ = e (ad e) oO Zz HH O jad xe) 2 e & am fom iL e ) O—O 10 20 FRACTION NUMBER Fic. 2. DNA synthetic activities of Balb/c 3T3 fibroblasts in 10 KDa fraction of the culture medium analyzed by DEAE Sephadex A-25 column chromatography. The fractions at 19-20 in Fig. 1 were dialyzed against 10 mM phosphate buffer, applied on a DEAE Sephadex A-25 column and eluted with 10 mM phosphate buffer containing various NaCl concentrations as shown in the figure. Open circles show the DNA synthetic activities of Balb/c 3T3 fibroblasts. Each fraction was previously dialyzed against PBS to remove the salts in the fraction and its activity was assayed. The horizontal bar shows the control activity by PBS. 280 Y. OKAI the fractions at 150-300mM NaCl from the other stimulating factors (Fig. 2): When the cell growth activities in the same fractions of DEAE Sephadex A-25 column chromatography were assayed, a similar elution profile was obtained: Three growth- promoting activities were eluted at the positions similar to those found in DNA synthesis assay and one strong inhibitory activity was eluted at 150- 300 mM NaCl (Fig. 3). This inhibitory activity was due to a cytotoxic effect: When examined micro- scopically, number of the adherent viable cells remarkably decreased and the most cells were lysed. This cytotoxic activity could not be detected in the fresh medium supplemented with FCS. A relatively weak activity was observed in the culture medium conditioned by normal human embryo fibroblasts (YH-1) (data not shown). Then it can be concluded that the cytotoxic activity is specific to the medium conditioned by the SV40- transformed cells. 50 mM A 4 ca ae (py (ES ad fea] {C) Mm wW = (ak a © Zn) ese reap (84 ea] {) CO fel = ca ig SS ~Q ry H "oO > od ~*~ 0 Fic. 3. DEAE Sephadex A-25 column chromatography. Next, the author analyzed the biochemical and physicochemical properties of the cytotoxic activ- ity. The activity was resistant to heat treatment (56°C for 30 min) and to freezing and thawing. It was also stable in an acidic (pH 4.0) or basic (pH 10.0) environment (Table 1). The activity was considerably inactivated by proteinase K, but slightly by trypsin (Table 1). In addition, the cytotoxic activity was separated into two fractions, pass-through and bound fractions by a Con A affinity column chromatography (Fig. 4). These results suggest that the cytotoxic activity is associ- ated with a heat stable protein or glycoprotein. At last, the author studied the effect of the factor on various mammalian cultured cells (Table 2). The SV40-transformed human embryo fibro- blasts which produce the factors were very sensi- tive to the factors and more than 90% of DNA synthesis was inhibited. A similar high sensitivity was found in YH-1 (untransformed human embryo 150 mM 300 mM 500 mM piles 20 FRACTION NUMBER Cell growth activities of Balb/c 3T3 fibroblasts in 10 KDa fraction of the culture medium analyzed by The effect by the same fractions in Fig. 2 on the number of viable cells in Balb/c 3T3 fibroblasts was assayed by a dye exclusion test with trypan blue. Open circles show the average number of viable cells per microscopic field and the horizontal bar is the value in the PBS control. Novel Cytotoxic Factors from Transformed Fibroblasts 281 TaBLE 1. Biochemical and physicochemical properties of the cytotoxic factor a eee [PH]TdR Incorporation (CPM) Control 2839 + 210 +Factor treated with None Seay) Heat (56°C, 30 min) 69+ 10 pH 4.0 (Sas) pH 10.0 Ag-ee 4 Freezing and thawing Stee 2 Trypsin Pte 29 Proteinase K 2080 + 268 eee eee ere Ot iP Rb el peafowl The radioactivity is shown as the mean and standard error of triplicate assays. : | ) -3 [2H] TaR INCORPORATION (CPM x 10 10 20 FRACTION NUMBER Fic. 4. The cytotoxic factors are eluted in the unbound and bound fractions of a Con A-Sepharose column chromatography. The active fractions at 14-17 in Fig. 2 were dialyzed against PBS, applied on a Con A-Sepharose column and eluted with PBS, and PBS with 0.3 M a-methyl- D-mannoside (a-mM), successively. The arrow in- dicates the addition of a-mM. The fraction with PBS containing a-mM was dialyzed against PBS and its activity was assayed. The horizontal bar is the activity of the PBS control. fibroblast), PC 10 (epimoid carcinoma) and SK 28 (melanoma). The next high sensitivity (50-90% inhibition) was found in KB (epimoid carcinoma), Flow 7,000 (foreskin fibroblast) and GM 258 TaBLE2. Effects of cytolytic factor on various cultured cells Cell strain Origin Sensitivity YH-1 human embryo fibroblast lalate SV40-transformed YH-1 Statats Syl murine fibroblast sits PE 10 human epidemoid carcinoma +++ SK 28 human melanoma sick: KB human epimoid carcinoma +H Flow 7000 human foreskin fibroblast SR GM 258 human embryo fibroblast s K 562 human erythroleukemia cell os IMR 90 human embryo fibroblast = B32 human embryo lung cell 35 L 929 murine fibrosarcoma = T3M-1 human carcinoma = Namarwa human lymphoblastoid cell = Wish human amniotic cell — DNA synthesis inhibition, +++: more than 90%, ++: 50-90%, +: less than 50%, —: no change (embryo fibroblast) cells. K 562 (erythroleukemia cell), IMR 90 (lung fibroblast) and L 132 (embryo lung cell) cells were relatively resistant (less than 50% inhibition). No significant inhibitory effect was observed in T3M-1 (carcinoma), Namarva (lymphoblast) and Wish (amniotic cell) cells. The cytotoxic activity derived from the culture medium of SV40-transformed human embryo fibroblasts was cytotoxic to the same cells (Table 2). The reason why the cells can grow and become confluent in the conventional culture was found in the fact that this cytotoxic activity was inactivated by the growth-promoting factors derived from the same transformed cells: As shown in Figure 5, the isolated cytotoxic factor was represented as a single inhibitory activity at about 10 KDa in Sephadex G-100 column chromatography (open circles). When the growth-promoting factors in Figure 2 were combined, mixed with the cytotoxic factor and applied on a Sephadex G-100 column, the cytotoxic factor was inactivated, and even reversely, some _ stimulating activities were observed at void volume, 30-70 KDa and about 10 KDa (closed circles in Fig. 5). The larger stimulat- ing activities were derived from the aggregates of 10 KDa activities. 282 Y. OKAI ) -3 [?H]TAR INCORPORATION (CPM x 10 10 20 30 FRACTION NUMBER Fic.5. The effect by the cytotoxic factor was inacti- vated by the growth-promoting factors. The active fraction of the cytotoxic factor in Fig. 2 was incubated with (closed circles) and without (open circles) the growth-promoting factors at 4°C for 30 min and applied on a Sephadex G-100 col- umn as performed in Fig. 1. The growth-promoting activities in Fig. 2 (Fractions at 6, 8, 11, 13, and 19) were combined, concentrated with 60% saturated ammonium sulphate and added to the cytotoxic factor. Upper arrows in the figure are the same as in Fig. 1. DISCUSSION The results mentioned above suggest that a novel regulating factor for cell growth is released from SV40-transformed human embryo fibro- blasts. This factor showed the cytotoxic effects on certain transformed and untransformed cell lines, but its activity was inactivated by the growth- promoting factors from the same transformed cells. Some cytotoxic or growth-inhibiting factors from mammalian cells have been reported. First of all, lymphotoxic (LT) and tumor necrosis factor (TNF) are released from the lymphocytes or monocytes and they exhibit the cytotoxic or cytostatic effects on certain transformed cell lines [S—8]. A considerable homology exists in their amino acids and DNA sequences [9, 10]. Their molecular weights are estimated to be 25 KDa and 17 KDa by means of their DNA clonings [9, 10] and aggregate as the forms larger than 40-50 KDa [5-8]. They show little or no anticellular activities on primary cell culture or normal cell strains such as primary human embryo fibroblasts or mouse 3T3 fibroblasts [5-8]. On the other hand, the factor obtained in this study was estimated to be about 10 KDa by a gel filtration chromatography under the conventional buffer condition and show the cytotoxic effect on some transformed and untransformed cell lines including normal human embryo fibroblasts and 3T3 fibroblasts. Therefore, the further analysis is required, the present factor is considered to be different from LT or TNF. Tumor cell growth-inhibiting factors (TIFs) were reported and they inhibit the growth of various tumor cell lines, but stimulate normal human foreskin fibroblasts and epithelial cells in monolayer cultures [11,12]. The effects by the factors are not cytotoxic, but reversible when the affected cells are no longer exposed to the factors. On the contrary, the factor in the present paper showed the irreversible cytotoxic effect on un- transformed and transformed cell lines and did not exhibit the stimulating activity for the proliferative response of human embryo fibroblasts. TGF is a potent stimulator of growth in soft agar of ancholage-dependent cells including hu- man fibroblasts and also a potent growth inhibitor for human foreskin keratinocytes and certain human cancer cell lines [13]. This inhibition is not cytotoxic in nature, but reversible which is differ- ent from the inhibition by the factor in this paper. Tumor degenerating factor (TDF) was found in the culture medium of normal human embryo fibroblasts [14]. It has a basic charge property and does not bind to DEAE Sephadex column. It causes a morphological change on KB, HeLa and other cell lines [14]. It can cause neither significant inhibition to DNA synthesis nor cytotoxic effect (personal communication by Dr. A. Tanaka, Kyoto Prefecture University of Medicine). The author reported the heterogeneous molecu- lar weight inhibitory factors for DNA synthesis from the serum-free culture medium conditioned by normal human embryo fibroblasts (YH-1 cells) [15]. Although they show a strong inhibition to Novel Cytotoxic Factors from Transformed Fibroblasts DNA synthesis in normal fibroblasts or lympho- cytes, the cell viability is not changed. In addition, the production of the cytotoxic factor in this paper was very weak in the culture medium of normal human embryo fibroblasts. Thus it may be concluded that a novel cytotoxic factor is released from the SV40-transformed human embryo fibroblasts which exhibits the cytotoxic effect on certain untransformed and transformed cell lines and that the action of this factor is counteracted by the growth-promoting factors from the some transformed cells. Although the more detailed study is required, the existence of this cytotoxic factor in tumor virus-transformed human embryo fibroblasts seems to serve an interesting problem concerning the cell survival, growth and death in untrans- formed and transformed cells. ACKNOWLEDGMENTS The author would like to thank Miss S. Kurata for her technical assistance and Dr. N. Fujiyoshi for his en- couragement in Tokyo Research Laboratories of Kyowa Hakko Kogyo Co. REFERENCES 1 Holley, R. W. (1980) Control of animal cell prolif- eration. J. Supramol. Struct., 13: 191-197. 2 Delarco,J.E. and Todaro,G.J. (1978) Growth factors from a murine sarcoma virus-transformed cells. Proc. Natl. Acad. Sci. USA, 75: 4001-4005. 3. Roberts, A.B., Frolik,C.A., Anzano, M.A. and Sporn, M.B. (1983) Transforming growth factors from neoplastic and nonneoplastic tissues. Fed. Proc., 42: 2621-2626. 4 Yanagisawa,K., Suenaga, Y., Nishio,K. and Gotoh, $.(1983) Establishment of human diploid cell strain. J. Univ. Occup. Eniron. Health (Kita- kyushu), 5: 49-54. 5 Aggarwal,B.B., Moffat,B. and Harkins, R.N. (1984) Human lymphotoxin - Production by a lym- phoblastoid cell line, purification, and initial charac- terization. J. Biol. Chem., 259: 686-691. 6 10 11 2 13 14 15 283 Ruddle, N.H., Powell,M.B. and Conta,B.S. (1983) Lymphotoxin, a biologically relevant model lymphokine. Lymphokine Res., 2: 23-31. Williamson, B.D., Carswell, E.A., Rubin, B. Y., Prendergast, J.S. and Old,L.J. (1983) Human tumor necrosis factor produced by human B cell lines : Synergistic cytotoxic interaction with human interferon. Proc. Natl. Acad. Sci. USA, 80: 5397- 5401. Ruff, M.R. and Gifford, G. E. (1981) Tumor necro- sis factor. Lymphokines, 2: 235-272. Gray, P. W., Aggarwal, B. B., Benton, C. V., Bring- man, T.S., Henzel, W.J., Jarrett, J. A., Leung, D. W., Moffat, B., Ng, P., Svedersky, L. P., Palladino, M.A. and Nedwin, G.E. (1984) Cloning and ex- pression of cDNA for human lymphotoxin, a lyoho- kine with tumor necrosis activity. Nature, 312: 721-724. Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P.H., Derynck,R., Palladino, M.A., Kohr, W.J., Aggarwal,B.B. and Goeddel, D. V.(1984) Human tumor necrosis factor : precursor structure, expression and homology to lymphotoxin. Nature, 312: 724-729. Iwata, K.K., Fryling,C.M., Knett,W.B. and Todaro, G. J.(1985) Isolation of tumor cell growth- inhibiting factors from a human rhabdomyosarcoma cell line. Cancer Res., 45: 2689-2694. Fryling, C.M., Iwata, K. K., Johnson, P. A., Knott, W.B. and Todaro, G. J.(1985) Two distinct tumor cell growth-inhibiting factors from a human rhab- domyosarcoma cell line. Cancer Res., 45: 2695- 2699. Moses; Hels lucker, Rees Lock EaB-,) Coffey. R.J., Halper, J.J. and Shipley, G. D.(1985) Type-£ transforming growth factor is a growth stimulator and a growth inhibitor. In “Cancer Cells, Vol. 3”. Ed. by J. Feramisco, B. Ozanne and C. Stiles, Cold Spring Harbor Lab., New York, pp. 65-71. Tanaka, A., Matsuoka, H., Uemura, H., Kakui, Y., Imanishi, T., Nishino, H. and Imanishi, J.(1985) Production and_ characterization of tumor- degenerating factor. J. Natl. Cancer Inst., 74: 575-581. Okai, Y.(1986) Heterogeneous molecular inhibitory factors for lymphocyte DNA synthesis from human embryo fibroblasts—two conversion pathways by a trypsin-like protease(s). Zool. Sci., 3: 271-276. 2 & etoc).” bet 9M Meal dg #4 pin 6 hour :evebot, otieigehide pitonccligor(d LEBUT de Poe ESS aeodaeny, 1 oie relayinn dl Yo nde A 3 Dewees cumuht (Par 0 BRS Gee 2S Ae umcroy “ior ipigssaved tamil ene Am. - iT eM OTT: rong tradi &., | tine be os and? (a SU bee Oe ime va Seo 27a eV SSE Se Re "> j aa J i: “ga oheereM, Jiant o3¢ ess2t / 3 eeteeGd- Be lointa lh mi ey ‘Vein? nue) . AL tag A teteh 27 nein ett ! rok. Rig lott telonh 4 hee asin’) (2B) 8 2 abetralt. bre a 4 SON 2} rye jon: damnit yan rit i> ee sees (ja sxe mre maw of . 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Yotpwinereaes " via £0 rer tela | Bis aot nfonhees cthaay tis 7 presse ye: c ; : il tna ikon si Tegal To" Mao. riage ean huigeaers jas ar rarre ay doe a i ‘ii av + joann eau: Sau pa Li oF Li bie DM, CHIT ‘ ercugar Rie ADT Au hee Mee | “Henke 5 x if ety een Uae “Ub afinenS Distal saubserithet | uo} meer ei) sed brn tcasipe} HURTS | : Ako Ati acy LF ata, bah Baa Gye fentont 7 eat ills APRA i) iperonntnegoaay Ayo ‘Praleeteite. fae eather repo ae a idl j ‘ AMT weight ceitettenry PRN ZOOLOGICAL SCIENCE 4: 285-292 (1987) © 1987 Zoological Society of Japan Two-dimensional Polyacrylamide Gel Analysis of Papilloma and Normal Skin Proteins in Newt KAZUNORI SHIMADA, HIROMICHI Koyama! and MAKoTo ASASHIMA~ Department of Biology, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236, and "Department of Anatomy, School of Medicine, Yokohama City University, Urafune-cho, Minami-ku, Yokohama 232, Japan ABSTRACT—The protein patterns of papilloma found in Japanese newt Cynops pyrrhogaster were compared with those of the nontumourous skin of the same animal by two-dimensional (2-D) gel electrophoresis method. There were 11 protein spots specific to the skin. Almost all of them were detected also in skin derived from different regions of normal adult male and female. On the other hand, 7 protein spots specific to papilloma were detected. The papilloma-specific spots were detected neither in the skin, lung and liver of normal adult newt, the skin of normal larvae, nor the presumptive ectodermal region of embryo. One of the 11 spots specific to adult skin coincided with one of the spots of normal larval skin. Two unique spots were identified in the larval skin. Such appearance of these tissue specific proteins suggests that the cell transformation involves a disdifferentiative but not dedifferentiative process. INTRODUCTION Little attention has been paid to the tumors of amphibians, as compared to those of the higher vertebrates. This is due to the fact that not much is known about the variety of tumors among this group, and also because amphibians are possessed of such unique characteristics as a strong regener- ating ability, hibernation and metamorphosis which are not shared by other vertebrates [1, 2]. We have been conducting a clinical laboratory study of papilloma-bearing Japanese newts, Cynops pyrrhogaster, collected in Niigata and Iwate prefectures. Differences in the geographical and seasonal abundance of the papilloma, and the effects of temperature on the growth of the papilloma have been reported [3, 4]. However, no comparison or analysis has been made as to the biochemical natures of papilloma and normal skin. Biochemical studies of Lucké renal carcinoma in the Rana pipiens frog revealed the appearance of tumor specific proteins after the transformation of Accepted November 7, 1986 Received August 19, 1986 2 To whom reprints should be requested. normal renal tissues [5,6]. As papilloma are thought to consist of transformed epidermal cells [7], it is possible that such tumor specific proteins are present also in the papilloma of newts. We, therefore, conducted the study on the protein pattern of Japanese newt papilloma by means of 2-D polyacrylamide gel electrophoresis. The pro- tein pattern of newt papilloma was determined and then compared with those of normal adult and larval skin, and those of embryonic presumptive ectoderm, in order to examine whether or not tumor specific protein exists in the papilloma of the newt. MATERIALS AND METHODS Sample preparation The material used was Japanese newt Cynops pyrrhogaster collected in Iwate prefecture in the autumn of 1984. Female newts, approx. 110mm in body length, with papilloma (ca.2mm in dia- meter) on their dorsal surface were selected. Each newt was anesthetized with 0.02% MS-222 in water, and its papilloma was removed with a fine scalpel (Fig. 1). The dissected segment of papillo- 286 K. SHIMADA, H. KOYAMA AND M. ASASHIMA Fic. 1. Papilloma Normal (control) Schematic diagram of source materials. Skins (or presumptive ectoderm) were taken as controls from shaded areas. A; normal adult (male and female). B; adult female newt bearing papilloma. C; larval newt (stage 55). D; early gastrula (stage 10). ma was cleaned at room temperature in Holtfre- ter’s solution (HS) [8]. Blood cells and muscle tissue were removed. They were twice washed with HS. Cleaned papilloma tissues were homoge- nized at 0°C in a lysis buffer containing 8M urea, 5% Ampholine pH 3-10 (LKB, Sweden), 5% 8-mercaptoethanol and 2% NP-40 (Sigma, USA) [9]. The homogenate was left standing at room temperature for 5 hr, and then centrifuged at 3,000 g for 30 min, and the supernatant was obtained as papilloma sample. For a control, papilloma-free skin with dermis was taken from the dorsal surface of the same newt from which papilloma had been removed (Fig. 1). At the same time, skin samples were obtained from the dorsal region of normal male newts, body length ca. 100mm, and from 4 different regions (head, dorsal, ventral and tail; Fig. 1) of normal female newts, body length ca. 110mm. Lung and liver tissue of normal female newts also were used. These samples were processed as described for the papilloma sample. Larvae at stage 55 [10] before metamorphosis were also anesthetized with MS-—222. Larval skin samples were prepared after removal of fins. A part of the body (Fig. 1) between the anterior and posterior limbs was removed with a fine scalpel and the skin was removed from it in HS, with forceps. Presumptive ectoderm was prepared from early gastrula after the jelly envelope and vitelline membrane were removed (Fig. 1). All the samples were stored at —15°C until use. The samples were thawed at room temperature, precipitated with trichloracetic acid (TCA), and then the concentration of protein was determined by the method of Lowry et al. [11]. 2-D Polyacrylamide gel electrophoresis The basic process followed the method de- scribed by O'Farrell [12, 13]. The first-dimension (1-D) featured polyacrylamide gel and isoelectric focusing, and SDS polyacrylamide gel was used in the second-dimension (2-D). The acrylamide stock solution contained 30% acrylamide in total concentration, i.e., 29.2% acrylamide (BIORAD, USA) and 0.8% _ bis-acrylamide (BIORAD, USA). 1-D Isoelectric focusing gels (8M urea, 3.5% acrylamide, 2% NP-40, 5% Ampholine pH 3-10, 0.015% ammonium persulfate, 0.1% TEMED: N, N, N’, N’-tetramethylethylenediamine) were cast to a length of 12cm in 2.5mm i.d. 13cm long glass tubes, overlayed with distilled water to form the uniformly flat surface, and allowed to polymer- ize at room temperature for 5 hr. After prerunning at 200 V for 15min, at 300 V for 30min and at Analysis of Newt Papilloma Proteins 287 400 V for 30min, in this sequence, each gel was loaded with a sample, usually containing 0.02 mg of proteins. Electric focusing was carried out at 400 V for 14 hr, followed by at 800 V for 30min. Gels were removed from the glass tubes and shaken in an SDS sample buffer (10% glycerol, 5% 6-mercaptoethanol, 0.0625M Tris-HCl pH 6.8, 2.3% SDS) for 60 min. The 2-D separation gel used was 12cm-wide, 11cm-high and 1mm-thick 7.5% acrylamide gel with 0.375M Tris-HCl, pH 8.8, 1% SDS, 0.078% ammonium persulfate and 0.075% TEMED. Af- ter polymerization of the separation gel, a 1 cm-high stacking gel (3% acrylamide, 0.125M Tris-HCl pH 6.8, 1% SDS, 0.1% ammonium per- sulfate and 0.1% TEMED) was added onto it. The SDS sample buffer was wiped off from each equalized 1-D gel and this was fixed onto polymer- ized 2-D gel with a hot agar mixture (1% Agarose in SDS sample buffer) containing a slight amount of bromophenolblue. The gels were set in an electrophoresis apparatus (Atto Co., Ltd., Tokyo) with a buffer (0.025 M Tris, 0.1% SDS and 0.192 M glycine) and was run for 270-300min at 20 mA/gel until the dye front reached 10mm from the bottom of the gel. As a standard protein, actin (Sigma, USA) was run in the 2-D gel. Silver staining The 2-D separation gel was fixed in a mixture of 50% methanol and 10% acetic acid, shaken for 1 hr, washed three times in distilled water for a total of 45 min, and then immersed into a silver stainer (0.776% AgNO3, 0.0756% NaOH and 0.392% NH,OH) [14] and shaken for 15min. The gel was then rinsed twice for 3 min each in distilled water and transferred to a developer (0.005% citrate and 0.019% formaldehyde). When spots became suf- ficiently visible, the developer was replaced with a double volume of a fixer to terminate staining. Photography and drying Stained gels were photographed with Neopan F ASA 32 (Fuji) using transmission light, and then dried for one day between sheets of cellophane. Both the photographs and the dried gels were used to analyze the protein spots. RESULTS In our modification of the method of O’Farrell, the pH4-9 range of the first dimension was broader than that in the original method [12, 13]. When actin monomer was subjected to elec- trophoresis, the isoelectric point was pH 4.8 and Rf value 0.64. As the constantly detectable spot of probable actin in our experiments showed similar values (isoelectric point pH 4.8; Rf value 0.65), it was reasonable to use this spot as an actin marker spot. Comparison between normal skin and papilloma As shown in Figure 2, the proteins of normal skin and papilloma were separated by the 1-D gel in the range of pH 4 to 9. The arrow followed by “A” indicates a probable actin spot, and this spot was used as a marker in the present analysis. There was an average of 300 spots detected in both normal skin and papilloma. At least 11 and 7 spots were detected only in normal skin and papilloma, respectively. The former will be referred to as skin specific spots (SSSs) and the latter as papilloma specific spots (PSSs). The staining intensity of each spot was diverse. Regional difference of protein patterns of skin Of the 11 SSSs, spot numbers 1, 2, 3, 4, 7, 8, 9 and 10 were detected in the dorsal, ventral, head and tail skin in normal adult female (Table 1), although some region specific spots were detected (spot patterns not shown in figures). No spot from these materials corresponded to any of the PSSs (Table 2). Difference by sex The 11 SSSs were also found in the dorsal skin of normal adult male, but no PSSs were detected (Tables1 and 2). There were no significant differences between spot patterns of males and females (data not shown). Comparison with larval skin There were fewer spots in larval skin than in adult skin, or an average of about 200 (Fig. 3A). One spot corresponding to SSSs was observed (Fig. 3A, 9). Two spots not detected in any other 288 K. SHIMADA, H. KoYAMA AND M. ASASHIMA A i we : ais we Fic. 2. 2-D Gel protein pattern of skin (A) and papilloma (B) of adult female. “A” indicates the spot of actin. Numbers 1 to 11 in (A) indicate skin specific protein spots (SSSs). Letters a to g in (B) indicate papilloma specific protein spots (PSSs). Analysis of Newt Papilloma Proteins TABLE 1. Spot number Materials Papilloma-bearing adult (f) papilloma _ _ — skin dorsal ab aE - Normal adult skin dorsal (f) ventral (f) - head (f) + tail (f) se dorsal (m) - + +++ +4 lung (f) are en liver (f) ae ee Larval skin (st. 55) aahW geae iet oe Ectoderm (st. 10) Sth ENE ra 1-11; skin specific spots, f; female, m; TABLE 2. Spot number Materials a b Papilloma-bearing adult (f) papilloma a at skin dorsal — — Normal adult skin dorsal (f) == — ventral (f) _ = head (f) _ = tail (f) — = dorsal (m) = = lung (f) = os liver (f) = = Larval skin (st. 55) = = Ectoderm (st. 10) = = ++ +++ male. 289 Comparison of skin specific spots among various materials | +++4+ + +++4+ + ++ +++ ++ +++ 1 + Comparison of papilloma specific spots among various materials c d e f g LSS actin | | | | | | + -— = =O FH | | | | | | ++ ++ ++4++4+ +4 a-g; papilloma specific protein spots, f; female, m; male, LSS; larva specific protein spots. samples were larva specific spots (Fig. 3A, L). Except for these larva specific spots, the spot pattern of larval skin corresponded to that of adult skin. Comparison with presumptive ectoderm This tissue demonstrated even fewer spots than larval skin, the average being 100 (Fig. 3B). The total protein content of this sample used was as 5 times (0.1 mg) as in Figures 2 and 3A. Except for the appearance of two large spots at pH 7-8 and extending from high MW to low MW, which is probably indicating yolk protein, all spots detected in presumptive ectoderm corresponded to those found in adult skin and no PSSs were found. Tables 1 and 2 summarize the occurrence of SSSs and PSSs in the subject materials. 290 K. SHIMADA, H. KOYAMA AND M. ASASHIMA eee st 7 6 4) v V v <@«o =0.96, P=0.0001) and female (Y(F)= --- with r?=0.95, P=0.001) Blattella germanica as a function of developmental stage. Fic.1B. Differential plasmatocyte counts of male (Y(PLM)=—with r°>=0.95, P=0.001) and female (Y(OLF)= ---with 1?=0.96, P=0.001) Blattella germanica as a function of developmental stage. Fic.1C. Differential granulocyte counts of male (Y(GRM) =—with 1r°=0.95, P=0.001) and female (Y(GRF)=—with r°=0.96, P=0.001) Blattella germanica as a function of developmental stage. another (P=0.05) (Table 1), were analyzed by the Linear Regression Analysis, and when plotted against stages, resulted into two straight lines for both male and female B. germanica (P=0.001) (Fig. 1A). This showed not only a gradual, but a linear increase of THCs (log THCs) during de- velopment. We beleive that a linear increase in hemocyte populations occurs probably concom- itantly with an increasing demand for nutrient supply during development, cellular defense, pro- duction of immunologic factors (such as hemagglu- tinins, antimicrobial, antiviral factors), and supply of tyrosine and phenoloxidase for sclerotization and recognition of nonself tissue [3]. The linear increase of hemocytes at a given time of each stage (e.g., “mid stage”, Fig. 1A), in addition to trimod- al in Gryllus [4] and bimodal in Periplaneta [5] or cyclic in Halys ({16] and for review see Shapiro [17]) is probably a widespread phenomenon in insects, yet to be explored. Differential hemocyte counts (DHCs) The mean DHCs and their log 10 values for PLs, GRs, prohemocytes (PRs), coagulocytes (COs), and oenocytoids (OEs) are shown in Tables 2 and 3. The log 10 (DHCs) values for all the above hemocyte types were analyzed by Linear Regres- sion Analysis; each mean log value, when plotted against the developmental stages, resulted into two straight lines, male and female, for each of the PLs (Fig. 1B), the GRs (Fig. 1C) and the OEs (Fig. 2). PRs and COs were not recognized to have any linearity because they had r?<0.75. The differential counts of PLs also were signi- ficantly different among developmental stages in both sexes, with the exception of the female 3rd- and 4th- and the male S5th- and 6th-instar nymphs (Tables 2 and 3). Like those of the THCs, the GR counts of each developmental stage not only increase linearly, but differ significantly from one another (Tabled 2 and 3). The mean differential counts of the PLs and GRs are slightly higher in the male Ist-, 2nd-, 4th-, and S5th-nymphal instars, but those of the female 3rd-, and 6th-nymphal instars and adults are significantly higher (P<0.05), compared with those in the male. The continuous and linear increases of PLs and GRs probably correspond to 310 L. K. HAZARIKA AND A. P. GupTa TABLE 2. Comparisons of DHCs in the developmental stages of female Blattella germanica Stage Hemocyte types CO GR OE PL PR 1 14.32* 179.94 Oa 258.74 189.08 aS) aE, / stella ase) +4.5 fet DDS) 0.96° 2.41" Urls 2 DMZ) 262.71 UL 2 519.62 34.78 21.3 +3.4— +1.4 aE U/)s) ats 220) E28 2.42) 1.02° Deli 1S Sy 3) 38.42 Dehskw) SIL. 1230357 92.86 steed aco) 0) aE Slt SE D).o a 62 2: Hoe JA 1.45" S07 12967 4 37.06 Woh Doms 1197.99 75.6 ac5))) SE 25) aco) aE S)//) ac Il3),Il eos) DAS e385 3.07° IL scopile 3) 428.62 3095.6 MOPS) 4374.61 380.62 aroha // +199.4 +28.4 aE 23)5)6 +60.5 2162? 3.488 ZOE 3.64° 2.540 6 718.71 5027.99 245.31 7774.18 618.81 +74.5 +74.5 t= S853 + 660.9 +63.1 2.84 3.68" 2.34™ Stor? Me dil” adult 482.46 9746.53 399.86 12347.98 USD). S7 ate ILO ae Uks\ile74 ae o)/all + 1210.8 ae Sled 2.60° 3.98 2.56' 4.08? JS} * First row of each stage represents the mean, the second row standard error, and the the third row the log transformed mean. Sample size=10. Log (mean DHCs) with the same letters are not significantly different from each other (Duncan’s Test, alpha=0.05). DHC=differential hemocyte count, CO=coagulocyte, GR= granulocyte, O0E=oenocytoid, PL=plasmatocyte, PR =prohemocyte. 4.50 7 A 4.25 Y(M) = 2.4646 + 0.2547x 7 od Y(F) = 2.3986 + 0.2815X LOG (THC) 6 ADULT DEVELOPMENTAL STAGES Fic. 2. Differential oenocytoid counts of male (Y(OEM) = — with r? = 0.80, P = 0.01) and female (Y(OEF) = --- with r?>=0.85, P= 0.01) Blattella ger- manica developmental stage. growing demand for cellular immunity as B. germanica progresses its development through various instars. In fact, Ragyalis [18] suggested that each ontogenic stage in Acantholyda posticalis has specific hemocyte composition to meet its developmental needs. Because the effectiveness of phagocytosis, encapsulation, and other related defense mechanisms, is primarily a result of the available circulating immunocyte population (PLs and GRs, [2, 3]), it is reasonable to suggest that larger the PL and the GR populations, the stronger the cellular defense. Apparently, the effectiveness of phagocytosis and encapsulation differs from one developmental stage to another (for review see Gupta [2]). We believe that this differential defense capability of an insect during development is largely due to the differential population of the circulating PLs and GRs, and possibly of the OEs. It is reasonable to assume that the early imma- ture instars (for example, 1st-instar nymphs of B. germanica) are immunologically less competent, because they have comparatively fewer PLs and GRs. They are likely to succumb more readily to pathogen attack as well as susceptible to biologi- Ontogeny and Hemocyte Populations in Cockroach TABLE 3. Comparisons of DHCs in the developmental stages of male Blattella germanica Hemocyte type ea CO GR OE PL PR 1 15/58" 207.04 16.74 264.04 147.8 Hellen sal PAM ate ae9 tue seat Wah IsSS 2.31) aise 2.42° Zale? 2 58.45 281.82 24.99 473.9 34.71 +8.0 se0.On ate +27.5 ney) eZ” 2.45’ 1.387 2.67 ESS: 3 63.65 398.92 23.98 801.67 67.37 SECs / t= O29 omer) toS Feil 59.1 1725 26n 1345 ASE J estirh + 63.65 765.47 27.4 1391.05 59:73 +13.4 +37 ate 28 SEIS) sei) 7/ 1722 2.888 1.42™ 3.14P boyy 5) 272.28 3129.59 94.69 4812.84 340.51 020 SE (28 SE //at) +149.3 +44.4 235% 3.49! 1.97! 3.68° 2.49" 6 389.52 3852.14 185.13 4783.68 484.59 +40.3 aE A/T) +24.1 + 183.0 at TOEL 27565 S255 Dg 3.68° 22005 adult 341.68 6414.77 209.48 8608.1 Ol723 ata l6 + 436.7 an /28)s) + 453.9 ae 113i). 1 2.48? 3289 Dor 593% iil 311 * First row of each stage represents the mean, the second row standard error, and the third row the log transformed mean. Sample size=10. Log (mean DHCs) with the same letters are not significantly different from each other (Duncan’s Test, alpha=0.05). DHC=differential hemocyte count, CO=coagulocyte, GR=granulocyte, OE=oenocytoid, PL=plasmatocyte, PR =prohemocyte. cally active substances, including insecticides and insect growth regulators (IGRs). For example, when the Ist- and 2nd-instar nymphs were allowed to feed on IGRs-treated food pellets, they died within a few days without molting ([19, 20] and unpublished observation), in comparison with controls (acetone-treated food pellets). Lower PL- and GR counts as well as THC in these instars may partly contribute to their mortality directly or indirectly by reducing their immunity or by failing to undergo ecdysis [19, 20]. As shown earlier, the linear OE counts seem to correlate with the higher demand for nutrient supply as B. germanica advances in its develop- ment; OEs supposedly carry proteins in the cock- roach, Gromphadorhina portentosa [21]. Compared with the male 5th and 6th instars, the corresponding female instars had _ significantly higher CO counts (Tables 2 and 3). The penulti- mate and ultimate female nymphal instars prob- ably require higher COs in comparison with those of males. Considering the presence of significant differences in CO counts among various develop- mental stages, it is possible that there is a differential coagulative capability and prophenol- oxidase-carrying capacity among them (Tables 2 and 3). This, however, needs confirmation. Although the differential counts of COs were lower in the female I1st-, 3rd-, and 4th-instar nymphs, those of the 2nd-instar male (P=0.0004) were significantly higher, compared with those in the female (Tables 2 and 3). This sexual dimorph- Bi L.K. HAZARIKA AND A. P. Gupta ism in CO counts in an early instar is hard to explain. However, we believe that maximum differentiation of hemocytes is presumably very pronounced in the 2nd-instar nymphs, including a dimorphic differentiation in this developmental stage. As shown in the subsequent discussion of the PR counts, a similar differentiation pattern of PRs was also observed. The circulating population of COs during development does not seem to be progressively linear during development; however, in the event of injury their numbers may increase to stop bleeding as a result of coagulation. In addition to increasing continuously only in the 5th- and 6th-instar nymphs and adults of both sexes (Tables 2 and 3), the PR counts were also significantly different from one another (among Sth-, 6th-instar nymphs and adults) and also among other developmental stages (P=0.0001). However, the Ist-instar nymphs had significantly higher PR counts (Tables 2 and 3) than the 2nd-, 3rd- and 4th-instar nymphs. The latter two had insignificantly different PR counts between them. The non-linear distribution of the PR counts suggests that perhaps there are three phases of PR differentiation during the development of B. germanica: 1) The relatively higher counts in the lst instar indicate that PRs are still being pro- duced. 2) The significantly lowest counts in the 2nd instar suggest that the PRs are undergoing trans- formation into PLs and other types. 3) The trans- formation process is completed in the 3rd- and 4th-instar nymphs, as suggested by the insignificant differences in the PR counts between these instars. These suggestions are also supported by the mean percent data. A steady increase in THC during the Sth- and 6th-instar nymphs, and adults (Fig. 1A and Tables 1, 2 and 3) contributes probably to a similar steady increase in PR counts during those developmental stages. Correlation between THC and DHC The rates of increase of PLs and GRs are almost identical to those of THCs, suggesting strong correlation between THCs and DHCs of PLs and GRs. With the exception of PRs and COs, there is a linear increase for PLs, GRs and OEs. The significant differences between the stages of B. germanica for GRs and PLs (with a slight variation in case of PLs) and also an exactly similar sexual dimorphism in THCs and DHCs of PLs and GRs, strongly suggest a positive correlation between the THCs and DHCs of the major hemocyte types. The PLs and GRs represent almost more than 70-80% of THCs in all the developmental stages; thus, they determine the total quantitative picture of the hemocyte populations in any of the seven developmental stages. ACKNOWLEDGMENTS This paper is the New Jersey Agricultural Experiment Station Publication No. D-08112-12-86, supported by state funds, U. S. Hatch funds, and Rutgers Research Council. LKH was on sabbatical leave from Assam Agricultural University, Jorhat, India, during this study on a Government of India fellowship. REFERENCES 1 Gupta, A. P. (1979) Insect Hemocytes, Cambridge Univ. Press, Cambridge. 2 Gupta,A.P. (1985) Cellular elements in the hemolymph. In “Comprehensive Insect Physiology, Biochemistry and Pharmacology”. Ed. by G.A. Kerkut and L. I. Gilbert, Pergamon Press, Oxford, Vol. 3, pp. 402-451. 3 Gupta, A. P. (1986) Arthopod Immunocytes: iden- tification, structure, functions, and analogies to the functions of vertebrate B- and T-lymphocytes. In “Hemocytic and Humoral Immunity in Arthro- pods”. Ed. by A.P. Gupta, Wiley & Sons, New York, pp. 3-59. 4 Tauber, O.E. and Yeager, J. F. (1934) On the total blood (hemolymph) cell count of the field cricket, Gryllus assimilis pennsylvanicus Burm. Iowa State Coll. J. Sci., 9: 13-24. 5 Smith, H.W. (1938) The blood of the cockroach, Periplaneta americana L. Cell structure and degen- eration, and cell counts. Studies on contact insecti- cides. Tech. Bull. New Hampshire. Agric. Exp. Stn., 71: 1-23. 6 Ross, M.H. and Cochran, D.G. (1960) A simple method for sexing nymphal German cockroaches. Ann. Entomol. Soc. Am., 53: 550-551. 7 Gupta, A. P. and Sutherland, D.J. (1968) Effect of sublethal doses of chlordane on the hemocytes and midgut epithelium of Periplaneta americana. Ann. Entomol. Soc. Am., 61: 910-918. _, 8 Gupta, A. P. (1979) Identification key for hemocyte types in hanging drop preparation. In “Insect Hemocytes.” Ed. by A. P. Gupta, PEE Univ. Press, Cambridge, pp. 527-529. 10 11 12 13 14 15 Ontogeny and Hemocyte Populations in Cockroach Tauber, O. E. and Yeager, J. F. (1935) On the total hemolymph (blood) cell counts of insects. I. Ortho- ptera, Odonata, Hemiptera, and Homoptera. Ann. Entomol. Soc. Am., 28: 229-240. Wheeler, R. E. (1963) Studies on the total hemocyte count and hemolymph volume in Periplaneta amer- icana (L.) with special reference to the last molting cycle. J. Insect Physiol., 9: 223-235. Arvy, L., Gabe, M. and Lhoste, J. (1949) Contribu- tion a l'étude morphologique du sang de Mantidea. Rev. Can. Biol., 8: 184-200. (original not seen) Webley, D.P. (1951) Blood cell counts in the African migratory locust (Locusta migratoria migra- torioides). Proc. R. Entomol. Soc. Lond., A26: 25-37. Kunkel, J.G. (1981) A minimal model of meta- morphosis: fat body competence to respond to juvenile hormone. In “Current Topics in Insect Endocrinology and Nutrition”. Ed. by G. Bhaskar- an, S. Friedman and J. Rodrigues, Plenum, New York, pp. 102-129. Tanaka, A. (1981) Regulation of body size during larval development in the German cockroach, Blat- tella germanica. J. Insect Physiol., 27: 587-592. Ross, M.H. (1929) The life history of the German cockroach. Trans. Illinois., St. Acad. Sci, 21: 84-93. 16 17 18 19 20 21 313 Bahadur, J. and Pathak, J. P.N. (1971) Changes in the total hemocyte counts of the bug, Halys dentata under certain conditions. J. Insect Physiol., 17: 329-334. Shapiro, M. (1979) Changes in hemocyte popula- tions. In “Insect Hemocytes.” Ed. by A. P. Gupta, Cambridge Univ. Press, Cambridge, pp. 475-523. Ragyalis, A. K. (1982) Physiological and biochemi- cal changes in Acantholyda posticalis hemolymph during ontogeny: 1. Cell morphology. Liet. Tsr. Mokslu Akad. Darb. Ser. C Biol. Mokslai., 0 (4): 69-78. (Abstract; original not seen) Das, Y.T. (1975) Effects of juvenile hormone analog on the German cockroach, Blattella germani- ca (L.) (Dictyoptera: Blattellidae). Ph. D. disser- taion in Entomology, Rutgers University, New Brunswick, NJ. Masner, P., Hangartner, W. and Suchy, M. (1975) Reduced titres of ecdysone following juvenile hor- mone treatment in the cockroach, Blattella germani- ca. J. Insect Physiol., 21: 1755-1765. Gupta, A. P. (1985) The identity of the so-called crescent cell in the hemolymph of the cockroach, Gromphadorhina portentosa (Schaum) (Dictyo- ptera: Blaberidae). Cytologia, 50: 739-746. 4 aT _— ‘i heat meting sharpened RNY: Line ti arene a te PTT eT mT ke eal nit Heonbanenp kad ea i ae pethce { che Ai iat None: ny LOGSR yy Mey AAT IMAD of i sone nt. a ane 3A. = ¢ eas +A va Ua Vahomtibt wane hi lee at erie ) PRE el See! bepnercaee & aed Bey a eas he OOD im Poebergek: +) OU fara ith Pryeerty, a Lunch apindh, | HY 0 Ah 2h ene = My, an tines, bmaes ae SSE Hay eA bay oe actA : Oe a ee a 8 Ay eo na tl AST ike Oe i, Yeoh died. a Le peal gfcrot? WY Pa: ay Avi ‘wand | ee; eo ye ea he a) ke EMAL t rete ete Rita ae Weriahs” salt ee Way OED dw ees i Si a pee bel ¢ ioaped Reman yea» ( ead nolan al TRANG 3) 9 - AL is at ies : dsiveuianrtl, | be (AT RDS. Wh dione Dae. a ane Lal ML erty ale igid-olinaval) gerald apangteyo, Wh, ean? bool 4) Mae Kadned iris Gal as Ta Pitsie ou stb BP > fee. mt PSE Oa ine aint S.A? he Pd Ween ut Ai’ ep truely pe Lehrer ry eae ae Re anata aes F _— \ Vsens ; CPL TR ak (PERS RITES Pip ky htt 146%, wn ca t os x * 4 4 , fie ¥¢\ ‘ee. 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"yj ‘Wesdakalog Sipe, if te Ty ual) a cite et 7 10 — es Siaiiib: - Pie ZOOLOGICAL SCIENCE 4: 315-321 (1987) © 1987 Zoological Society of Japan Growth and Maturation of Ovaries in Isolated Abdomens of Bombyx mori: Response to Ecdysteroids and Other Steroids Ei OHNISHI Department of Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464, Japan ABSTRACT—Studies on ovaries in isolated pupal abdomens of Bombyx mori revealed that they remained in an immature state, but could be stimulated to develop by injection of ecdysteroids. The stimulatory activity of ecdysone was dose-dependent and the process of vitellogenesis of ecdysone- stimulated isolated abdomens, as judged from electrophoretograms of major egg proteins, was comparable with that in normal pupae. Various ecdysteroids differing in the numbers and positions of their hydroxyl groups were examined for their stimulatory activities in comparison with that of ecdysone or 20-hydroxyecdysone. From the results it was concluded that the hydroxyl group at C-2, C-20, and C-22 has an enhancing effect, whereas that at C-24, C-25 and C-26 has an inhibitory effect. Several Cig, Cio and C,, steroids of vertebrate hormone type and their related compounds were also examined for possible ovary stimulatory activity. However, no activity was shown following the injection of these compounds. INTRODUCTION The growth and maturation of insect ovaries are under hormonal control. In the majority of species, Ovaries are immature in adults at emer- gence and become functional after a period rang- ing from several days to several months. In insects of this type, juvenile hormone seems to play a central role in the maturation of the ovaries [1]. In species belonging to the Saturniidae and Bombyci- dae, ovaries maturate during the pupal period and adults are able to copulate and lay eggs immediate- ly after emergence. In insects of these families, ecdysteroids seem to replace the function of juvenile hormone, which in itself plays no role [2]. In the commercial silkworm, Bombyx mori, it has been shown that ovaries in the isolated abdomens remain in an immature state, but can be stimulated to develop upon injection of ecdyster- oids [3, 4]. Such isolated abdomens seem to be a versatile model system for the study of ecdysteroid action on ovarian maturation. In the present study, the stimulatory effect of Accepted December 22, 1986 Received December 9, 1986 ecdysone was re-examined in more detail and the effect of various ecdysteroids and steroids of vertebrate hormone type were compared for their activity. MATERIALS AND METHODS Isolation of pupal abdomens, injection of steroids and dissection of ovaries The method of preparing isolated pupal abdo- mens was the same as that reported previously [4]. Steroids or steroid derivatives were dissolved in warmed isopropanol and the solution was diluted 5 to 10 times with deionized water just before injection. In cases of strictly water-insoluble materials, a 1/10 volume of olive oil was included in the the isopropanol solution and the emulsion resulting upon dilution with water was injected immediately after vigorous agitation. Seven days after injection, ovaries were dissected out into 1% NaCl solution containing 10-°M sodium diethyl- dithiocarbamate. The latter compound was in- cluded in order to suppress melanization of iso- lated ovaries. The isolated ovaries were placed on wetted tissue paper in a plastic container and kept 316 E. OHNISHI in cold until weighed. SDS-polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the pres- ence of sodium dodecyl sulfate was performed according to the method of Laemmli [5]. Several oocytes were removed from mature ovaries and homogenized with 10 parts of Tris-buffered saline (20mM Tris-HCl, pH7.4, containing 150mM NaCl). The homogenate was centrifuged at 10,000 xg for 10min and an aliquot of the supernatant was placed on the top of the gel. The elec- trophoresis was done at a current of 25mA per slab for 2 hr. The proteins in the gel were stained with Coomassie brilliant blue [6]. Chemicals Ecdysone was obtained from Simes s.p.a and 20-hydroxy-ecdysone was a gift from Dr. T. Takemoto, Tokushima Bunri University. Ponas- terone A was from Takeda Pharmaceutical Co. and inokosterone from Rhoto Pharmaceutical Co. Abutasterone was sent by Dr. A.I. Da Rocha, University of California at San Diego. 2-Deoxyecdysone, 2-deoxy-20-hydroxyecdysone and 2, 22-dideoxy-20-hydroxyecdysone were ex- tracted and purified from the ovaries of Bombyx mori [7]. 22(R)-Hydroxycholesterol, 5-pregnen- 38, 208-diol, 178-estradiol-3-(-p-glucoside), and 17-estradiol-17-(a-p-glucoside) were provided by Drs. N. Ikekawa and Y. Fujimoto. 5-Pregnen-3/, 206-diol was also given by Dr. K. Suzuki of the National Institute of Radiological Sciences. Pregnenolone, progesterone, estradiol-17a, estra- diol-178, 178-estradiol benzoate, estrone, and tes- tosterone were the products of Sigma Chemical (CO), RESULTS Dose-dependency of ovarian development on the injected ecdysone and the maturity of the ecdysone- stimulated ovaries The increase in the fresh weight of a pair of ovaries, removed from each injected abdomen after 7 days’ incubation, was taken as a measure of the development of stimulated ovaries. As shown in Figure 1, ecdysone was effective at a dose of 0.5 yg and the effect was dose-dependent up to a level of 5 ug per abdomen. When ecdysone was injected in amounts exceeding 10 ug per abdomen, the injected abdomens were filled with mature ovaries after 7 days and scanty fat body remained. S oO o o E U) 300 uJ < > Oo u. 200 Oo [I= ac © uJ 100 = 0 ° ce Cl Ok i} 2 5 ECDYSONE (ug) Fic. 1. Dose-response curve of ecdysone. Ecdysone was injected into isolated pupal abdomens and the fresh weight of a pair of ovaries in each abdomen was weighed after 7 days’ incubation. Each point represents an average value of 7 to 10 samples, with standard deviation. Ordinate: weight of a pair of ovaries in milligrams. Abscissa: dose of injected ecdysone in micrograms. C: control. In order to clarify the process of vitellogenesis in the ecdysone-stimulated ovaries, the compositions of the major egg proteins were analyzed by SDS-gel electrophoresis. The electrophoreto- grams (not shown) of the ecdysone-injected ova- ries Showed major bands of heavy and light chains of vitellogenin, 30 K-proteins and egg-specific pro- tein [8]. This pattern was essentially the same as that found in normal ovaries. Thus, vitellogenesis in the ecdysone-stimulated isolated pupal abdo- mens seemed to be comparable to that in normal mature pupae. In further attempt to gain an insight into the degree of maturity of the stimulated ovaries, the ecdysone-stimulated ovaries were heat-treated (46 +0.5°C, 18min) to induce artificial parthenogene- sis [9]. After the heat-treatment, eggs were maintained at 20+0.5°C to allow embryonic de- Ovaries in Bombyx Isolated Abdomens S17 velopment to occur. About half of the treated eggs exhibited red to purple coloration about 3 days after the treatment, indicating the initiation of embryonic development. Most of them became first instar larvae with fully pigmented setae. However, none of them hatched out from their chorions, indicating that their embryonic develop- ment had been impaired. Stimulatory activity of Bombyx ooecdysteroids The ovaries of Bombyx mori accumulate several ecdysteroids (ooecdysteroids) characteristic to the ovaries and eggs [7]. These ecdysteroids were isolated from the ovaries and purified to a chroma- tographically pure state. Concentrations of the ecdysteroids were estimated on the basis of their optical densities at 243 nm in ethanol. In each experiment, ecdysone or 20-hydroxyecdysone was injected at several doses into the isolated abdo- mens of the same batch and the dose-response curves were compared in order to estimate the stimulatory activities of the test samples relative to that of ecdysone or 20-hydroxyecdysone. 2-Deoxyecdysone, one of the major ooecdyster- oids in Bombyx, had about 1/5 the activity of that of ecdysone (Fig. 2A). 2, 22-Dideoxy-20-hydroxy- ecdysone, which has been identified only in Bom- byx ovaries [10], has negligible activity up to the dose of 36 ug per abdomen (Fig. 2B). 2-Deoxy-20- hydroxy-ecdysone, most abundantly found in Bombyx ovaries, had about 1/3 of the activity of eedysone, Or 1/5 of the activity of 20- hydroxyecdysone (Fig. 2C). Stimulatory activity of phytoecdysteroids Ponasterone A (20-hydroxy-25-deoxyecdysone) exhibited quite high activity, being about twice as active as ecdysone (Fig. 3A). Inokosterone (20, 26-dihydroxy-25-deoxyecdysone) showed an activ- ity comparable with that of 20-hydroxy-ecdysone (Fig. 3A), indicating that the hydroxyl group at C-26 has a similar effect to that at C-25. Abutas- terone (20, 24-dihydroxyecdysone) [11] had a slightly lower activity than 20-hydroxyecdysone (Fig. 3B). 300 200 ECD 2DE 100 w to) fo) (mg) 200 100 WEIGHT OF OVARIES Cc OSa nit ez SHO) 200 740 ECDYSTEROID (ug) Fic. 2. Dose-response curves of Bombyx ooecdyster- oids. A: 2-deoxyecdysone (2DE) and ecdysone (ECD). B: 2, 22-dideoxy-20-hydroxyecdysone (2, 22DD) and ecdysone (ECD). C: 2-deoxy-20- hydroxyecdysone (2D20E), ecdysone (ECD) and 20-hydroxyecdysone (20E). For other explanations, see legend for Fig. 1. Effect of vertebrate steroid hormones and related compounds The following steroids and steroid conjugates were examined for their possible stimulatory activ- 318 (mg) WEIGHT OF OVARIES Fic. 400 300 wee 20E ABU 100 01 Osan! (ie) 15) BeCDY STEROID Guig)) 3. Dose-response curves of phytoecdysteroids. A: ponasterone A (PON), inokosterone (INOK) and 20-hydroxyecdysone (20E). B: abutasterone (ABU) and 20-hydroxyecdysone (20E). For other explana- tions, see legend for Fig. 1. TABLE 1. Effect of injection of steroids of compounds on ovary development Injected compounds and doses of injection ( 4g) Expt. 7Au6 estradiol-17 40 20 10 testosterone 40 20 10 estrone 40 20 10 E. O#NISHI ity at doses up to 40 ug per abdomen: 22(R)- hydroxycholesterol, progesterone, pregnenolone, 5-pregnen-3/, 208-diol (2 samples), estradiol-17a, estradiol-17f, estrone, testosterone, 17P-estradiol benzoate, 17{-estradiol-3-(@-p-glucoside) and 17£-estradiol-17-(a-D-glucoside). As summarized in Table 1, all of these compounds gave negative results. DISCUSSION The gonadotropic activity of prothoracic gland hormone was first demonstrated in the classic experiment performed by Williams using isolated abdomens of the saturniid Hyalophora cecropia [12]. By transplantation of the activated protho- racic glands into the isolated abdomens, he was able to show that the recipients not only expressed imaginal characteristics but also laid several eggs. This clearly indicated that the ovaries in the abdomens had become fully mature. In Bombyx mori, Sakurai and Hasegawa [3] showed that ovaries in isolated pupal abdomens could be stimulated by injection of ponasterone A and that the fresh weights of the stimulated ovaries roughly parallelled the doses of the injected ecdysteroid. We also observed, using isolated pupal abdomens, that the injection of 20- hydroxyecdysone not only stimulated ovarian de- velopment, but also induced the accumulation of vertebrate hormone type and their related Weight of ovaries (mg) mean + S.D. Mie) as (U9 IME) ae Id) OS )=taltel Bae eT WS) seit LOS MO)3) a2 (0), 7/ 11.4+0.9 10.4 + 1.3 Ovaries in Bombyx Isolated Abdomens 319 TABLE 1. (Continued) Injected compounds and Weight of ovaries (mg) doses of injection ( “g) mean +S.D. estradiol-17a 40 1-8=-1-0 20 11.4+1.0 10 10.5+1.0 178-estradiol benzoate 40 12.0+1.8 20 LINES Feneyds7) 10 10:25 1-0 control e087, Expt. 2Jn6 estradiol-17a-glucoside 40 G02 1S 20 (cram let 10 Ojos tee: 4 oh Jia 5-pregnen-3f, 20-diol* 40 shlvasdls) 20 8.9+ 0.6 10 10.4+ 1.6 control S:6203 Expt. 12Jn6 estradiol-3@-glucoside 40 10.6 +0.9 20 10.6 + 0.7 10 OFS ae 0 5-pregnen-3/, 208-diol** 40 9.4+1.4 20 Solis lal 10 10.2+2.4 22 (R)-hydroxycholesterol 40 9.4+0.9 20 OFS 0X7 10 weasitel! control 9:3 a= Os; Expt. 26Jn6 pregnenolone 40 L0SEe2-9 20 8.0+ 0.6 10 9:4 ate tt progesterone 40 10.0+ 0.8 20 8.5+ 0.6 control 8.4+1.1 eS The effect was determined by examining the fresh weight of a pair of ovaries isolated 7 days after injection. * sample from Dr. K. Suzuki. ** sample from Dr. Y. Fujimoto. 320 E. ONISHI ecdysteroids in the ovaries [4,13]. Upon re- examination of the dose-effect of ecdysteroids, we found a quite typical dose-response relationship. Thus, comparison of stimulatory activities among various ecdysteroids differing in the number and position of their hydroxyl groups was shown to be possible. 2-Deoxyecdysone exhibited about 1/5 of the activity of ecdysone, indicating that the hydroxyl group at C-2 has a significant effect. Also, 2, 22-dideoxy-20-hydroxyecdysone, which is also one of major ooecdysteroids in Bombyx, exhibited quite low activity, showing that the hydroxyl group at C-22 is requisite for the activity. These results seem to suggest that these major ecdysteroids lacking hydroxyl group at C-2 are not the active forms, but rather the precursors of active com- pounds. 20-Hydroxyecdysone, which is a rather minor component in Bombyx ovaries, was twice as active as ecdysone, indicating that the hydroxyl group at C-20 has an enhancing effect. Consistent with the report of Sakurai and Hasegawa [3], and with the results of other studies employing cul- tured cells [14] or imaginal disks of Drosophila [15], ponasterone A was more active than ecdy- sone and 20-hydroxyecdysone. Together with the results of the experiments using inokosterone and abutasterone, it can be concluded that the hy- droxyl group at the termini of the side chain of ecdysteroids has some inhibitory effect. These differences in activity among the ecdysteroids should be expected to be a reflection of the structure of the binding site(s) of the target cells in the isolated abdomens. Steroids of vertebrate hormone type, and their related compounds, were tried for possible ovarian stimulatory activity. Estradiol-176, a vertebrate female sex hormone, has been identified in the ovaries of Bombyx mori [16]. In an in vitro system of isolated Bombyx ovaries, estradiol has been shown to be converted into several metabolites including its two glucosides, 17@-estradiol-3-(f-D- glucoside) and 17/-estradiol-17-(a-p-glucoside) [17]. However, estradiol and its derivatives includ- ing these two glucosides exhibited no ovary- stimulatory activity in the present study. In the tobacco hornworm, Manduca sexta, Thompson and his colleagues [18] demonstrated the efficient conversion of ['*C] cholesterol into the glucoside of 5-pregnen-3f, 20@-diol and further showed the presence of this glucoside in intact pupae. These facts seemed to suggest the presence in insects of metabolic activities yielding C5, steroids from cholesterol. However, in the present study, ex- amination of the ovary-stimulatory activity of several C,, steroids including 5-pregnen-3/, 20/- diol, produced negative results. Thus the signif- icance of the presence of C;g and C>; steroids in insect Ovaries remains for future study. The hormonal regulatory mechanism of insect ovarian maturation seemed to be variable among species. In most species, maturation is stimulated by juvenile hormone with or without concomitant action of neurohormones. In Bombyx mori, the scheme seems to be rather simple. Mizuno [19] has shown through his experiments using debrained pupae and isolated pupal abdomens, that protho- racic gland hormone secreted in the early pupal period stimulates ovarian maturation and that neurohormone seems to play no role. The present experiments have thus confirmed the above scheme and extended the previous findings. ACKNOWLEDGMENTS The author wishes to thank Dr. S. Y. Takahashi and Mr. K. Soma for their skilful technical assistance and to Dr. O. Yamashita for his valuable advice. He is also indebted to Dr. T. Takemoto, Tokushima Bunri Uni- versity, to Drs. N. Ikekawa and Y. Fujimoto, Tokyo Institute of Technology, Dr. A. I. Da Rocha of Universi- ty of California at San Diego, and to Dr. K. Suzuki, National Institute of Radiological Sciences, for their gift of steroids. The present study was supported by grants Nos. 60304009 and 61540517 from the Ministry of Education, Science and Culture. REFERENCES 1 Koeppe,J.K., Fuchs,M., Chen, T.T., Hunt, L. H., Kovalick, G. E. and Briers, T. (1985) The roles of juvenile hormone in reproduction. In “Compre- hensive Insect Physiology, Biochemistry and Phar- macology”. Ed. by G. A. Kerkut and L.I. Gilbert, Pergamon Press, Oxford, New York & Toronto, Vol. 8, pp. 165-204. 2 Hagedorn, H.H. (1985) In “Comprehensive Insect Physiology, Biochemistry and Pharmacology”. Ed. by G. A. Kerkut and L. I. Gilbert, Pergamon Press, 10 11 Ovaries in Bombyx Isolated Abdomens Oxford, New York & Toronto, Vol. 8, pp. 205-262. Sakurai, H. and Hasegawa, K. (1969) Response of isolated pupal abdomens of silkworm, Bombyx mori L., to injected ponasterone A (Lepidoptera: Bom- bycidae). Appl. Entomol. Zool., 4: 59-65. Chatani, F. and Ohnishi, E. (1976) Effect of ecdy- sone on the ovarian development of Bombyx silk- worm. Dev. Growth Differ., 18: 481-484. Laemmli, U.K. (1970) Cleavage of structural pro- teins during the assembly of the head of bacteri- ophage T,. Nature, 227: 680-685, Weber, K., Pringle,J. and Osborn,M. (1972) Measurement of molecular weight by elec- trophoresis on SDS acrylamide gel. In “Methods in Enzymology”. Ed. by C.H.W. Hirs and N. Timasheff, Academic Press, New York & London, Vol. 26, pp. 3-27. Ohnishi, E., Mizuno, T., Ikekawa, N. and Ikeda, T. (1981) Accumulation of 2-deoxyecdysteroids in ovaries of silkworm, Bombyx mori. Insect Biochem., 11: 155-159. Takahashi, S. Y. (1986) Studies on the phosphoryla- tion of ovarian proteins from the silkworm, Bombyx mori: Identification of band 2 protein as egg-specific protein. Insect Biochem., 17: 141-152. Sakaguchi, B. (1978) Method for artificial parthe- nogenesis. In “The Silkworm: An _ Important Laboratory Tool”. Ed. by Y. Tazima, Kodansha, Tokyo, p. 21. Ikekawa, N., Ikeda, T., Mizuno, T., Ohnishi, E. and Sakurai, S. (1980) Isolation of a new ecdyster- oid, 2, 22-dideoxy-20-hydroxyecdysone from the ovaries of the silkworm, Bombyx mori. Chem. Commun., 1980: 448-449. Pinheiro, M.L.B., Filho, W.W., Da Rocha, A.I., Porter, B. and Wenkert, E. (1983) Abutasterone, 13 14 15) 16 d/ 18 19 321 an ecdysone from Abuta velutina. Phytochem., 22: 2320-2321. Williams, C.M. (1952) Physiology of insect di- apause. IV. The brain and prothoracic glands as an endocrine system in the cecropia silkworm. Biol. Bull., 103: 120-138. Ohnishi, E. and Chatani, F. (1977) Biosynthesis of ecdysone in the isolated abdomen of the silkworm, Bombyx mori. Dev. Growth Differ., 19: 67-70. Maroy, P., Dennis,R., Beckers,C., Sage, B.A. and O’Connor, J.D. (1978) Demonstration of an ecdysteroid receptor in a cultured cell line of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA, 75: 6035-6038. Yund,M.A., King,D.S. and _ Fristrom, J. W. (1978) Ecdysteroid receptors in imaginal disks of Drosophila melanogaster. Proc. Natl. Acad. USA, 75: 6039-6043. Ohnishi, E., Ogiso, M., Wakabayashi, K., Fuji- moto, Y. and Ikekawa, N. (1985) Idetification of estradiol in the ovaries of the silkworm, Bombyx mori. Gen. Comp. Endocrinol., 60: 35-38. Ogiso, M., Fujimoto, Y., Ikekawa, N. and Ohnishi, E. (1986) Glucosidation of estradiol-17@ in the cultured ovaries of the silkworm, Bombyx mori. Gen. Comp. Endocrinol., 61: 394-401. Thompson, M.J., Svoboda,J.A., Lusby, W.R., Rees, H.H., Oliver, J. E., Weirich, G. F. and Wil- zer, K.R. (1985) Biosynthesis of a C, steroid conjugate in an insect. The conversion of ['*C] cholesterol to 5-['*C] pregnen-3f, 208-diol gluco- side in the tobacco hornworm, Manduca sexta. J. Biol. Chem., 260: 15410-15412. Mizuno, T. (1982) Control of accumulation of ecdysteroids in ovaries of the silkworm, Bombyx mori. Zool. Mag., 91: 12-17. af. ai ar yy AME a9 Cadre Ges Ae r ail mar gry \ iy é aw ve i tite ze ae * ijt ay tf ‘1 re y iy Vil? dp A i ay “TA is avid 4 ay ™ 2 Ter F453 vous i 19 ey apt bee Dy tare 4 | me i Ail ‘ heme re ed SEPA iS WORST YT) 4) 2) Sift , PTR MA tte (Ay WD teh vot i r o = s rad oe L bie: Me a yay’ Rata aT. vallsarg hy ik ve Pr j peel Bios viet Oh VRE TY fo Serio Sig Oe FREES a Et A a iV NS Pi Mey) 1) eagle ae Pee) + maa if _ eee. f pew Abe SH ‘. re # 3 er sitions’ fi esa bol buiwt ehupy Sioa eapiam hele diane {iow'ys ha ear ue VP pots: scab ‘ mE REN hdl Tae ee a haetenst’s eet ute ev her (fee bere il Maly j hd ede eats BGR Le A ato, ae “if ja ny ane ly}” Khe Ao a A a Ww ~ " ” 5 * , iw a ‘ (! aN a ae beat boiery ties TO at ae 1a bare ei b { sf ‘ BU Ty AAU oe a, ts Lo Orb PIT * ¥ are vS : Ah iat "tiri . ot it yi Sr ps eay at ; or pul TA , \esTeny ore Ay ee: pepper Baty UNM whlta) pei | } ‘ \ F Y f meena sit oat. +; fi Jt '. ‘ AG ‘ ‘a diirer’ dui ie AF li : es wie \ yee f ie Y He Oye d 7 4 | 4 ry ‘a Lveyi hie PN nae Pal 4 a tox) f t Pu ’ tg aT 4 ik = ek ert .: ew cy ‘ tg PAPER Cc iamNT sie ; WV REE SiR Cpe er ‘ if ; ¢ sara tT ty iy oe g uy erie Wb i ‘Par BVA } Me iA t 4 t i hg é vf ae Pv ea by 5 :; LY my J i ; ? oe 4 1 A hy Wh aN \ I %, i one i oben lw : dpi oe - e a ™ 1 “i . 1" an oes an rl on i an Ve 7 7 a aa a ae ay Sy. ee , , tf CHa coh Fie OT Cw yh ne he phe: rer if ayy VOR by ES, ea cB by « ec et | , P| ry net tahpisaredtiaten par 7 “WLC td Cate de bY ROPE eine bet LR, ocala tive cote NERA aU Ma see Rs MARR i Vie Ly hang ag 5 a uly . Bey tes shit : tes y arin) B) ze hg Pause Sie Ma: ight" Shs QAP Opty ea ai ap! aii. idabiine dal nl peer aes me = i aye | janeehiy AAP ad HANG appr ib. tite CG. Rae } bot bai, Waves ute bs Anite ti “Slot e see ta Ary MTC SAT orsign oni herd throne), his : eo CR | aL yo cian onthanes Patyity (abel ; ait sng OD Wee ARS Ah} Ok eat patie) tr ii cna i | Wey iy rive ra oiler Ae Fe pala tel theron Lote Pi tro wae i Tera ne iremaptay h vile The baa a \ A idemat?..T, oqursiNt 7 ing HOTU SUD WA ales Aavelgely { (GRE PRS ALE aes PY Wa IE 1 + acd (ait, oirpaihoe sathi pert Tite) IN! ; he nt Pen th } eo. one ik pti 1 wep a apes! (eat 4 yh ep is = i es Z - “Vee mais 1 a, i ae Ban Of ty cviculipe Sk dalladd factinlivyy Lome ZOOLOGICAL SCIENCE 4: 323-329 (1987) © 1987 Zoological Society of Japan Annual Changes in Plasma and Testicular Androgen in Relation to Reproductive Cycle in a Japalura Lizard in Taiwan Ruat-Hue! CHEN, JuN-Y1 Lin, Yuu-Lin Yu’, and HsiEN Yu CHENG” Department of Biology, Tunghai University, Taichung, ‘Institute of Zoology, Academia Sinica, Taipei, and *Department of Zoology, National Taiwan University, Taipei, Taiwan, R.O.C. ABSTRACT—Male Japalura mitsukurii formosensis are reproductively active from emergence in March through July. Plasma and testicular androgen levels in association with all other reproductive activities decline dramatically in July-August to a minimum in August-September. A sudden surge of testicular androgen level dissociated from plasma androgen level observed near the end of sperma- togenic cycle appears to be involved in the control of decline of the spermatogenesis. After the regression, spermatogenesis, interstitial cells and Sertoli cells began to recrudesce and peak prior to hibernation. However, accessory sex organs weights, testicular androgen level and plasma androgen level still remained low after the regression until the emergence in March. The plasma androgen level was more positively correlated with recrudescence of spermatogenic activity. The increased testicular androgen level appeared more coincided with high spermiogenesis and also with high mating activity. INTRODUCTION Numerous studies of the male reproductive cycle in lizards demonstrate changes in size and histolo- gy of the testes and accessory sexual organs, and androgen production [1-4]. However, the correla- tion between androgen production and the testicu- lar cycle is less well documented [5-8], and such fundamental research is necessary to answer ques- tions on the nature of endocrine physiology in nonmammals and the evolution of vertebrate hormone structure [9, 10]. The objectives of the present study on Japalura mitsukurii formosensis are to provide information on seasonal changes of androgen levels in plasma and testis and to correlate annual androgen levels in plasma and testis with the phases of the spermatogenic cycle. MATERIALS AND METHODS Lizards were collected monthly from the woods of mainly Acacia confusa, on the campus of Accepted November 11, 1986 Received September 22, 1986 Tunghai University, Taichung, Taiwan (24°10'N, 120°35’E; elevation 180m), from March 1982 to March 1983. Only adult males with snout-vent length (SVL) greater than 65 mm were used for the analysis [11, 12]. All individuals were anesthetized with ether within three hours of capture. Their weight and SVL were measured to the nearest 0.1g and 0.1mm, respectively. Blood samples (0.4-0.7 ml) were drown from the inferior vene cava near its entry in the heart, then placed into heparinized tubes. The blood was immediately centrifuged and plasma (0.2-0.45 ml) was frozen and stored at —20°C for androgen analysis. The testes and the accessory sexual organs including epididymis, vas deferens and kidney were removed and weighed to the nearest 0.1mg. The right testis was fixed in Bouin’s solution for histological examination, and the left testis was frozen and stored for the androgen analysis in the tissues. Androgen concentrations in plasma and testis were measured by radioimmunoassay methods modified from Wingfield and Farner [13] and Anderson et al. [14], as described previously by Yu et al. [15]. The modified procedure quantitated 324 total androgen, since a chromatographic separa- tion of androgens was omitted. The assay was sensitive to 10 pg of testosterone per assay tube. Standard and samples produced parallel dis- placement of tritiated testosterone. The between- assay coefficient of variation was 13.4% and the within-assay coefficient of variation was 3.8%. Recovery of tritiated testosterone and unlabelled testosterone standards added to plasma or testis homogenates was about 94%. The specificity of testosterone antiserum used for this assay was described previously [14]; it cross-reacted with dihydrotestosterone, andros- tenedione, and androstenediol at 90%, 12%, and 11%, respectively, relative to testosterone (100%), and had negligible cross-reactivities with other test steroids. The concentration of androgen in the sample was expressed as testosterone equivalent extrapolated from the standard curve. Paraffin-embedded testes were sectioned at 6 ym and stained with Harris’s hematoxylin and eosin. The diameters of seminiferous tubules, the thickness of germinal epithelium, Sertoli cell nu- clear diameter and interstitial cell nuclear diameter were measured with ocular micrometer to the nearest 0.1 ~m. The number of cellular layers of germinal epithelium, Sertoli cells around the peripheral regions of the seminiferous tubules and maximal amount of interstitial cells in the inter- R.-H. CHEN, J.-Y. LIN et al. tubular area were estimated from counts directly under the microscope. Values presented for each variable are the averages of the 10 measurements for each testis. Reproductive stages were judged from the spermatogenetic process outlined in Table 1. RESULTS A total of 146 adult males were used in this study. The monthly mean SVL of the samples was similar in all months (ANOVA, p=0.87) and ranged from 75.0mm to 79.1 mm (Table 2). In 1982, first emergence was observed on March 12 and was March 19 in 1983. Individuals entered hibernation in late November or early December in 1982. Males emerge from hibernation about 2 weeks before females [11, 16]. In 1982, during the 12 days following the first emergence, 124 males and 21 females were observed (5.9:1). In 1983, during the same period, 62 males and 24 females were observed (2.5:1). After March in both year, sex ratio appeared to be 1:1. The monthly data on the testis weights, acces- sory sex organ weights and histological observa- tions of the testes including the diameters of seminiferous tubules, the thickness of germinal epithelium, Sertoli cell nuclear diameter and in- terstitial cell nuclear diameter, the number of TABLE 2. Monthly means (+SEM) of the weights in testis and accessory sex organs, androgen epithelium, germinal layer number, and nuclear diameters and numbers in interstitial cells and Androgen concentration Seminiferous Snout-vent : : Month N length be ay 1982 Mar 1 79 Oo 25. 0see des Apr IG HHeSseUO ew W3S.tsae S55) May Wh TU Ose) Sanu M22 ates Abe) Jun I 15.92 IZ heise OS) Jul IS) yp Sse lls, MileSse eS) Aug Iss. (HS SseWsts Weller 27) Sep 14 76.5+0.9 I). Sees eI) Oct (Mb Ta sete 74.8+ 5.6 Nov 127 7835-088 15674 -E 104 1983 Mar 10 76.9+1.0 143.3+ 6.6 Accessory Ser OTE “Tests Blood plasma diameter (ng/testis) (ng/ml) ( um) 119.54+4.7 3.50+0.70 2.41+0.43 301.84 7.8 114.84+3.6 6.55+1.35 2.27+0.79 279.2+ 8.1 115.643.1 7.00+0.91 1.18+0.26 292.0+ 7.1 112.9+5.9 19.42+4.80 1.58+0.44 273.8+ 8.4 106.5+6.2 29.27+5.47 0.35+0.09 292.8+ 7.6 91.5446 2.45+0.45 0.1640.03 99.7+ 5.9 73.04+4.2 1.33+0.25 0.1740.02 134.74£11.5 76.14+3.0 2.38+0.29 0.4040.09 247.04 5.8 90.2+4.4 0.62+0.16 0.63+0.42 317.8+ 8.1 119.9+6.1 5.7441.58 7.10+2.35 298.84 7.6 Androgen and Reproduction in Lizard 325 TABLE 1. Spermatogenic stages during reproductive cycles in Japalura mitsukurii formosensis Stage Testicular spermatogenic condition NNN fF WN Fe cellular layers of germinal epithelium, the number of Sertoli cells and of interstitial cells are summa- rized in Table 2, and they all showed a similar corresponding pattern of reproductive cycle. Adult males of this species showed a distinct spermatogenic cycle (Table 3), and were reproduc- tively active from March (right after emergence) to July. The testis weight decreased in August and September, then recrudesced in October and reached the maximum size in November. The diameters of seminiferous tubules, the thickness of germinal epithelium, Sertoli cell nuclear diameter and interstitial cell nuclear diameter also dropped in August but their recrudescence occurred in September, one month ahead of the recrudescence of testis weights. The accessory sex organ weights declined in September, one month later than the regressoin of testis weight and remained low or all stages of spermatogenesis present and abundant. all stages of spermatogenesis present and abundant. only spermatogonia, few or no spermatocytes, no lumen. primary spermatocytes present, few or no secondary spermatocytes, few or no lumen. secondary spermatocytes abundant, few or no spermatids. all stages of spermatogenesis present, spermatids abundant, few or no spermatozoa. No or few spermiogenesis. Spermiogenesis occurs. spermatozoa abundant but spermatids and spermatocytes greatly reduced. increased slightly in the pre-hibernation period (October to November). The male reproductive pattern of J. m. formosensis in this study was in close agreement with the previous finding by Cheng and Lin [11]. Monthly changes of plasma androgen concentra- tion showed a distinct cycle with the highest concentration in late March, immediately after emergence (Table 2). Thereafter, the plasma androgen level declined, although remaining re- latively high (greater than 1.1ng/ml) until June, then dropped sharply to below 0.5ng/ml in July, and reached the minimum level (0.16ng/ml) in August. Plasma androgen level remained low during the pre-hibernation period. Testicular androgen level, measured per testis, was moderate at emergence and increased to the maximum level in July (Table 2). The July peak of testicular concentrations in testis and plasma, diameters of seminiferous tubules, thickness of germinal Sertoli cells in Japalura mitsukurii formosensis Sertoli cell Interstitial cell Germinal Germinal es — No./tubule Nuclear No./intertu- Nuclear tbickaicss (yam). number (N) (N) diameter (sm) bular area(N) diameter ( um) GET = 3.1 UikGse.s) 1329 027 5.06+ 0.13 4.8+0.2 4.95+0.10 59-25 1.6 11.6+0.5 nOP ste Oa7, 4.90+0.12 5.0+0.3 4.74+0.11 Sy NS ayy 11.8+0.4 20.4+0.5 4.88+0.08 Shs Ore 4.88+0.15 58.5+4.9 Bey a-0:9 1662077 5.070223 4.9+0.4 4.71+0.29 52-64. 1.2 9.5+0.4 17.1+0.6 Se Stae\) ails) 5.0202 4.87+0.17 io 1.6 2.4+0.2 7.4+0.8 4.31+0.18 SHliee(I)7 529 90.05 40.7+3.6 G:32-025 AES Bae N We 4.49+0.11 SD Oe 4.02+0.17 74.142.4 13:02-0:5 16.4+0.6 5.02+0.10 Dats One 4.18+0.10 Soe LE 3:0 16.5+0.5 222820. 7) 528s 0:12 3.05502 4.43 +0.13 63.8+1.8 iPass Ved 17.3 20:6 S12 EROL 7 4.6+0.2 S12 O16 326 R.-H. CHEN, J.-Y. LIn et al. TABLE 3. Percent distribution of spermatogenic stages in Japalura mitsukurii formosensis Stages Month N 1 7 4 5 6 7 Mar 14 == = — = 100 a Apr 16 — — — —- 100 a Mar 17 — — — = 100 = Jun 16 — — = — 68.8 SZ Jul iI5) — — — — 40.0 60.0 Aug 18 100 —- == ue Sl en Sep 14 28.6 71.4 — a= BS -_ Oct 14 — Tal 85.8 7.1 — — == Nov 12 — — 58.3 41.7 — — TABLE 4. The means (+SEM) of testis weight, accessory sex organs weight and androgen concentrations in testis and plasma at different spermatogenic stages in Japalura mitskurii formosensis Androgen concentration : Accessory sex Testis weight Stage N (mg/pair) organs weight Testis Plasma (mg) (ng/testis) (ng/ml) i 22, NOs. 277 87.8+4.4 2.10+0.40 Oris 0702 2 11 25.34 4.4 74.24+4.3 1.70+0.26 0.23+0.04 3 12 74.8+ 6.3 TM Ase 35h 2.37+0.34 0.38+0.10 4 8 144.1+16.6 87.2+7.0 0.94+0.34 0.82+0.63 5 5 ISs Hae D5 92.4+2.4 0.57+0.65 0.43+0.12 6 64 124.2+ 4.7 116.8+4.4 11.34+4.95 1.92+0.34 7 14 SB /SSE) SD) 102.8+6.8 21.90+4.48 0.83+0.31 androgen concentration dropped sharply to the DISCUSSION minimum in August, and remained low during the pre-hibernation period (Table 2). Both plasma and testicular androgen level were higher at the time of emergence than prior to hibernation period (Table 2). Changes in testicular weight, accessory sex organ weights, testicular and plasma androgen concentrations were correlated with sperma- togenic stages (Table 4). Plasma and testicular androgen levels were relatively high during the peak of spermatogenesis and spermiation (stage 6) in the breeding season. Testicular androgen level reached the highest level when the germinal epithelium started to regress (stage 7) in June and July. However, plasma androgen concentration regressed to a relatively low level (Tables 1, 3, and 4). | The concordance of the present data with the previous finding [11] indicates a consistent timing of the reproductive cycle in male J. m. for- mosensis. Males emerge from hibernation in March with the testis and accessory sex organs ready for further spermiogenesis. In March plas- ma androgen concentration is at the highest level but testicular androgen concentration level is still relatively low (Table 2). Matings occur mainly from April through June [16] as the testicular androgen concentration is at increasing phase (Table 2). The plasma androgen declined steadily, coincided with the regression of testicular weights during mating period (March through June, Table 2, Fig. 1). In contrast, testicular androgen rised markedly with an increase in spermiogenesis Androgen and Reproduction in Lizard 327 high Te TESTIS WEIGHT ACCESSORY ORGANS WEIGHT FAT BODY WEIGHT ON TESTICULAR ANDROGEN LEVEL PLASMA ANDROGEN LEVEL - < 4 & wl {-°] = AGGRESSIVE BEHAVIOR COURTSHIP BEHAVIOR MATING FEMALE REPRODUCTION medium low Pore JF MAMJ JA S ON D Fic. 1. MONTH A summary of annual reproductive pattern of Japalura mitsukurii formosensis. The variables are: testis weight, high >90 mg, medium<90 mg and >30 mg, low<30 mg; accessory sex organs weights, high > 100 mg, medium<100 mg and >80 mg, low<80 mg; fat body weight (male), high >200 mg, medium <200 mg and>100 mg, low<100 mg; testicular androgen level, high>10 ng/testis, medium<10 ng/testis and>3 ng/testis, low<3 ng/testis; blood plasma androgen level, high >2 ng/ml, medium<2 ng/ml and>1 ng/ml, low<1 ng/ml; aggressive behavior, courtship behavior, and mating, high=active months, low=non-active months; female reproduction, high=occurrence of enlarged follicles or oviducal eggs, low=absence. (Tables 2 and 4). During June-July, some indi- viduals possessed regressing testes (spermatogene- tic stage 7, Tables 1 and 3). Thereafter, all the reproductive activities drop dramatically in July— August, and reached the minimum in August- September (Table 2). All individuals collected in August were spermatogenetically inactive (stage 1, Tables 1 and 3). During pre-hibernation, the activities of testicular tissues including interstitial cells and Sertoli cells start to accelerate (Tables 2, 3, and 4). Accessory sex organs weights, testicular androgen and plasma androgen remain low after the regression (Table 2, Fig. 1). Interstitial cells and Sertoli cells have been proposed as the major sites of steroid biosynthesis in the reptilian testis [17, 18]. In general, during the breeding season, high plasma and testicular androgen levels were accompanied with a numeri- cal increase and large nuclear diameter of intersti- tial cells and Sertoli cells (Table 2). However, plasma androgen and testicular androgen were at the lowest level during the whole pre-hibernation period as testicular activities began to increase in September-October and reached the highest level before entering hibernation in November (Table 2). The evidence shows that both testicular and plasma androgen level are not associated with the recrudescence phase of the testicular activities. This disassociation pattern is not concordant with the findings of the previous studies on other lizards [5,6]. The functional meaning of the hyper- trophication of interstitial cells and Sertoli cells during this pre-hibernation period is obscure. The main cause responsible for the resumption of the spermatogenesis is still an open question. The level of androgen needed in the initiation of spermatogenic recrudescence may be quite low (about 10% of the maximum) in this lizard [18, 19]; or like epitestosterone, other than androgen, may involve in this testicular recrudescence [10]. In reptiles, unlike endotherms, the ambient temperature may play a key role in modulating the pattern of the annual reproductive rhythm [5, 20- 22]. A marked drop in testicular androgen level 328 (in November) prior to hibernation may be due to a progressive inactivity of the steroidogenic tissue in response to decreasing temperature and, perhaps, also to shortening photoperiods [5]. The unexpected drop of androgen concentration in testicular and plasma tissues as the spermatogene- sis reached relatively high level (stage 5; Tables 1 and 4), may be explained owing to their occur- rence in November prior hibernation (Table 3). Discrepancies between testicular and plasma androgen levels have been observed and discussed in other lizards [5-7]. A similar finding with a sudden surge of testicular androgen level dissoci- ated with plasma androgen level in this lizard during the termination of spermatogenesis (Table 2, Fig. 1) indicates further that steroid hormones might play an important part in the control of decline of spermatogenesis [7, 23]. Increase in plasma androgen was observed to be related to mating season [5,24]. Increase in testicular androgen was observed to be associated with recrudescent spermatogenesis [5, 6]. However, our data indicated that it was the plasma androgen level which was more positively associ- ated with recrudescence of spermatogenic activity (Table 4). The testicular androgen level showed depressed as the spermatogenic activity recrudes- cent, although the testicular androgen concentra- tion dramatically increased as the spermatogenic activity reached the peak (stage 6). Furthermore, the androgen level continually surged even as the spermatogenic activity had started to regress (Table 4). In this lizard, the increase of testicular androgen level appeared more coincided with high spermiogenesis activity and also with high mating activity (Table 4, Fig. 1). ACKNOWLEDGMENTS Thanks are to the Department of Laboratory Medi- cine, University of Washington, Seattle, USA, for the supply of testosterone antiserum, and to the Institute of Botany, Academia Sinica, for the use of the liquid scintillation spectrometer. The manuscript was com- pleted while HYC held a Smithsonian postdoctoral fellowships. For critical comments on the manuscript, we are greateful to George Zug. 10 fet 12 13 R.-H. CHEN, J.-Y. LIN et al. REFERENCES Fox, H. (1977) The urinogenital system of reptiles. In “Biology of the Reptilia”. Ed. by C. Gans, Academic Press, New York, pp. 1-157. Callard,I.P., Callard,G.V., Lance, V., Bolaffi, J.L. and Rosset, J.S. (1978) Testicular regulation in nonmammlian vertebrates. Biol. Reprod., 18: 16-43. Crews, D. (1979) Endocrine control of reptilian reproductive behavior. In “Endocrine Control of Sexual Behavior”. Ed. by C. Beyer, Raven Press, New York, pp. 167-222. Crews, D. (1979) Neuroendocrinology of lizard reproduction. Biol. Reprod., 20: 51-73. Arslan, M., Libo,J., Zaidi, A.A., Jalali,S. and Quzi, M.H. (1978) Annual androgen rhythm in the spiny-tailed lizard, Uromastix hardwicki. Gen. Comp. Endocrinol., 36: 16-22. Coutry, Y. and Dufaure, J.P. (1979) Levels of testosterone in the viviparous lizard (Lacerta vivi- para Jacquin). Gen Comp. Endocrinol., 39: 336- 342. Courty, Y. and Dufaure, J.P. (1980) Levels of testosterone, dihydrotestosterone, and andros- tenedione in the plasma and testis of lizard (Lacerta vivipara jacquin) during the annual cycle. Gen. Comp. Endocrinol., 42: 325-333. McPherson, R.J., Roots, L.R., McGrege, R. III, and Marion, K. R. (1982) Plasma steroids associated with seasonal reproductive changes in a multiclutch- ed freshwater turtle, Sternotherus odoratus. Gen. Comp. Endocrin., 48: 440-451. Licht, P., Papkoff, H., Farmer, S.W., Muller, C. H., Tsui, H. W. and Crews, D. (1977) Evolution in gonadotropin structure and function. Rec. Prog. Horm. Res., 33: 169-248. Bourne, A.R., Taylor,J.L. and Watson, T.G. (1985) Identification of epitestosterone in the plas- ma and testis of the lizard Tiliqua (Trachydrosaurus) rugosa. Gen. Comp. Endocrinol., 58: 394-401. Cheng, H. Y. and Lin, J. Y. (1977) Comparative reproductive biology of the lizards, Japalura swinho- nis formosensis, Takydromus septentrionalis and Hemidactylus frenatus in Taiwan. I. Male reproduc- tive cycle. Bull. Inst. Zool., Academia Sinica (R.O.C.), 16: 107-120. Cheng, H. Y. and Lin, J. Y. (1978) Comparative reproductive biology of the lizards, Japalura swinho- nis formosensis, Takydromus septentrionalis and Hemidactylus frenatus in Taiwan. II. Fat body and liver cycles of the males. Bull. Inst. Zool., Academia Sinica (R.O.C.), 17: 67-74. Wingfield, J.C. and Farner, D.S. (1975) The deter- mination of five steroids in avian plasma by radioim- munoassy and competitive protein-binding. Ster- 14 15 16 17 18 19 Androgen and Reproduction in Lizard oids, 26: 311-327. Anderson, P.H., Fukushima, K. and Schiller, H.S. (1975) Radioimmunoassay of plasma testosterone, with use of polyethylene glycol to separate antibody- bound and free hormone. Clin. Chem., 21: 708-714. weetoy.b., Chang, T. Y., Hsu, H.K., Liao, C.F. and Wan, W.C.M. (1981) Androgen/testosterone synthesis by the dissociated testicular cells from mice of different ages in response to rat LH stimulation in vitro. Bull. Inst. Zool., Academia Sinica (R.O.C.), 20: 57-65. Lin, J. Y. (1979) Ovarian, fat body and liver cycles in the lizard Japalura swinhonis formosensis in Taiwan (Lacertillia:Agamidae). J. Asian Ecol., 1: 29-38. Lofts, B. (1972) The Sertoli cell. Gen. Comp. Endocrinol., Suppl., 3: 636-648. Lofts, B. and Bern, H.A. (1972) The functional morphology of steroidogenic tissues. In “Steroids in Nonmammalian Vertebrates”. Ed. by D.R. Idler, Academic Press, New York, pp.6—36. Lofts, B. (1968) Patterns of testicular activity. In “Perspectives in Endocrinology”. Ed. by E.J.W. 20 21 22 23 24 329 Barrington, and C. Barker-Jorgensen, Academic Press, New York. Licht, P. (1972) Envrionmental physiology of repti- lian breeding cycles: Role of temperature. Gen. Comp. Endocrinol., Suppl., 3: 477-488. Pearson, A.K., Tsui, H.W. and Licht, P. (1976) Effect of temperature on spermatogenesis, on the production and action of androgens and on the ultrastructure of gonadotropic cells in the lizard Anolis carolinensis. J. Exp. Zool., 195: 291-304. Cheng, H. Y. (1986) Internal rhythms and the mechanisms in the regulation of annual reproductive and energetic patterns in lizards. Tunghai Journal, 27: 565-590. Johnson, L. and Jacob, J.S. (1984) Pituitary activity and reproductive cycle of male Cnemidophorus sexlineatus in West Tennessee. J. Herpetol., 18: 396-405. Bourne, A. R. and Seamark, R. F. (1975) Seasonal changes in 17-hydroxysteroids in the plasma of a male lizard (Tiliqua rugosa). Comp. Biochem. Physiol., 50B: 535-536. “ee, Ni be A cH RT ERR das oiiniiaa Hebron Ges finite Ai SR tte ol * bib av brake iy. mi oH. pala waned ABST h “, Ley ie , ~ tartan’ Bg a) a an ti luis We ee “iere'd ' re, i "6 bes < MEM beathed abt i pa ak ae Be a read ar He ee Seah Saale ave iiytalyo wvhawp lass he iodine cise aeRO eu eH MA tene tah SAN nt OS, RN oh wei L sghiorraceng ore cpuenehy, ry he fies =e ae ese OF 5 ath ATCA PERRET Te isin (iit i ih H ih ais a: bon yi erie 4 SAAD Peay tr) tga ivehingst rs aul} is at} er >it! PERS = ' ‘ | ait i vi met abit 7. ia a tet :, tiihas ns oer eg ARS: VON ata PA CE RD ee tee eee POE Peal lhs take A RL Ts Se RE ae Hemi a « ES We 4 = ' ml oe ee es Td 7 won ind Lean iat aie ee ie oh Die AMRIT Ap, Arete Ss, 5 SES. ED Nip Fen?, Chery t > in oe sane 4-4 4 Whayigee ny had "4 ol (CUls F Waren hee A oro F Now Me) | Ce Pe oe mee eee ie, seieneie SEUt Fan eee OO ed OE a et i Li Man yO miei). 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Pere ai] *% | od 4! ay *F bu) Tibede sy.” | ca GAT: |g ’ : r ’ it dubict wher hematite} ¥ i ae ; a ou th unten: ee basal’ 14, felch Chae is, Prepay iaedt ih div cage y bib ear Wh Fe FFAs mines jh saltiness ‘oi a) STi) “el, ee: inate, Tat aaa: | Sine GELS Ve Gea ti ie "Pend: a ~ wre aphir i see ‘ “iii wml bal treet ag * i Y , hh @ ery i Tt beans ikiah hyvag aN hecteherpye ZOOLOGICAL SCIENCE 4: 331-337 (1987) © 1987 Zoological Society of Japan Effects of Intracranially Implanted Cholecystokinin and Substance P on Serum Concentrations of Gonadotropins, Prolactin and Thyroid Stimulating Hormone in the Rat Haruko UEmurA, ATSUHIKO Hatrori’, Masaru Wapa! and HipEsHI KoBAYASH? Biological Laboratory, Kanagawa Dental College, Yokosuka, Kanagawa 238, ‘Department of General Education, Tokyo Medical and Dental University, Ichikawa, Chiba 272, and *Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan ABSTRACT—A cannula containing a mixture of cholecystokinin-4 (CCK-4) and cholesterol (1:1 or 5:3 by weight) and one containing a mixture of substance P (SP) and cholesterol (1: 1) were implanted into the median eminence of male rats. CCK-4 reduced serum LH but not FSH, PRL and TSH concentrations; SP reduced serum TSH but not LH, FSH and PRL concentrations, according to measurements made 4—5 days following the implantations. CCK-4 implanted into the ventromedial or arcuate nucleus failed to have any effect on serum LH concentration. It is thus concluded that CCK-4 inhibits the release of LH, and SP inhibits the release of TSH, probably by inhibiting LHRH and TRH secretion from their respective axon terminals in the median eminence. INTRODUCTION The presence of the gastrin-cholecystokinin family of peptides (G-CCK) and substance P (SP) has been demonstrated by radioimmunoassay (RIA) in the hypothalamus of mammals (G-CCK: [1,2]; SP:[3]) and pigeons (SP: [4]). Further- more, the axon terminals of these substances have been shown immunohistochemically in the median eminence (ME) of mammals (G-CCK: [5]; SP: [6, 7, 10]) and lower vertebrates (G-CCK: [8]; SP: [9-11]). Thus, G-CCK and SP may possibly be essential to the regulation of the secretion of hypothalamic releasing or release-inhibiting hor- mones from the ME. To gain confirmation of this, various studies have been conducted on the effects of G-CCK and SP on the release of adenohypo- physial hormones. Intraventricular injections of COOH-terminal octapeptide of CCK (CCK-8) [12] and gastrin [13] inhibited release of luteinizing hormone (LH), but CCK-8 stimulated it when implanted in the medial preoptic area in rats [14]. Accepted December 15, 1986 Received November 20, 1986 Further, intraventricularly injected SP stimulated LH and prolactin (PRL) secretion in rats [15] but had no influence on the release of LH or follicle- stimulating hormone (FSH) in the rhesus monkey [16]. It is thus apparent that these findings are at variance with each other. Intraventricularly administered substances dif- fuse into nervous tissue and affect neurons near the ventricle, thus making it difficult to identify the site of action of the substances. In the present study, in order to find a particular site that is affected, a cannula containing the COOH-terminal tetrapeptide of CCK (CCK-4) or SP was chronical- ly implanted into the ME. In addition, a cannula containing CCK-4 was implanted into the arcuate (AR) or hypothalamic ventromedial (HVM) nu- cleus, since G-CCK is found in these nuclei [2, 5, 21]. Then the serum concentrations of LH, FSH, PRL and thyroid-stimulating hormone (TSH) were estimated by RIA. MATERIALS AND METHODS Male rats of the Wistar-Imamichi strain, 8 to 10 weeks of age (220-290 g), were used. The animals 382 H. Uemura, A. Hatrori et al. were housed in an air-conditioned room at about 24°C under a 12 hr photoperiod (07:00-19:00) and had free access to water and food obtained from a commercial source. CCK-4 and SP (both from Peptide Institute, Inc.) were used for the implantation. CCK-4 was chosen among G-CCK for implantation, since it is known that CCK-4 interacts with CCK receptors in the rat brain [17]. For their implantation into the brain, CCK-4 and cholesterol were mixed well in a small glass-mortar with a pestle at a ratio of 1:1 or 5:3 by weight. SP and cholesterol were mixed in the same way at a ratio of 1:1. About 7-10 mg of each mixture or cholesterol alone was packed into a steel cannula of 0.35mm in inner diameter. Each rat was anesthetized with Nembutal and fixed to a stereotaxic instrument. A cannula was then implanted stereotaxically into the brain, guiding its tip so that it would contact the desired hypothala- mic sites, with the aid of X-rays. In the ME, the tip of a cannula containing CCK-4, SP or cholesterol was placed to contact with or just beneath the ependymal layer (Fig. 1). Furthermore, a cannula containing CCK-4 was implanted so that its tip would be in the center of the right AR or HVM (Fig. 1). It has already been demonstrated that substances mixed with cholesterol packed into a cannula, which is then implanted into the brain, diffuse out from the tip of the cannula for a certain period of time [18-20]. Rats implanted with a cannula containing either cholesterol or a piece of nylon thread of 0.3mm in diameter served as cholesterol or blank controls. They were im- planted into the same sites as those of the experimental rats. Animals without any implanta- tion were also killed to serve as intact controls. The details of the implantation technique have been described in an earlier paper [19]. Four or 5 days after the operation, the rats were decapitated and blood samples were collected from the trunk between 10:00 and 11:00. Serum was separated by centrifugation at 2,000 rpm for 20min and stored at —80°C until hormone assay. At the time of decapitation, a piece of hypothalamic tissue hit by the tip of the cannula was dissected out and fixed in Bouin’s fluid. Paraffin sections were cut at 14 um in thickness and stained with hematoxylin and eosin. The loci of the cannula tips in the Fic. 1. Frontal section of basal hypothalamic region of a rat implanted with a cannula containing cholester- ol into the median eminence (ME). The tissue was fixed in Bouin’s fluid after the cannula was removed. The tip of the cannula was placed just beneath the ependymal layer. AR: arcuate nucleus, B: blood, E: ependymal layer, HVM: hypothalamic ventromedial nucleus, T: a tissue mass consisted mostly of epen- dymal, glial and some phagocytic cells. x45. hypothalamus were examined microscopically. Data were collected only from rats in which the cannula tip was situated at the desired position. Serum concentrations of LH, FSH, PRL and TSH were determined in triplicate with RIA kits sup- plied by the National Hormone and Pituitary Program. Reference standards for the assays were NIADDK rat LH-RP-1, rat FSH-RP-2, rat PRL- RP-1 and rat TSH-RP-1. Concentrations of these hormones were expressed in terms of LH-NIH-S1, FSH-RP-2, PRL-RP-1 and TSH-RP-1, respective- ly. The data were statistically analyzed by the Student’s t-test and Cockran-Cox method. Fic. FiG. CCK, SP and Pituitary Hormones 1.2 | | = | | £ | | > 0.8 | * = | oi | = | = | = | = 0.4 | = ® | a | | 0 16 | e oy . } / Oy i>) ~/ 6 oO e ® @ Oo e C ME HVM 2. Serum LH concentrations 4 or 5 days following the implantation of a cannula containing a piece of nylon thread (Blank), cholesterol (Chol), a mixture of CCK-4 and cholesterol at 5:3 by weight (CCK) or a mixture of SP and cholesterol at 1:1 (SP). Each column with vertical line shows mean and SE. Number of rats of each group is shown at the bottom of the column. ME: median eminence, HVM: hypothalamic ventromedial nucleus. * Significant (p<0.05) compared with the cholesterol control rats (Student’s f-test). 5 1.0 0 0.5 Serum LH (ng/ml) ME AR 3. Serum LH concentrations 4 or 5 days following the implantation. AR: arcuate nucleus. In this experiment, a mixture of CCK-4 and cholesterol at 1: 1 (CCK) was used. Other abbreviations are the same as those in Fig. 2. * Significant (P<0.05) compared with the cholesterol control rats (Cockran-Cox method). 333 334 H. Uemura, A. Hartrori et al. RESULTS blank controls Were not significantly different from intact controls, irrespective of implantation sites Effects of operation: Concentrations of serum _ (Figs. 2 to 6). These findings indicate that im- LH, FSH, PRL and TSH in rats of cholesterol and plantation of a cannula or cholesterol into the Fic. Fic. 15 | : = | 3 cS 10 ! x | “Y | LL. | Esa 18 | ~) | © | “0 | | O ; Se he > ~y, ) oy y oy y ME HVM 4. Serum FSH concentrations 4 or 5 days following the implantation. Abbrevia- tions are listed in Fig.2. No effects were observed on FSH levels by any implantation. 500 | = | E | = | = | a as | — 250 | ce | ; & | SS] | ~~ | ® | (p) | | O . y KS y, A y I ~y, ® Os ¢e ME HVM 5. Serum PRL concentrations 4 or 5 days following the implantation. Abbrevia- tions are the same as those in Fig. 2. No effects were observed on PRL levels by any implantation. CCK, SP and Pituitary Hormones 335 12 oe) 00 Serum TSH (pg/ml) o) p | | ! ! | : | ! | | ! | | | | | O Oo” oy” ME @ YO 5 ® 'y ®& % O HVM Fic. 6. Serum TSH concentrations 4 or 5 days following the implantation. Abbrevia- tions are the same as those in Fig. 2. ** Significant (P<0.01) compared with the cholesterol control rats (Student’s f-test). brain has no effect on the release of LH, FSH, PRL and TSH. Therefore, data obtained from experimental rats were compared only with cholesterol controls. Serum LH concentration: Two series of experi- ments were carried out: (1) implantation of CCK-4 plus cholesterol (5:3) in the ME and HVM (Fig. 2) and (2) implantation of CCK-4 plus cholesterol (1:1) in the ME and AR (Fig. 1). The serum LH concentration of rats implanted with CCK-4 into the ME was lower than that of cholesterol controls (P<0.05) (Figs. 2 and 3). Implantation of CCK-4 into the HVM (Fig. 2) or AR (Fig. 3) has no effect on serum LH concentration. In rats implanted with SP in the ME, the serum LH concentration was not significantly different from that of cholesterol controls (Fig. 2). Serum FSH and PRL concentrations: Im- plantations of CCK-4 _ plus cholesterol (5:3) in both the ME and HVM and SP in the ME caused no change in the serum concentrations of FSH and PRL, compared with cholesterol controls (Figs. 4 and 5). Serum TSH concentration: The implantation of a cannula containing CCK-4 plus cholesterol (5:3) had no effect on the concentration of serum TSH, whereas SP implantation into the ME reduced remarkably the serum TSH level (P< 0.01), compared with cholesterol controls (Fig. 6). DISCUSSION In the present study, serum LH concentration decreased when CCK-4 was implanted into the ME. Vijayan et al. [12] reported that LH secretion was inhibited by CCK-8 injected intraventricularly in ovariectomized rats. This reduction of LH secretion may possibly be explained by the assumption that CCK-8 acted on the ME. This explanation is also supported by the observation that CCK-immunoreactive fibers are present in the ME [5]. Two possible mechanisms may be considered for the inhibitory effects of CCK-4 implanted in the ME on serum LH. (1) CCK-4 affects nerve terminals containing LH-releasing hormone (LHRH) in the ME causing reduced LHRH secretion, and (2) CCK-4 drains into the capillaries of the primary plexus, reaching the adenohypo- physis to inhibit LH release. However, the second possibility seems unlikely, since CCK-8 had no effect on the release of LH from the hemipituita- 336 H. Uemura, A. Hatrori et al. ries of rats in vitro [12]. Rather, it is probable that CCK acted on LHRH axons at least in the ME in such a way as to inhibit the release of LHRH, resulting in reduced LH concentration in the serum. Since gastrin and CCK-8 share biologically active COOH-terminal tetrapeptide (CCK-4), they may have similar actions to that of CCK-4 within the ME. CCK-8, CCK-4 and gastrin have been demonstrated by RIA to be present in the porcine hypothalamus [1]. Further, intraventri- cularly administered CCK-8 and gastrin were actually found to reduce LH secretion [12, 13]. It thus remains to be determined which peptides physiologically functions to reduce LHRH release, CCK-8, CCK-4 or gastrin. In our experiments, CCK-4 implanted in the HVM and AR at the right side of the hypothala- mus had no effect on serum LH concentration, although the presence of many G-CCK im- munoreactive fibers in the HVM [5] and relatively high concentration of G-CCK in the AR [2] have been reported. It is probable that the HVM and AR at the left side may have compensated for possible inhibition of LHRH release by CCK-4 or G-CCK in these regions may not be involved in the release of LHRH. Serum concentrations of FSH, PRL and TSH were not affected by CCK-4 implantation in the HVM or ME of male rats. However, according to Vijayan et al. the intraventricular injection of gastrin reduced serum concentrations of PRL and TSH [13] and the intraventricular injection of CCK-8 reduced TSH secretion, but not FSH and PRL secretion [12] in ovariectomized rats. The differences between their results and ours may possibly be ascribed to differences in sites of action, duration of action, concentrations of sub- stances administered and animal sex. Implantation of SP into the ME caused a remarkable decrease in serum TSH in the present study. Vijayan and McCann [22], however, failed to observe such an inhibitory effect of intraventri- cular SP on TSH release in rats. This discrepancy may be explained by differences in experimental design, especially the mode of SP administration. The present data indicate SP implanted into the ME may possibly interfere with release of TSH- releasing hormone (TRH) through interactions that may occur between SP and TRH axons within the ME. SP immunoreactive fibers have been shown to be present in the internal and external layers of the rat ME [23, 24]. Electron microscopic examination has demon- strated SP terminals projecting into the perivas- cular space of the capillaries of the primary plexus of the rat ME [24]. Thus, SP may be released into the portal vessels and reach the adenohypophysis to bring about a reduction in TSH release, as suggested by Tsuruo ef al. [24]. In the in vitro experiments, however, SP did not alter the release of TSH from the hemipituitaries of ovariectomized rats in vitro [22]. On the basis of the present data, it appears most likely that SP acts on TRH fibers in the ME to reduce TRH release. SP implanted into the ME had no effect on the serum concentrations of LH, FSH and PRL in the present study. The absence of any effect of intraventricularly injected SP (100g) on the release of LH and FSH has been observed in normal female rhesus monkeys [16]. However, SP (2 ~g) injected into the third ventricle stimulated LH and PRL secretion in ovariectomized rats [15]. Differences in reported data may be due to particular sites of action, dose amounts of SP and animal hormonal status. To achieve greater con- sistency in the results obtained, further experi- mentation on such aspects as implantations of mixtures of SP and cholesterol at different ratios should be carried out. ACKNOWLEDGMENTS Prof. K. Wakabayashi (Institute of Endocrinology, Gunma University) is gratefully acknowledged for his valuable advice in conducting the hormone measure- ments and permitting use of his laboratory facilities. We thank Dr. Y. Okawara for help in the preparation of the manuscript. The present research was supported by a Grant-in-Aid for Scientific Research from the Japan Ministry of Education, Science and Culture. REFERENCES 1 Rehfeld, J.F (1978) Localization of gastrins to neuro- and adenohypophysis. Nature, 271: 771-773. 2 Beinfeld, M.C., Meyer, D.K., Eskay,R.L, Jen- sen, R.T. and Brownstein, M.J. (1981) The dis- 10 11 12 13 CCK, SP and Pituitary Hormones tribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res., 212: 51-57. Brownstein, M.J., Mroz, E.A., Kizer, J.S., Palko- vits, M. and Leeman, S.E. (1976) Regional dis- tribution of substance P in the brain of the rat. Brain Res., 116: 299-305. Reubi, J.C. and Jessell, T.M. (1978) Distribution of substance P in the pigeon brain. J. Neurochem., 31: 359-362. Lorén, I., Alumets, J., Hakanson, R. and Sundler, F. (1979) Distribution of gastrin and CCK-like peptides in rat brain. An immunocytochemical study. Histochemistry, 59: 249-257. Hokfelt, T., Pernow, B., Nilsson, G., Wetterberg, L., Goldstein, M. and Jeffcoate, S. L. (1978) Dense plexus of substance P immunoreactive nerve termi- nals in eminentia medialis of the primate hypothala- mus. Proc. Natl. Acad. Sci. USA, 75: 1013-1015. Stoeckel, M.E., Porte, A., Klein, M. J. and Cuello, A.C. (1982) Immunocytochemical localization of substance P in the neurohypophysis and hypothala- mus of the mouse compared with the distribution of other neuropeptides. Cell Tissue Res., 223: 533- 544. Rehfeld, J.F., Goltermann,N., Larsson, L.-I., Emson, P.M. and Lee, C.M. (1979) Gastrin and cholecystokinin in central and peripheral neurons. Fed. Proc., 38: 2325-2329. Gaudino, G. and Fasolo, A. (1980) Substance P- related peptides in the hypothalamus of Amphibia. Cell Tissue Res., 211: 241-250. Ho, R.H. and DePalatis, L.R. (1980) Substance P immunoreactivity in the median eminence of the North American opossum and domestic fowl. Brain Res., 189: 565-569. Mikami, S. and Yamada, S. (1984) Immunohisto- chemistry of the hypothalamic neuropeptides and anterior pituitary cells in the Japanese quail. J. Exp. Zool., 232: 405-417. Vijayan, E., Samson,W.K. and McCann, S.M. (1979) In vivo and in vitro effects of cholecystokinin on gonadotropin, prolactin, growth hormone and thyrotropin release in the rat. Brain Res., 172: 295-302. Vijayan, E., Samson, W.K. and McCann, S.M. (1978) Effects of intraventricular injection of gastrin on release of LH, prolactin, TSH and GH in 14 15 16 17 18 19 20 21 af 23 24 337 conscious ovariectomized rats. Life Sci., 23: 2225- 2232, Kimura, F., Hashimoto, R., Kawakami, M. (1983) The stimulatory effect of cholecystokinin implanted in the medial preoptic area on luteinizing hormone secretion in the ovariectomized estrogen-primed rat. Endocrinol. Jpn., 30: 305-309. Vijayan, E. and McCann, S.M. (1979) In vivo and in vitro effects of substance P and neurotensin on gonadotropin and prolactin release. Endocrinology, 105: 64-68. Eckstein, N., Wehrenberg, W.B., Louis, K., Car- mel, P. W., Zimmermann, E. A., Frantz, A. G. and Ferin, M. (1980) Effects of substance P on anterior pituitary secretion in the female rhesus monkey. Neuroendocrinology, 31: 338-342. Saito, A., Sankaran, H., Goldfine,I.D. and Wil- liams, J. A. (1980) Cholecystokinin receptors in the brain: characterization and distribution. Science, 208: 1155-1156. Matsui, T. (1967) Effects on the rat estrous cycle of implants of norepinephrine placed in the median eminence. Annot. Zool. Japon., 40: 74-81. Uemura, H. and Kobayashi, H. (1971) Effects of dopamine implanted in the median eminence on the estrous cycle of the rat. Endocrinol. Jpn., 18: 91-100. Uemura, H. and Kobayashi, H. (1974) Effects of norepinephrine and dibenamine implanted in the median eminence on the estrous cycle of the rat. Horm. Res., 5: 89-111. Larsson, L.-I. and Rehfeld, J.F. (1979) Localiza- tion and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system. Brain Res., 165: 201-218. Vijayan, E. and McCann, S.M. (1980) Effects of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro. Life Sci., 26: 321-327. Ljungdahl, A., Hoékfelt, T. and Nilsson, G. (1978) Distribution of substance P-like immunoreactivity in the central nervous system of the rat—I. Cell bodies and nerve terminals. Neuroscience, 3: 861-943. Tsuruo, Y., Kawano, H., Nishiyama, T., Hisano, S. and Daikoku,S. (1983) Substance P-like im- munoreactive neurons in the tuberoinfundibular area of rat hypothalamus. Light and electron micro- scopy. Brain Res., 289: 1-9. 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Department of Aquaculture and Biology, Shimonoseki University of Fisheries, Shimonoseki, Yamaguchi 759-65, and ‘Faculty of Applied Biological Science, Hiroshima University, Fukuyama, Hiroshima 720, Japan ABSTRACT— A new stenopodid shrimp, Spongicola levigata sp. nov., is described and illustrated from specimens associated with hexactinellid sponges in the East China Sea.-It is a small species, measuring only 8.3-11.8 mm in body length of the mature female.—This species is unique in the entirely smooth carapace and abdomen, and the lack of telsonal dorsal spines near the articulation with the sixth abdominal somite.-Its affinities with related species and sexual distinctions are discussed. INTRODUCTION Six unusual specimens of the stenopodidean shrimp were recently examined. They were found in spongocoels of a certain species of the hexac- tinellid sponge collected from the East China Sea in about 200m. This type of sponge is usually known to house a commensal shrimp, Spongicola venusta De Haan; in fact, that commensal was collected together during the cruise. The present unusual shrimps are referred to a species of the genus Spongicola but their size is much smaller, only 1/3 or 1/4 the length of S. venusta, and it apparently represents an undescribed species. The type-series has been deposited in the collection of the Shimonoseki University of Fisheries, Shimo- noseki. MATERIALS TYPE: Holotype, male, East China Sea, 30°44.7'N, 127°48.3’E, about 200 m deep, 14 June 1978, 13:44-16:30, otter trawl, Koyo Maru, O. Tabeta and K. Hayashi leg. Paratypes, 1 male, 3 ovigerous females and 1 female, collected with holotype. Accepted October 21, 1986 Received September 9, 1986 DESCRIPTION Spongicola levigata sp. nov. (Figs. 1-4) Diagnosis: Rostrum completely smooth on dor- sal and ventral margins, or provided with 1-4 dorsal and 1 ventral processes, all step-like. Carapace glabrous and smooth, lacking grooves and spines. Abdominal terga entirely smooth. Pleura of first to fifth somites broadly rounded without marginal spines. Telson lacking dorsal spines near base, but 3 pairs present on dorsal longitudinal ridges. Chela of third pereopod broad with serration or irregularity on upper and lower margins. Dactyli of fourth and fifth pereopods biunguiculate. Description: Shell of soft, somewhat mem- braneous texture. Body slender and glabrous, abdomen rather depressed (Fig. 1). Rostrum short, falling short of end of first antennular segment (Fig. 2a); directed downward or slightly curved downward; triangular in dorsal view, its base considerably wide; distally pointed obtusely; dorsal margin bearing few step-like processes, and ventral margin with one similar process or occasionally entirely smooth. Carapace 4 times as long as rostrum; glabrous and smooth, without any grooves and spines on surface, ante- 368 K.-I. HAYASHI AND Y. OGAWA Fic. 1. Spongicola levigata sp. nov. from the East China Sea. a, Holotype, male, 2.8mm in cl; b, paratype, ovigerous female, 3.2 mm in cl. Scales 2.0 mm. rior margin also smooth; suborbital or antennal angle obtusely pointed; pterygostomial angle rounded or rectangular, not spiniform (Fig. 2b). Abdomen smooth; first to fifth terga glabrous, sixth tergum with few setae; pleura of first to fifth somites broadly rounded without marginal spines; no spiniform process on sixth somite. Telson broadly lance-shaped, 1.5 times as long as broad, slightly narrowed at base; dorsal surface with pair of longitudinal ridges, each bearing 3 large, pos- teriorly directed spines; lateral margin with 2-4 pairs of distinct spines; posterior margin fringed with long setae and provided with 3 tiny spines, 2 subterminal flanking median terminal (Fig. 2c). Eyes comparatively large, eyestalk mesially with few spinules near cornea (Fig. 2a). Antennular peduncles simply elongate; basal segment more than twice as long as second segment; stylocerite small, ending in sharp point. Antennal scale broad and semi-circular, about twice as long as broad; outer margin almost straight, with 4 or 5 spinules on anterior half, terminal one largest; inner margin A New Commensal Stenopodid Shrimp 369 fg e C Fic. 2. Spongicola levigata sp. nov. a, c, e, male holotype, 2.8 mm in cl; 5, d, f, paratype, ovigerous female, 3.2 mm in cl. a, Anterior part of body, dorsal view; b, anterior part of body in lateral view; c, tail fan, right uropod removed; d, left antenna; e, right first pleopod; f, right second pleopod. Scales 1.0 mm. strongly convex; long setae on inner and distal margins; basicerite dorsomesially with quadrate flap, ventromesially with conspicuous spine near base of carpocerite (Fig. 2d). Mandible with 3-segmented palp; incisor and molar processes fused with each other (Fig. 3a, b). Palp of first maxilla slender and simple with 2 apical and few subapical setae (Fig. 3c). Palp of second maxilla long and slender; 2 endites each bilobed (Fig. 3d). Palp of first maxilliped unilobed with feeble median notch; epipod large and bilobed; distal endite large and unequally bilobed; proximal endite small and unilobed (Fig. 3e). Second maxilliped with 5-segmented endopod; epipod bilobed, small globular podobranch pre- sent (Fig.3f). Third maxilliped rather stout, especially merus and ischium, setae on distal three segments rigid; epipod slender and partly twisted, exopod extremely short, only bud-like (Fig. 3g). First pereopod slender and chelate; fingers finely pectinate on distal 1/4 of cutting edge; palm about 1.5 times as long as fingers; carpus slightly longer than merus, bearing small subterminal spine on inner side (Fig. 4a, b). Second pereopod similar in shape to first pereopod but larger and longer; palm twice as long as fingers; carpus 1.5 times as long as merus (Fig. 4c). Third pereopod strongest, sparse- ly provided with short setae on chela, slightly shorter than body length; lower margin of chela serrate on distal 2/3 of length; upper margin smooth or faintly serrate; fingers distally curving inward, crossing, each ending in sharp point; opposable margin of movable finger with promi- nent median tooth fitting to large opposing con- 370 K.-I. HAYASHI AND Y. OGAWA Fic. 3. Mouthparts of Spongicola levigata sp. nov. (right side), paratype, ovigerous female, 3.2 mm in cl. a, mandible, ventral view; b, same, dorsal view; c, first maxilla, ventral view; d, second maxilla, ventral view; e, first maxilliped, ventral view; f, second maxilliped, ventral view; g, third maxilliped, outer view. Scale 1.0 mm. cavity; cutting edges distal to snapping part armed with several quadrangular teeth; palm rather massive, roughly quadrate or slightly longer than broad, width as long as dactylus; carpus triangular in lateral view, anterior margin pointed at upper and lower ends; merus armed with 5 spines on posteromedian margin in male, spineless in female; ischium with small spine at anterodistal end (Fig. 4d,e). Fourth and fifth pereopods similar, long and slender, sparsely setose; dactylus short, simply biunguiculate, penultimate tooth articulated; propodus 2.5 times as long as dactylus and half as long as carpus, bearing 8-12 spines on posterior margin; carpus slightly longer than merus, armed with 2 small but sharp spines on posterodistal end (Fig. 4f, g). Branchial formula as shown below. Epipods absent from fifth pereopods; exopods well de- veloped on first and second maxillipeds, rudimental on third maxillipeds, absent from pereopods. Maxillipeds Pereopods ie P23 1 9120833" AS Pleurobranchs — 1 1 Le PE ee Arthrobranchs — 1 2 2 2 2a Podobranchs —- 1l—- ——~— — ~ — Epipods i? bss Lee ti Exopods llr ~~~ ~ — First pleopods in male small, composed of 2 segments and feebly setose (Fig. 2e), those in female largely unilobed, and fully setose (Fig. 2f). Second to fifth pleopods bilobed, each fringed with long setae. Endopod and exopod of uropod each with weak longitudinal dorsal ridge, bearing long setae on inner and distal margins; endopod armed with 4-7 spines on distal 2/3 of outer margin, terminal spine larger; exopod with 6-10 spines on distal half of outer margin, terminal spine relative- ly large (Fig. 2c). Color: brown. Body almost transparent; cornea light A New Commensal Stenopodid Shrimp 371 Fic. 4. Pereopods of Spongicola levigata sp. nov. a—d, g, paratype, ovigerous female, 3.2 mm in cl; e, f, male holotype, 2.8 mm in cl. a, right first pereopod, outer view; b, chela and anterior part of carpus of same, inner view; c, right second pereopod, outer view; d, left third pereopod, outer view; e, right third pereopod, outer view; f, right fourth pereopod, outer view; g, right fifth pereopod, outer view. Scales 1.0 mm. Measurements: Holotype, male, 10.0mm in _ ovigerous female, 8.3mm in BL. Subglobular body length excluding rostrum (BL), 2.8mm in _ eggs, 0.4-0.6 mm in diameter. carapace length excluding rostrum (CL), 9.8 mm in length of right third pereopod. Paratypes, 1 male, 8.8mm in BL, 2.9mm in CL; 4 females, 8.3-11.8 mm in BL, 2.7-3.4 mm in CL; smallest In spite of having the glabrous smooth integu- DISCUSSION 372 K.-I. HAYASHI AND Y. OGAWA ment, the present species may be referred to a species of the genus Spongicola. As mentioned by Holthuis [7] and Bruce and Baba [4], Spongicola bears well-developed exopods in the first two maxillipeds but rudimental one in the third maxil- lipeds. Recently Saint Laurent and Cleva [15] slightly modified this definition to include S. inflata, which has a normal, well-developed ex- opod on the third maxillipeds. The gill formula of the present species is exactly the same as shown by Holthuis [7] for Spongicola and differs from those of the related genera, Spongicoloides [11], Spon- giocaris [4] and the recently erected Paraspongico- la [15]. The glabrous, smooth integument unique to the present species is, however, shared with some species of Spongicoloides [7, 11]. The chela of the third pereopod in this species also represents the feature of Spongicola. The known species and subspecies of the genus Spongicola retain several teeth or spines on the rostrum and carapace, with rather fair regularity. In most of the species, the rostrum bears a few to several dorsal and a single ventral spines; the carapace is also provided on each side with 1 postrostral, 1 hepatic, 1 antennal, and some small spines on both the anterior margin and the pterygostomial region directly inside of it. The present new species, however, is entirely devoid of the carapacial spines and grooves. Even the antennal or suborbital angle is reduced to an obtuse process. The dorsal margin of the rostrum bears a few step-like processes, none of which ends in a sharp point; occasionally they are absent, even the ventral marginal one. In almost all species of Spongicola, the abdom- inal pleura are more or less serrated marginally, and telson bears 2 pairs of spines near the articulation with the sixth abdominal somite, in addition to the dorsal pairs on the longitudinal carinae [1, 7, 14, 15]. In the present new species such spines or processes are barely recognizable, only excepting the last mentioned ones. The sexual distinctions in this species are ap- parent in the third pereopods and the first pleopods. In the male about 5 spines are distinct on the posteromedian margin of the third pereopods, while they are absent in the female. The first pleopod is a single lobe without articula- tion in the female, instead of being composed of 2 segments in the male; the marginal setae in the female are longer and more numerous in the female. Like the other members of this genus as well as the related genera [2, 4, 7, 9, 15], the present new species is a symbiont of the deep-sea glass sponge. All of the present type series were obtained from the spongocoels of a hexactinellid sponge, prob- ably Euplectella oweni Herklots and Marshall. Four of them were found in a pair in 2 different host sponges. The female is larger than the paired male; in one pair the female is 11.8 mm in body length and the male is 10.0 mm and in the other pair the female is 10.8 mm and the partner is 8.8 mm. Other examples of this sponge found in the same trawl haul accommodated the other common stenopodid, S. venusta De Haan; a total of 18 (6 pairs and 6 singles) specimens were collected. S. venusta was recorded from Japan, Korea, the East China Sea and the Philippines at the depths of 174-315 m [3, 5-8, 10, 12, 13, 15, 16]. S. levigata and S. venusta may be readily distinguished from each other at the field by the size difference. The former is only 1/3 or 1/4 of the latter and is probably the smallest among the species of this genus, being only 8.3 mm in body length of the smallest ovigerous female. ACKNOWLEDGMENTS We thank Dr. K. Baba of the Kumamoto University, Kumamoto, Japan, for his critical review of the manu- script. REFERENCES 1 Alcock, A. (1901) A descriptive catalogue of the Indian deep-sea Crustacea Decapoda Macrura and Anomala, in the Indian Museum. Being a revised account of the deep-sea species collected by the Royal Indian Marine Survey Ship Investigator. pp. 1-286, i-iv, pls. 1-3. 2 Baba, K. (1983) Spongicoloides hawaiiensis, a new species of shrimp (Decapoda: Stenopodidea) from the Hawaiian Islands. J. Crust. Biol., 3: 477-481. 3 Bate, C. S. (1888) Report on the Crustacea Macrura collected by H.M.S., Challenger during the years 1873-76. Rep. Voy. Challenger, Zool., 24: i-xc, 1- 10 A New Commensal Stenopodid Shrimp 942. pls. 1-150. Bruce, A. J. and Baba, K. (1973) Spongiocaris, a new genus of stenopodidean shimp from New Zealand and South African waters, with a descrip- tion of two new species (Decapoda, Natantia, Stenopodidea). Crustaceana, 25: 153-170. Fujino, T. and Miyake,S. (1970) Caridean and stenopodidean shnmps from the East China and the Yellow Seas (Crustacea, Decapoda, Natantia). J. Fac. Agr.. Kyushu Univ., 16: 237-312. De Haan, W. (1833-1850). Crustacea. In “P. F. von Siebold; Fauna Japonica sive descriptio animalium, quae in itinere per Japoniam, jussu et auspiciis superiorum, qui summum in India Batava Imperium tenent, suscepto. annis 1823-1830 collegit, notis, observationibus et adumbrationibus illustravit”. i-XVil, I-xxxi, 1-244, pls. 1-55, A-Q, 1, 2. Holthuis, L. B. (1946) The Decapoda Macrura of the Snellius Expedition. 1. The Stenopodidae. Nephropsidae, Scyllaridae and Palinuridae. Biolo- gical results of the Snellius Expedition XIV. Tem- minckia, 7: 1-178. Kim, H. S. (1977) Macrura. Illustrated Flora and Fauna of Korea, 19: 1-414. pls. 1-56. Kubo, I. (1943) A new commensal shrimp, Spongi- cola japonica n. sp. Annot. Zool. Japon. , 21: 90-94. Miers, E. J. (1878) On species of Crustacea living within the Venus’s flower-basket (Euplectella) and in Meyerina claviformis. J. Linn. Soc. Lond., Zool.., 13: 506-512, pl. 24. 11 i4 16 373 Milne Edwards, A. and Bouvier, E. L. (1909) Les peneides et Stenopodides. Reports on the results of dredging, under the supervision of Alexander Agas- siz, in the Gulf of Mexico (1877-78), in the Caribbean Sea(1878-79) and along the Atlantic coast of the United States (1880), by the U. S. Coast Survey Streamer “Blake”. XLIV. Mem. Mus. Comp. Zool., Harvard, 27: 177-274, pls. 1-9. Ortmann, A. (1890) Die Decapoden-Krebse des Strassburger Museums, mit besonderer Berucksich- tigung der von Herrn Dr. Doderlein bei Japan und bei den Liu-Kiu-Inseln gesammelten und z. Z. im Strassburger Museum aufbewahrten Formen. I. Theil. Die Unterordnung Natantia Boas. Zool. Jahrb., Syst., 5: 437-542, pls. 36, 37. Parisi, B. (1919) I Decapodi Giapponesi del Museo di Milano. VII. Natantia. Atti Soc. Ital. Sci. Nat., 38: 59-99. pls. 3-6. Rathbun, M. J. (1906) The Brachyura and Macrura of the Hawauan Islands. Bull. U.S. Fish. Comm., 23: 827-930, pls. 3-24. Saint Laurent, M. de and Cleva, R. (1981) Crus- taces decapodes: Stenopodidae. In Resultats de la Campagne Musorstom. 1. Philippines (18-28 Mars 1976), 1 (7). Mem. ORSTOM, 91: 151-188. Yokoya, Y. (1933) On the distribution of decapod crustaceans inhabiting the continental shelf around Japan, chiefly based upon the materials collected by S.S. Soyo-Maru, during the years 1923-1930. J. Coll. Agr., Tokyo Imp. Univ., 12: 1-126. 2c (CORD abedbaptedl dibs 1 10. elas A) pays RO. aA pabibor iy = tee. er Taga aes) ‘a ig BAY Mearet wits af 4) : 3 SHY" He atest er) gp i Sta) Lam OR wi Na ee aera yh Fann tah Sg Oita’? Wate eas chic ok ORS “seb to pot lisll Teh vay) Li (idaatoua: Bed Caepate, ny 4 Dork ther BL | Vis < emest EOS, ed wk pusertal bors me | ae ‘SRT ; A pnetsyG 0 = Avspowiedl 1stobrioesd Jen eae TS roti fee ne” Bit abit fod ished Bog Pied | Week feds pietngh}" " ; iSong ey “ipntaer rae Lean PHT irra wena: potinmanaty BA, ¢ Cyaan apelacaa ny Giger me OS Cea » ‘ y ae Les peta leet al a gibt MY he 2 Ava wert, ies) ¥ ‘ad ie (eee. ey Rhy 3B ot ee Puls. } Ba f y* rere iy ”) pel fot Seas rae) pe vats 1 VIE) Si re ts oh €] LN z iS : iy ; ‘ were) 7AeT Hae Wee sd bers i ee ie re re Bitp NMR A Lh Wi ct ae nnrteiai hh gil) bth ny, in? Loe ae Si * UY tT Pe § wei tee ae orth) va ah) pe uk BIG 34 ha aha itst wr ni ne ete raise eben: “yb 208) Nie a 7) isvadiyty rihiey': Pou en karale } Ai wer o PALTRY he GT EG GTee! brew H Genrer aA tn bp) CER eae tela (aieemiey oil) dich) Koto ean by ue sit Aca, egld tmepcr liean() witty POH Ptyve v ve et ay ON, SRTAIO ITtREY): Bae - pene | Hh? Gent OV eAt ah lod : q ii : - ibis t [ eM V dpe 1 “) i ™ 5 ia 3) o t} | ¢ ie §)) 4 {OTIS } ihe yued OF hs? haed- y wy the { ‘ ey ‘ , whe yy MEITUVAL? ULES TCTSED qnctie Bee ne | nals thi ne Leivnw Hbset qe eT ERT ae ORR ecu “ane count fy? ey ory ‘akin .aboquxsCh) int end Like THAT DARAE heeboerepta inde beens tess (PWR). R ‘panei | bag: : Ly (ainsi: phogassG gostaui Pag one Ney’ a ee ai acti cahvshe sian binigead (scavemy: ners berveregel re aioe qearvonsel sary te nity ord ay LET an WOH i} ae, AS fis f ae ane s Twi i sul “ibhwou rari, 19 7UGHES aL ite ae Ay hl ia) fi +. ae ' Life. pony aes IRA tbat! Sy alt SAP MR aight } Vmbibxwjonste sdT i. nee rien. hath oop bien pled bhevoabpellentyy ; wad -isynquitaand ai Hane fs hy a ¥ wings eset she ket I 2 HEAR Weihihe LER Hat aniced ee a DEIN IT ndigk Look hae mea Liivit? haomnaiy > Recwsktind 2) ME dans Oo \wrnelpard ioteoleed: “wee ie te ite AOS, hood 208 Mbt hy See a ‘yi lin ue UF ri i lh Gash 2 rua’ r sf 3 te CRNOWLI EON, ; er ig. K spc. “4 igen} eerie of, ie toned cet wh), CES n icelgytive [valet sheer tenn ae rade a PPE Trt hia x a be ~~ ne Ai: Miyso ar "eect thas bee ft: Pr nae iy Love) ti bias, ls rete Suche ah FTG tet pgs he, Eh to x con janay gaasavalall Ma i potion of Phen (Decapoda: | tee Lg an ab es ZOOLOGICAL SCIENCE 4: 375-378 (1987) © 1987 Zoological Society of Japan Notes on the Genus Sasanychus Ehara, New Status, with Description of A New Species from Hokkaido (Acarina, Tetranychidae) SHOz6 EHARA and Tetsuo Gorton! Biological Institute, Faculty of Education, Tottori University, Tottori 680, and ‘Laboratory of Applied Entomology, Faculty of Agriculture, Ibaraki University, Ami, Ibaraki 300-03, Japan ABSTRACT—The subgenus Sasanychus Ehara in the genus Panonychus Yokoyama was elevated to a full genus. A mite, so far referred to “stipeless-egg form” of P. (S.) akitanus Ehara, was described under the name S. pusillus n. sp. from Sasa apoiensis Nakai in Tomakomai, Hokkaido. INTRODUCTION The subgenus Sasanychus was created by Ehara [1] in the genus Panonychus Yokoyama to accomodate a new species akitanus taken from Sasa sp. in Akita Prefecture. Recently, it was reported by Gotoh [2, 3] that in P. (S.) akitanus there were two forms differing in egg shape, micro-habitat preference, overwintering stage, di- apause characteristics and voltinism. Moreover, the two forms were experimentally confirmed to be reproductively isolated [4]. On close examination we have come to the conclusion that one of the two forms, “stipeless- egg form”, is not assigned to akitanus but to a new species. In this paper the new species is described and illustrated from Sasa apoiensis Nakai in Tomakomai, Hokkaido. Further, the subgenus Sasanychus is raised here to genus. Genus Sasanychus Ehara, new status Panonychus (Sasanychus) Ehara, 1978 [1], p. 88. Type-species: Panonychus (Sasanychus) akita- nus Ehara. Two pairs of para-anal setae present. Dorsal idiosomal setae not set on tubercles. Hysterosoma with transverse striae on the dorsocentral area. Empodium claw-like, with 3 pairs of prox- Accepted December 6, 1986 Received November 8, 1986 imoventral hairs that are similar in length and set at a nearly right angle with claw. Two sets of duplex setae on tarsus I adjacent to each other. Tibia I with 9 tactile setae; tibia II with 8 tactile setae. Sasanychus akitanus Ehara Panonychus (Sasanychus) akitanus Ehara, 1978 [1], p. 88, Figs. 1-11. Sapporo population of Panonychus akitanus: Gotoh, 19867 (25 pel25; 1986) [3] ips ls7- Stiped-egg form of Panonychus akitanus: Gotoh, 1986 [3], p. 149; 1986 [4], p. 153. Female Dark green. Measurements in sm: body length (including rostrum) 463, body width 273; lengths of setae? PR; 80:7 22015", P5130.5= 1.8, Ps 92.022 MOG OES iC, M4 stl. Cc Seba) (Ca Ws ae HG. We A Shae ae JO es.) Hall 3, ba MIB iOse 1kSs 9477 +026; O'86.8220!8. CL 64.5+0.7, PA 50.4+1.1; distances: P,;-P,; 81.4+ 0.6, P2>—P2 91.6+0.7, Cy-C, 78.7+1.9, C>-C, 81.5 +1.1, C3;-C3 58.4+1.3. Length ratio P3;/body= 0.197+0.005, H/body=0.229 +0.005, L,/body= 0.256+0.005, L3/body=0.245+0.004. The num- ber of setae and solenidia (in parentheses) on leg podomeres: femora 10-—7-4-3, genua 5-5-4-4, tibiae 9(1)—-8-6-7, tarsi 14(1)+2 dupl. —13(1)+1 dupl. —10(1)—10(1). Means. Seb ni — 10) 376 S. EHARA AND T. GOTOH 100 um A) Fics. 1-8. Sasanychus pusillus n. sp. 1: Dorsum of female. 2: Lobes on dorsal hysterosomal striae (schematic). 3: Distal segment of palpus of female. 4: Distal segment of palpus of male, showing variation of terminal sensillum. 5-6: Peritremes of female. 7-8: Aedeagi. Male Measurements in ym: body length (in- 66.0+1.1, O 56.6+1.0, CL 35.3+1.0, PA 31.7+ cluding rostrum) 344, body width 160; lengths of 1.6. The number of setae and solenidia (in setae: P; 67.1+0.7, P, 92.6+1.0, P3 75.3+1.3,H parentheses) on leg segments: femora 10-—7-4-3, 82.7+1.0, C, 86.341.2, C, 83.1+0.9, C3 75.34 genua 5-5-4-4, tibiae 9(3)-8-6-7, tarsi 13(3) 0.5, L; 81.5+0.9, Ly 88.5+1.0, L; 84.040.8, 1 +2 dupl. —13(1)+1 dupl. —10(1)—10(1). Mites of Sasanychus 307. Specimens examined In addition to the type- series (type locality: Nishi-senboku, Akita Pref., Honshu) , the following specimens from Hokkaido have been examined: Four ¢¢ and 5 ?%, Hok- kaido Univ. campus, Sapporo, 9-IX-1983 (S. Ehara and T. Gotoh), on Sasa senanensis (Fran- chet et Savatier) Rehder; 4 @¢ and 4 #4, 9-IX- 1983 (S. Ehara and T. Gotoh), bred in Inst. of Appl. Zool., Hokkaido Univ., originally collected on Hokkaido Univ. campus, V—1983 (T. Gotoh), on Sasa senanensis; 5 2% and 13 ¢%, Hokkaido Univ. campus, VII~VIII-1985 (Y. Sait6), on Sasa senanensis. Distribution WHokkaido, Honshu. Remarks The redescription is based on the type-series, but the measurements nearly accord with those for the specimens from the Hokkaido University campus, Sapporo. Sasanychus pusillus Ehara et Gotoh, n. sp. (Japanese name: Hime-midori-hadani) (Figs. 1-12) Tomakomai population of Panonychus akitanus: Gotoh, 1986 [2], p. 125; 1986 [3], p. 137. Stipeless-egg form of Panonychus akitanus: Gotoh, 1986 [3], p. 149; 1986 [4], p. 153. Female Body blackish green. Idiosoma with dorsal setae slender, pubescent, longer than dis- tances between consecutive setae. Dorsal hyster- osomal striae with lobes rounded apically. Peri- tremes dilated distally. Genital flap with mostly transverse striae; area immediately anterior to flap with longitudinal striae. Palpus with terminal sensillum approximately twice as long as wide; dorsal sensillum much smaller, slender. Measure- ments in yum: body length (including rostrum) 416, body width 250; lengths of setae: P; 64.7+0.7, P> LOSe/ete HOSEA 67202055 Ei 7 OiGsil ONE OOS VO. (Cy ler Ase Ca eyed) ae ADS Mer OW seOlr3).. Jks S828 ade 2, 13 87 02201951 73222. 0:9 OOF = 078) CL 45.6+0.6, PA 38.9+0.8; distances: P,;—P, 70.2 +0.8, P,—P, 77.2+0.6, Cy-C, 71.34+1.2, C,-C, 71.8+0.8, C3-C3 53.5+0.9. Length ratio P3/body =().161+0.004, H/body=0.170+0.005, L,/:body =0.214+0.005, L;/body=0.210+0.004. The number of setae and solenidia (in parentheses) on Fics. 9-12. Sasanychus pusillus n. sp. 9: Tarsus and tibia I of female. 10: Tarsus and tibia II of female. 11: Tarsus and tibia I of male. 12: Tarsus and tibia II of male. 378 S. EHARA AND T. GOTOH leg segments: femora 10—7—4-3, genua 5-5—4-4, tibiae 9(1)—8-6-7, tarsi 14(1)+2 dupl. —13(1)+1 dupl. —10(1)—10(1). Tarsus I with 5 tactile setae and 1| solenidion proximal to duplex setae; tarsus II with 3 tactiles and 1 solenidion proximad of duplex setae. Male Aedeagus bent dorsad; the shaft with a prominent process near basilar lobe, the distal upward part nearly straight, terminating in a round tip. Palpus with terminal sensillum slender; dorsal sensillum shorter, very slender. Empodium I with the middle pair of hairs strong, the ventral pair weak, and the dorsal pair similar to the middle or ventral one. Measurements in um: body length (including rostrum) 318, body width 158; lengths Oi Meas Ie) D3). ae I, 125 isos 4h ley Sts \Wse ili. leloyfef/ ae 1, (C7 Coie) aellella (O5(C0. /ac Wes), On OF. Oar AQ, Wen COLDS 9), Ie Citra LW, Ws SSE Ih.3), 50.3+1.0, O 41.2+0.6, CL 24.0+0.7, PA 22.1+ 0.9. The number of setae and solenidia (in parentheses) on legs: femora 10—7—4-3, genua 5- 5-4-4, tibiae 9(3)-—8-6-7, tarsi 12(3)+ 2 dupl. — 11(1)+ 1 dupl. —10(1)—10(1). Tarsus I with 3 tactile setae and 3 solenidia proximal to duplex setae; tarsus II with 1 tactile seta and 1 solenidion well proximal to duplex setae. Type-series Holotype: $, Takaoka, Tomako- mai, Hokkaido, 7—VIII-1982 (T. Gotoh), on Sasa apoiensis Nakai. Paratypes: 6 ¢¢ and 19 ?, with the above data; 6 ¢¢ and 30 ?%, 9-IX-1983 (S. Ehara and T. Gotoh), bred in Inst. of Appl. Zool., Hokkaido Univ. [host in laboratory, S$. apoiensis and occasionally §$. senanensis ( Franchet et Savatier ) Rehder], originally collected at Takaoka, Tomakomai, 13—VII-1983 (T. Gotoh), on S. apoiensis. The type-series is deposited in the Biological Institute, Faculty of Education, Tottori Uni- versity. Distribution Hokkaido. Remarks The female of Sasanychus pusillus closely resembles that of S. akitanus but differs from the latter in having the dorsal idiosomal setae noticeably shorter (t-test, P<0.001 for each seta). The former is smaller in body size than the latter, but the length ratios of setae P3, H, L, and L; to body are distinctly smaller in pusillus (t-test, P< 0.001). | Further, the male of pusillus is characterized by having tarsus II with one tactile seta and one solenidion proximal to duplex setae; in the male of akitanus tarsus II has three tactiles and one solenidion on the area. In addition, the male tarsus I of pusillus has three tactiles and three solenidia proximal to the duplex setae, instead of four tactiles and three solenidia. The aedeagus of pusillus is distinctive in that the distal upward part is not undulate and ends in a round tip, and the dorsal margin of the shaft bears a prominent process, whereas in akitanus the distal part is slightly sigmoid and has a truncate tip, and the shaft is smooth dorsally. S. pusillus overwinters as eggs on Sasa apoiensis Nakai in Tomakomai, while akitanus overwinters both as eggs and female adults on Sasa senanensis (Franchet et Savatier) Rehder in Sapporo [2, 3]. The eggs of pusillus are deficient in a dorsal stipe, but those of akitanus have a distinct dorsal stipe [3]. Moreover, there is the obvious post-mating reproductive isolation between the two species [4]. ACKNOWLEDGMENTS We wish to express our gratitude to the following gentlemen for their kind and much help: Dr. H. Mori and Dr. Y. Sait6 (Institute of Applied Zoology, Hok- kaido University), Dr. K. Ishigaki (Tomakomai Ex- perimental Forest of Hokkaido Univ.), Dr. K. Takahashi (Hokkaido Institute of Public Health), Dr. Y. Ohta (School of General Education, Tottori Univ.), and Dr. S. Okuyama (Laboratory of Plant Pathology, Ibaraki Univ.). This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 61560054). REFERENCES 1 Ehara, S. (1978) A new genus and a new subgenus of spider mites from northern Japan (Acarina: Tetrany- chidae). J. Fac. Educ. Tottori Univ., Nat. Sci., 28: 87-93. 2 Gotoh, T. (1986) Life-cycle variation in Panonychus akitanus Ehara (Acarina: Tetranychidae). Exp. Appl. Acarol., 2: 125-136. 3. Gotoh, T. (1986) Local variation in overwintering stages of Panonychus akitanus Ehara (Acarina: Tet- ranychidae). Exp. Appl. Acarol., 2: 137-151. 4 Gotoh, T. (1986) Reproductive isolation between the two forms of Panonychus akitanus Ehara (Acarina: Tetranychidae). Exp. Appl. Acarol., 2:153-160. ZOOLOGICAL SCIENCE 4: 379-380 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Lamprey Connectin YOSHIHARU IToH, D1 HUA Hu, KAzuyo OHASHI, SUMIKO KIMURA and KoscAK MARUYAMA Department of Biology, Faculty of Science, Chiba University, Chiba 260, Japan ABSTRACT—In lamprey skeletal muscle, only a- connectin, mother molecule of connectin, but not £- connectin was detected in an SDS gel electrophoresis. Lamprey connectin was identified by an immunoblot technique using antisera against chicken breast muscle connectin. Immunofluorescent observations showed that connectin is located at A-I junction regions of a sarcomere as in chicken or rabbit skeletal muscle. Connectin is an elastic filamentous protein of a huge molecular weight (3000 kDa) in striated muscle, linking myosin filaments to Z discs in a sarcomere (for a review, see [1]). In a comparative survey, it has been shown that connectin is widely distributed among vertebrate striated muscles from fishes to mammals [2, 3]. However, cyclo- stomes were not investigated. In the present study, connectin was also de- tected in lamprey skeletal muscle by SDS gel electrophoresis and by immunoblot and im- munofluorescence techniques using anti-chicken connectin. Interestingly, only a-connectin, mother molecule of connectin, was present in freshly sacrified lamprey muscles. In all the muscles of other vertebrates so far examined, #-connectin coexisted together with a-connectin [2]. MATERIALS AND METHODS Live lamprey was obtained in a local market. Skeletal muscles were cut out, immediately ground in an SDS solution and electrophoresed using Accepted November 26, 1986 Received November 13, 1986 1.8% polyacrylamide as described before [2]. Immunoblot and immunofluorescence procedures were carried out as reported previously, using antisera against chicken breast muscle connectin [4]. RESULTS AND DISCUSSION An SDS gel electrophoresis pattern clearly showed that there is only one main band corre- sponding to connectin in lamprey breast muscle A B a-con == — B-con = N =e on Fic.1. SDS gel electrophoresis pattern of lamprey skeletal muscle proteins. A total SDS extract was elctrophoresed as described by Hu et al. [2], using 1.8% polyacrylamide gels. A, chicken breast mus- cle; B, lamprey skeletal muscle. a-, B-con, connec- tin; N, nebulin; M, myosin heavy chain. 380 Y. Irou, D.H. Hu et al. (Fig. 1B). Note that there are distinct doublet bands (@-, and #’-connectins) in chicken breast muscle in addition to a-connectin (Fig. 1A). The electrophoretic mobility of lamprey connectin was almost the same as that of chicken breast muscle a-connectin, suggesting the molecular weight of approximately 3 million [5]. It is to be added that nebulin was recognized in lamprey skeletal muscle as well as in chicken (Fig. 1). To identify the lamprey connectin, an immuno- blot technique was adopted. The electrophoresed bands were transferred onto a nitrocellulose sheet followed by the treatment with antisera against chicken breast muscle connectin. The connectin band was detected by the binding of fluorescent antisera against chicken immunoglobulin. As shown in Figure 2, only the slowest band corre- sponding to a-connectin reacted with anti- connectin. It should be emphasized that anti- chicken connectin crossreacts with lamprey con- nectin. Therefore, there is not any specificity in anti-connectin so far as vertebrate muscles are concerned. Previously, it was reported that anti- chicken connectin reacts with frog connectin [6]. It has been repeatedly described that polyclonal antibodies against connectin strongly bind the A-I junction region of a sarcomere [1, 4]. An indirect immunofluorescence microscopy confirmed this Fic. 2. Immunoblot pattern of lamprey skeletal muscle connectin. A total SDS extract was used. Left, Coomasee brilliant blue stained pattern; Right, stained with an antiserum against chicken breast muscle connectin [6]. ; localization of connectin in cyclostomes striated muscle (Fig. 3). As seen in Figure 3, both edges of an A band were fluorescent. Central region, 1 um long, of an A band (1.5 um ), and also Z disc area were not stained with anti-connectin. These staining patterns were due to specific locations of antigenic sites on connectin filaments in register in a relaxed sarcomere (cf. [1]). The present work demonstrated that there is a-connectin alone in lamprey (cyclostomes) skele- tal muscle. This is very unique, because [- connectin, proteolytic product of @-connectin, is always present togther with a-connectin in other vertebrate striated muscles including a variety of fishes [7]. Immunofluorescent pattern of lamprey skeletal myofibrils treated with antisera against chicken breast muscle connectin. A, phase contrast micro- scopic image; B, fluorescent microscopic image. Bar, 10 um. Fic. 3. ACKNOWLEDGMENT This work was supported by a grant from the Ministry of Education, Science and Culture to “Molecular Biolo- gical Studies on Marine Animal Muscles” at Chiba University. REFERENCES 1 Maruyama, K. (1986) Int. Rev. Cytol., 104: 81-114. 2 Hu, D.H., Kimura, S. and Maruyama, K. (1986) J. Biochem., 99: 1485-1492. 3 Locker,R.H. and Wild, D. J.C. Biochem., 99: 1473-1484. 4 Maruyama, K., Sawada, H., Kimura, S., Ohashi, K., Higuchi, H. and Umazume, Y. (1984) J. Cell Biol., 99: 1391-1397. 5 Maruyama, K., Kimura, S., Yoshidomi, H., Sawada, H. and Kikuchi, M. (1984) J. Biochem., 95: 1423- 1433. 6 Maruyama, K., Yoshioka, T., Higuchi, H., Ohashi, K., Kimura, S. and Natori, R. (1985) J. Cell Biol., 101: 2167-2172. 7 Seki, N. and Watanabe, T. (1984) J. Biochem., 95: 1161-1167. (1986) J. ZOOLOGICAL SCIENCE 4: 381-383 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Disappearance of Immunoglobulin G in Endodermal Cells in Non-Immunized Murine Yolk Sac Placenta towards Parturition KIYOSHI SHIMIZU and MAsAmI HOKANO Department of Anatomy, Tokyo Medical College, Shinjuku-ku, Tokyo 160, Japan ABSTRACT—Immunoglobulin G in the endodermal cell of yolk sac placenta of non-immunized mouse exisited on day 16 of pregnancy but most of it dis- appeared on day 18 of pregnancy. Disappearance of immunoglobulin G in mouse was similar to that in rat. INTRODUCTION Immunoglobulin G (IgG) is the only immuno- globulin that is incorporated into the circulating system of fetus. The yolk sac placenta is the transporting site of passive immunity from mother to fetus in mouse and rat [1]. In mouse, IgG production is stimulated by substance or factor from fetus [2] and increases towards parturition [3]. A transmission of IgG from mother to fetus increases towards parturition [4]. Antibodies produced after immunization with horseradish peroxidase in female rat before mating are uptaken by visceral endodermal cells of yolk sac placenta and stored in it’s apical vacuoles during mid pregnant period [5-7]. Most of these antibodies in the vacuoles, however, is degraded by lysosomal enzymes towards parturition and thus disappears [7]. Since similar disappearance of IgG in the murine endodermal cells towards parturition has been unknown, we observed IgG in the endodermal cells of non-immunized mouse with immunohistochemical method. Accepted December 2, 1986 Received October 23, 1986 MATERIALS AND METHODS Animals used were female mice of the IVCS strain. They were reared under 12L:12D and given food and water ad libitum. At 8 weeks of age they were mated. They were examined every morning for the presence of a copulatory plug and day when the pulg was found was desingned as day 0 of pregnancy. In this strain the pregnant period was 19 days. The uterine horn was removed, fixed in 10% buffered formalin, embedded in paraffin and cut in sections 3 um thick. The anti-mouse IgG (heavy and light chains), anti-sheep y-globulin, sheep-peroxidase anti- peroxidase (PAP) and sheep whole serum was obtained from Cappel laboratory. Abbreviations of the antibodies are as follows; anti-mouse IgG, sheep antiserum to mouse IgG; anti-sheep /y- globulin, rabbit antiserum to sheep y-globulin; PAP, sheep anti-peroxidase IgG conbined with horseradish peroxidase. Endogenous peroxidase in the sections was destroyed by exposure to absolute alcohol with contained H,O, (10%, V/V). The sections were exposed to sheep whole serum (1:200) or anti- mouse IgG (1:200) for 30 min at room tempera- ture. Then they were washed three times in phosphate-buffered ‘saline (PBS, pH/7.2), incu- bated in anti-sheep 7-globulin (1:10) for 30 min at room temperature. After three-fold washing in PBS, the sections were reincubated in PAP (1:50) for 30 min at room temperature. The sites of antibody were determined histo- chemically, using 3, 3’-diaminobentizine solution 382 K. SHIMIZU AND M. HoKANo [8] for 5 min and counterstained with hematoxylin. When exposed to sheep whole serum (control RESULTS AND DISCUSSION staining), the specific stain was not found in the On day 16 of pregnancy, IgG was found in the sections. endodermal cells, being heaviest in the apical vacuole (Fig. la). IgG positive cells were located & Fic. 1. IgG in the endodermal cells on day 16 (a), 17 (b), or 18 (c) of pregnancy (PAP method). (a) IgG positive endodermal cells (arrows) scattered in the visceral endoderm. IgG exsisted in apical large vacuoles (530). (b) Most of IgG in apical vacuoles of the endodermal cells disappeared. A little IgG remained in a rim of apical vacuoles (arrows). There were some IgG positive cells (double arrows) in the bottom of villi. (x 530). (c) There were no IgG positive cells in the bottom of villi. Disappearance of IgG in apical vacuoles of the endodermal cells became clearer from that on day 17 of pregnancy. (530). mes; mesothelium. ul; uterine lumen. IgG in Murine Yolk Sac Placenta 383 in scattered groups of the visceral endoderm. On day 17 of pregnancy, villi became tall and the endodermal cell hypertrophied. Most of IgG in the apical vacuoles disappeared and a little IgG remained in a rim of vacuoles (Fig. 1b). In bottom of villi, there were some IgG positive cells. The endodermal cells hypertrophied more on day 18 than on day 17 of pregnancy. IgG in the apical vacuoles disappeared more on day 18 than day 17 of pregnancy (Fig. 1c). There was no IgG positive cell on day 18 of pregnancy. These observations indicated that IgG in the endodermal cells of murine yolk sac placenta disappeared towards parturition and that this disappearance of IgG was similar to that in rat [7]. Both biochemical [9] and histochemical [10] methods show that activity of lysosomal enzymes in the endodermal cells becomes high towards parturition. Since large quantities of materials uptaken by the endodermal cells are degraded by lysosomal enzymes for a source of amino acid for protein synthesis by fetus [11, 12], IgG in the endodermal cells may be degraded as materials for protein synthesis by fetus. The transporting route that conveys endocytosed materials from apical to deep portion within the endodermal cells estab- lishes towards parturition [13, 14], suggesting that IgG is transported by this route and thus IgG is not degraded by lysosomal enzymes towards parturi- tion when a transmission of IgG becomes high [4]. REFERENCES 1 Brambell, F. W. R. (1970) Front. Biol., 18: 80-101. 2 Carter, J. and Dresser, D. W. (1985) J. Reprod. Fertil., 74: 535-542. 3 Carter. J. and Dresser, D. W. (1983) Immunology, 49: 481-490. 4 Appleby, P. and Catty, D. (1983) J. Reprod. Im- munol., 5: 203-213. 5 LaLiberté, F., Mucchielli, A., Ayraud,W. and Masseyeff, R. (1981) Am. J. Reprod. Immunol., 1: 345-351. 6 Mucchielli, A., LaLiberté, F. and LaLiberté, M.-F. (1983) Placenta, 4: 175-184. 7 Jollie, W. P. (1985) J. Reprod. Immunol., 7: 261- 274. 8 Graham,R.C. and Karnovsky, M.J. (1966) J. Histochem. Cytochem., 14: 291-302. 9 Beck, F. F., Lloyd, J. B. and Griffiths, A. (1967) J. Anat., 101: 461-478. 10 Padykula, H. A. (1958) J. Anat., 92: 118-129. 11 Freeman, S. J., Beck, F. and Lloyd, J. B. (1983) J. Embryol. Exp. Morphol., 73: 307-315. 12 Freeman, S. J. and Lloyd, J. B. (1983) J. Embryol. Exp. Morphol., 73: 307-315. 13. Lambson, R. O (1966) Am. J. Anat., 118: 21-52. 14 Seibel, W. (1974) Am. J. Anat., 140: 213-236. ve avid G0 Heriot mirag Sh aires GaerNS: hepa neat: irene #9) (miebobs> haraneiy art toe Fe gpeinenenee © ait 0 nie sty bn Hie Sinbad hy 8 caf he ate to.giin | feoieptaamagont ie bso a a Lig tha tile bo veetpgd eb aabou . aA Wi SAFRTS Va sii ay) eae ' pee ‘e Tat-0R Qt ihe Jeort 0b) Bee Newtons ; ot bi baer hed t wep yf (ft ; } ae Me ae net te “gehetie x FOES ier a ts 4% T Josgnel te Ld a2 . Pins ince ae ’ moe i Meet) fe iactenie ato eT ae fe wh way or ogee ‘ at so (ta. als ee RGR IETY: Sree wit ¥ ; pli sirinuli G2 for 10-15 min at room temperature. To examine the specificity of the immunoreaction by AII antiserum, immunostaining with AII anti- serum preabsorbed by Asn'-Val°-AII (20 ng/ml antiserum; Hypertensin Ciba) was performed. RESULTS There are 4 to 6 CS in the rainbow trout. CS belong to Type IV as defined by Krishnamurthy and Bern [16] and are comprised of many small lobes. Each lobe consists of a number of incom- plete lobules (Fig.1). Cells showing AII- immunoreactivity were observed singly (Fig. 1) or in clusters (Fig. 2) among the cells of the lobules (Fig. 2) . Some AII-immunoreactive cells were observed to have cytoplasmic projections (Fig. 3). The intensity of All-immunoreactivity and num- ber of AlIlI-immunoreactive cells varied with the individual. The AII-immunoreactivity observed in the rainbow trout was abolished when AII anti- serum was preincubated with AII. Thus, the immunoreaction is thought to be specific to AII. In some trout specimens, no AIl-immunoreactive Fic. 1. Corpuscles of Stannius of the rainbow trout, Salmo gairdneri, containing an AII-immunoreactive cell (IRAII). L, lobule. CT, connective tissue between lobes. Scale, 20 um. Fic. 2. AII-immunoreactive cells (IRAII) in loose clus- ters in the CS of the rainbow trout. Scale, 20 um. Fic. 3. Some AlIJ-immunoreactive cells (IRAII) with cytoplasmic projections (arrow heads). Scale, 20 um. cells “were detected ink GS: No AITI- immunoreactivity was detected in the CS of the All in the Corpuscles of Stannius 389 four other species. DISCUSSION In the present study, immunoreactive AII was first demonstrated in the CS of the rainbow trout, Salmo gairdneri. However, in the biwa trout, the Japanese char, the carp and the goldfish, no All was detected immunohistochemically. It is not clearly understood why the CS of these fishes fail to show AlIl-immunoreactivity, but there is the possibility that the amount of AII-like substance in CS cells is too small to be detected by the present immunohistochemical techniques in these four species. Also, the antiserum used in the present study may possibly have been unable to cross-react with AIl in the CS of the four species of fish. By using different fixatives, sectioning techniques and antisera or radioimmunoassay, All-like substance may be detected. It is important to clarify whether All is present in the cells of CS in fish in general. Since AlIJ-immunoreactive cells in CS of the rainbow trout have cytoplasmic projections, they apparently belong to the type 2 cells with projec- tions described by Wendelaar Bonga et al. [17] in the threespined stickleback, Gasterosteus aculeatus and also correspond to AF-negative cells in the rainbow trout [16]. However, Aida er al. [18, 19] consider type 2 cells likely to be different physiolo- gical stages of type 1 cells in the Coho salmon, Oncorhynchus kisutch. It is thus difficult to confirm whether AIl-immunoreactive cells belong to type 1 or type 2 at the present time. Cells containing All-like substance observed in the rainbow trout were not many in number and most of the cells in CS were not immunoreactive to the AIl antiserum used. Thus, these immunoreactive cells with AII antiserum may possibly be a cell type distinct from type 1 or 2. A renin-like enzyme in CS has been suggested on the basis of histological [see 1] and pharmacolo- gical findings [6-8]. Recently, angiotensins were chemically found to be produced by incubating extracts of CS with plasma in the chum salmon [9] and the Japanese goosefish [10]. In the present study, ATI itself was immunohistochemically found in the cells of CS in the rainbow trout. The presence of AII-like substance in the CS of species other than the rainbow trout is now being ex- amined using the same and different techniques. The physiological significance of immunoreactive AII in CS is not clear at the present time. However, the AlIlI-like substance produced in CS likely functions locally in CS rather than systemi- cally, since its amount in CS may be very small, based on a report that in the carp, the crucian carp and the Japanese goosefish, amount of renin in CS is far less than that in the kidney [7]. AII in CS cells may regulate intracellularly the secretion of CS hormones. The CS of the eel were recently found to synthesize and secrete a mammalian parathyroid- like hormone called parathyrin of CS (PCS), by immunofluorescence techniques, to be located in the cytoplasm of all cells in CS [20]. Whether the CS of the rainbow trout contain PCS or not is not known at the present. If they do, it would be of interest to examine the distribution of AlIlI-like substance and PCS in CS. ACKNOWLEDGMENTS The authors are grateful to Dr. Ken-ichi Yamaguchi, Department of Physiology, Niigata University School of Medicine, for a supply of AII antiserum. This study was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 Oguri, M. and Sokabe, H. (1974) Bull. Jpn. Soc. Sci. Fish., 40: 545-549. 2 Oguri, M. (1966) Bull. Jpn. Soc. Sci. Fish. , 32: 903- 908. 3 Fujita, H. and Honma, Y. (1967) Z. Zellforsch., 77: 175-187. 4 Ogawa, M. (1967) Z. Zellforsch., 81: 174-189. Cohen, R.S., Pang,P.K.T. and Clark,N. B. (1975) Gen. Comp. Endocrinol., 27: 413-423. 6 Chester Jones, I., Henderson, I. W., Chan, D. K. O., Rankin, J. C. and Mosley, W. (1966) J. Endo- crinol., 34: 393-408. 7 Sokabe, H., Nishimura, H., Ogawa, M. and Oguri, M. (1970) Gen. Comp. Endocrinol., 14: 510-516. 8 Ogawa,M. and Sokabe,H. (1982) Gen. Comp. Endocrinol., 47: 36-41. 9 Takemoto, Y., Nakajima, T., Hasegawa, Y., Wata- nabe, T. X., Sokabe, H., Kumagae, S. and Sakaki- bara, S. (1983) Gen. Comp. Endocrinol., 51: 219- POA E Nn 10 11 2 13 14 iS 390 Hasegawa, Y., Watanabe, T. X., Nakajima, T. and Sokabe, H. (1984) Gen. Comp. Endocrinol., 54: 264-269. McKenzie, J. C., Naruse, K. and Inagami, T. (1985) Anat. Rec., 212: 161-166. Yamaguchi, K. (1981) Acta Endocrinol., 97: 137- 144. Hayashi, T., Nakayama, T., Nakajima,T. and Sokabe, H. (1978) Chem. Pharm. Bull., 26: 215- 219: Hasegawa, Y., Nakajima, T. and Sokabe, H. (1983) Biomed. Res., 4: 417-420. Sternberger, L. A., Hardy, P. H., Cuculis, J. J., Jr. and Meyer, H. G. (1970) J. Histochem. Cytochem., 16 17 18 19 20 C. YAMADA AND H. KOBAYASHI 18: 315-333. Krishnamurthy, V. G. and Bern, H. A. (1969) Gen. Comp. Endocrinol., 13: 313-335. Wendelaar Bonga, S.E., Greven,J. A.A. and Veenhuis, M. (1976) Cell Tissue. Res., 175: 297- 32, Aida, K., Nishioka, R.S. and Bern, H. A. (1980) Gen. Comp. Endocrinol., 41: 296-304. Aida, K., Nishioka, R.S. and Bern, H. A. (1980) Gen. Comp. Endocrinol., 41: 305-313. Lopez, E., Tisserand-Jochem, E-M., Eyquem, A., Milet, C., Hillyard, C., Lallier, F., Vidal, B. and MacIntyre, I. (1984) Gen. Comp. Endocrinol., 53: 28-36. ZOOLOGICAL SCIENCE 4: 391-394 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Phosphocalcic Response of Stannius Corpuscles Extract in the Freshwater Snake, Natrix piscator N. Hasan, S. Das, Asal K. Srivastav and KrisHNA Swarup! Department of Zoology, University of Gorakhpur, Gorakhpur-273009, India ABSTRACT—The effect of i.p. injection of aqueous extract of Stannius corpuscles (2.5, 5 and 10 mg/100 g b. wt.) on serum calcium and inorganic phosphorus levels was investigated in the freshwater snake, Natrix piscator over a period of 8 hr. This treatment evokes hypocalce- mia and hypophosphatemia with a return to control. INTRODUCTION The corpuscles of Stannius(CS) are putative endocrine glands located on the kidneys of holos- tean and teleostean fishes. Recently, they have aroused considerable interest for their role in calcium homeostasis. Stanniectomy in teleosts brings forth hypercalcemia [1-3]. The hypocalce- moc factor (s) released from CS are hypocalcin [4] and teleocalcin [5]. A PTH-like substance (para- thyrin) has also been localized immunocytochemi- cally in the eel CS [6]. Recently, it has been reported that CS extract induces hypocalcemia in non-piscine vertebrates i.e. mammal [7], bird [8] and amphibia [9, 10]. So far, there exists no information regarding the effect of CS extract on the serum calcium and inorganic phosphorus levels of reptiles. This tempted us to undertake such a study in the freshwater snake, Natrix piscator. MATERIALS AND METHODS The CS used in this study were surgically Accepted October 6, 1986 Received June 30, 1986 ‘ To whom reprints should be requested. removed from both sexes of the adult freshwater catfish, Heteropneustes fossilis. These glands were stored in ice and used almost immediately. The glands were weighed wet and homogenized in ice-cold saline (0.9% sodium chloride solution). The homogenate was centrifuged (5000 rpm for 10 min) and the supernatant was collected. Forty eight individuals of the freshwater snake (Natrix piscator, b. wt. 150-200 g ) were main- tained under laboratory conditions for 1 week prior to use. They were then divided into 4 numerically equal groups — Group A, injected with saline only (0.2 ml/100 gb. wt.); Group B, in- jected with extract of 2.5mg CS/100g b. wt.; Group C, injected with extract of 5 mg CS/100 g b. wt.; Group D, injected with extract of 10 mg CS/100 g b. wt. All injections were giveni.p. In all the groups the volume of the injection was the same (0.2 ml/100g b. wt.). The blood samples were always collected after slight ether anesthesia. An initial sample of blood (zero hour) was taken from group A. Multiple sampling of blood from groups A-D were taken serially after 0.5, 1,2, 4, 6 and 8hr following the injection. There are 12 individuals in each group. From 6 individuals (at each interval 6 determinations from separate individual) blood for the analysis of serum calcium level was taken, similarly from the other 6 indi- viduals (at each interval 6 determinations from separate individual) blood for the analysis of serum inorganic phosphorus level was taken. The serum calcium and inorganic phosphorus levels were measured according to Trinder’s [11] and Fiske and Subbarow’s [12] methods, respectively. 392 N. HASAN Differences between the values of serum cal- cium and inorganic phosphorus levels among the snakes injected either with saline (group A) or CS extract (groups B, C and D) were evaluated using Student’s t-test. RESULTS The initial values (zero hour) of serum calcium ©—— © Saline-injected , S. DAs et al. and inorganic phosphorus are 11.7+0.10 and 6.9 +0.40 mg/100 ml, respectively. The serum calcium levels of saline injected specimens (group A) at different time intervals have been shown in Figure 1. The serum calcium level of the specimens of group B (extract of 2.5mg CS/100g b. wt.) exhibits no change at 0.5 hr. At 1 hr a perceivable hypocalcemic effect is noticed which continues up @----O 25mg/100gb wt. e—e 50 mg/100g5b wt. @----@ 10:0mg/ 100g b wt. CS extract-injected 13 = (@) S) ~ io) / ‘E / = if / & 9 / 5 / rs) if _ / o / O / / S / 3 *)/ IL, o 7 op) > me Pee \ eee \ 7 \ / of Ne N * 5 0 Z 4 6 Bh TIME AFTER TREATMENT Fic. 1. mean+SD of 6 determinations. respectively. Changes in the serum calcium level after administration of saline and CS extract. Each point indicates * and ** represent significant responses, p<0.001 and p<0.01, Effects of CS Extract in Snake 393 to 2hr. There after, the values show a tendency towards normocalcemia (Fig. 1). In group C (extract of 5mg CS/100g b. wt.) hypocalcemia is perceived ealier i.e. at 0.5 hr which progressively increases till 4 hr following the treatment (Fig. 1). Thereafter, the serum calcium level becomes normal at 6 and 8 hr. In group D (extract of 10 mg CS/100g b. wt.), the hypocalcemia is perceived after 0.5 hr. This response gets exaggerated up to 2hr. This is followed by a rise in the serum calcium level ultimately approaching normocalcemia between 6 and 8 hr (Fig. 1). The serum inorganic phpsphorus levels of the specimens of group A (saline injected) at different time intervals have been shown in Figure 2. In groupB hypophosphatemic effect of CS extract has been noticed at 0.5 hr which gradually increases till 1 hr following the administration. From 2 hr onwards the hypophosphatemic effect decreases till the close of the experiment ( Fig. 2). In group C the hypophosphatemic effect in- creases progressively from 0.5 hr up to 4 hr follow- ing the treatment (Fig. 2). Thereafter, the values tend to become normal. However, the hypophos- phatemic effect of CS extract is significant till the close of the experiment. In group D, the hypophosphatemic effect has been noticed at 0.5 hr. This response persists throughout the experiment (Fig. 2). CS extract-injected O©——©O Saline-injected O-—- 2025 mg /1DO'g b-wt. @——®@ 50mg/100g bwt. @----@ 10-0mg/ 100g bwt. 9 £ | 2 i Mm E = 5 T° rae we he oir o oe £ 1 \ ae re < 2 5 \ : ue oh w ae = \' Pa * E oe 3 F—-85 4 6 TIME AFTER TREATMENT 8h Fic. 2. Changes in the serum inorganic phosphorus level after administration of saline and CS extract. Each point indicates mean+SD of 6 determinations. *, ** and *** represent significant responses p<0.001, p< 0.01 and p<0.02, respectively. 394 DISCUSSION The present study shows that CS extract induces hypocalcemia in freshwater snake, Natrix piscator. The hypocalcemic effect in N. piscator derives support from the observations on non-piscine vertebrates - mammal [7], bird [8] and amphibia [9, 10]. However, reports of the effect of CS extract administration on serum calcium of mam- mals are contradictory. Pang (cited by Leung and Fenwick[7]) did not find any hypocalcemic action in rats in the CS from cod or killifish and Copp (cited by Leung and Fenwick[7]) obtained similar results when he tested salmon CS in rats. More- over, CS extract of bullhead failed to induce hypocalcemia in rats although eel CS extract is effective on rats [7]. Ma and Copp [13] have reported the ineffectiveness of teleocalcin on rats. On the contrary, Milet et al. [14] have reported hypercalcemia after CS extract administration. There seems to be no report regarding the effect of CS extract administration on the serum inorga- nic phosphorus from non-piscine vertebrates ex- cept that of Milet et al. [10] which reports hypophosphatemia in Xenopus laevis. The present study describes hypocalcemic and hypophosphatemic effects of CS extract in N. piscator which is the first report from reptiles. ACKNOWLEDGMENTS One of us (KS) is thankful to UGC, New Delhi for providing financial assistance for this study. 14 N. Hasan, S. Das et al. REFERENCES Fontaine, M. (1964) C.R. Acad. Sci., 259: 875-878. Chan, D. K. O. (1972) Gen. Comp. Endocrinol., Suppl., 3: 411-420. Kenyon, C. J., Chester Jones, I. and Dixon, R.N. B. (1980) Gen. Comp. Endocrinol., 41: 531-538. Pang, P.K.T, Pang,R.K. and Sawyer, W. H. (1974) Endocrinology, 94: 548-555. Ma, S. W. Y. and Copp, D. H. (1978) In“Compara- tive Endocrinology”. Ed. by P. J. Gaillard and H. H. Boer, Elsevier/ North-Holand Biomedical Press, Amsterdam, pp. 283-286. Lopez, E., Tisserand-Jochem, E. M., Eyquem, A., Milet, C., Hillyard, C., Lallier, F., Vidal, B. and MacIntyer, I. (1984) Gen. Comp. Endocrinol., 53: 28-36. Leung, E. and Fenwick, J.C. (1978) Can. J. Zool., 56: 2333-2335. Srivastav, Ajai K. and Swarup, K. (1982) Experien- tia, 38: 869-870. Pandey, A. K., Krishna, L., Srivastav, Ajai K. and Swarup, K. (1982) Experientia, 38: 1314-1315. Milet, C., Buscaglia, M., Chartier, M. M., Martel- ly, E. and Lopez, E. (1984) Gen. Comp. Endocri- nol., 53: 497. Trinder, P. (1960) Analyst, 85: 889-894. Fiske, C. H. and Subbarow, Y. (1925) J. Biol. Chem., 66: 375-400. Ma, S. W. Y. and Copp, D. H. (1982) In “Compa- rative Endocrinology of Calcium Regulation”. Ed. by C. Oguro and P.K.T. Pang, Japan Scientific Societies Press, Tokyo, pp. 173-179. Milet, C., Lopez, E., Chartier, M. M., Martelly, E., Lallier, F. and Vidal, B. (1979) In “Molecular Endocrinology”. Ed. by I. MacIntyre and M. Szelke, Elsevier/North Holland Biomedical Press, Amsterdam, pp. 341-348. ANNOUNCEMENT Optical Approaches to the Dynamics of Cellular Motility Marine Biological Laboratory Centenary Symposium A Symposium in Honor of Robert D. Allen October 5-8, 1987 at Marine Biological Laboratory, Woods Hole, MA Monday, October 5: (8:00 P.M.-) Wine and cheese party and demonstrations of optical equipment by manufacturers. Tuesday, October 6: Morning: Introductory remarks: N. Kamiya (Okazaki). Optical Techniques in Cell Biology: Chairmen: D. L. Taylor and S. Inoué G. Nomarsky (Antony, France): Contrast En- hancement and Imaging in Light Microscopy ; S. Inoué (MBL, Woods Hole): Video Microscopy; D. L. Taylor (Carnegie-Mellon, Pittsburgh) : Optical Studies of the Chemistry of Living Cells; D. A. Agard (UC Med, San Francisco): Optical Sectioning Microscopy ; T. Yanagida (University of Osaka): Direct Observation of Molecular Motility by Light Microscopy. Afternoon: Amoeboid Movement and Cytoplas- mic Streaming: Chairman: J. Condeelis N. Kamiya (Okazaki): Cytoplasmic Streaming ; J. Condeelis (Einstein, NYC): Amoeboid Chemotaxis; M. Schliwa (UC, Berkeley): Regulation of Directionality of Cell Locomotion; J. Hartwig (Mass. General, Harvard) : Pseudopod Extension in Macrophages; G. Oster (UC, Berkeley): Biophysics of the Leading Lamella. Evening: Talks and demonstrations by equipment manufacturers (Swope Center). R. Wicks (PMI Corp.) Ultralow Light Imaging Optical Microscopy; Zeiss Representative: Conformal Laser Scanning Microscopy (remainder of prog- ram to be arranged). Wednesday, October 7: Morning: Mitosis: Chairman: L. I. Rebhun Y. Hiramoto (Tokyo): Micromanipulation Stu- dies of Dividing Cells; E. W. Salmon (UNC, Chapel Hill): Microtubule Dynamics and Chromosome Movement; A. Bajer (UO, Eugene): Comparison of Turnover of Plant and Animal Tubulin in Higher Plant Cells; L.I. Rebhun and_ R. Palazzo (U. Va., Charlottesville): In Vitro Approaches to Spindle Function; W.C.Cande UC, Berkeley): In Vitro Studies of The Mechan- ism of Anaphase Spindle Elongation. Afternoon: Poster Session. Evening: Motility in Other Systems; Chairman: K. Edds N.S. Allen (Wake Forest, NC): Intracellular Particle Motions in Plant Cells; R. Hard (SUNY-Buffalo) : Control of Ciliary Metachrony in Newt Lungs; J. Hayden (Sien- na College, NY): Regulation of Organelle Translocation in Fibroblasts; J. Lafountain (SUNY, Buffalo): Chromosome Movements During Meiotic Prophase; C. Izzard (SUNY, Albany): Cell-Substrate Interactions and Cellular Movement in Fibroblasts. Thursday, October 8: Morning: Microtubule Dynamics: Chairman: R. Sloboda R. Sloboda and _ §.Gilbert (Dartmouth, Hanover): Vesicle-Microtubule Interactions During Axoplasmic Transport; J. Travis (Vassar, Poughkeepsie) and S. Bowser (N.Y. State Dept. Health, Albany): Microtubule- Associated Motility in Allogromia (Foraminifera); 7. 7. Chen (Wayne State, Detroit): Analysis of Particle Translocation in Goldfish Xanthophores; P. Satir (Enstein, INAG)E Dynein As a _ Microtubule Translocator; H. Hotani (Kyoto University) : Darkfield Studies of Microtubule Assembly and Disassembly. Afternoon: Axoplasmic Transport: Chairman: D. Weiss D. Weiss (Munich) and G. Langford (UNC, Chapel Hill): Microtubule-Based Particle Transport in Squid Axoplasm; Richard Smith (Edmonton, Canada): Motion Analysis of Bidirectional Organelle Movement; Anthony Breuer (Cleveland Clinic, Cleveland): In- traaxonal Organelle Traffic Analysis in Hu- man Nerve; Bruce Schnapp (MBL, Woods Hole): Mechanochemistry of Microtubule Translocators; Richard Weisenberg (Temple, Philadelphia) : Microtubule __Gelation- Contraction In vitro: A Model for Slow Axoplasmic Transport. Evening: Dinner and Concert. Registration and Housing: The registration fee is $75. Housing is limited and will be assigned in order of receipt of application. Deadline for housing and meal plan is July 1, 1987. Forms and information may be obtained by writing: Robert Day Allen Symposium P. O. Box 477 Woods Hole, MA 02543 USA Abstracts may be submitted for inclusion in the poster session and will be published in Cell Motility and Cytoskeleton. Information on prepa- ration of abstracts will be sent with application forms for registration. Final date for abstract submission will be June 15, 1987. Published by Gi V1 £ Defe ; ; Via fic ) " the Japanese Society of Developmental Biologists The journal is devoted to the publication of original papers dealing with any aspects of developmental phenomena in all kinds of organisms, including plants and micro-organisms. Papers in any of the following fields will be considered: developmental genetics, growth, morphogenesis, cellular kinetics, fertilization, cell division, dormancy, germination, metamorphosis, regeneration and pathogenesis, at the biochemical, biophysical and analytically morphological levels ; reports on techniques applicable to the above fields. At times reviews on subjects selected by the editors will be published. Brief complete papers will be accepted, but not preliminary reports. Members of the Society receive the Journal free of charge. Subscription by institutions is also welcome. Papers in DGD, Vol. 29, No. 2. (Apr. 1987) 1. REVIEW: H. Fusisawa: How do retinal axons arrive at their targets?: Cellular and molecular approaches. 2. P.ANDREUCCETTI and M. CARRERA: The differentiation of the zona pellucida (vitel- line envelope) in the lizard Tarentola mauritanica. 3. Y.HRAo and J. Hiraoka: Surface architecture of sperm tail entry into the hamster oocyte. 4. H.Suemort and N. Nakatsus!: Establishment of the embryo-derived stem (ES) cell lines from mouse blastocysts: Effects of the feeder cell layer. 5. H.TAKASAKI: Fates and roles of the presumptive organizer region in the 32-cell embryo in normal development of Xenopus laevis. 6. I. NisHryAMA, T. Matsui, Y. Fusimoto, N. IKEKAwWA and M. Hosur: Correlation be- tween the molecular structure and the biological activity of Co-ARIS, a cofactor for acrosome reaction-inducing substance. 7. S.A. Watrs and J.M. Lawrence: The effects of 17@-estradiol and estrone on intermediary metabolism of the pyloric caeca of the asteroid Luidia clathrata (Say) maintained under different nutritional regimes. 8. I. NisHtyvAMA, T. Matsui and M. Hosni: Purification of Co-ARIS, a cofactor for acrosome reaction-inducing substance, from the egg jelly of starfish. 9. M.I. BUHLER, T. PETRINO and A. H. LEGNAME: Sperm nuclear transformation and aster formation related to metabolic behaviour in amphibian eggs. Development, Growth and Differentiation (ISSN 0012-1592) is published bimonthly by The Japanese Society of Developmental Biologists, Department of Biology, School of Education, Waseda University, Tokyo 160, Japan. 1987: Volume 29. Annual subscription U. S. $ 110.00 including air speed delivery except Japan. Application to mail at second class postage rate is pending at Jamaica, NY 11431, U.S. A. 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Yi lw” _. a Y (Photo: by courtesy of Olympus Optical CO., LTD) SOME FEATURES of MO-102 and MO-103: * The manipulator head is so small that it can be mounted directly on the microscope stage. There is no need for a bulky stand. * Hydraulic remote control ensures totally vibration-free operation. * 3-D movements achieved with a single joystick. Micromanipulators Microelectrode pullers Stereotaxic instruments NARISHIGE SCIENTIFIC INSTRUMENT \====7 LABORATORY CO.,LTD. 4-9-28, Kasuya, Setagaya-ku, Tokyo 157 JAPAN Telephone: 03-308-8233 Telex: NARISHG J27781 (Contents continued from back cover) nius of the rainbow trout, Salmo gairdneri (COMMUNICATION) Hasan, N., S. Das, A. K. Srivastav and K. Swarup: Phosphocalcic response of Stan- nius corpuscles extract in the freshwater snake, Natrix piscator (COMMUNICATION) Taxonomy Matsuoka, N.: Biochemical study on the tax- onomic situation of the — sea-urchin, IPSCUAOGeMiTOlUS GEPTeSSUS — «0... eee ne 339 Konishi, K. and R. Quintana: The larval sta- ges of Pagurus brachiomastus (Thallwitz, 1892) (Crustacea: Anomura) reared in the laboratory Hayashi, K.-I. and Y.Ogawa: Spongicola levigata sp. nov., a new shrimp associated with a hexactinellid sponge from the East China Sea (Decapoda, Stenopodidae) __... 367 Ehara, S. and T. Gotoh: Notes on the genus Sasanychus Ehara, new status, with descrip- tion of a new species from Hokkaido (Acar- ina, Tetranychidae) ZOOLOGICAL SCIENCE VOLUME 4 NUMBER 2 APRIL 1987 CONTENTS REVIEW Nagahama, Y.: Gonadotropin action § on gametogenesis and steroidogenesis in teleost gonads ORIGINAL PAPERS Physiology Khan, M.M. and Y.K.Ip: Effect of host myo-inositol deficiency on Hymenolepis diminuta (Cestoda) ¥ Tsukamoto, Y.: Morphometrical features of rod outer segments in relation to visual acui- ty and sensitivity in the retina of Rana cates- beiana Kasukawa, H., N.Oshima and_ R. Fujii: Mechanism of light reflection in blue dam- Selfishmotilemndophone 5.442 -nee ee 243 Cell Biology Mesa, A. and B.Goni: Meiosis in the Japanese gryllacridid Anoplophilus acu- ticercus Karny, 1931 (Orthoptera Saltatoria, Rhaphidophoridae): Amphitelic orientation of the X and supernumerary chromosome(s) Itoh, T.J., H. Sato and A. Kobayashi: Ex- aminations of spindle structure modified by T-1, the mitotic arrester Itoh, Y., D. H. Hu, K. Ohashi, S. Kimura and K. Maruyama: Lamprey connectin (COMMU- INTC AMITION)) © ce 2 5 ee aa ee Glee eS) Shimizu, K. and M. Hokano: Disappearance of immunoglobulin G in endodermal cells in non-immunized murine yolk sac placenta to- wards parturition (COMMUNICATION) _ ..381 ' Genetics Ota, H., M. Matsui, T. Hikida and T. Hidaka: Karyotype of a gekkonid lizard, Cosymbotus platyurus, from Sabah, Borneo, Malaysia (COMMUNICATION) Biochemistry Okai, Y.: Novel cytotoxic factors from tumor virus-transformed human embryo fibroblasts Shimada, K., H. Koyama and M. Asashima: Two-dimensional polyacrylamide gel analy- sis of papilloma and normal skin proteins in NEW) ascii ess seas ck eens tee 285 Developmental Biology Nakajima, Y.: Localization of catechola- minergic nerves in larval echinoderms’... 293 Shimizu, T.: Jn vitro spermatids formation from diapausing pupal spermatogonia of the cabbage armyworm, Mamestra brassicae L. (Lepidoptera; Noctuidae) Hazarika, L. K. and A. P. Gupta: Variations in hemocyte populations during various de- velopmental stages of Blattella germanica (L.) (Dictyoptera, Blattellidae) Endocrinology Ohnishi, E.: Growth and maturation of ova- ries in isolated abdomens of Bombyx mori: Response to ecdysteroids and other steroids Chen R.-H., J.-Y. Lin, Y.-2> Yur andmieae Cheng: Annual changes in plasma and tes- ticular androgen in relation to reproductive cycle in a Japalura lizard in Taiwan Uemura, H., A. Hattori, M. Wada and H. Kobayashi: Effects of intracranially im- planted cholecystokinin and substance P on serum concentrations of gonadotropins, pro- lactin and thyroid stimulating hormone in the Tat esi. te dS ome oe emer ee 6B) Yamada, C. and H. Kobayashi: Immunoreac- tive angiotensin II in the corpuscles of Stan- (Contents continued on inside back cover) INDEXED IN: Current Contents/LS and AB & ES, Science Citation Index, ISI Online Database, CABS Database Issued on April 15 Printed by Daigaku Printing Co., Ltd., Hiroshima, Japan OLOGICAL SCI An International Journal. ZOOLOGICAL SCIENCE The official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Hideshi eebayasti (Tokyo) Toshin-building, Hongo 2—27-2, Bunkyo-ku b ariagan or Tokyo 113, Japan. Tel. (03) 814-5675 Sclichiro aad, ie. ima “«(Hiroshima) ie ~ ( “Saehnl Editors: . \ President: Nobuo Egami (Tsukuba) Pakeo Machida’ (Hiro: ima) Secretary: Yasuto Tonegawa (Urawa) Sumio Takahashi (Ejj#oshima) Treasurer: Tadakazu Ohoka (Tokyo) SY at Se Librarian: Shun-Ichi Uéno (Tokyo) copying nae Editorial Board: Howard A. Bern (Berkeley) Walter Bock (New York) Aubrey Gorbman (Seattle) Horst Grunz (Essen) Robert B. Hill (Kingston) Yukio Hiramoto (Tokyo) Susumu Ishii (Tokyo) Yukiaki Kuroda (Mishima) Koscak Maruyama (Chiba) Roger Milkman (Iowa) Hiromichi Morita (Fukuoka) Kazuo Moriwaki (Mishima) Tokindo S. Okada (Okazaki) | Andreas Oksche (Giessen) Hidemi Sato (Nagoya) Hiroshi Watanabe (Shimoda) | Mayumi Yamada (Sapporo) Ryuzo Yanagimachi (Honolulu) ZOOLOGICAL SCIENCE is devoted to publication of original articles, reviews and communications in the broad field of Zoology. The journal was founded in 1984 as a result of unification of Zoological Magazine (1888-1983) and Annotationes Zoologicae Japonenses (1897-1983), the former official journals of the Zoological Society of Japan. ZOOLOGICAL SCIENCE appears bimonthly. An annual volume consists of six numbers of more than 1000 pages including an issue containing abstracts of papers presented at the annual meeting of the Zoological Society of Japan. MAMMSCRIPTS OFFERED FOR CONSIDERATION AND CORRESPONDENCE CONCERN- ING EDITORIAL MATTERS should be sent to: Dr. Seiichiro KAWASHIMA, Managing Editor, Zoological Science, Zoological Institute, Faculty of Science, Hiroshima University, 1-1—-89 Higashisenda-machi, Naka-ku, Hiroshima 730, Japan, in accordance with the instructions to authors which appear in the first issue of each volume. Copies of INSTRUCTIONS TO AUTHORS will be sent upon request. SUBSCRIPTIONS. ZOOLOGICAL SCIENCE is distributed free of charge to the members, both domestic and foreign, of the Zoological Society of Japan. To non-member subscribers within Japan, it is distributed by Business Center for Academic Societies Japan, 6—16-3 Hongo, Bunkyo-ku, Tokyo 113. Subscriptions outside Japan should be ordered from the sole agent, VNU Science Press BV, Europalaan 93, 3526 KP Utrecht, (postal address; P.O. Box 2093, 3500 GB Utrecht), The Netherlands. Subscription rates will be provided on request to these agents. New subscriptions and renewals begin with the first issue of the current volume. All rights reserved. No part of this publication may be reproduced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1987, The Zoological Society of Japan Publication of Zoological Science has been supported in part by a Grant-in-Aid for Scientific Publication from the Ministry of Education, Science and Culture, Japan. | ZOOLOGICAL SCIENCE 4: 395-410 (1987) © 1987 Zoological Society of Japan REVIEW The Variety and Distribution of the FMRFamide-Related Peptides in Molluscs Davip A. PriceE, NoEL W. DAviEs, KAREN and MICHAEL J. GREENBERG! C. V. Whitney Laboratory, University of Florida, Box 121, St. Augustine, FL 32086, U.S.A. INTRODUCTION Peptide families, like societal kinships, are perceived in terms of morphological, functional, and geneological similarities. Moreover, both sorts of families are poorly delimited, since they comprise a network of relationships extending, from an elementary unit, outward to other existing individuals (or peptides), and backward in time through ancestral lineages. Therefore, although peptide families are often discussed, the under- lying concepts remain variable and vague. Since this review is a description of a peptide family, we thought to introduce it with a clarification of our hierarchical terminology. First, the fundamental unit in peptide associa- tions is the intragene family, a set of peptides with similar sequences, all processed from one precur- sor encoded by a single gene. The sets of enkephalin-containing (EC-) peptides processed from pre-pro-enkephalins A and B are each examples of such fundamental families [1]. Second, within any species, a number of pep- tides may be clearly related structurally, but be derived from two or more precursors. Such groups are intraspecific families, and examples include: the set of EC-peptides generated from 3 precursors in any mammal [1]; the pancreatic polypep- tide-related peptides (PP-RPs; including PP, NPY and PYY) in pig [2]; and the ELH-like peptides from two precursors in Aplysia californica [3]. Received February 14, 1987 " To whom reprints should be requested. Third, homologous peptides from closely, related species usually have very similar sequences, but as species and their ancestries become dissimilar, so often do their peptides. Thus, bovine and porcine PP differ by only one residue out of 36, whereas bovine and anglerfish PP have only 16 residues in common [4]. Still, intraphyletic peptide families are readily recognized, even when the number of residues common to all members is relatively small. A case in point is the family of 8 peptides related to pigment concentrating hormone of prawns and adipokinetic homone of insects which has only three invariant common residues {pGlu', Phe*, and Trp®) [5]. Interphyletic, or evef@@niver- sal families have been proposed [6], but are very difficult to define. Finally, the functions of peptides are not a consideration in these definitions for, although members of intragene families may have a com- mon function, the functions attributed to related peptides become increasingly heterogeneous at higher hierarchical levels. In this review, we consider an emerging intraphyletic family of molluscan peptides related to FMRFamide (Phe-Met-Arg-Phe-NH,). We summarize the major and minor components of this family and their phylogenetic distribution; we describe a methodological key to the characteriza- tion of the member peptides, and present some new, exemplary, data about the family in the snail Helix aspersa. In the end, we speculate about the ancestry of this peptide family and about its extension to other phyla. 396 D.A. Price, N. W. DAvIEs et al. MAJOR FMRFamide-RELATED PEPTIDES (FaRPs) FMRFamide FMRFamide had its origin in several observa- tions that ganglion extracts from various molluscs would increase the force of contraction of isolated hearts of venerid clams, and that this excitation was not inhibited by the serotonin antagonists methysergide and 2-bromo-d-lysergic acid dieth- ylamide [7-9]. Frontali et al. [10] began to purify this cardioactivity by gel chromatography with Sephadex G-15. Four cardioexcitatory peaks were separated from the well-studied molluscan neurotransmitters, and one of them—peak C, which was retained on the column and eluted just after the salt volume —occurred in every species tested in all of the major groups of molluscs; and it was a potent cardioexcitor and antiarrhythmic agent [8, 9]. The source of this activity was sought, with persistance and vigor, in the ganglia of Macrocallista nimbosa, the sunray venus clam, and was finally identified as the tetrapeptide, FMRFa- mide [11]. FMRFamide has since proven to be ubiquitous in Mollusca and an invariably major component of FMRFamide-like immunoreactivity in that phylum (Table 1). The pulmonate heptapeptides Studies on the FMRFamide-like activity in the ganglia of Helix aspersa started soon after the isolation of FMRFamide, and the first was an attempt to localize FMRFamide to identifiable neurones using bioassay [12]. This investigation led to the preliminary conclusion that FMRFamide itself is not present in Helix, but is replaced by a novel FMRFamide analog with a modified N- terminal and considerably more potency on the Helix heart than the parent peptide [13]. Using HPLC for fractionation and radioimmunoassay for detection, three major peaks of FMRFamide immunoreactivity were detected in Helix ganglia [14]. Attention was focused on the most retained (i.e., non-polar) one, which was identified, finally, as a heptapeptide with an N-terminal pyroglutamyl residue [14]. The amino acid sequence of this peptide is: pGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH, (pQDPFLRFamide), the methionyl residue of FMRFamide being replaced by leucine, and it is, indeed, much more potent than FMRFamide on the Helix heart [15]. The remaining two major peaks in Helix are FMRFamide and a partially characterized analog of pOQDPFLRFamide, obser- vations that will be discussed below in some detail. Several additional species of pulmonate snails and slugs have since been examined. In each case, the extracts contain three major FaRPs in roughly equal quantities. One of these is always FMRFa- mide, as mentioned above; the others are all heptapeptides analogous to pQDPFILRFamide (Table 1). The common heptapeptide sequence is X-DPFLRF-NH), and peptides with pGlu, Gly, Ser and, probably, Asn as the N-terminal residue have been idetified thus far (Table 1). Although only a small number of species has been sampled, a clear difference has emerged between the heptapeptides of the two major orders of Pulmonata— the Basommatophora (mostly lim- nic snails) and the Stylommatophora (mostly terrestrial snails and slugs). First, in the stylo_.ama- tophoran pulmonates—represented in Table 1 by Helix, three other snails from disparate families, and the slug Limax maximus —one of the pair of heptapeptides is always pODPFLRFamide; the other is indeterminate (Table 1). Moreover, the chromatographic peaks of stylommatophoran hep- tapeptides are easily distinguished (see section on Methodology, below). Among the basommatophorans, two species have been studied in some detail: the false limpet Siphonaria pectinata, a primitive member of the order [16], and the more advanced snail Lymnaea stagnalis [17]. These two species representing the upper and lower ends of the phylogenetic range, as well as other lymnaecean snails that have been tested, all contain the Gly’ analog GDPFLRFa- mide as one of their two heptapeptides (Table 1). Thus, GDPFLRFamide appears to be character- istic of the Basommatophora. As to the second heptapeptide in this pulmonate order, the Ser! and the Asn!, have been identified; but pODPFLRFa- mide has never been detected in a limnic snail (Table 1). The basommatophoran heptapeptides are also characteristically difficult to resolve, and thus to characterize (see section on Methodology, Molluscan FMRFamide-Related Peptides 397 TABLE 1. Major and minor FMRFamide-related peptides (FaRPs) in molluscs CLASS SUBCLASS Order Species POLYPLACOPHORA Acanthopleura granulata BIVALVIA PTERIOMORPHIA Geukensia demissa HETERODONTA Macrocallista nimbosa GASTROPCDA PROSOBRANCHIA Mesogastropoda Pomacea_ paludosa Neogastropoda Busycon contrarium OPISTHOBRANCHIA Aplysia brasiliana Aplysia californica PULMONATA Basommatophora Siphonaria pectinata Lymnaea _ stagnalis Stagnicola palustris Helisoma sp. Stylommatophora Strophocheilus oblongus Succinea campestris Limax maximus Helix aspersa Cepaea nemoralis CEPHALOPODA Octopus vulgaris Octopus bimaculoides Major FaRPs Minor FaRPs FMRFamide —Heptapeptides* (X-DPFLRFamide) ii 2. FLRFamide Others Refs. ap —ni— ni d == —ni— == [19] a= ni [11] =F —ni— + SGFLRF [19] =F —ni— == [19] +e —ni— ni [20] =F —ni— ae nt-GYLRFa* [21] - Gly Asn ni [16] + Gly Ser ni [17] - —c— ni [19] + Gly ni ni d aT pGlu c ni d + pGlu c ni [19] _ pGlu c ni [18] + pGlu Asn? ni [15], d ss pGlu Cc ni [19] a —ni— +e YGGFMRFamide [26, 43] + —ni— + ni [46] + Unambiguous identification. ni Sought but not identified. * Predicted from gene sequence. @ Where heptapeptides were identified, the N-terminal residue (X-) is listed. The primary heptapeptide (1°) is characteristic of the order; the 2° heptapeptide is not. b Prediction based on amino acid composition and homology. c Unresolved peak eluting on HPLC (ACN/TFA system) with Ser-, Asn, and Gly-DPFLRFamide. d Previously unreported. 398 D.A. Price, N. W. Davis et al. below). MINOR FaRPs A number of minor immunoreactive peaks al- ways occur in chromatograms of ganglion extracts. Some appear to be FMRFamide analogs secreted in small amounts, some may arise as artifacts of purification, and some may be processing in- termediates. FLRFamide This peptide was discovered in the mesogastro- pod, Pomacea paludosa, the apple snail. Pomacea contains very large quantities of FMRFamide-like immunoreactivity in its ganglia: several hundred picomoles per snail. Most of this immunoreactivity is due to FMRFamide, but the Leu’ analog FLRFamide is also present at 10-20% the level of the parent peptide [19]. FLRFamide has since been identified in several other molluscs, including bivalves, cephalopods, and always at the same low levels (Table 1). This tetrapeptide is difficult to identify in most animals, not only because it is present in small amounts, but also because it is less reactive in the radioimmunoassay (see Methodolo- gy section below). Although FMRFamide was easily characterized in the opisthobranch Aplysia braziliana, FLRFa- mide was not detected [20]. Yet the FMRFamide precursor in Aplysia californica does appear to generate one copy of FLRFamide [21]. FLRFa- mide has also never been detected in a pulmonate, but nothing is yet known about the gene or precursor in these organisms. These gaps in our information will be discussed toward the end of this review. SGFLRF This is the tentative sequence for another, very small peak of immunoreactivity from Pomacea. Notwithstanding the low immunoreactivity, the amino acid levels were high enough to determine a composition —Ser, Pro, Phe,, Leu, Arg— so this peak is probably not amidated [19]. YGGFMRFamide Met-enkephalin-Arg®’-Phe’ (YGGFMRF) was first discovered in the adrenal chromaffin granules and in the striatum of the ox [22], and has been reported to occur in molluscs [23]. Although YGGFMREF has no effect as an agonist on the standard bioassay for FMRFamide, its C- terminally amidated analog is approximately equipotent with FMRFamide [24, 25]. A peak of FMRFamide-like immunoreactivity eluting with YGGFMRFamide was detected in ganglion extracts from Octopus vulgaris [26]. Its identification as YGGFMRFamide was based on its immunoreactivity with various antisera, its HPLC retention times in various solvents, and its activity in an opiate binding assay (Table 1). Artifacts of purification FMRFamide is susceptible to oxidation, but the oxidized peptide has only slight biological activity and may not be detected by bioassay. However, oxidized FMRFamide is still immunoreactive, and can be separated from the unoxidized peptide on HPLC, if gradient elution is used. Deliberate oxidation of Helix ganglion extracts with hydrogen peroxide showed that the FMRFamide peak be- haved as expected for authentic FMRFamide; 1.e., the peak at the normal elution time for FMRFa- mide disappeared, and the peak at the position of oxidized FMRFamide increased [15]. This shift of the peak with oxidation, or the appearance of both peaks as a doublet, are good, practical indicators of the occurrence of FMRFamide. In an analysis of ganglion extracts from the pulmonate Lymnaea stagnalis, Ebberink et al. [17] detected the unamidated forms of the two major heptapeptides in a single batch of material. A conservative interpretation of this observation is that the heptapeptides were enzymatically deami- dated during the purification. This notion is supported by the diminished height of the im- munoreactive heptapeptide peak in the same preparation. Another peak that might be expected to arise during the purification of pulmonate extracts is one containing Pro-Phe-Leu-Arg-Phe-NH,. This pep- tide could arise from the cleavage of the extremely acid-labile Asp-Pro bond of the heptapeptides. Molluscan FMRFamide-Related Peptides 399 THE INTRAPHYLETIC FAMILY The phylogenetic distribution of major and minor FaRPs within the Phylum Mollusca is summarized diagrammatically in Figure 1. The heterogeneity of the intraphyletic family, due primarily to the taxonomic restriction of the pulmonate heptapeptides, is evident. Also clear, however, is that the picture in Figure 1 is incom- plete in two ways. First, some minor classes —Aplacophora, Poly- placophora, and Scaphopoda— have been ignored or inadequately studied, primarily because of practical considerations such as supply or size. More critical is the absence of information about peptides in taxa closer to the divergence of the three gastropod subclasses. For example, a study of the families Onchidiidae and Veronicellidae, which are considered systellommatophoran pul- / 7 << \ ee \ ~ N GD POLYPLAC SON St \ toaimarophore e Ae AS \\e H 2 y af ; aS OPISTHOBRANCHIA “ monates by some authors [28] and gymnophilan opisthobranchs by others [29] should be particular- ly rewarding. Second, although the characterizations of SGFLRF in Mesogastropoda, and of YGGFMRFamide in Cephalopoda, are not com- plete, their occurrence does suggest that a variety of minor FaRPs are widely distributed in molluscs and await discovery. Some new information about minor FaRPs in Helix is set out below (and Table 1). METHODOLOGY The identification of a particular peptide de- pends, not only upon the species and tissue examined, but also upon the methods of extraction and purification followed and the bioassay used to monitor these procedures. Therefore, we review ~ x XN N \ PULMONATA \ \ GOPFLRFa © SDPFLRFa EX? FMRFa ! LGD I FMRFa Ks FLRFa GDPFLRFa He YGGFMRFa ~, NDPFLRFa ,/ Se FMRFa _.’ BIVALVIA MONOPL ACOPHORA Fic. 1. The distribution of FMRFamide-related peptides in the Mollusca, showing, especially, the ubiquity of FMRFamide and the delimited occurrence of the pulmonate heptapeptides (general sequence, X-DPFLRFamide). The phylum is assumed to radiate from primitive molluscs similar to tryblidian monoplacophorans. An archeogastropod is pictured to provide continuity, but no member of this subclass has been examined for its peptides. a, amide. 400 D.A. Price, N. W. Davies et al. in brief the specific steps involved in characterizing the FMRFamide-related peptides of molluscs. Assay systems: bioassay The classical clam heart assay [30] was used throughout the identification of FMRFamide, but the isolated radula protractor of Busycon contrar- ium, prepared as first described by Hill [31], was also introduced and increasingly relied upon. The radula protractor muscle is not only very sensitive to FMRFamide, but is also much more selective than the clam heart [32]; e.g., this muscle is contracted by FMRFamide and its analogs, but by few other substances, and especially not by 5- hydroxytryptamine (5-HT), a ubiquitous major component of molluscan ganglion extracts. Moreover, the radula protractor muscles from small Busycon can be isolated in a microbath and used to assay very small volumes of material [33]. Other molluscan hearts, muscles, muscular organs, and nerve cells respond to FMRFamide, and the structure-activity relations (SAR) of the peptide have been studied on some of them [34- 36]. But most of these preparations are neither sufficiently sensitive nor convenient to serve as bioassays. In any event, radioimmunoassay (RIA) has become the system of choice for monitoring the purification of FMRFamide-like peptides. Assay systems : radioimmunoassay Several RIAs for FMRFamide, using different antisera, were developed soon after the tet- rapeptide was sequenced [e.g., 37-41], but only one of them has been used to identify all of the new molluscan FMRFamide-like peptides [41]. This antiserum was raised to a conjugate of thyroglobulin with YGGFMRFamide. The rationale for doing this was that, since the peptide could only be conjugated through its N-terminal, the glycyl residues would serve as a spacer, extending the C-terminal FMRFamide sequence away from the protein carrier and allowing more specificity for the peptide hapten. The expected result was realized: the antiserum, in contrast to ones made to conjugates of FMRFamide itself, recognizes (though not equally) all of the residues in the FMRFamide sequence. The cross-reactivity of the antiserum has been studied in detail and compared with those of other antisera [41, 42]. Three of its features have especially contributed to the analysis of chromato- graphic data (illustrations in Table 2): 1) The antiserum is unusually specific for the N-terminal phenylalanyl residue; even the substitution of a tyrosine in this position drastically lowers the cross-reactivity. 2) Analogs extended at the N-terminal are more reactive in the RIA than is FMRFamide itself, especially if the extension has a glycyl residue analog. Other antisera, and most bioassays, react with extended peptides about as strongly as with FMRFamide, or slightly worse [41]. 3) Substitutions for the methionyl residue tend to be relatively benign; in particular, oxida- tion of the methionine or its replacement with a leucyl residue has relatively little effect on immunoreactivity. TABLE 2. Immunoreactivity of selected synthe- tic peptides in an RIA for FMRFamide Peptide sequence Cross-reactivity* FMRFamide 1.00 FLRFamide ws) YMRFamide .007 FMR Yamide .002 YGGFMRFamide 50.0 YGGFLRFamide 10.0 pODPFLRFamide 2.0 SDPFLRFamide 6 GDPFLRFamide 6 * Relative to FMRFamide taken as 1.00. Extraction In most peptide investigations, an early purifica- tion step can be achieved through the judicious choice of a species from which very large ganglia (or other tissues) can be dissected cleanly and conveniently. In contrast, phylogenetic studies (e.g., on the evolution of a peptide family) place special demands on extraction methods; the proce- dures must apply to any relevant species, no matter how small, heavily shelled, or otherwise inconveniently constructed. Acetone has proven to be a very salutory extraction medium. If the ganglia can be dissected out, the FaRPs will be extracted with a sufficient Molluscan FMRFamide-Related Peptides 401 degree of purity that gel filtration can be eschewed, and high perfomance liquid chromato- graphy (HPLC) performed directly (details be- low). More important, significant quantities of immunoreactive FMRFamide can be recovered simply by steeping whole animals, with their shells, in acetone at —4°C. Recently, we were able to characterize the three major FaRPs in the primi- tive pulmonate snail Siphonaria pectinata by ex- tracting thousands of these abundant, but very small organisms [16]. Notwithstanding the success of acetone marina- tion, other methods such as acid extraction have also been used to isolate FMRFamide-like pep- tides with good results [43]. Gel chromatography Although this methodology would seem to have been rendered obsolete by HPLC, fractionation on Sephadex G-—15 remains a useful component in the purification of FaRPs (Fig.2A). The major advantage is that all of the known peptides in this family elute together, with good recovery, in the classic peak C of Frontali et al. [10], thereby effecting an early and impressive purification. Still, in instances when a very clean dissection has been possible, gel filtration is omitted. In such cases, the extracts are injected directly onto an HPLC column (e.g., Waters C18 Radial Pak) and eluted with an ammonium acetate/n-butanol buf- fer system. If the extract is clean enough, this chromatographic step can separate out several major and minor peaks (method and example in Fig. 3A). HPLC: a chromatographic key The identification of the components of peak C by HPLC may require two steps, with the proce- dure and results dependent on the taxon of the sample species. The possibilities and their out- comes are summarized in Figure 2, a kind of methodological key. The first step is reverse-phase HPLC with elution in a gradient system (ACN/TFA) compris- ing 0.072% trifluoroacetic acid (TFA, the aqueous solvent) and 80% acetonitrile with the same concentration of TFA (ACN, the organic solvent; details of the ACN/TFA system in Fig. 2 and [16]). When extracts of non-pulmonate molluscs are chromatographed in this system, FMRFamide and oxidized FMRFamide appear as a pair of immunoreactive peaks, and FLRFamide occurs in a very small peak (Fig. 2Bi), sometimes only a shoulder. A similarly unambiguous result obtains when a stylommatophoran extract is chromato- graphed in the ACN/TFA system: the im- munoreactive peaks of pQDPFLRFamide and the second heptapeptide are well separated and both are equal in height to the FMRFamide peak (Fig. 2Biii). Basommatophoran extracts in the ACN/TFA system, however, yield a single immunoreactive peak in addition to, and about twice as large as, that of FMRFamide plus oxidized FMRFamide (Fig. 2Bii). This result is caused by the virtual coelution of GDPFLRFamide (i.e., the basomma- tophoran characteristic) and all other hep- tapeptides except pODPFLRFamide which is nev- er found in basommatophorans. The two hep- tapeptides can be resolved by HPLC with elution in another gradient system (ACN/PO,) compris- ing 5mM sodium phosphate, pH 7.0, and acetonit- rile (details in Fig. 2 and [16]). The ACN/PO, system clearly separates GDPFLRFamide from its Asn! analog in Siphonaria (Fig. 2Ci), or from its Ser! analog in Lymnaea (Fig. 2Cii). THE FMRFamide-RELATED PEPTIDES OF HELIX The ganglion extracts of Helix aspersa were the first to be examined after FMRFamide was dis- covered, and it was in this snail that the first pulmonate heptapeptide to be sequenced— pQDPFLRFamide— was identified. Neverthe- less, the second heptapeptide in Helix has not yet been characterized, and most of the minor im- munoreactive peaks have never been analyzed. Recently, however, we purified 400 circum- oesophageal ganglionic rings, dissected out of snails in St. Andrews, Scotland. The results of this study, reported here, have given us further insight into the FMRFamide-like peptides of Helix and the evolution of the peptide family. 402 D.A. Price, N. W. Davies et al. A. Gel filtration (Sephadex G-15) MOLLUSCA B. HPLC (i) NON-PULMONATE gf he PULMONATA (ACN/TFA) MOLLUSCA (ii) Basommatophora (iii) Stylomatophora lo} toy to) [oyfe) fo} (o} fe)ite! fo} fo) {o) lo} {o) fo} fo){o} fo) ve vere lee [re Ve ole Wee (ir ve eve lee ve GQTacaca fos (oAlad (oclaci (od aaacacae & 2 eo) Sj 4 SS So) SS SSS 2 but wu boue we bubueu Ww S oo a as oa a ae oa a o CoO 0 5 oOaQ iS ©O a ialeve: | ire miata: ; eae ddendoetty Hy 1 1 O O 5). 10 1: 2O 5 IO 1 2O iain C. HPLC (ACN/POq) (i) Siphonaria (ii) Lymnaea = NDPFLRFa = GDPFLRFa =< SDPFLRFa = GDPFLRFa 5S (© i 2o 5 10 15 20 min Fic. 2. A diagrammatic scheme for purifying and characterizing FMRFamide-related peptides (FaRPs) from molluscan ganglion extracts, including a set of idealized chromatographic profiles characteristic of particular taxa. The profiles are plots of FMRFamide-like im- munoreactivity with time. A: The extract is applied to G-15 and eluted with 0.1M acetic acid. B: Immunoreactive peak C, comprising fractions that elute at one column volume (V~), is applied to reverse-phase HPLC, and eluted with a solvent system including acetonitrile (ACN) and trifluoroacetic acid (TFA). The nonpulmonate FaRPs (Bi), and those of stylommatophoran pulmonates (Biii), are resolved in the ACN/TFA system; basommatophoran heptapeptides are not (Bii). C: The complex immunoreactive peak of basommatophoran heptapeptides can be resolved by HPLC in a solvent system containing acetonitrile and phosphate buffer (Ci and Cii). The buffer systems are described in the text and the legend for Fig. 3, as well as [16]. Molluscan FMRFamide-Related Peptides 403 ) re a = re x ° ——_ F MRFa —#\— FLRFa +— YGGFMRFa +— SDPFLRFa + pQDPFLRFa O = ox.FMRFa oa Ue lect <————_ FiiERFa <+— YGGFMRFa +— SDPFLRFa + pQDPFLRFa <«~— ox.F MRFa + FMRFa FLRFa =— YGGFMRFa + pQDPFLRFa nmoles <—— ox, EMREG y—— FMRFa eek a <— YGGFMRFa <— SDPFLRFa < pQDPFLRFa ee OEMREG ee pra ete Serpe <— YGGFMRFa <—SDPFLRFa DQDPFLRFa Time (min) O 10 20 30 40 Fraction no. Fic. 3. Purification and chromatographic identification of an acetone extract of snail (Helix aspersa) ganglia. A: HPLC fractionation on a Waters C18 Radial-Pak column; elution with a solvent system consisting of 0.5M ammonium acetate, in 0.1M acetic acid (pH 5.5), and n-butanol. The butanol was first held constant at 4% (10 min), and was then increased to 8% over 20 min. The solvent was pumped at 4ml/min; 0.5 min fractions were collected. The FMRFamide-like immunoreactivity in each fraction (plotted on the ordinate) was determined from radioimmunoassay (RIA) of an aliquot. The six major peaks of im- munoreactivity are labeled (1-6), and these designations are used in B-D: The six peaks were separately pooled and lyophilized and re-chromatographed. HPLC was on a Waters C18 Microbondapak column; elution with a gradient (12-40% over 30 min) of acetonitrile in 6mM trifluoroacetic acid. Solvent flow, 2 ml/min; fractions, 0.5min. Immunoreactivity was measured by RIA, and UV absorbance was monitored at 210nm. The positions of several standards, determined in separate but similar runs, are indicated. 404 D. A. Price, N. W. Daviess et al. Extraction Each ganglionic ring was dropped into acetone as soon as it was removed from the animal in Scotland, and the mixture was kept frozen until it was brought to St. Augustine, Florida, for further processing. The well-travelled acetone was de- canted, centrifuged, and the supernatants saved. The ganglia were then homogenized (Polytron) in aqueous acetone (80%), the mixture centrifuged, and the acetone removed from the combined supernatants on a rotary evaporator. The small volume of aqueous solution left in the flask was forced through a C18 cartridge (Waters Sep-Pak) which was washed with water and the active material then eluted with methanol. The methanol was also removed by rotary evaporation, and the residue was taken up in water for injection directly onto the HPLC column (ammonium acetate/ butanol system). This, then, is an instance of a clean dissection obviating a preliminary fractiona- tion on Sephadex G-15. Purification Six, clear, immunoreactive peaks (labeled 1-6 in Fig. 3A) were the major features in the elution pattern of this first HPLC step. Peak J eluted at the expected position of oxidized FMRFamide and was not further characterized. Peaks 2 and 3 both eluted near the position of FMRFamide itself. Either peak could have con- tained FMRFamide, but they were well-resolved, so we kept them separate and re-chromatographed each with the ACN/TFA buffer system (Fig. 3A, B). This new separation showed that the bulk of the material eluting with FMRFamide is in Peak 3 (Fig. 3C), but there also appears to be some in Peak 2 (Fig. 3B). The fractions constituting the two peaks were hydrolyzed and the amino acid composition of each fraction analyzed. The fractions that appeared to contain FMRFamide in peak 3 had, in fact, the composition expected of the tetrapeptide (e.g., 3-19 in Table 2). In fact, this composition completes the identification of FMRFamide in Helix aspersa; previous characterizations had been based only on the elution times of the oxidized and unoxidized peptides [15, 44]. The results of re-chromatographing peak 2 were not so clear (Fig. 3B). The largest peak, at about 10 min, included the small amount of FMRFamide occurring in peak 2, but contained very low levels of amino acids. In contrast, the minor peak of immunoreactivity, comprising fractions 23 and 24, which eluted near the position of FLRFamide, contained high levels of a small number of amino acids. The composition, Asx, Pro,, Tyr,, Leu,, Arg,, Phe, (Table 2; 2-24), however, is not consistent with the minor peak being FLRFamide. Moreover, the immunoreactivity observed was low: only 2% of that to have been expected were FLRFamide present at the level suggested by the amino acid analysis. Finally, since FLRFamide elutes after FMRFamide in the ammonium ace- tate/butanol system, it should have appeared (had it been present) in peak 3, rather than in peak 2. When peak4 from the butanol system was re-chromatographed with the ACN/TFA buffer system, a large peak of immunoreactivity resulted (Fig. 3D), but its component fractions contained no corresponding peak of amino acids. Thus, this peak could be an RIA artifact [44], but it might also represent a substance with considerably more immunoreactivity than FMRFamide. Peak 5, upon re-fractionation, yielded a clear peak of immunoreactivity (Fig. 3E) with a coinci- dent peak of UV absorbance (not shown) and high amino acid levels (Table 3, 5-32). The analysis is not unambiguous, but the most reasonable com- position —Asx,, Pro,, Phe,, Leu,, Arg;— is identical to that determined for an analogous heptapeptide peak in Siphonaria pectinata [16]. Moreover, it is also similar to the composition of the minor peak in peak 2, except that, in the latter, a tyrosinyl residue replaces one of the phenylala- nines (compare 5-32 with 2-24 in Table 3). Finally, when the most retained peak in the ammonium acetate/butanol system —peak 6— was re-chromatographed in the ACN/TFA system, the elution time corresponded well to the previously characterized heptapeptide, pQDPFLRFamide (Fig. 3F). Summary of Helix FaRPs Price [14] had previously shown that Helix ganglia contain three major peaks of FMRFamide- Molluscan FMRFamide-Related Peptides 405 TABLE 3. Fraction 3-19* Amino acid nmol ratio Phenylalanine 1.543 1.83 (2) Arginine .873 1.03 (1) Aspartic Acid 141 .17(0) Proline Ih) .14(0) Leucine .205 .24(0) Methionine 845 1.00(1) Tyrosine .072 .09 (0) Glycine 129 .15(0) Serine 150 .18(0) i) Amino acid analysis of peak immunoreactive fractions from Helix aspersa Fraction 2-24? Fraction 5-32? An extract of ganglia was fractionated on HPLC (ammonium acetate/butanol system). peaks were then rechromatographed (TFA/ ACN system) (see Fig. 3). nmol ratio nmol ratio BrlS2 1.18(1) 3.505 1.70(2) 2.654 1.00(1) 2.082 1.01(1) 5.124 1.93 (2) 2.910 1.41 (1-2) 2.622 .99(1) 2.057 1.00(1) 22832 1.07(1) 2.450 1.19(1) .192 .07 (0) 435 .21(0) 1.940 73h) .192 .09 (0) 977 .39 (0) 507, .28 (0) eh .29 (0) 1.144 .56(0) The 6 Fractions 19, 24 and 32, from rechromatographed peaks 3, 2 and 5, respectively, were hydrolyzed, and the amino acids analyzed. like immunoreactivity. One of them is FMRFa- mide and another is the classic heptapeptide pQDPFLRFamide [15]. The remaining major peak (peak 5, Fig. 3E) still eludes full characteriza- tion. But we hypothesize that it is the hep- tapeptide Asx-DPFLRFamide, a sequence also proposed for its analog in Siphonaria pectinata which was discussed earlier in this review. The two minor peaks in Helix ganglia differ from the major peaks in that their levels of immunoreac- tivity are not equivalent to the levels of peptide determined by amino acid analysis. One of these minor peaks (fractions 23 and 24 in re-chromatographed peak 2; Fig. 3) is much less immunoreactive than FMRFamide although the peptide levels are similar. In contrast, peak 4 contains little peptide, and thus seems to be more immunoreactive than FMRFamide. The charac- teristics of the antiserum used in the RIA (Table 2) suggest that a decrease in immunoreactivity would reflect some change in the tetrapeptide core of FMRFamide. An increase in immunoreactivity would be brought about by changes in the mole- cule (e.g., N-terminal extension) that would make it more like YGGFMRFamide, the antigen to which the antiserum was raised. If the minor peak of peak 2 had the sequence: Asx-DPYLRF-NH)p, suggested by its composition (Table 3; 2-24), it would (in spite of the N-terminal elongation) surely be less immunoreactive due to the substitu- tions of the leucyl and, especially, the tyrosinyl residues (see Table 2). Peak4 is considered further, below. FMRFamide GENES AND PRECURSORS The Aplysia precursor The inhomogeneity evident in the intraphyletic family of FMRFamide-related peptides should be explicable in terms of the precursors of the constituent intragene families. To date, however, only one FMRFamide precursor, that from Aply- sia californica, has been studied. The gene encoding this precursor has been cloned and its sequence determined [21]. The precursor (Fig. 4A) is about 600 amino acids long and contains: 28 putative copies of the FMRFamide sequence, one copy of FLRFamide, and one copy of another peptide ending in -Gly-Tyr-Leu-Arg-Phe-NH), (de- signated nt-GYLRFa to indicate that it is at the n- terminal of the precursor and is amidated). The number of functional copies of FMRFamide arising from this precursor is still in question because many of the sequences would have to be processed out by cleavage at a single lysine residue, a mechanism unknown for other peptide precursors [45]. However, the report of Lehman et al. [20], that FMRFamide was the only identifiable FaRP in Aplysia ganglion extracts, is consistent 406 D.A. Price, N. W. Davies et al. with FLRFamide and nt-GYLRFa being very : . : The basic molluscan pr minor components of the intraspecific family. precursor One striking feature of the Aplysia gene (and the precursor it encodes) is its high content of CX AA AAASSAI VT 000000000: OO) A Mererorererereneses: Start \) NH» NH>5 CXS AAA 155525252505 eel 12020s0205020205020e: Molluscan FMRFamide-Related Peptides 407 repetitive sequences (Fig. 4a; [21]). Long stretch- es of the precursor are composed of repeated 15 or 16 amino acid segments, each containing one FMRFamide sequence. Many of the repeats are identical at the amino acid level, and several are even completely identical at the nucleotide level. These repeats must represent relatively recent iterative events, and we can therefore approximate the ancestral gene by simply deleting the segments containing most of the similar repeats (Fig. 4B). This gene encodes a truncated precursor contain- ing FLRFamide and nt-GYLRFa, but fewer than ten copies of FMRFamide. Such a precursor would account reasonably well for the ratio of FMRFamide to FLRFamide seen in both a prosobranch, Pomacea paludosa |19], and in two species of octopods, Octopus bimaculoides [46] and Octopus vulgaris [43]. This correlation therefore supports our proposition that the back- extrapolation in Figure 4B resembles the basic molluscan precursor, which is conserved in mod- ern bivalves, cephalopods and most gastropods, and is ancestral to the opisthobranch and pulmon- ate precursors. The number of FMRFamide-related peptides actually processed from the putative basic mollus- can precursor—e.g., three distinct peptides as in Aplysia, or only the two (FMRFamide and FLRFamide) detected to date—remains un- known. However, Price [19] reported the occur- rence, in Pomacea, of a minor peptide which appeared to be SGFLRF (Table 1); and K.H. Voigt has found other FLRFamide-related pep- tides in Octopus (personal communication). We conclude, therefore, that the proposed basic pre- cursor probably contains at least as many distinct peptides as that of Aplysia. The pulmonate precursor If we assume that our analysis of the minor peaks of Helix is both correct and applicable to all pulmonate species, and if we add those data to our information about the major immunoreactive peaks of pulmonates (Table 3), we find that the family of FMRFamide-related peptides in the Subclass Pulmonata falls into two branches, as follows. The heptapeptide branch contains three FLRFamide analogs, all present at about equal levels: XDPFLRFamide, X*DPFLRFamide, and X*DPYLRFamide (e.g., in Helix, X is pGlu, and X”* is Asn). As described above, the first two are much more immunoreactive than the third which is the minor peak of fraction 2 of Helix (Fig. 3B). What we will call the basic branch of the family contains FMRFamide and a highly immunoreac- tive peptide present at very low levels (Helix peak 4, Fig.3D). Of these two branches, it is the heptapeptide one that is novel and that distin- guishes the pulmonate FaRP family from those of the other molluscs. The precursor proteins in the pulmonates, and the genes that encode them, are completely unknown at present. Nevertheless, a reasonable conjecture about the molecular basis for the pulmonate family is possible. We propose that the pulmonates contain two distinct genes and precur- Fic. 4. Hypothetical representation of the FMRFamide precursors in molluscs and their derivation by back-extrapolation from the known Aplysia precursor. A: The FMRFamide precursor of Aplysia redrawn from [21]. The initiation methionine at the N-terminal is labeled “Start,” and the following hydrophobic signal sequence is solid black. The large arrow shows where the signal sequence is cleaved from the precursor, and the nt-GYLRFa peptide follows immediately (cross-hatched). The bold black bars are basic amino acid residues, potential processing points. Simple cleavage points are at the small arrows; cleavage sites with amidation signals (glycyl residue) are indicated by NH). The single copy of FLRFamide is vertically hatched. Copies of FMRFamide in the unique (basic) region of the precursor are horizontally hatched; those in the iterative regions are diagonally hatched. The bends in the diagram have no physical meaning. B: Hypothetical basic molluscan FMRFamide precursor. The most iterative region of the Aplysia precursor has been deleted, leaving FLRFamide, and about 10 copies of FMRFamide. (All symbols are as in A.) C: Two hypothetical precursors accounting for the FMRFamide-related peptides of Helix and other pulmonates. One precursor is presumed to be similar to the basic molluscan precursor (upper diagram). It yields FMRFamide, FLRFamide and an analog of nt-GYLRFa (possibly peak 4 of Helix). The second precursor, unique to the pulmonates, produces three heptapeptides, two immunoreactive (in Helix, pQDPFLRFamide and putative NDPFLRFamide) and one poorly immunoreactive (putative NDPYLRFamide from Helix peak 2). (All symbols as in A.) 408 D.A. Price, N. W. Davies et al. sors giving rise to the two branches of the peptide family (Fig. 4C). (The alternative notion, that a single precursor gives rise to all of the pulmonate peptides, ignores both the minor peaks of im- munoreactivity and observations that the relative sizes of the various peaks vary markedly from tissue to tissue [44]). First, a precursor resembling the conserved basic molluscan precursor will produce multiple copies, about ten, of FMRFamide. We suggest that it also produces a peptide with the partial sequence -GFLRFamide. This peptide —a close analog of nt-GYLRFa in the Aplysia precursor— would have a greatly enhanced immunoreactivity and, therefore, though its concentration would be about one tenth that of FMRFamide, it would appear to be more abudant. This peptide would account for fraction 4 of Helix (see Fig. 3A, D). Finally, the basic precursor should also give rise to a single copy of FLRFamide, a peptide not yet detected due to its scarcity and low immunoreac- tivity. Clear demonstrations of FLRFamide at a level one tenth that of FMRFamide, and of the proposed -GFLRFamide, would go far toward confirming this portion of the hypothesis. The origin of a distinct heptapeptide gene is not at all clear. Two bits of evidence—that the heptapeptides are all FLRFamide analogs, and that the phenylalanyl residue is replaced with a tyrosine in both the pulmonate heptapeptide of low immunoreactivity (X*DPYLRFamide) and the nt-GYLRFa peptide of Aplysia—suggest that the heptapeptide gene could have arisen by du- plication from the N-terminal portion of the basic molluscan gene. Alternatively, the heptapeptide gene, rather than being unique to the pulmonates, may occur in all molluscs. Its products might not have been detected for two reasons: they might be expressed at low levels, or they might be processed in a non-immunoreactive form (i.e., unamidated). An example of the latter possibility might be the putative peptide SGFLRF in Pomacea [19]. THE INTERPHYLETIC FAMILY FMRFamide, sensu stricto, has been unambi- guously identified in a variety of molluscan species (Table 1), but never convincingly in any non- mollusc. Indeed, until very recently, the few non-molluscan FMRFamide-like peptides sequ- enced have only had the final two residues of the C-terminal [47, 48] —or at most the final three [49, 50]— in common with either FMRFamide or FLRFamide. Within the last several months, however, three very FMRFamide-like peptides have been sequ- enced in non-molluscs, and they are all N- terminally extended analogs of FLRFamide. One of these peptides is pGlu-Asp-Val-Asp-His-Val- FLRF-NH, (leucomyosuppressin), isolated from extracts of cockroach heads [51]. Two other peptides, characterized from the pericardial glands of the American lobster, are: Ser-Asp-Arg-Asn- FLRF-NH, and _ Thr-Asn-Arg-Asn-FLRF-NH, [52]. That these closest non-molluscan analogs of FLRFamide occur in the Arthropoda, a protosto- mous phylum with close affinities to the molluscs, is strongly suggestive of an interphylum peptide family. The novel arthropodan peptides most resemble the two molluscan peptides that are processed out of the N-terminus of the Aplysia precursor; i.e., nt-GYLRFa and FLRFamide. Moreover, the N-terminus is especially lacking in obvious redun- dancies and would thus appear to be the oldest part of the precursor. We therefore speculate that the primeval “FMRFamide” precursor present in the common ancestor of the molluscs and arthro- pods had only FLRFamide-related sequences. ACKNOWLEDGMENTS This work was supported by NIH grant HL28440 to M.J.G. The assistance of Louise MacDonald, Lynn Milstead and Jim Netherton in preparing the manuscript is gratefully acknowledged. We would like to thank all the kind people who supplied us with animals: Y. Grimm-Jgrgensen, T. Audesirk, Harvey Blankespoor, and especially R. A. Koch. We are even more in debt to collaborators who supplied us with dissected tissue, including: A. G. M. Bulloch, C. P. Jaeger and G. A. Cot- trell. We thank E.A. Kravitz for sending us pre- publication copies of manuscripts from his laboratory, and K.H. Voigt for keeping us informed of progress in his laboratory on the peptides of Octopus. This is contribution 258 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association. 14 15 16 7 18 19 20 21 22 Molluscan FMRFamide-Related Peptides REFERENCES Douglass, J., Civelli,O. and Herbert, E. (1984) Ann. Rev. Biochem., 53: 665-715. Emson, P.C. and De Quidt, M. E. (1984) Trends in Neurosci., 7: 31-35. Scheller, R.H., Jackson, J.F., McAllister, L. B.., Rothman, B.S., Mayeri, E. and Axel, R. (1983) Celles297=22. Andrews, P. C., Hawke, D., Shively, J. E. and Dix- on, J.E. (1985) Endocrinol., 116: 2677-2681. Gade, G. and Rinehart, K. L., Jr. (1986) Biochem. Biophys. Res. Comm., 141: 774-781. LeRoith, D., Shiloach, J., Heffron, R., Rubinovitz, C., Tanenbaum, R. and Roth, J. (1985) Can. J. Biochem. Cell Biol., 63: 839-849. Hill, R.B. and Welsh, J.H. (1966) In “Physiology of Mollusca”, Vol. 2. Ed. by K. M. Wilbur and C. Y. Yonge. Academic Press, New York, pp. 125-174. Agarwal, R.A., Ligon, P. J.B. and Greenberg, M. J. (1972) Comp. Gen. Pharmacol., 3: 249-260. Greenberg, M.J., Agarwal, R.A., Wilkens, L. A., and Ligon, P.J.B. (1973) In “Neurobiology of Invertebrates, Tihany 1971”. Ed. by J. Salanki, Akademiai Kiado, Budapest, pp. 123-142. Frontali, N., Williams, L. and Welsh, J. H. (1967) Comp. Biochem. Physiol., 22: 833-841. Price, D. A. and Greenberg, M. J. (1977) Science, 197: 670-671. Cottrell, G. A., Price, D. A. and Greenberg, M. J. (1981) Comp. Biochem. Physiol., 70C : 103-107. Greenberg, M.J. and Price, D. A. (1980) In “Pep- tides: Integrators of Cell and Tissue Function”. Ed. by F.E. Bloom, Raven Press, New York, pp. 106- 127. Price, D. A. (1982) Comp. Biochem. Physiol., 72C 325-328. Price, D. A., Cottrell, G. A., Doble, K. E., Green- berg, M.J., Jorenby,W., Lehman, H.K. and Riehm, J.P. (1985) Biol. Bull., 169: 256-266. Price, D. A., Cobb, C.G., Doble, K.E., Kline, J. K. and Greenberg, M.J. (1987) Peptides, 8: (in press). Ebberink, R.H.M., Price, D.A., van Loenhout, H., Doble, K.E., Riehm, J. P., Geraerts, W. P.M. and Greenberg, M. J. (1987) Peptides, 8: (in press). Krajniak, K.G., Greenberg, M.J., Doble, K.E. and Price, D. A. (1985) Amer. Zool., 25: 15A. Price, D. A. (1986) Amer. Zool., 26: 1007-1015. Lehman, H.K., Price, D. A. and Greenberg, M. J. (1984) Biol. Bull., 167: 460-466. Taussig, R. and Scheller, R.H. (1986) DNA, 5: 453-462. Stern, A.S., Lewis, R. V., Kimura, S., Rossier, J., Gerber, L. D., Brink, L., Stein, S. and Udenfriend, 23 24 245) 26 Dh 28 29 30 Sil 3) 33 34 2p) 36 3) 38 39 40 41 42 43 409 S. (1979) Proc. Natl. Acad. Sci. USA, 76: 6680- 6683. Leung, M.K. and Stefano, G. B. (1984) Proc. Natl. Acad. Sci. USA, 81: 955-958. Greenberg, M.J., Painter,S.D. and Price, D.A. (1981) Nuropeptides, 1: 309-317. Greenberg, M.J., Lambert, S.M., Lehman, H. K. and Price, D. A. (1986) In “Handbook of Compara- tive Opioid and Related Neuropeptide Mecha- nisms”, Vol. 1. Ed. by G.B. Stefano, CRC Press, Boca Raton, pp. 93-101. Voigt, K.-H. and Martin, R. (1986) In “Handbook of Comparative Opioid and Related Neuropeptide Mechanisms”, Vol. 1. Ed. by G. B. Stefano, CRC Press, Boca Raton, pp. 127-138. Walsh, K. A., Ericsson, L.H., Parmelee, D.C. and Titani, K. (1981) Ann. Rev. Biochem., 50: 261- 284. Solem, A. (1978) In “Pulmonates, Vol. 2A, Sys- tematics, Evolution and Ecology”. Ed. by V. Fretter and J. Peake, Academic Press, New York, pp. 49- Oi: Keen, A.M. (1971) Sea Shells of Tropical West America, 2nd ed., Stanford Univ. Press. Welsh, J.H. and Taub, R. (1948) Biol. Bull., 95: 346-353. Hill, R. B. (1958) Biol. Bull., 115: 471-482. Greenberg, M.J. (1983) In “Molluscan Neuroen- docrinology”. Ed. by J. Lever and H. H. Boer, North Holland Publ. Co., New York, pp. 190-196. Nagle, G.T. and Greenberg, M.J. (1982) Comp. Biochem. Physiol., 71C: 101-105. Painter, S.D., Morley, J.S. and Price, D. A. (1982) Life Sci., 31: 2471-2478. Kobayashi, M. and Muneoka, Y. (1986) Comp. Biochem. Physiol., 84C : 349-352. Muneoka, Y. and _ Saitoh, H. Biochem. Physiol., 85C : 207-214. Dockray, G.J., Vaillant,C. and Williams, R.G. (1981) Nature, 293: 656-657. Weber, E., Evans, C. J., Samuelsson, S. J. and Bar- chas, J.D. (1981) Science, 214: 1248-1251. O’Donahue, T. L., Bishop, J. F., Chronwall, B. M., Groome, J. and Watson, W. H. (1984) Peptides, 5: 563-568. Boer, H.H., Schot, L.P.C., Veenstra, J.A., and Reichelt, D. (1980) Cell Tissue Res., 213: 21-27. Price, D. A. (1983) In “Molluscan Neuroendocri- nology”. Ed. by J. Lever and H.H. Boer, North Holland Publ. Co., New York, pp. 184-190. Dockray, G. J. (1985) J. Neurochemistry, 45: 152- 158. Voigt, K.-H., Hirt, R., Kiehling, C. and Martin, R. (1987) In “Neurobiology, Molluscan Models”. Ed. by H.H. Boer, W.P.M.Geraerts and J. Joose, (1986) Comp. 44 45 46 47 410 Mon. Kon. Ned. Akad. Wetensch., North Holland Publ. Co., Amsterdam, (in press). Lehman, H.K. and Price,D.A. (1987) J. Exp. Biol., (in press). Schaefer, M., Picciotto, M.R., Kreiner, T., Kal- dany, R.-R., Taussig, R. and Scheller, R. H. (1985) Cell, 41: 457-467. Price, D. A. (1987) In “Neurobiology, Molluscan Models”. Ed. by H. H. Boer, W. P. M. Geraerts and J. Joose, Mon. Kon. Ned. Akad. Wetensch., North Holland Publ. Co., Amsterdam, (in press). Yang, H.-Y.T., Fratta, W., Majane, E. A. and Cos- ta, E. (1985) Proc. Natl. Acad. Sci. USA, 82: 7757- 7761. 48 49 50 51 5 D.A. Price, N. W. Davies et al. Grimmelikhuijzen, C.J.P. and Graff, D. Proc. Natl. Acad. Sci. USA, 83: 9817-9821. Dockray, G. JF) Reeves JgRo) Jrey a shively see Gayton, R.J. and Barnard, C.S. (1983) Nature, 305 : 328-330. Nachman, R.J., Holman, G.M., Haddon, W. F. and Ling, N. (1986) Science, 234: 71-73. Holman, G.M., Cook, B.J. and Nachman, R. J. (1986) Comp. Biochem. Physiol., 85C : 329-333. Trimmer, B.A., Kobierski, L.A. and Kravitz, E. A. (1987) J. Comp. Neurol., (submitted). (1986) ZOOLOGICAL SCIENCE 4: 411-425 (1987) © 1987 Zoological Society of Japan REVIEW Tumors in Amphibia Makoto ASASHIMA, TsuToMU O1NuUMa! and V. BENNO MryYER-RocHow7 Department of Biology, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama 236, ‘Department of Anatomy, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan, and *Department of Biological Sciences, University of Waikato, Hamilton, New Zealand INTRODUCTION Amphibians, together with reptiles and fishes, are poikilothermic (or ectothermic) animals and are often referred to as lower vertebrates. The principal body structures and organs of lower vertebrates, however, are the same as those of the higher vertebrates such as birds and mammals. It is not surprising, therefore, that tumors of amphi- bians with regard to the organs affected by the tumor, the morphology of the tumors and the relationships between factors of tumorigenesis and tumor formation, are basically the same as those of other vertebrates, including the human being. Many investigators have been studying tumors mainly in mammals and this, no doubt, has increased our knowledge concerning tumors; but at the same time aspects of comparative oncology within different vertebrate classes have been somewhat neglected despite a _ certain amount of solid fundamental works [1-4]. Recognising the unique position of amphibians as animals of both aquatic and terrestrial habitats we and others before us [5-16] have been looking for tumors, their causes, their growth patterns, etc. in these animals for quite some time now. Although amphibians have long been used as suitable material for various lines of research within the subject of biology, e.g. experimental embryology, biochemistry, genetics and cell biolo- gy, the number of reports on tumors in amphibia Received February 9, 1987 is relatively small. In mammals, birds and fishes, on the other hand, many different kinds of tumors have been studied and the number of reports is larger. There are two major explanations for the small number of reports on amphibian tumor. One is that investigators, who have used amphibians as experimental material for all kinds of research, may not specifically have looked for tumors and, thus, may not have reported them [17]. Tumors could have been overlooked and if investigators, dealing with amphibians, had carefully examined the internal organs of amphibia, various types of tumors could possibly have been found. The other explanation is that amphibians do suffer less from tumor, because they may have some specific tumor-repelling system which makes it especially hard for a tumor to form in their bodies. In short, they may be different from all other vertebrates with regard to tumors [18, 19]. If such a charac- teristic or something in the nature of amphibians is present that prevents tumors from forming or proliferating we have to identify these forces and relate them to the characteristic way of life of amphibians. For example, amphibians can live both in water and on land; they highly depend on the changes of the environment; as adults they are entirely carnivorous, etc. In addition to the ability to adapt to the environment, they have other characteristics such as metamorphosis dur- ing development and, especially in urodela, a remarkably strong ability to regenerate lost or damaged parts of the body. When we consider 412 M. ASASHIMA, T. OINUMA AND V.B. MEYER-ROCHOW tumors of amphibians in relation to their unique biological characteristics, on the one hand, and their similarity to human neoplasms on the other [5]. Amphibians may perhaps turn out to be particularly suitable material for tumor investiga- tion. Already Khudoley has been calling Rana temporaria “a new experimental animal in cancer research” [20]. The one amphibian tumor that has been well investigated and has been known for decades is renal adenocarcinoma in Rana pipiens first reported by Lucké in 1934 [21]. In addition to furthering our knowledge of tumor generally, the purpose of our study on tumor of amphibians is to recognise common problems in cancer research and to study the biological nature TABLE 1. Species Tumors (number of animals) of tumor by making use of the unique biology of one group of vertebrates: amphibians. Recently, papillomata in the newt Cynops pyr- rhogaster and tumors in Xenopus laevis have been found and investigated. In this review we intend to summarize what is known about tumors in amphibia, concentrating on studies of renal ade- nocarcinoma in Rana, papilloma in Cynops and tumors in Xenopus. REPORTS ON SPONTANEOUS TUMORS The number of reports on spontaneous tumors in amphibians is smaller than that dealing with tumors in mammals, birds and fishes. For exam- List of spontaneous tumors in anurans (—1986) Sites Rana pipiens osteogenic sarcoma (1), adenocarcinoma (many) carcinoma (6), teratoma (1), lymphosarcoma (3), liposarcoma (2), epithelioma (1), hepatoma (2), mesothelioma (2), carcinosarcoma (1), rhabdomyosarcoma (1), plasmacytoma (1), cystoadenocarcinoma (1), thigh, kidney, lung, fat body, muscle, ovary, spleen, bladder, viscera, skin, liver, dermal glands, squamous cell carcinoma (7), papilloma (1) Rana esculenta carcinoma (1), fibroma (1), adenoma (1), hepatoma (1), adenocarcinoma (1), buccal cavity, kidney, ovary, leg, liver hypernephroma (1), sarcoma (1) Rana catesbeiana adenoepithelioma (2) Rana clamitance myxosarcoma (1) Rana temporaria adenocarcinoma (2), neurosarcoma (1) melanoma (1), epithelioma (1) skin, sacral plexus, kidney tail skin cystadenocarcinoma and cystadenopapilloma (7) Rana arvalis adenoma (1) Rana ridibunda Rana chensinensis Bufo bufo Bufo calamita Bufo marinus Bufo boreas Ceratophrys ornata adenocarcinoma (1) adenoma (1) fibroma (1) fibrosarcoma (1) Dendrobates pumilio erythrophoroma (1) Hyla meridionalis guanophoroma (1) Hyla arborea xanthophoroma (1) Xenopus laevis neuroma (3) Xenopus fraseri lymphosarcoma (2) © cystadenocarcinoma and cystadenopapilloma (16) tumor-like displasias (many) capsulated tumor (1), fibroma (8), lipoma (1) lymphosarcoma (3), carcinoma (1), fibroma (1), adenocarcinoma (3), fibromata (1), nephroblastoma (1), lipoma (1), papilloma (1), adenoma (1), melanoma (8), skin skin limb kidney, skin, bladder lung parotid gland muscle leg, kidney viscera, skin skin skin kidney, pelvis, face, under skin, viscera, head, skin, liver, orbit viscera Tumors in Amphibia 413 TABLE 2. Species Andrias japonica Ambystoma opacum mixed tumor (1) Ambystoma tigrinum Tumors (number of animals) fibroma (2), carcinoma (1), fibroma (1) List of spontaneous tumors in urodeles (—1986) Sites limb, testis, under skin skin papilloma (1), fibroma (2), melanoma (1), skin melanocytoma (1), myxofibroma (1) Ambystoma mexicanum melanoma (4), lymphosarcoma (3), melanosarcoma (1), epithelioma (1), skin, mouth, tail, testis adenocarcinoma (1), neuroepithelioma (2) teratoma (1), testicular tumor (16) Amphiuma tridactylum leiomyoma (1) Necturus maculosus adenocarcinoma (1) Triturus cristatus Triturus alpestris Triturus vulgaris Cynops (=Triturus) pyrrhogaster Notophthalmus viridescens Cryptobranchus adenoma (1) alleganiensis adenocarcinoma (1), melanoma (1) papilloma (many), nephroblastoma (1) mesenchymal tumor (1), neuroblastoma (1) lung kidney skin gland, skin carcinoma (4), epithelioma (1) skin chondroma (1), fibroma (25) skin lymphosarcoma (1), sarcoma (5), viscera, liver, skin, kidney skin, under skin, neck testis ple, Effron et al. examined tumors by necropsy and by histology in various species of wild animals which had died in the San Diego Zoological Garden and in the Wild Animal Park region from 1964 to 1976 [22]. They found tumors in 2.75% of 3,127 mammals, 1.89% of 5,957 birds and 1.90% of 1,233 reptiles, but they did not detect any tumors in amphibia (0% of 198). The number of reports on spontaneous tumors in amphibians known to us up until 1986 is about 491 cases involving 18 species of anurans and about 253 cases involving 12 species of urodeles. Relevant data are listed in Tables 1 and 2. The known amphibian tumors are dealt with in some excellent reviews [8, 11, 17, 22-28]. The number of reports in anurans is greater than that in urodeles which according to Brunst [29] is merely a reflection of the greater extent to which anurans are used in research. In some anuran species like Rana pipiens, Rana esculenta and Xenopus laevis several kinds of tumor were reported; the same holds true for the urodele species Ambystoma tigrinum, Ambystoma mexicanum and Cynops pyrrhogaster. These species are frequently used as experimental material in biology. Amongst the tumors listed in Tables 1 and 2, there are some reports dealing with precancerous changes and tumors in Rana. Tumors in intersubspecific and interspecific hybrids have also been reported, e.g. in Xenopus laevis laevis X Xenopus laevis victor- lanus and Rana pipiens < Rana palustris. Lym- phosarcoma has been found in the viscera of the former frog, which was produced by nuclear transplantation. The tumor in the latter was teratocarcinoma in the testes. The reported tumors were classified into six types; epithelioma, mesenchymal tumor, pigment tumor, blood cell tumor, central nervous system tumor and repro- ductive organ tumor. Epithelial tumors such as adenocarcinoma, adenoma, and_ papilloma accounted for nearly half of all reports. Epithelial tumors are thought to be related to the fact that amphibians have many glands in the skin and that the skin is covered by a thin stratum corneum. Mesenchymal tumors, such as fibroma, lipoma and smooth muscle tumor, made up about a quarter of the total. Tumors of pigment cells, hematopoietic cells, and gonads could also be encountered. Tumors of the central nervous system were reported once each in anurans and urodeles. It is very rare that the same type of tumor has been found in many animals of the 414 M. ASASHIMA, T. OINUMA AND V.B. MEYER-ROocHOW same species, but renal adenocarcinoma in the leopard frog Rana pipiens (Lucké renal tumor) and papilloma in the Japanese newt Cynops pyrrhogaster (newt papilloma) are exceptions and have been found in very large numbers. These tumors will be discussed later. Recently, a few reports on the incidence of spontaneous tumors in amphibians have been published. Khudoley and Mizgireuv collected 320 specimens of Rana temporaria and 978 Rana ridibunda in the Leningrad region and found tumors in 7 and 16 frogs, respectively [30]. There were one to seven tumors in each frog and the tumors were all cystadenopapillomata or cyst- adenocarcinomas originating from mucous glands. Infiltrations were not found in most animals, but a metastasis was found in one frog. Mizgireuv et al. collected many frogs and toads in three regions of Southern Sakhalin [31]. They found tumor-like dysplasiae of osteochondrous tissue of hind limbs in Rana chensinensis. With 11.5% out of 1,095 frogs the highest incidences of the dysplasiae were observed in point A, a place polluted with the sewage effluent of a paper factory; in points B and C the figures were 5.5% of 3,651 and 0% of 1,614, respectively. Oinuma et al. observed large tumors in the dorsal region of four adult females of the African clawed frog (Xenopus laevis) [32]. The tumor-bearing frogs were found in amongst about 20,000 adults which were bred in artificial ponds. Surprisingly, no tumors were seen in about 10,000 larvae and 5,000 juvenile frogs. The tumors of the four frogs were similar to each other and were thought to be melanomas and neuromas. In urodeles, Counts et al. reported a mixed tumor in a male Ambystoma opacum [33]. The number of collected animals was not clear. The tumor was composed of epithelial cells and mesenchymal cells and it appeared rather benign. Khudoley and Eliseiv found a melanoma in the skin of one out of 272 axolotls (Ambystoma mexicanum) of 5 months old [34]. This tumor proliferated and metastasized during breeding. Counts et al. captured about 300 newts (Notoph- thalmus viridescens) and found a neuroblastoma in one newt [35]. A cyst of connective tissue was observed and the tumor occurred under the skin. Deformations and abnormal growth in amphi- bians have repeatedly been reported [36, 37]. In 1969 Rostand and Darré suggested that deforma- tions such as brachymelia, polymelia, and poly- dactylism in Rana esculenta could have been caused by a teratogenic virus, which was carried by certain species of fish like tench and eel [38]. These two authors, thus, appear to have been one of the first to recognise the connection between virus and abnormal regeneration. Other possible causes of abnormal growth must, of course, not be completely discounted [37]. FREQUENCY OF TUMOR OCCURRENCE We have seen in the preceding section that some kinds of tumor in anurans and urodeles are nothing new and have been known for quite a while. However, the types of tumor which could be used or have been used as detailed ex- perimental material for detailed investigations are very few in number. The three kinds among them which we shall discuss one by one are Lucké renal adenocarcinoma in anurans, melanoma and neuroma in the African clawed frog Xenopus, and newt papilloma in urodeles. An important point to consider is also the artificial tumorigenesis in amphibians using carcinogenic materials. 1. Lucké renal tumor Lucké renal tumor is an adenocarcinoma in the kidney of the leopard frog (Rana pipiens) and was first reported by Lucké in 1934 [21]. Since then, this tumor has been investigated as a model of tumors in lower vertebrates. Lucké renal tumor appears spontaneously at a relatively low frequen- cy (less than 13%) in one kidney alone or on both sides. Almost no metastases to other organs were observed. However, when the frogs were kept in a laboratory (at about 22°C) for a long period the incidence of spontaneously appearing tumors in- creased by about 50%, and metastases to lung or liver became recognisable. The origin of the tumor cells is considered to be the epithelium of the urinary tubule, since microvilli were often observed in the tumor cells. The transplantation to healthy frogs is possible. When the tumor fragment was implanted into the anterior chamber of the eye, the transplants proliferated very rapid- Tumors in Amphibia 415 ly [39, 40] and might induce formation of a tumor in the kidney of the host. McKinnell et al. examined seasonal fluctuations of the Lucké renal tumor from 1965 to 1968 [41, 42]. They collected a total of 3,367 frogs from the wild in spring, summer and autumn, and found that the tumor-bearing frogs were most numerous in spring and autumn (average 5.0% and 4.4%, respectively), but considerably less so in summer (0.14%). As for the reason of the rare appear- ance of tumorous frogs in summer, McKinnell et al. considered that the tumor-bearing frogs were easily captured by their predators and, thus, showed a lower survival rate, but they could not exclude the possibility that death of sick animals Or spontaneous regression of the tumor were additional, important factors involved. McKinnell et al. [42] collected a total of 1,363 frogs at 15 localities in Minnesota and it became obvious that the frequency of the tumorous frogs depended not only on seasons as described above, but also on the specific region in Minnesota from which the frogs came. In spring or autumn collections, tumor-bearing frogs made up 0.9-14.0% in 9 out of 15 regions, but no tumor-bearing frog was found in any of the other 6 regions. Thereafter, McKinnell et al. turned towards the phenomenon of decreasing numbers of tumor- bearing frogs in Minnesota [43,44]. In the regions where many tumor-bearing frogs had appeared in the springs and autumns of the years 1966-1975, they captured 685 frogs in 1977 and 1,216 in 1978 and 1979, but not one tumor- bearing frog was found. Nowadays it appears to be difficult to find renal adenocarcinoma in R. pipiens from the wild, but a full explanation why this should be so remains to be put forward. 2. Tumor in Xenopus laevis Throughout the world the African clawed frog (Xenopus laevis) has been used as an ex- perimental animal for a wide range of biological investigations. However, as stated in Table 1, reports on tumors number only 24 cases. This figure seems to indicate a very low incidence. Oinuma et al. examined the frequency of tumor in Xenopus bred in an artificial pond in 1983 and 1984 [32]. In the first examination, they found four tumor-bearing frogs in a population of about 20,000 frogs (0.02%). But following re- examination 7 months later, no tumor-bearing frog could be found at all. Compared with the cases of Lucké renal tumor or newt papilloma the frequency of tumor in Xenopus is really consider- ably depressed. Considering Lucké renal tumor and newt papilloma, normal frogs or newts kept with tumor-bearing animals of the same species in the same water tank for a long period (about a year) developed the tumor with high regularity (more than 50%). However, as for the tumor of the African clawed frog, it did not occur in normal animals for at least a year. It seems that the infection is related to a virus and that the path of the infection takes in Xenopus is different from that of Lucké tumor or newt papilloma. In tissue sections of the tumor in Xenopus, many mature pigment cells were observed among the tumor cells. The DOPA test was carried out on the tumorous tissue, and positive results were obtained [28]. Based on these results and the long term-cultivation of these cells [45], the tumors in Xenopus were thought to be mela- nomas and neuromas. 3. Newt papilloma Papillomata are occasionally found in the skin of the Japanese newt, Cynops pyrrhogaster. The tumors may be found anywhere on the body surface but preferably occur on the tail, back, and limbs. This tumor, which is also called epithe- lioma, was first reported from one case each by Honma and Murakawa [46] and Bryant [47]. Since then newt papilloma has been found in large numbers in a variety of species (e.g. Diemic- tylus viridescens: [48]; Triturus alpestris: [49]; T. cristatus: [10,50]) and presently newt papilloma as well as Lucké renal tumor is considered to represent important material for the investigation of amphibian tumor [51-56]. As newt papilloma often regresses and disappears during breeding, it may turn out to be very suitable material to analyse the mechanisms involved in the spon- taneous regression of the tumor. Newt papilloma grows by proliferation of epithelial cells of the tumor region, but metastases have not been observed in any organs other than the skin. 416 M. ASASHIMA, T. OINUMA AND V.B. MEYER-ROCHOW Asashima et al. collected newts at specific localities in Nigata prefecture from autumn of 1979 to autumn of 1983 and have examined the seasonal changes of the papilloma [53, 54]. The total number of newts collected was 28,630 and the frequency of papilloma-bearing newts among the collected newts was monitored for every season. Papilloma-bearing newts were numerous only in autumn (1.9-7.9%) whereas in spring, summer and winter (0.16—-0.32%, 0.47- 0.50% and 0.48-0.50%, respectively) they were far less frequent. Though males tended to be affected more commonly than females, it is not clear if there really is a sex-related difference in the abundance of papilloma. Seasonal peaks in papilloma-bearing newts not only occurred in Niigata but in definite regions of other prefectures (Yamagata and Iwate), too. The incidence of Lucké renal tumor was high in two seasons per annum, namely, spring and autumn, but that of newt papilloma was high only in autumn. The reason for this difference is not easy to understand. Besides seasonal changes in the abundance of papilloma-bearing newts, geographical variations of the tumor frequency in autumn from 1980 to 1985 have also been examined [53,54]. Newts from 16 prefectures in Japan were collected and examined whether they had papillomata or not. Papilloma-bearing newts were numerous in Aomori, Iwate, Yamagata and Niigata prefectures (1.3-7.9%), less numerous in Yamanashi, Gifu, and Shimane (0.7-2.6%), and least numerous in Chiba, Shizuoka, Aichi, Kochi, Okayama, Naga- saki, Saga, Kumamoto and Miyazaki (0-0.6%). More newts from the North and West side of Japan than from Southern and Eastern parts suffered from papillomas. To complicate the picture, there are prefectures like Kochi and Shizuoka in which papilloma-bearing newts were found in some years but apparently seemed absent in others. THE RELATION BETWEEN TUMOR AND ONCOGENETIC FACTOR 1. Oncogenic virus The existence of viruses in amphibian tumors has been proved in some cases. It became evident, for example, that Lucké renal tumor was caused by a virus [3,8]. In newt papilloma, too, virus particles were detected [51, 53] and spontaneous tumors have successfully been transmitted by experiment (e.g. in Pleurodeles waltii: [57]). In Xenopus lymphosarcoma virus particles were de- tected and infection experiments gave equally positive results. Particles resembling viruses were also present in Xenopus melanoma. Fish-born viruses were thought to be involved in abnormal growth of appendages in Rana esculenta [38]. For Lucké renal tumor there no longer exists any doubt that it is caused by a virus [35, 58, 59]. Lucké on the basis of the following observations had already suggested that the tumor was caused by a virus; the acidophilic inclusion bodies existed in the nucleus of the tumor cells and resembled those agents responsible for herpes infection [21]. When glycerinated or dried tumor was inoculated to another, healthy leopard frog, the tumor appeared regularly soon after [58]. At present, this virus is known as Lucké herpes virus. Though the virus was present in most instances, there were cases in which it was not found. Rafferty noticed that the virus was found in tumors of frogs captured in winter, but not in summer material [40]. Subsequently the virus was observed by electron microscopy in the renal tumor of a frog captured in winter, and in the tumor of a frog kept at low temperature in the laboratory. Actually, when seven tumor-bearing frogs in hibernation were exposed to a higher temperature (20-22°C), many inclusion-body containing cells were observed at first but later they were broken and the residues of cells and virus particles were flushed out into the urinary tubules. Seven days later, cells that did not contain any inclusion- bodies were to be observed in the tumor [60]. On the other hand, when frogs were captured before they entered a lake for hibernation and they were put in a cage and experimentally immersed in the Tumors in Amphibia 417 lake [61], the virus was not found in the tumor before the frogs entered the lake, but began to appear seven days later so that more than a month later all the tumors contained viruses. The water temperature in the lake was 5-9°C. Even though tumor-bearing frogs were kept at low temperature in the laboratory, a similar result could not be obtained. Thus, the maturation of the virus appears to occur at low temperature while the proliferation of the tumor cells requires higher temperature. The encapsulation of Lucké herpes virus is a hibernation-related phenomenon in nature. Though the virus is not found in tumors of the summer-type, it is believed that the virus genome is contained in the tumor cells in a masked or latent state [40]. When frog larvae were reared segregated from early stages of development, the frequency of tumor formation was almost the same as that found in the field. The infection by virus from a tumor-bearing frog to another healthy animal may occur at an early period of development [40]. Furthermore, such an infection seems to occur perhaps in spring, the season of spawning and embryonic development. Horizontal infections might well represent one pathway for the spread of the disease, because experiments with transmitting infections have been successful. Generally, to know whether a virus is a tumor agent, transplantation experiments have to be carried out successfully. Cultured cells will have to be infected with the virus and their transplantabil- ity has to be affirmed. In Lucké tumor, it has been proved that the virus was the etiological agent on the basis of virus isolation, transplantation and cell culture studies [96]. Tweedell separated the tissue of frog renal tumor into cell organelle fractions which were kept under low temperature [62]. Each fraction was injected to sterilized early embryos or hatching larvae. In embryos injected with the mitochond- rial fraction, tumors were formed in the pro- nephros or mesonephros in large numbers (13- 92%) during or after metamorphosis. In the embryos injected with the microsomal fraction, tumors were also induced in the metamorphosing larvae or in juvenile frogs (0-50%). These newly created tumors were proliferating renal adenocar- cinomas. Furthermore, Mizell separated the mitochondrial fraction of the tumor into several fractions by the zonal centrifugation method and obtained a fraction which readily induced the growth of tumors when it was injected into early embryos [63]. The establishment of cell lines originating from the virus-induced pronephric tumor was performed by Tweedell and Williams [64]. Primary explants obtained from normal pronephri of larvae were cultured in vitro and the cells were infected with herpes virus obtained from adult tumors. These cells were cultured for three passages and the mitochondria-herpes virus frac- tion was obtained from these cells. Then frog embryos were inoculated with the fraction. When the embryos developed and became tadpoles, the tumor was formed in pronephros or mesonephros. Dissociated cells were obtained from this tumor, cultured, and two cell lines PNRT 4 and PNRT were established. More than 85% of these cells were epitheloid and the rest was fibroblastic. Naegele et al. examined whether Lucké herpes virus fulfilled Koch-Henle postulates [65]. Accord- ing to Koch-Henle postulates, the experiment was separated into 4 steps. (1) Herpes virus was associated with kidney tumor of frog adult. (2) A cell fraction, containing virus, was obtained from the tumor. Tail-bud embryos were infected with this fraction and allowed to develop until tadpoles. Tumors were induced in_ pronephros_ or mesonephros of these tadpoles at a high frequency (about 62%). (3) Then the tissue fragments of the induced tumor were cultured. If the tissue was kept at 7.5°C, herpes virus was detected in the nucleus, but if kept at 22°C, the virus was not found. (4) Cultured tissue fragments were homogenised and centrifuged. Early embryos were inoculated with the supernatant and were allowed to develop. Tumors were not induced in animals inoculated with the cell extract kept at 22°C, but readily so (64.7%) when they received the cell extract kept at 7.5°C. Herpes virus was detected in the newly formed tumor. It had oncogenic activity and it was the same virus isolated from the tumor of wild adult frogs. Nace et al. found an antigen “X” by im- munoelectrophoresis and fluorescent antibody techniques, which was contained in normal cells 418 M. ASASHIMA, T. OINUMA AND V.B. MEYER-ROcHOW but was absent from tumor cells. The antigen X was identified as a lysozyme [66]. There were at least eight isozymes in the normal kidney of adult frogs. One of them always existed, three were absent from the tumor and others were variably distributed. Since frog lysozyme was thought to possess activity against frog herpes virus [67], the hypothesis was advanced that the absence of an isozyme of lysozyme was linked to the virus infection and the subsequent initiation of the growth of the tumor. Next, as for the newt papilloma, the existence of virus particles was confirmed in the tumor by Pfeiffer et al. [51] and Asashima et al. [53]. Which type of virus group this virus belonged to has not been clearly established yet. This virus resembles the herpes virus and Lucké herpes virus in regard to size and morphology but it is entirely possible that newt papilloma virus belongs to the group of iridoviridae [68]. Because the virus is often observed to proliferate in the cytoplasm of papillo- ma cells, the core of the virus particle must be large and the form of the virus a 6-edged body. This has to suffice to determine the nature of this virus and the nature and size of nucleic acid of this virus. Isolation of it will be required. In newts papilloma transplantation experiments were successfully carried out [69]. When tumor fragments were implanted under the skin of healthy newts, tumors were formed in the skin of 17% of the hosts within a year. The tumorous tissue was then homogenized. When this homoge- nate was used for inoculation in newts collected at Kumamoto prefecture, a region where tumor- bearing newts had not been found previously, tumors did occur in 10.0-15.8% of these newts. In these transplantation studies, the frequency of tumor formation was higher than that of the control experiments and that of newts captured from nature. During the study of tumor homoge- nate injection, it became obvious that we did not know whether all the cells of the tumor were destroyed completely, but at least it appears to be the generally accepted view now that some fac- tor(s) contained in the tumor cells induce the tumor. Differences in the protein patterns of papilloma and normal skin tissues of the Japanese newt Cynops pyrrhogaster were studied by the two- dimensional (2-D) gel electrophoresis method. Also compared were protein patterns of skin derived from different regions of the same body and that from male and females. Groupings of 11 protein spots specific to normal skin and 7 protein spots specific to papilloma were detected [70]. The papilloma specific protein spots were not detected in the normal skin of adult newts, the skin of larvae, the presumptive ectodermal region of embryos, or such organs as the lung and liver. There were some differences regionally, but none by sex. One of the 11 spots specific to normal skin of adult newts was found to coincide with one in the spot grouping of larval skin. Two unique spots were identified in normal larval skin. The possibil- ity exists that the appearance of papilloma specific proteins indicates the presence of virus associated proteins. Virus particles were also seen in at least two cases of tumors of Xenopus laevis. In the first case, virus particles were detected in lymphosarcoma by electron microscopy [71]. A cell free extract containing the virus of this tumor could induce tumor formation. This infectious virus was about 0.05 wm in size. In the second case, a virus was found in a melanoma of Xenopus laevis [32]. The virus particles were often found in large number in the nucleus of a tumor cell, and in some cells, it was observed, that the inside of the nucleus was filled with virus, which would actually spill over into the cytoplasm. The virus found in the melanoma of Xenopus was considered to be herpes virus judging by its morphology, size and situation of the core. The existence of virus associated with tumor in amphibians must, therefore, be regarded as fac- tual. From now on amphibian virus-associated tumors could provide material for the analysis of tumorigenesis, cell transformation and cell dif- ferentiation and for the comparative study of the virology of tumor. 2. The effects of carcinogens A large number of abiotic causes for tumorous growths have become recognised, notably ionising radiation and chemicals. The latter may exert their effectiveness directly or indirectly through food Tumors in Amphibia 419 uptake and the production of carcinogenic break- down-products. When a carcinogenic chemical, known to induce tumor in mammals and fish following exposure to a small quantity or dose of it, is given to amphibians, the probability of inducing tumor is generally low [27, 28, 72]. Methylcholanthrene (MC), which is known to induce skin tumor when applied to the skin of mice and which induces sarcoma when injected sub- cutaneously, exerts an effect that is broadly the same in both anurans and urodeles. 3, 4-Benzo- pyrene (BP) showed similar results. BP induces only skin tumors in urodeles but tumors of internal organs in anurans. Though hepatoma was induced with dimethylnitrosoamine (DMNA), diethyl- nitrosoamine (DENA), benzidine and aflatoxin in anurans, no hepatoma was formed with these carcinogens in urodeles. | It is thought that urodeles have strong internal resistance to the hepatoma-causing carcinogens. The authors examined newts (Cynops pyrrhogas- ter) which were bred in water containing a carci- nogen such as 4-nitroquinoline-1-oxide (4-NQO), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) or DMNA for a long period (a year), but no tumor formation was found in any organ of these newts (data not shown). It is suggested that urodeles possess some kind of system which minimises the effect of the carcinogens. Regeneration phenomena are known to occur in urodeles following amputation of a limb or the tail. Though a carcinogen such as MNNG or 4-NOQO was administered into the regeneration blastema, no tumor formation was observed and the dif- ferentiation itself proceeded normally, though various abnormalities (=morphological malforma- tions) occurred. This problem will be discussed later. It seems certain that amphibians, and in particu- lar urodeles, are less sensitive towards carcinogens than other vertebrates. Since urodeles are the least sensitive, they could possibly serve in con- junction with their strong regeneration capacity as a convenient experimental animal in the study of the mechanisms of cellular resistance against carci- nogens. BIOLOGY OF TUMOR CELLS The last published work of the Nobel laureate Hans Spemann, the discoverer of the organiser in the amphibian embryo, dealt with the tumor problem [73]. He transplanted the organiser region of gastrula stage embryos onto the liver of the adult newt Triton taeniatus and finally found the tumor cells resembling those of teratocarcino- ma near the transplanted area of the newt. Following this experiment, many other investiga- tors have used the amphibian tumor cells to study aspects of cell differentiation. 1. The relation between regeneration and tumor cells In amphibians, the number of reports on spon- taneous tumors is less than that for other verte- brates, and the resistance of amphibians towards chemicals, which in other animals are known to possess strong carcinogenic activity, seems to be very high. If it is true that in amphibians tumors have greater difficulty to form, then this may well be related to the strong regeneration potential of amphibians. Urodeles, in particular, have an enormous capacity for regeneration. When limbs, tail, or lens are amputated or removed, the powers of regeneration in each amputated area begin to work to reconstruct the original morphology. Dedifferentiation, proliferation, redifferentiation and morphogenesis occur in the amputated region, which eventually is restored to the former state. Tumor cells, however, escape from the contact of normal cells and from the control in normal tissues, and they proliferate independently and abnormally ignoring the order of the surrounding tissue. Though the cells in the “regeneration field” proliferate abnormally, their degree of freedom is minimised by the effect of the “regeneration field” in the rest of the amputated region, which is also involved in the process of normal cell differentia- tion. It is known that in urodeles, such as newts, normal tissue dedifferentiates first before the new specific structure is reconstructed in the regenera- tion process [74]. Perhaps this was one of the reasons why Jonas [75] argued, though not totally unopposed [76], that the process of regeneration 420 M. ASASHIMA, T. OINUMA AND V. B. MEYER-RocHOW could be seen as a kind of cancerous growth. Whether true tumor cells can be converted to normal cells if placed in the “regenerating field” is, of course, an interesting and important problem. Rose and Wallingfold transplanted the tissue fragment of frog renal tumor into the limb of the newt Triturus viridescens [77]. After the trans- plant took hold and infiltrated the newt limb, the limb of the host was amputated leaving the tumor. In subsequent histological observations, frog cells were distinguished from newt cells by the size of the nucleus and the difference in stainability with haematoxylin. The results showed that regeneration occurred in the normal way in all cases, and that transplanted cells originating from frog tumor differentiated into muscle, cartilage and fibrous connective tissue and freely mingled with and spread in the host tissues. The cells of the renal tumor were epithelial in origin, but they apparently differenti- ated into many other directions. The results, however, remain controversial, since a reinves- tigation, carried out by Ruben [78] gave negative results, i.e., the transplants kept their own char- acteristic tubuli renalis (=or uriniferous tubule) structures during regeneration and had not mixed with other tissues. There was an interesting report that urodele epithelial tumor induced by treatment with MC differentiated into normal tissue spontaneously [79] but it is not clear to what extent the tumor disappeared or became differentiated. Recently, Tsonis [80] using newt papilloma, examined the effects of the presence of a tumor on the process of limb regeneration and the behaviour of the tumor cells in the regenerated tail. He observed that although the differentiation proceeded nor- mally, the formation of the regenerative cone became retarded in tumor-bearing newts. When the tail with a tumor was amputated through the tumor, the tumor cells covered the surface of the wound but did not mix with normal epithelium and did not invade the blastema. The problem of redifferentiation of tumor cells into normal tissue in the regenerating field needs to be reinvestigated. A clear distinction of transplanted tumor cells from host cells in trans- plantation studies is paramount for the correct interpretation of the results. Furthermore we need to give attention to the question whether tumor cells can be incorporated at all in the regeneration process. The effects of chemical carcinogens on regen- eration have been examined. Urodela obviously have a high resistance against carcinogens, and malformations rather than tumors occurred as a response. Tsonis and Eguchi treated forelimb blastema with a crystal of various carcinogens (about 5 ug), e.g. MNNG, 4-NQO, MC or BP for 7 days after amputation [81-83]. These carci- nogens were not able to arrest the regeneration completely, but abnormal bones were formed or the regeneration was delayed. Abnormal limb regeneration can be classified into several types. For example, complete deficiency of both ulna and radius; abnormal regeneration and poly- morphism of carpal bones, metacarpal bones and phalanges of fingers; hypertypic limb regenera- tion, and incomplete limb regeneration. Normal- ly newts have four fingers in the forelimb and five in the hindlimb. When the left hindlimb was amputated and a small crystal of 4-NQO was put into 7-day blastema, the regenerated limb, de- veloped an abnormal 6-fingered polymorphism, but a tumor was not formed. The effects of carcinogens such as 4-NQO or MNNG on the cells of newt blastema are different from those on normal cells of other vertebrates. In newts the carcinogens show no carcinogenic activity, but only cause changes in cell movement or cell behaviour in the blastema. The blastema cells of newt are very resistant and stable against carcinogens. The relationship between the action mechanism of carcinogens in the regeneration process of urodeles and the reaction of blastema cells in the regeneration field provides the tumor scientist with a unique experimental system. It is known that the iris in an eye also has strong regeneration power. When the lens is removed from an eye of a newt, cells of the upper part of the iris dedifferentiate, proliferate, form a lens vesicle, and bring about the regeneration of the lens. At this time, a strong carcinogen was administered into an eye ball after the removal of the lens. But, once again, no tumor was formed Tumors in Amphibia 421 as in the case of limb regeneration. Though only one lens is regenerated in normal regeneration, several lenses were regenerated following the administration of a carcinogen. The lens, re- generated while being treated with a carcinogen, is normal with regard to lens differentiation and transparency, but it seems almost impossible for the eye to function properly, considering there are several lenses in an eye [84]. This ex- perimental system is very interesting if one desires to study the mechanisms of cell differentiation and cell reaction affected by carcinogens in lens regeneration. 2. Nuclear transplantation and potency of cell differentiation Studies have been performed by means of the nuclear transplantation technique to determine whether the nucleus of a tumor cell possessed latent pluripotency and whether it had genes which enabled it to differentiate into various types of normal cells, tissues and organs. King and DiBerardino transplanted a nucleus from a Lucké renal tumor cell into an anucleated egg [85]. The nucleus was obtained from either proliferated tumor cells of primary tumorous tissue which were implanted into the anterior chamber, or from cells which were cultured in vitro for a short period. Out of the eggs with nuclear transplants, 1-5% reached the normal blastula state. In some of them development proceeded to late neurula and even larval state. On the other hand, when the donor nuclei originated from normal kidney cells 3% of the eggs reached the blastula. The capacity to con- tinue the process of cleavage was similarly de- veloped in the nuclei from the tumor. However, when nuclei from blastula or gastrula were used as donors, 37% of all embryos reached the state of blastula and 40% of them developed into normal larvae. As for the arrest of the develop- ment, at some stage, of the embryos with nuclear transplants, it was shown that the chromosomes divided abnormally during the early stages of development and that the embryos, thereafter, failed to develop normally [86]. Although abnor- mal chromosomes occur frequently in embryos which developed from nuclei that originated from a differentiated cell, abnormal chromosomes were relatively rare in embryos which originated from nuclear transplants of undifferentiated cells. From these observations it was concluded that abnormal chromosomes in embryos coming from nuclear transplants reflected the degree of dif- ferentiation of donor nuclei prior to transplanta- tion. Then, to unambiguously show pluripotency of renal tumor nuclei, the nuclei were marked and made identifiable by being triploid [87, 88]. Since the nuclei of the host cells were diploid, the distinction between host nuclei and transplanted tumor nuclei was possible. First, triploid early embryos were obtained by the technique of low temperature treatment. These triploid embryos were infected with Lucké herpes virus. Embryos which proceeded development and yielded tumor cells in the pronephros were obtained. Next, the triploid tumor cell nuclei were injected into anucleated eggs. Then the eggs that had received triploid tumor nuclei proceeded development, but no tumor at all was formed. Moreover, to clarify these results, the nuclei from cultured triploid renal carcinoma cells were also transplanted [88]. Eggs with transplanted nuclei developed in such a way that 47% of them became blastulae, 17% and 20% became gastrulae and neurulae, respec- tively, and 3% developed into swimming larvae. The nuclei of these embryos were all triploid, and histologically, the cells differentiated into all organs of the body such as brain, spinal cord, optic cup and lens, somites, pronephros, midgut and so on. These results suggest that the nucleus of a tumor cell is genetically multipotent and can differentiate reversibly. Thus, using the nucleus of Lucké renal tumor cell, normal cloned larvae could be obtained. This is not only an important result, which agrees with observations on plant tumors [89] but also demonstrates a convenient experimental approach to study the expression of pluripotency of tumor cells and their ability to redifferentiate. 3. Effects of temperature It has already been described that both in Lucké renal tumor and in newt papilloma, the- abundance of the tumor depended notably on the 422 M. ASASHIMA, T. OINUMA AND V.B. MEYER-RocHOW season [41-44, 52-56]. One of the causes of the seasonal change in tumor appearance is thought to be temperature. Especially, since amphibians are poikilothermic animals, they represent a con- venient material to study the changes of tumor cells by means of alteration of temperature. In homoiothermic animals such as mammals, although it is possible to change the temperature regionally or even that of the whole body for a short period, it is difficult to change the body temperature from higher temperature to lower for any length of time. Amphibians allow such experiments to be performed and it is then possible to investigate the effects of temperature on the tumor, the regulatory mechanisms of the body and the properties of the virus. Newts with papillomata of moderate size (2.5- 3.5mm in diameter) were chosen. They were divided into five experimental groups of different temperature conditions (4, 10, 13,25 and 30°C) and bred under these controlled temperatures, while the diameters of the tumors were measured weekly [55,56,90]. As a control, papilloma- bearing newts were bred outdoors in the shade. The size of the tumor tended to increase gradually at 10°C and 13°C which are temperatures similar to those present in autumn, but it decreased notably both at 4°C (lower temperature) and at 25 and 30°C (higher). Newts have the ability to attenuate the tumor in their bodies; depending on changes in environmental temperature, they may even possess the ability to cure themselves and have the tumor regress. Interestingly, the tumor regression occurred at both lower and higher temperatures, but it was found by histological examination that the way the regression occurred was different at both temperatures. At the higher temperature end the regression occurred more vigorously at 30°C than at 25°C. The size of the papilloma began to decrease soon after the animal was placed in the higher temperature. Cells of the upper layer of the tumor keratinized more actively than that of normal epidermis, i.e., shedding off tumor cells and size-reduction of tumor mass took place simultaneously. On the other hand, under condi- tions of the lower temperature movement of cells into the dermal layer was observed though tumor cells necrosed in part. The movement of cells was found at the earlier period soon after the change in temperature (within 4 weeks). However, the apparent size of the tumor did not become reduced in this early period. The reduction in size began after two months, and the tendency of the tumor to regress became more intense than at the higher temperature condition. Generally, epithelial cells proliferate in the papilloma, but important changes in the dermal and pigment layers are lacking. However, at lower temperature, the number of cells in the epithelium decreased and simultaneously, that of the pigment layer increased. A down-growth of the epithelioma cells was observed at 4°C (low temperature treatment) [91]. The effects of temperature became evident much earlier. The mitotic index was strongly affected within as early as one week after the onset of temperature treatment. In the newts kept at 4°C or 30°C, the mitotic indices remained low (0.03-0.27) through- out the experimental periods, whereas under mild temperature conditions (10°C), the mitotic index of papilloma cells became significantly higher than at other temperatures. It is evident that the mitotic indices are closely related to the size of the newt papilloma [91]. When newts with regressing papillomata, caused by exposing them to lower or higher temperatures, were Once again maintained at middle temperatures (10°C or 13°C), the tumor cells began to proliferate and increase the size of the tumor again. The growth of this tumor can be controlled or regressed reversibly by the effects of temperature. In addition to this effect, it has occasionally been observed that newts are likely to possess another method for curing a tumorous growth. Generally, the tumor regresses gradually by depending on the change of temperature, but in some newts the mass of tumor disappeared almost abruptly from their bodies. This phenomenon may represent a form of “spontaneous therapy of tumor” in newts. It may be called “tumor cut-off” and could be comparable to the casting off of the tail in a lizard [74], and may be the most efficient method, and certainly fastest, to deal with a tumor. Practically, the process is achieved by Tumors in Amphibia 423 blood vessels being clogged up by blood cells and the subsequent prevention of the blood flow through the tumorous tissue. As a result, tumor cells necrose entirely and the mass of the tumor is removed from the root. When it became obvious that newt papilloma is influenced by changes in temperature, it was thought that amphibians could serve as ex- perimental material to study tumor regression, especially aspects of change and movement in tumor cells, and the contribution of virus in tumorous growth. However, to date almost no study has been performed with such objectives. For biochemical analysis, diamine and polyamine levels in spontaneous skin papilloma of newt were determined. In the papilloma putrescine was most abundant among the polyamines being near- ly 5 times higher than that in the control skin [92]. Tumor bearing leopard frogs and the virus are not found in nature during summer. However, the latent existence of a virus can be confirmed by cold temperature treatment. Lucké renal tumor is, therefore, also thought to be a convenient system to analyse the effects of temperature on tumor cells and the role that the virus plays. Back to newts, it was pointed out earlier that they had papillomata predominantly in autumn, whereas Lucké renal tumor was present in large numbers twice a year, namely in spring and in autumn. If papillomas in newts would be controlled by means of temperature as the only causation factor, newt tumor ought to appear both in spring and in autumn. However, as the frequency of the tumor is actually low in spring, it suggests that factors promoting the tumor are not only temperature alone but others like, for example, hormones, growth factors or properties of the virus, too. As yet the different roles of these factors are not fully understood. There is proof of the close relationship between temperature and cell motility and the cell move- ment of tumor cells. McKinnell et al. observed the distribution of microtubules in the cytoplasm of Lucké renal tumor cells, the established cell line PNKT-4B, the primary culture of renal tumor and, as a control, normal kidney cells of tadpole [93]. The cultures were kept at 20°C or 28°C and the microtubules were observed by the immunofluorescence method. In all of the three cultures, the microtubules were distributed reg- ularly from the centre of the cell to the periphery, but when the cultures were kept at 7°C the distribution of microtubules in the tumor cells became irregular while normal cells remained unaffected. The microtubules of tumor cells quite unlike those of certain dermal cells in fish [94] become disordered by low temperature treatment similar to the disorder of microtubules seen in normal cells after application of a microtubule inhibitor. In Lucké renal tumor, metastasis formation occurs commonly at 28°C (77%), but much more rarely at low temperature (6%). It has also been suggested that the collagenase secreted by Lucké tumor in vitro explants degra- dated type 1 collagen at 30°C and was having an effect similar to that of temperature during metas- tasis formation [95]. However low levels of collagenase were also released at room tempera- ture. In those studies meant to illuminate the effects of temperature in newt papilloma the observed cell movements did not always agree with the results obtained on Lucké renal tumor. Nonethe- less, amphibian tumor cells are excellent material to study the mechanisms of tumor formation, the complicated movement of cells at tumor growth and the phenomenon of regression. We are, therefore, convinced that human cancer research can only gain from the work, presently under- taken in various labs around the world, on amphibian tumor and regeneration. ACKNOWLEDGMENTS We are grateful to Professor Emeritus G. Nace of Michigan University for his suggestions. REFERENCES 1 Krontovsky, A. (1916) Comparative and Ex- perimental Pathology of Tumors, Kiev, Bacteriol. Inst., (In Russian). 2 Sheremetieva,E.A. (1938) Rep. Inst. Zool., Acad. Sci. Ukr. S.S.R., 12: 37-61 (In Russian). 3 Lucké, B. and Schlumbarger, H. G. (1949) Physiol. Rev., 29: 91-126. 4 Duryee, W.R., Long, M.E., Taylor, H.C., McKel- is 16 17 18 19 20 21 D ep} 24 25 26 Pai} 424 way, W.P. and Ehrmann, R. L. (1960) Science, 131: 276-280. Brunst, V.V. and Roque, A.L. (1967) J. Natl. Cancer Inst., 38: 193-204. Hadji-Azimi, I. and Fischberg,M. (1967) Suisse Zool., 74: 641-645. Hadji-Azimi, I. and Fischberg, M. (1971) Cancer Res., 31: 1594-1599. Mizell, M. (1969) Biology of Amphibian Tumor, Springer, Berlin, Heidelberg & New York, pp. 1- 484. McKinnell, R.G. and Ellis, V.L. (1972) Cancer Res., 32: 1154-1159. Wirl, G. (1972) Arch. Geschwulstforsch., 40: 111- SY Balls, M. and Clothier, R. H. (1974) Oncology, 29: 501-519. Khudoley, V. V. and Eliseiv, V. V. (1979) J. Natl. Cancer Inst., 63: 101-104. Khudoley, V. V., Anikin, I. V., Sirenko, O. A. and Pliss, G. B. (1979) Vopr. Onkol. (Leningrad), 25: 70-75 (In Russian). Eliseiv, V.V. and Khudoley, V. V. (1980) Vopr. Onkol. (Leningrad) 26: 70-71 (In Russian). Rev. Jaenisch, W. and Schmidt, T. (1980) Arch. Geschwulstforsch., 50: 253-265. McKinnell, R.G., DiBerardino,M.A., Blumen- feld, M. and Bergad,R.D. (1980) Results and Problems in Cell Differentiation, 11: Differentiation and Neoplasia, Springer-Verlag, Berlin, Heidelberg & New York, 310 pp. Schlumberger, H.G. and Lucké, B. (1948) Cancer Res., 8: 657-753. Waddington, C. H. (1935) Nature, 135: 606-608. Needham, J. (1936) Proc. R. Soc. B, 29: 1577- 1626. Khudoley, V. V. (1982) Bull. Exp. Biol. Med., 92: 1084-1085. Lucké, B. (1934) Am. J. Cancer, 20: 352-379. Effron, M., Griner, L. and Benirschke, K. (1977) J. Natl. Cancer Inst., 59: 185-198. Balls, M. (1962) Cancer Res., 22: 1142-1154. Reichenbach-Klinke, H. and Elkan, E. (1965) II. Diseases of Amphibian, Acad. Press, New York. Dawe, C.J., Harshbarger, J. C., Kondo, S., Sugi- mura, T. and Takayama, S. (1981) Phyletic Approaches to Cancer. Proc. 11th Int. Symp. Princess Takamatsu Cancer Res. Fund., Japan Sci. Soc. Press, Tokyo. Okada, T.S. (1979) Cancer Cells, UP Biology Ser. 36, Tokyo Univ. Press, Tokyo, pp. 1-128 (In Japanese). Kimura, I. (1984) Current Encyclopedia of Patholo- gy, 9c: 304-318, Nakayama-shoten, Tokyo (In Japanese). 28 29 30 Sil 32 33) 34 35 36 Si) 38 Sy) 40 41 42 43 44 45 46 47 48 49 50 Sil 52 53 54 55 M. ASASHIMA, T. OINUMA AND V.B. MEyYER-RocHow Asashima, M. and Oinuma, T. (1985) Oncologia, 13: 99-114 (In Japanese). Brunst, V. V. (1968) Exp. Cell Res., 53: 401-409. Khudoley, V. V. and Mizgireuv, I. V. (1980) Neo- plasma, 27: 289-293. Mizgireuv, I. V., Flax, N. L., Borkin, L. J. and Khu- doley, V. V. (1984) Neoplasma, 31: 175-181. Oinuma, T., Seki,M. and Asashima,M. (1984) Proc. Jpn Acad., 60B: 265-268. Counts, C. L. II., Wilson, C. T. and Taylor, R. W. (1975) Herpetologica, 31: 422-424. Khudoley, V. V. and Eliseiv, V. V. (1979) J. Natl. Cancer Inst., 63: 101-103. Counts, C. L. III. (1980) Herpetologica, 36: 46-50. Woitkewitsch, A. A. (1959) Nattirliche Mehrfach- bildungen an Froschextremitaten. VEB Gustav Fischer Verlag. Meyer-Rochow, V.B. and Koebke, J. (1986) Zool. Anzeiger, 217: 1-13. Rostand, J. and Darré, P. (1969) C.R. Soc. Biol., 163: 2033-2034. Lucké, B. and Schlumbarger, H. (1936) J. Exp. Med., 70: 257-268. Rafferty, K. A. Jr (1964) Cancer Res., 24: 169-185. McKinnell, R. G. and McKinnell, B. K. (1968) Can- cer Res., 28: 440-444. McKinnell, R. G. (1969) In “Biology of Amphibian Tumors”. Ed. by M. Mizell, Springer-Verlag, Ber- lin, pp. 254-260. McKinnell, R.G., Gorham, E., Martin, F.B. and Schaad, J. W. IV. (1979) J. Natl. Cancer Inst., 63: 821-824. McKinnell, R.G., Gorham, E. and Martin, F.B. (1980) Am. MidI. Nat., 104: 402-404. Asashima, M., Sasaki, T. and Takuma, T. (1986) Proc. Jpn. Acad., Ser. B, 62: 307-310. Honma, Y. and Murakawa, S. (1967) Annot. Zool. Japon., 40: 211-214. Bryant, S. V. (1973) Cancer Res., 33: 623-625. Burns, R. E. and White, H. J. (1971) Cancer Res., 31: 826-829. Darquenne, J. and Matz, G. (1971) Bull. Soc. Zool. Fr., 96: 352-353. Rose, F. L. and Harschbarger, J.C. (1977) Science, 196: 315-317. Pfeiffer, C.J., Nagai, T., Fujimura, M. and Tobe, T. (1979) Cancer Res., 39: 1904-1910. Asashima, M, and Komazaki, S. (1980) Proc. Jpn. Acad., Ser. B., 56: 638-642. Asashima, M., Komazaki, S., Satou, C. and Oinu- ma, T. (1982) Cancer Res., 42: 3741-3746. Asashima, M. (1983) Animal and Nature, 13: 15- 19. Asashima, M., Oinuma,T., Matsuyama, H. and Nagano, M. (1985) Cancer Res., 45: 1198-1205. 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 13 74 i) Tumors in Amphibia Asashima, M. and Koyama, H. (1986) Hyperthermic. Oncol., 2: 359-370. Placais, D. (1974) Bull. Soc. Zool. Fr., 99: 283- 295. Lucké, B. (1938) J. Exp. Med., 68: 457-468. Mizell, M., Stackpole, C. W. and Halpern, S. (1968) Proc. Soc. Exp. Biol. Med., 127: 808-814. Zambernard, J. and Vatter, A.E. (1966) Cancer Res., 26: 2148-2153. McKinnell, R.G., Ellis, V.L., Dapkins, D.C. and Steven, L. N. Jr (1972) Cancer Res., 32: 1729-1733. Tweedell, K. S. (1967) Cancer Res., 27 : 2042-2052. Mizell,M. (1969) In “Biology of Amphibian Tumors”. Ed. by M. Mizell, Springer-Verlag, Ber- lin, pp. 1-25. Tweedell, K.S. and Williams, D.C. (1976) J. Cell Soi, 22: 385-395. Naegele, R.F., Granoff, A. and Darlington, R. W. (1974) Proc. Natl. Acad. Sci., 71: 830-834. Nace, G. W. and Ostrovsky, D.S. (1977) J. Natl. Cancer Inst., 58: 453-454. Rubin, M.L. and Nace, G. W. (1966) Am. Zool., 6: 510. Hirayasu, T., Iwamura, Y. (1987) (to be submitted). Asashima, M. and Oinuma, T. (1982) J. Fac. Sci. Univ. Tokyo, Sec IV., 15: 151-158. Shimada, K., Koyama, H. and Asashima, M. (1987) Zool. Sci., 4: 287-294. Balls, M. and Ruben, L.N. (1968) Tumors Res., 10: 238-260. Balls, M. and Ruben, L. N. (1964) Experientia, 21: 241-296. Spemann, H. (1942) Wilhelm Roux’ Arch. Entwick- lungsmech. Org., 141(4): 693-769. Alibardi, L. and Sala, M. (1983) Atti Mem. Acad. Rativitae sci. Lett. ed Atti, 95 (Pt II: Scienze Matematiche e Naturali): 101-151. Jonas, A.D. (1985) Orientierungshilfen zur Psychotherapie in der Allgemeinpraxis-archaische Relikte in Psychosomatischen Symptomen (pp. 92- pn J. and Asashima, M. Prog. sExp: 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ot 92 93 94 95 96 425 97). Edition Materia, Medica, Verlag Socio-medico, Grafelfing. Meyer-Rochow, V.B. (1985) Selecta, 27, 2309. Rose, S. M. and Wallingford, H. M. (1948) Science, 107: 457. Ruben, L.N. (1956) J. Morphol., 98: 389-404. Seilern-Aspang, F. and Kratochwil, K. (1962) J. Embryol. Exp. Morphol., 10: 337-353. Tsonis, P. A. (1984) Can. J. Zool., 62: 2681-2685. Tsonis, P.A. and Eguchi, G. (1981) Differentia- tion, 20: 52-60. Tsonis, P. A. and Eguchi, G. (1982) Dev. Growth Differ., 24: 183-190. Tsonis, P. A. and Eguchi, G. (1983) Dev. Growth Differ., 25: 201-210. Eguchi, G. and Watanabe, K. (1973) J. Embryol. Exp. Morphol., 30: 63-71. King, T.J. and DiBerardino, M.A. (1965) Ann. N.Y. Acad. Sci., 126: 115-126. DiBerardino, M.A. and King, T.J. (1965) Dev. Biol., 11: 217-242. McKinnell, R.G., Deggins, B. A. and Labat, D. D. (1969) Science, 165: 394-396. DiBerardino, M. A., Mizell, M., Hoffner, N. J. and Friesendorf, D. G. (1983) Differentiation, 23: 213- PANG Braun, A.C. (1965) Sci. Am., 213: 75-83. Asashima, M. (1984) Oncologia, 11: 147-150. Asashima, M., Seki, M., Kanno, H. and Koyama, H. (1986) Proc. Jpn. Acad., Ser. B., 62: 83-86. Matsuzaki, S., Kurabuchi, S. and Inoue, S. (1985) Zool. Sci., 2: 131-134. McKinnell, R.G., DeBruyne, G.K., Mareel, M., Tarin, D. and Tweedell, K.S. (1984) Differentia- tion, 26: 231-234. Obika, M. and Meyer-Rochow, V.B. (1986) Cell Tissue Res., 244: 339-343. Ogilvie, D.J., McKinnell,R.G. and _ Tarin, D. (1984) Cancer Res., 44: 3438-3441. Lunger, P.D., Darlington, R.W. and Granoff, A. (1965) N.Y. Acad. Sci., 126: 289-314. i Rab ete a APART: oie i WY : \ioctoan , CE abd ee ea 8 DP gS ddan Wi 44 Sty ig a ‘ - BAe ee ne Sel aS Pt ay, a ah 6 ERE ft se, /3 tos } oh nie ase bel : » f “age ut | An ere FP Sar rs aed jal + | + ty tiy a r 5 : ; yea Mt a os PY weal HAG NARA Ur eh Ree AL free, We DER. fis: Pe bin) vet frag Ne dak BR ee VE vw 7 age Py puee rary ? We Ki ee its (On AE EET SEU a Lee oa oe A sh OONT by mm \ eight here i ane Pam, ee eee iy Pe ee PP AA ite "s OU TE a tees Bh RBA, iy math eer thy 4 ¥ * ‘ Ca mae b PA bute sie ym eerie ee Tiel b ‘ rat OF bode hy to feria OID (ive ie A Mian Rint \ ae m aati. = pe ¥ ages eee Es bs ed a ‘ at co Vane fe) ere ye Ree Bact ea ge { ; a ee *} } é :" i Pha Fi ‘ paz 7 + oe F ie N yale we Lae 4 LHe cee [euler ' poe Pies f 7+ ws Hy pk be as Pee ATAVEY 5a: hoe Te Sees ce aa Dard ae eke, = eS ~ BF aka ey eee Be. Chen kc) ee Sere mt G4 dig Lee any 4 = a te ar Bh ) fi. “| eat , i, i” ef A 5 - ; LSD mf Oy, Loti t Y, §. 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Hy ne 7 m4 ‘i Aa r ae | ets Me ean * hs eh ~ 4 i ie U ' ' Ke 4 yi ' et ea Pa, ; A 7 why Oe ) sm ; Y 7 wer C= , if ha », noe i > nr r j hee, ‘ " t 4 t in An ‘4 oF a) I ' ’ i) & : a ZOOLOGICAL SCIENCE 4: 427-431 (1987) © 1987 Zoological Society of Japan Proposed Tertiary Olfactory Pathways in a Teleost, Carassius auratus KEN OHNISHI Department of Physiology, Nara Medical University, Kashihara, Nara 634, Japan ABSTRACT—Projection fields of the secondary olfactory terminal fields in the goldfish (Carassius auratus) were studied using retrograde axonal transport of horseradish peroxidase (HRP). The cell bodies within the ipsilateral lateral and posterior secondary olfactory terminal fields were labeled retrogradely after the injection of HRP into the medioposterior region of area dorsalis telencephali pars dorsalis (mpDd). The cell bodies within the hypothalamic secondary olfactory terminal field were labeled retrogradely after the injection of HRP into the posterior region of area dorsalis telencephali pars medialis (pDm). These anatomical results suggest that the mpDd and pDm in the goldfish probably form part of the tertiary olfactory projection fields. INTRODUCTION In the teleostean olfactory systems, the neural pathways of the secondary olfactory fibers have been studied by many investigators not only anatomically [1-10] but also electrophysiologically [11] for understanding of the evolution of the vertebrate central nervous systems. The projec- tion fields of the fibers revealed in these studies are essentially very similar among different species. In the goldfish, Carassius auratus, Ichikawa [5], Bartheld et al. [1] and Levine and Dethier [7] have pointed out that the secondary olfactory fibers project bilaterally to three main telencephalic regions: (1) ventral area surrounding the dorsal aspects of the medial olfactory tract (medial terminal field of Ichikawa [5]); (2) medioventral region of area dorsalis telencephali pars lateralis (lateral terminal field of Ichikawa [5]); (3) pos- teroventral region of area dorsalis telencephali _ pars lateralis (posterior terminal field of Ichikawa [5]) and one diencephalic region: nucleus posterior tuberis, NPT, (hypothalamic terminal field of Bartheld et al. [1]). However, projections of the secondary olfactory terminal fields in the telen- cephalic hemispheres of teleosts have not been Accepted January 21, 1987 Received December 25, 1986 investigated as yet except for Murakami et al. [8]. To examine whether the higher olfactory terminal fields exist or not in teleosts, axon terminal fields of the neurons within lateral, posterior and hypothalamic terminal field were examined using retrograde labeling with horseradish peroxidase (HRP). MATERIALS AND METHODS Sixty goldfish (Carassius auratus), 10 to 13cm in length, obtained from a commercial dealer were used. The animals were anesthetized by putting them in a small tank containing 0.1% tricaine methanesulfonate (MS 222) and then positioned in the stereotaxic apparatus slightly modified from that of Peter and Gill [12]. During surgery, the animals were respired by circulating aerated tap water containing 0.05% MS 222 through the gill. The dorsal part of the skull was removed with a dental drill and the brain was exposed under a dissecting microscope. HRP injection into the surface layer of the telencephalon was performed by the insect pin method. An insect pin (No. 000) coated with a small amount of HRP paste was manually inserted into the desired site at the surface layer of the telencephalon [13]. After survival times of 3 days in a water tank at 20-25°C, the animals were anesthetized with MS 222 and 428 K. OHNISHI then perfused through the conus anteriosus with 0.6% saline followed by a solution of 4% glutaral- dehyde in 0.1 M phosphate buffer (pH 7.4). The brain was removed from the skull and postfixed in the same buffer solution containing 10% sucrose for 24 hr. Frontal 50 um thick sections were cut on a cryostat while referring to the atlas of Peter and Gill [12]. The present terminology was after this reference [12]. The HRP reaction with tetra- methylbenzidine was processed according to the procedure of Mesulam [14]. The sections mounted on the gelatin-coated slide were counterstained with 1% neutral red. RESULTS AND DISCUSSION HRP injections were performed in twelve re- gions of the telencephalic surface layer divided into the anterior, anteromedial, medioposterior and posterior region of area dorsalis telencephali pars lateralis(Dl), dorsalis(Dd) and medialis (Dm), respectively. At the injection site, the diffusion of HRP was confined to a region of about 300 um in diameter. After the injection into the mpDd, a large number of labeled cells were observed densely in the ipsilateral medioventral (Fig. 1 C and D) and posteroventral region of Dl (Fig. 1 E and F) and precommissural mediopos- terior region of area dorsalis telencephali pars centralis (Dc), (Fig. 1 B and C). The labeled cells in the posteroventral region of DI were 5 to 7 um in diameter (Fig. 3a and b). A few labeled cells were also observed sparsely in the ipsilateral mediopos- terior region of DI (Fig. 1 B), Dd (Fig. 1 C) and Dm (Fig. 1 B—D) and posterior region of Dm (Fig. 1 E and F). No labeled cells were observed in the ABCDEF Fic. 1. A-F. The distribution of labeled cells in the telencephalon after injection of HRP into the mpDd. Blackened area and surrounding stippled area indicate the injection site and diffusion area of HRP, respectively. Each solid circle marks the location of several labeled cells. AC, anterior commissure; Dc, area dorsalis telencephali pars centralis; Dd, area dorsalis telencephali pars dorsalis; Dl, area dorsalis telencephali pars lateralis; Dld, area dorsalis telencephali pars lateralis dorsalis; Dlv, area dorsalis telencephali pars lateralis ventralis; Dm, area dorsalis telencephali pars medialis; NAPYy, nucleus anterioris periventricularis; NPO, nucleus preopticus; NPP, nucleus preopticus periventricu- laris; NT, nucleus tenia; OT, optic tract; Vd, area ventralis telencephali pars dorsalis; VI, area ventralis telencephali pars lateralis; Vp, area ventralis telencephali pars postcommissuralis; Vs, area ventralis telencephali pars supracommissuralis; Levels of A-F. Vv, area ventralis telencephali pars ventralis. G. Tertiary Olfactory Pathways in Goldfish 429 A BCDEF i C Fic.2 A-F. The distribution of labeled cells in the telencephalon and diencephalon after injection of HRP into the pDm. Filled triangles indicate labeled axons. C, cerebellum; LFB, lateral forebrain bundle; MFB, medial forebrain bundle; NAT, nucleus anterior tuberis; NDL, nucleus dorsolateralis thalami; NDM, nucleus dorsomedialis thalami; NLTi, nucleus lateralis tuberis pars inferioris; NPPv, nucleus poterioris periventricularis ventralis; NRL, nucleus recessus lateralis; NVM, nucleus ventromedialis thalami; OTec, optic tectum. G. Levels of A-F. See Fig. 1 for further explanations. medial terminal field. The medioventral and the posteroventral region of DI in the goldfish has been reported to receive the secondary olfactory fibers from the olfactory bulb [1, 5, 7]. Conse- quently, the labeled cells in the medioventral and posteroventral region of Dl probably make a connection with the secondary olfactory fibers. After the injection into pDm, retrogradely labeled cells were mostly observed in the NPT (Fig. 2 E and F) and few labeled cells in the nucleus preopticus periventricularis (Fig. 2 B and E) and nucleus ventromedialis thalami (NVM, Fig. 2 C and D). The labeled cells in the NPT were very small and they were less than 3 4m in diameter (Fig. 3c and d). Labeled axons were also observed in the medial forebrain bundle running ventral area of nucleus preopticus periventricularis and NVM (Fig.2 A-E) and some in the lateral forebrain bundle (Fig.2 A and B). No labeled cells were observed in the medial, lateral or posterior terminal field. The NPT receives secon- dary olfactory fibers from the olfactory bulb in the goldfish [1, 7]. Thus, the labeled cells in the NPT probably connect with the secondary olfactory fibers. HRP injections into other regions scarcely led to retrograde labeling of cells in the medial, lateral, posterior or hypothalamic terminal field. Mammalian central olfactory neural pathways can be divided into two main routes, direct projection from the secondary olfactory terminal field to the tertiary olfactory terminal field in the telencephalon [15-19] and another nondirect pro- jection via diencephalon to the telencephalon [19- 23]. However, in teleosts only direct projection is 430 K. OHNISHI eo U. : : a and b. Labeled cells 1 wes pes Fic. 3. HRP injection into medioposterior region of area dorsalis telencephali pars dorsalis (mpDd). n posteroventral region of area dorsalis telencephali pars lateralis (pvDl) after c and d. Labeled cells in nucleus posterior tuberis (NPT) after HRP injection into posterior region of area dorsalis telencephali pars medialis (pDm). The outlined area of a and c is shown at a higher magnification in b and d, respectively. Scale bar=300 um (a and c), 75 um (b and d). known [8]. In the present study, nondirect projection via diencephalon was revealed for the first time in the teleostean olfactory system. This suggests that not only in mammals but also in teleosts two main olfactory routes exist. In the nondirect projection in the monkey, the hypothal- amus receives the tertiary olfactory fibers from the secondary olfactory terminal field (piriform cor- tex) and projects to the telencephalic area (later- oposterior orbitofrontal cortex) [22]. Compared with this pathway, the route via the hypothalamus in the goldfish is more simple. The hypothalamus receives directly fibers from the olfactory bulb not via the secondary olfactory terminal field and projects to the telencephalic area. These results lead to the proposition that the central olfactory nervous systems become more complex with evolution. This view will be confirmed when olfactory projections of olfactory bulb-hypothala- mus-telencephalon are detected in lower verte- brates such as amphibians and reptiles. 3 Murakami et al. [8] have detected in the rockfish, Sebastiscus marmoratus, retrogradely labeled cells in the ipsilateral posterior terminal field after HRP injection into ventral region of the Dm and concluded the region as the tertiary olfactory terminal field. The projection field of the posterior terminal field in the goldfish was the ipsilateral mpDd. This discrepancy may result from method differences. In present study, since HRP injections were strictly limited to the surface layer of the telencephalon, the projections to the inner parts of the telencephalon, i.e. ventral region of Dm, Dc, medial region of DI and area ventralis telencephali, were not examined. Thus, the possibility of the projections to the inner parts from the secondary olfactory terminal fields, espe- cially the medial terminal field whose projection area are not detected, remain unclear. Further- more, compared with projections of the secondary olfactory fibers, those of the tertiary olfactory fibers may differ greatly with the species. To prove Tertiary Olfactory Pathways in Goldfish this hypothesis, investigations on the higher olfac- tory projections, i.e. projections of the secondary olfactory terminal fields in other teleostean spe- cies, are required. 10 11 REFERENCES Bartheld,; C.S., Meyer, D.L., Fiebig, E. and Ebbesson, S.O.E. (1984) Central connections of the olfactory bulb in the goldfish, Carassius auratus. Cell Tissue Res., 238: 475-487. Bass, A. H. (1981) Olfactory bulb efferents in the channel catfish, /ctalurus punctatus. J. Morphol., 169: 91-111. Ebbesson, S.O.E., Meyer, D.L. and Scheich, H. (1981) Connections of the olfactory bulb in the piranha (Serrasalmus nattereri). Cell Tissue Res., 216: 167-180. Finger, T.E. (1975) The distribution of the olfac- tory tracts in the bullhead catfish, [ctalurus nebulo- sus. J. Comp. Neurol., 161: 125-142. Ichikawa, M. (1975) The central projections of the olfactory tract in the goldfish, Carassius auratus. J. Fac. Sci. Univ. Tokyo, 13: 257-263. Ito, H. (1973) Normal and experimental studies on synaptic patterns in the carp telencephalon, with special reference to the secondary olfactory termina- tion. J. Hirnforsch., 14: 237-253. Levine, R.L. and Dethier, S. (1985) The connec- tions between the olfactory bulb and the brain in the goldfish. J. Comp. Neurol., 237: 427-444. Murakami, T., Morita, Y. and Ito, H. (1983) Extrinsic and intrinsic fiber connections of the telencephalon in a teleost, Sebastiscus marmoratus. J. Comp. Neurol., 216: 115-131. Oka, Y. (1980) The origin of the centrifugal fibers to the olfactory bulb in the goldfish, Carassius auratus: An experimental study using the fluorescent dye primuline as a retrograde tracer. Brain Res., 185: 215-225. Scalia, F. and Ebbesson, S. O. E. (1971) The central projections of the olfactory bulb in a teleost (Gym- nothorax funebris). Brain Behav. Evol., 4: 376-399. Fujita, I., Satou, M. and Ueda, K. (1984) A field-potential study of centripetal and centrifugal connections of the olfactory bulb in the carp, 12 13 14 15 16 17 18 19 20 21 jaf 23 431 Cyprinus carpio (L.). Brain Res., 321: 33-44. Peter, R.E. and Gill, V.E. (1975) A stereotaxic atlas and technique for forebrain nuclei of the goldfish, Carassius auratus. J. Comp. Neurol., 159: 69-102. Finger, T. E. (1978) Cerebellar afferents in teleost catfish (Ictaluridae). J. Comp. Neurol., 181: 173- 182. Mesulam, M.-M. (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: A non- carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and effe- rents. J. Histochem. Cytochem., 26: 106-177. Allison, A.C. (1953) The morphology of the olfactory system in the vertebrates. Biol. Rev., 8: 195-244. Gerfen, C.R. and Clavier, R.M. (1979) Neural inputs to the prefrontal agranular insular cortex in the rat: Horseradish peroxidase study. Brain Res. Bull., 4: 347-353. Harberly, L. B. and Price, J. L. (1978) Association and commissural fiber systems of the olfactory cortex of the rat. I. Systems originating in the piriform cortex and adjacent areas. J. Comp. Neurol., 178: 711-740. Kretteks J2 B-vand= Price, J_ E (1977) Erojections from the amygdaloid complex to the cerebral cortex and thalamus in the rat and cat. J. Comp. Neurol., 172: 687-722. Potter, H. and Nauta, W. J. H. (1979) A note on the problem of olfactory associations of the orbitofron- tal cortex in the monkey. Neuroscience, 4: 361-367. Giachetti, I. and MacLeod, P. (1977) Olfactory input to the thalamus: Evidence for a ventroposter- omedial projection. Brain Res., 125: 166-169. Powell, T.P.S., Cowan, W.M. and Raisman, G. (1965) The central olfactory connexions. J. Anat., 99: 791-813. Tanabe, T., Yarita, H., Iino, M., Ooshima, Y. and Takagi, S. F. (1975) An olfactory projection area in orbitofrontal cortex of the monkey. J. Neuro- physiol., 38: 1269-1283. Yarita, H., Iino, M., Tanabe, T., Kogure, S. and Takagi, S.F. (1980) A transthalamic olfactory pathway to orbitofrontal cortex in the monkey. J. Neurophysiol., 43: 69-85. ZOOLOGICAL SCIENCE 4: 433-439 (1987) Gonad Response to y-Aminobutyric Acid in the Sea Urchin NoBUAKI TAKAHASHI Marine Biomedical Institute, Sapporo Medical College, Higashirishiri, Hokkaido 097-01, Japan ABSTRACT—In the sea urchin, Strongylocentrotus intermedius, gonad response to y-aminobutyric acid (GABA) was examined by indirect measurements of gonad contraction. (1) Mechanical response was noted to increase with gonad growth. Young growing gonads showed small response but for maturing, ripe and post-spawning specimens, the response was large. (2) Two different GABA responses on the part of maturing, ripe and post-spawning gonads, and young and growing gonads were thus in evidence. In the former, there were both phasic and rhythmical contractions but in the latter, only phasic contraction. (3) GABA response was also noted to vary according to sex. In the female, the appearance of rhythm was high, but low in the male. (4) The duration of GABA response was about 20 min in maturing and ripe gonads. (5) The threshold concentration of GABA response was 0.01mM. The 50% effective and maximal doses were 0.1mM and 10mM, respectively. (6) The sites of action of GABA injected at a concentration of 10mM were examined. GABA acted directly © 1987 Zoological Society of Japan on the gonads. The generator inducing phasic and rhythmical contraction is discussed. INTRODUCTION Several studies for obtaining gametes from sea urchins have already been performed, such as those involving the use of mechanical damage [1], potassium [1-3] and calcium [1] ions, electrical shock [4-7], acetylcholine [8], and the radial nerve factor [9, 10]. However, no report has so far appeared on a _ physiological triggering mechanism for gamete shedding in sea urchins. The injection of y-aminobutyric acid (GABA) into sea urchins was recently found to induce gamete release [11]. The threshold concentration of GABA for spawning was noted to be about 0.01mM and the optimal for 100% spawning, to exceed 0.1mM. The initiation time of release following the injection was about 12 sec. Thus GABA spawning is a rapid phenomenon. In the present research, this matter was examined in greater detail by measuring the mechanical re- sponse of gonads toward GABA. Portions of this work recently appeared in abstract form [12]. Accepted January 8, 1987 Received November 15, 1986 MATERIALS AND METHODS Animals Sea urchins (Strongylocentrotus intermedius) were collected from the coast of Rishiri Island off northern Hokkaido throughout the year. The animals were kept at our laboratory in an aquarium provided with running seawater until use. Seawater Modified van’t Hoff seawater (ASW) (462 mM NaCl; 9mM KCl; 9mM CaCl; 36mM MgCl; 17mM MgSO,; 20mM Tris-HCl, pH 8.2) was used as the basal incubation medium. K*-rich ASW contained KCl at the concentration of 500mM. NaCl was removed. The composition of other ions was the same as ASW. GABA ASW was made by adding y-aminobutytic acid (Nakarai Chemicals Ltd.) at various concentrations to ASW. Apparatus Okada et al. [13] reported the central part of gonads at the time of spawning to increase in height and termed this height change as potential 434 N. TAKAHASHI difference (PD). The present study also uses this term as well as some of their procedures. The sea urchins were first fixed with large forceps. On the oral side of each specimen, a hole 3cm in diameter was opened by cutting the shell test with solid scissors. A small hole 5mm in diameter on a portion of the remaining test was made by a dental drill. The animal was fixed by placing one foot of the forceps into a small hole and pinching the test (Fig. 11). The PD of the gonads was measured by a strain guage (Fig. lc; SB-1TH, Nihon Koden). The strain guage was horizontally set and connected to a Straw. At a certain point on the horizontally set straw, it was then placed perpendicularly and cut vertically in half with its top made rectangular. It was placed perpendicularly on the central part of the gonad (Fig. 1j). The PD measurement was amplified (Fig. 1b; RP-3, Nihon Koden) and recorded on an ink-writing oscillograph (Fig. 1d; Nihon Koden). Solution exchange was carried out by withdraw- ing the old solution through the straw inserted in Fic. 1. Apparatus for measuring potential differences in sea urchin gonads. the body cavity (Fig. 1h) and then introducing the new solution through a 10 ml pipette (Fig. 1g). Recording The recording was performed at room tempera- ture which varied from 18 to 25°C. The Aristot- le’s lantern and oesophagus were removed from the sea urchin. In experiments on the action site of GABA, the oral and aboral intestines were also removed by forceps. The animal was fixed and immersed up to the equator into filtered natural sea water in a large beaker (Fig. li and e). Soon after the animal had been fixed, its body cavity was filled with coelomic fluid or ASW by pipette. The straw was placed on the gonad and the siphon in the central portion of the body cavity. In that state, the sea urchin was left for 30min. When the gonad was immersed in ASW, exchange of ASW was carried out two or three times during this period. After the 30min, the gonads were treated with GABA for 10 min (Fig. 2). The PD induced by GABA was designated as PDg and the GABA was removed. The gonad [Potential difference | a, main amplifier; b, carrier amplifier; c, strain guage; d, ink-writing oscillograph; e, beaker; f, lab jacks; g, pipette; h, siphon; i, forceps; j, magnification of straw cut vertically in half, with the top made rectangular. Gonad Response to GABA in Sea Urchin 435 + PDx — af PDc : WER chey ea cccees baa ASW (10 min) ASW or (30 min) CF (30 min) Fic. 2. Method for recording. ASW, artificial sea wa- ter; CF, coelomic fluid; PD, potential difference induced by GABA; PDx, potential difference by 500 mMK~*; RR, relative response. was washed two or three times in ASW and then left for 30min. This was followed by treatment with K*-rich ASW. The resulting PD was desig- nated as PDx. The relative value of PDg against PD x was designated as the relative response, RR. Histology To observe gonoduct and gonopore structures, the sea urchin was first placed in decalcified solution for about one week to adequately soften the test. The solution contained 5 ml nitric acid, 10ml formalin and 85ml distilled water. The decalcified sea urchin was cut into small pieces, which were then washed in running water. The gonaoduct and gonopore segments were embed- ded in paraffin according to the usual method and sectioned and stained with hematoxylin-eosin. The silver-staining method for nerve cells was carried out as follows: small pieces of gonad were fixed with 10% formalin for 6hr followed by washing in running water for 4hr. Five drops of ammonia water were added to the solution con- taining the pieces. Twenty-four hours later, they were transferred to a 1.5% silver nitrate solution at 38°C and left for 5 days. The pieces were then placed in a reducing solution consisting of 100 ml distilled water, 15ml formalin and 1g _hydro- quinone. This was followed by embedding in paraffin by the usual method. After sectioning and removal of the paraffin, they were dehy- drated and observed without stain. RESULTS Mechanical response Contraction mode and_ sex differences Mechanical response to GABA was generally of two types. The response in young and growing gonads showed smooth phasic contraction (Fig. 3a). That in maturing, ripe and post-spawning gonads indicated oscillating rhythmical contrac- tion superimposed on phasic contraction (Fig. 3b). Response to GABA ceased at about 20 min (Fig. 4). The time from the start of contraction to its peak was approximately 40 sec. Both small and large oscillations in rhythmical contraction were noted. On some occasions, rhythm could be observed to a small degree near the peak of the phasic contraction (Fig. 3a). Usually, large 200 um 2 min (a) (b) | le aaa | 2mM GABA ar Fic. 3. Response to 2mM GABA. a, a phasic con- traction in growing ovary; b, a rhythmical contrac- tion in addition to phasic contraction in the descending phase of a mature ovary; c, a phasic contraction in a testis. 436 N. TAKAHASHI 400 ym 2min ] | | 2 mM GABA 10 (min) 20 Fic. 4. Mechanical response induced by 2mM GABA, in a mature ovary. Gonad response ceased at 20 min. rhythm could be easily detected in females with mature ripe gonads. Males displayed only phasic contraction in response to GABA (Fig. 3c). Thus, response to GABA varies according to sex. Seasonal variation Seasonal variation of GABA response was observed. RRs in January (1mM GABA), May (2mM) and July (2mM) were 0.89+0.08 (mean+SEM), 0.60+0.11 and 1.03+0.08, respectively. RR in April through June was usually low. In Rishiri Island, the average weight of one lobule from a gonad was 0.86g in May and 1.39 in July in sea urchins 4— 5cm in diameter. Since one lobule in post- spawning and young gonads is usually 0.32 g, the gonads in May were apparently in the growing stage, thus accounting for the response in game- togenic gonads. RR in July was significantly 1.0—- ® : ae a (5) a NN OQ 05— (5) : a « rd (4) (5) 0o— _————— | | | 0.001 0.01 0.1 1 10 Concentration of GABA, mM Fic. 5. Dose-response curve of gonad contraction by GABA. Abscissa, concentration of GABA (mM); ordinate, relative response (RR). Points and ver- tical bars are means+SEM. The number of prepa- rations examined is shown in parentheses. 10mM GABA corresponds to 0.96 RR. higher than in May (P<0.01). This high RR exceeding 1.0 may possibly have been due in part to the abundant release of gametes during the time when the gonads were treated with GABA. Dose-response Dose-response in the present study is repre- sented as the response of PD toward GABA relative to that toward 500mM K*. The experi- ment was performed in February, when the gonads appeared to be in the post-spawning stage and the average weight of one lobule was 0.32 g for sea urchins 4-Scm in diameter. PD peaks were measured as RR. In Figure 5, the threshold concentration is 0.01mM. The 50% effective dose appeared near 0.1mM and maximal one, 10mM. The RR in 10mM GABA was 0.96. Site(s) of action Aboral nerve system The aboral nerve sys- ia ' J “hte ‘ gets £ i : GP =: 300 pm bD | ee “an ete : . - oe 4 ie é : < 7 ce es ; : oe ue : i 5 ee ; { ee Me™ : o 3 . : a4 ». nis 2 Z A ~ 2 4 P iJ- i . a a ii f 4 . > Ss i ii he . f : . & Se 100um Fic. 6. (a) Histology of male sea urchin reproductive system. The test is decalcificated. ANR, aboral nerve ring; GD, gonoduct; GP, gonopore; Te, test. Dotted line indicates a supposed place of aboral nerve ring. (b) Testis stained with silver. Both layers in the gonad wall are stained (two arrow heads). Gonad Response to GABA in Sea Urchin tem appears to be composed of nerve cells and fibers in the nerve ring surrounding the anus, those in the gonoduct and those in the gonad wall. The gonad wall in this animal is composed of three layers, outer coelomic epithelium (viscer- al peritoneum), middle connective tissue with muscle cells and nerve cells, and an inner germi- nal layer [14]. Silver-staining indicated two layers of nerve cells, one just beneath the outer coe- lomic epithelium and the other in the middle connective tissue (Fig. 6b). Some of the nerve cells in the gonad are also possibly associated with those in the gonoduct. As can be seen from Figure 6a, the gonoduct passes through the test and opens to the outside of the gonopore. At the point where the gonoduct enters the test, the nerve process in the gonoduct divides into two branches. By conjugating circularly with branches originating from each of the five lobules in the gonad, the aboral nerve ring is formed and surrounds the anus. Site(s) of action The experiment was per- formed in March, at the time gonads were in the (b) RR (PDc/ PDx«) recs 1.0 0.5 0 i4 437 resting stage. The RR for 10mM GABA was 0.8 (Fig. 7, control in b). To study the site(s) of action of injected GABA, three different experi- ments were carried out. The first experiment was conducted to determine if a supposed action site was the aboral nerve ring. One lobule was separated from the other four in a gonad by cutting nervous branches at both sides of the gonoduct with a surgical knife (Fig. 7, I in a). If the site is actually the aboral nerve rng, no GABA response should occur. In the second experiment, since the gonoduct consisted of several kinds of cell, one lobule was removed from the gonoduct with surgical knife (Fig. 7, II in a). In this experiment, the supposed site was found to be the gonoduct. In the third experi- ment, since the gonad was in contact with the peritoneum, one gonad was separated from the surroundings by cutting the peritoneum and bor- der of the gonoduct (Fig.7, III in a). The supposed site was the gonad. The RR of three different experiments exhibited no significant dif- ference with that of the control (Fig. 7b), indicat- Y WAY WY Ny WN MQ \S YYZ Wf \ WK \\ . N \N \ Control (1) (IN) (ID Site(s) of action of injected GABA. (a) Three different experiments (I, II and III) were carried out. Arrows indicate surgical knife cut. A, anus: ANR, aboral nerve ring; G, gonad; GP, gonopore. (b) The results obtained by (a) are shown in the columns. The number of preparations examined appears in parentheses. Abscissa, results for control and each experiment (I, II and III); ordinate, relative response (RR). 438 N. TAKAHASHI ing GABA to likely act directly on the gonad. When separating the gonad from the peritoneum and gonoduct (Fig. 7, III in a), small rhythmical contractions but no large ones were noted by gonad. DISCUSSION It is well established that starfish spawning is triggered by the release of gonad-stimulating substances (GSS) from supporting cells in nervous tissue containing radial nerves [15]. Recently, Shirai and co-workers [16] found two kinds of peptides with GSS action in Asterias amurensis. A water extract of radial nerves in the sea cucumber is capable of inducing the spawning of the same species and is possibly a peptide [17]. In the sea urchin, radial nerve water extract has also been reported to bring about gamete shedding [9, 10]. Iwata and Fukase [8] found that acetylcho- line, as a neuro-transmitter, induces sea urchin spawning. Recently, gamete shedding in the sea urchin has been observed to occur by injecting GABA [11]. The inhibitory substances depress- ing spawning activity are known to be present in coelomic fluid [18].. The sea urchin thus begins releasing gametes by washing ceolomic fluid with natural seawater. These substances have been identified as L-glutamic acid and aspartic acid [19], possible neuro-transmitters. Thus, sea urchin spawning appears to be associated particu- larly with a certain substance(s) from nervous tissue. The final step in gamete shedding is a contrac- tion of smooth muscle in the gonad wall. Since a ripe gonad in the sea urchin is easily injured in the course of an operation, the extent of gonadal contraction can be determined indirectly by measuring PD. Along with its development and maturation, the mechanical response of a gonad to GABA increases. Moreover, maturing and ripe ovaries display both phasic and rhythmical contraction while young and growing ovaries, only phasic contraction. GABA response is likely to vary according to sex differences. The time required for mechanical response due to GABA in maturing sea urchins is about 20min. On the dose-response curve, the threshold, 50% effec- tive, and maximal concentrations are 0.01 mM, 0.1mM and 10mM, respectively. Our surgical experimental data indicated that GABA acted directly on the gonad and induced contraction and spawning. Thus, the presence of GABA- responding nerve cells in the gonad appears to induce sea urchin spawning. Recently, it has been reported that the five lobules of sea urchin gonads contract synchro- nously and rhythmically through the aboral nerve system [13]. Okada et al. [13] suggested that the gonoduct generated this rhythm. From the data of the present study, it is evident that a lobule separated from the surroundings is also capable of inducing small but not large rhythm. This in turn appears to result in contraction of the gonad itself and vibration of the rhythmical contraction of the other four lobules. The generator of this rhythm should be studied cytologically. In conclusion, gonad response to GABA appears to be both phasic and rhythmical contrac- tions, each possibly originating from a different generator. That is, phasic contraction was noted to occur throughout the year, and rhythmical contraction, at the time of gonad maturation. Gonad contraction and spawning appear to be explained quite well if at least two different generators are assumed to be operative. At any rate, GABA appears to act on both these differ- ent generators. ACKNOWLEDGMENTS The author is grateful to Dr. M. Takahashi, Sapporo Medical College, and Professor M. Yoshida, Okayama University, for their valuable comments. He also thanks Professor K. Kikuchi, President of Sapporo Medical College, and Professor K. Takahashi, Sapporo Medical College, for their encouragement throughout the course of this work. This work was supported by a grant from the Hokkaido Newspaper Office for Social and Natural Scientific Research. REFERENCES 1 Palmer, L. (1937) The shedding reaction in Arbacia punctulata. Physiol. Zo6l., 10: 352-367. 2 Harvey, E.B. (1939) A method of determining the sex of Arbacia, and a new method of producing twins, triplets and quadruplets. Biol. Bull., 77: 312. 10 11 12 Gonad Response to GABA in Sea Urchin Harvey, E. B. (1940) A note on determining the sex of Arbacia punctulata. Biol. Bull., 79: 363. Iwata, K.S. (1950) A method of determining the sex of sea urchins and of obtaining eggs by electric stimulation. Annot. Zool. Japon., 23: 39-42. Harvey, E. B. (1952) Electrical method of “sexing” Arbacia and obtaining small quantities of eggs. Biol. Bull., 102: 284. Harvey, E. B. (1953) A simplified electrical method of determining the sex of sea urchins and other marine animals. Biol. Bull., 105: 365. Iwata, K.S. (1962) A simplified electrical method of determining the sex of sea urchins. Zool. Mag., 71: 301-302. Iwata, K.S. and Fukase, H. (1964) Artificial spawn- ing in sea urchins by acetylcholine. Biol. J. Okayama Univ., 10: 51-56. Cochran, R. C. and Engelmann, F. (1972) Echinoid spawning induced by a radial nerve factor. Scierice, 178: 423-424. Cochran, R.C. and Engelmann, F. (1976) Charac- terization of spawning-inducing factors in the sea- urchin, Strongylocentrotus purpuratus. Gen. Comp. Endocrinol., 30: 189-197. Takahashi, N. (1986) The spawing of the sea- urchin, Strongylocentrotus intermedius, by _ jy- aminobutyric acid. Bull. Japan. Soc. Sci. Fish., 52: 2041. Takahashi, N. (1986) Gamete shedding by sea 13 14 15 16 17 18 19 439 urchins in response to y-aminobutyric acid. Dev. Growth Differ., 28: 384. Okada, Y., Iwata, K.S. and Yanagihara, M. (1984) Synchronized rhythmic contractions among five gonadal lobes in the shedding sea urchins: coordina- tive function of the aboral nerve ring. Biol. Bull., 166: 228-236. Kawaguchi, S. (1965) Electron microscopy on the ovarian wall of the echinoid with special references to its muscles and nerve plexus. Biol. J. Okayama Univ., 11: 66-74. de Angelis, E., Viglia, A. Watanabe, T., Shirai, H., Kubota, J. and Kanatani, H. (1972) Presence of granules containing gonad-stimulating substance in starfish radial nerve. Annot. Zool. Japon., 45: 16- SAL Shirai, H., Bulet,P., Kanatani,H., Kondo,N., Imai, K., Isobe, M., Goto, T.and Kubota, I. (1985) Purification of gonad-stimulating substance in starfish. Zool. Sci., 2: 912. Maruyama, Y.K. (1985) Holothurian oocyte maturation induced by radial nerve. Biol. Bull., 168: 249-262. Okada, Y. and Iwata, K.S. (1985) A substance inhibiting rhythmic contraction of the gonad in the shedding sea urchin. Zool. Sci., 2: 805-808. Nogi, H. and Yoshida, M. (1984) Inhibitory sub- stance of rhythmic contraction in sea urchin gonad. TASS, SOb5 1 i/3 _ (h201). VE chaste, Rone A ah Bie wi i 4 staal pens “utd Hovis. ah to. bord a é + | Lege tee ofeadivdy the aii niletel dca ae | Tpitoste ak a3" 5 eae . Riise pairs iiiiens a ctor UAT ori Pench Lee Wee v2 | Se Cue apie HoH i ight ane orind Tete od 15 ait Aant wi tyaohan Wh ius bare atin . | ete sO ai fe aOR CRON Ee Mi Rae aie : opal y ‘ re ol ‘ ; ; uae. 7 Pay | | 2 Wawenwe + elt NEAL Tmo ye rin: carr ’ > ; } ; : f" yy ; aL by badtara, Jatin! puke fe sitiganly, A &e i ‘ Oh FAL, ant COE PT ) Rae } Nr : WEA Na ey ‘ ee ek { Hie) etsy: ‘agi lta qe ' t, my ei ro gihe oa0 th ip iow | ae " 4 2 tel) : WA Te #D Beha) tn hs ieee) Bit Ale qe ie | 3 ; » | Mi ; : a heh: looks earn ey ese is ; ‘ay r i / y ihe ou AM UR At! ot te ae SAR es SPT e toy ie WeeTLA 3 ial ee eh MELE a, mL fh Paro st He ti (rib) re (per Ue ae MG ans 8 ¢ ‘ } , i . at . vis L iA lode yorulitieh ice. ie r ve ur he { ; if: orca. ees : Pe LEB Me : poy LES f y ‘ 4 sy | tt?) ia Oped } MRE bea fh Gr ayy fi paces Misi ; reds Capit ¢ iy @ en r ‘ps dy ns * ¥ ie yn i A eet Put hat wr Bap ” Are 7.0 AA hw 48 ey dre f id scab ee ones a td \ Chie hha tes Po bia ted Bah V5, i i & At ' ; Wi “ { } j ; i j A | A , { ie Le 1 ye Rt iT} Ce yh Ps 77 Tha hk Bs’ RRA ALLONEE Sh: eb ee ee, 7D wee eee rae oni Tp Eh phate ot) oh oF, os wih Ps +a he Dal on ay ia TF hy : age we #I Vee ves anys 't ee § A ae 4 ‘ia. ban foal i -r ere le 4 I ah Ut tM bie ae yy ate CORE EE ts Fi ai erarit. 2; i) } .) 4 y WT, | * \ fe oi { Pacd i Lt Vy ' on i i Fe j . ra e, r Py ry \ ‘ ae a . ? fi ; a i s 4 ; 1 Te rs i vn t ih vo t 1) Ugh 4 Fh aD eae ‘ ' ¥ ? , ‘ - a9 , ’ : wes aey x :% 1S ek a jive st si hi a rr dea le f ] i ty pee? ‘ ZOOLOGICAL SCIENCE 4: 441-446 (1987) Gonad Response to Calcium and a Comparison of the Effects of Calcium, Potassium, Acetylcholine and y-Aminobutyric Acid on the Sea Urchin Gonad NoBUAKI TAKAHASHI AND MASAKI TAKAHASHI Marine Biomedical Institute, Sapporo Medical College, Higashirishiri, Hokkaido 097-01, Japan ABSTRACT— Gonad response to calcium and the effects of calcium, potassium, acetylcholine (ACh) and y-aminobutyric acid (GABA) on the gonads of the sea urchin, Strongylocentrotus intermedius were studied on the basis of measurements of the potential difference (PD). Rhythmical contraction due to Ca** occurred at a concentration exceeding 50mM and its frequency increased as the concentration rose above this. In mature gonads, rhythmical contraction lasted for over an hour. Among four stimuli, ACh initiated threshold-contraction at the lowest concentration, 10~*° or 10~’M. In relative response (RR) indicating the PD of a certain stimulation against that of 500mM K”, the RR of GABA and K* reached 1.0 but Ca** and ACh saturated near 0.8. Ca** and GABA induced both rhythmical and smooth phasic contraction while K* and ACh, only smooth long lasting contraction. The response to 50mM K* and 50mM Ca** persisted for more than an hour but that © 1987 Zoological Society of Japan toward 2mM GABA and 2mM ACh, only about 20 min. INTRODUCTION Palmer [1] indicated that, on dropping potas- sium chloride directly onto a gonad surface, visible steady contraction of the gonad wall resulted and following the injection of this salt, a genital substance was released with force from all five pores. At the time of this observation, he devised a procedure for obtaining sea urchin gametes. He also found that a drop of isotonic calcium chloride on a gonad surface appeared to cause the wall to contract and relax in a rhythmical manner and the injection of this salt to cause characteristic rhyth- mical shedding followed by the complete release of genital substance from the gonads. In the preced- ing papers, y-aminobutyric acid (GABA) was found to induce rhythmical as well as phasic contraction of the sea urchin gonad and gamete shedding [2,3]. This prompted the authors to examine again gonad rhythmical contraction using calcium. In the present study, measurements were made of rhythmical contraction induced by cal- cium. Calcium, potassium, acetylcholine (ACh) Accepted January 8, 1987 Received November 15, 1986 and GABA were compared for their ability to bring about gonad response. MATERIALS AND METHODS Animals Sea urchins (Strongylocentroturs intermedius) were collected from the coast of Rishiri Island off northern Hokkaido during all seasons of the year. The animals were kept at our laboratory in an aquarium provided with running seawater until use. Seawater Modified van’t Hoff seawater (ASW) (462mM NaCl; 9mM KCl; 9mM CaCl; 36mM MgCl; 17mM MgSO,; 20 mM Tris-HCl, pH 8.2) was used as the basal incubation medium. Ca**-rich ASW was prepared at various concentration of Ca**, using CaCl, in place of NaCl to maintain isotonic- ity. K*-rich ASW was prepared in a similar manner. ACh (Sigma Chemical Co.) and GABA (Nakarai Chemicals Ltd.) seawater solutions were made by the addition of various concentrations of each chemicals to ASW. 442 N. TAKAHASHI AND M. TAKAHASHI Apparatus The apparatus used in the present study is described in the preceding paper [2]. Recording The recording was carried out at room tempera- ture which varied from 18 to 25°C. To measure gonad contraction, namely potential difference (PD) devised by Okada et al. [4], a hole 3cm in diameter was made on the oral side of each sea urchin with solid scissors followed by the removal of Aristotle’s lantern and the oesophagus by forceps. The animal was subsequently fixed by pinching the test with large forceps and immersed up to its equator in filtered natural seawater in a beaker and soon after, the body cavity was filled with ASW. The straw previously cut vertically in half and connected to a strain guage (SB-1TH, Nihon Koden) was placed on a gonad and the siphon in the central portion of body cavity. The gonad was allowed to remain in this state for 30 min; during which time the ASW was changed two or three times. The gonad was first treated with a stimulant at a concentration of x mM until immediately following a peak of induced contrac- tion (Fig. 1). The gonad was washed, placed in ASW solution and 30min later, the same treat- ment was repeated but at a stimulant concentra- tion of y mM (Fig. 1). This was followed by washing the gonad and placing it in ASW for 30min. It was finally treated with 500mM K* SED! PDx ee nak RR ek PDx PDy PDx / \ (30 min ) (30) (30) Sin) SOD y mM St. 500 mM K* ASW Fic. 1. Method for measuring relative response (RR). In a gonad, two RRs were measured at different concentrations of a stimulant. Immediately after PD peak was reached, the gonad was washed and immersed in ASW for 30 min. ASW, artificial sea water; PD, potential difference; St., stimulant. ASW. Mechanical response by 500mM K* was designated as PDso9x o6,x The relative value of PDg; (stimulant) against PD, was represented by relative response (RR). In this way, the two RRs of one preparation were measured. RESULTS Ca** effects Dose-response experiment This experiment was conducted in March. The average weight of one lobule in a gonad was 0.28 g and from external observation, it appeared to be in the resting state. The first recording was generally performed at a low Ca** concentration and the second, at a high concentration. The filled circles in Figure 2 10> (6) (6) ¢ a RR (PDca/ PDk ) it (6) fe) ee 06 O [ | Mien 9 15 25 50 100 Concentration of Ca’", mM Fic. 2. Dose-response curve of potential difference (PD) by Ca’t. Filled circles indicate rhythmical contraction. Points and vertical bars are means+ SEM. The number of preparations assayed appears in the parentheses. Abscissa, concentration of Ca** (mM); ordinate, relative response (RR). indicate the occurrence of rhythmical contraction superimposed on phasic one. The highest peak of rhythmical contraction was measured. The Ca*t concentration inducing a_threshold-contraction was about 20mM. At about 100mM, the dose- response curve indicated saturation of gonad response to Ca”* with an RR of 0.74. The 50% effective dose ranged from 30mM to 40 mM Ca’". For generation of rhythm, the _ threshold- contraction was induced at a concentration of 40mM Ca’*. Contraction mode Figure 3 a and b were recorded using the same ovary. The sea urchin Ca?*,K*, ACh and GABA on Sea Urchin Gonad 443 (a) (b) 50 mMCa™ 100 mM Ca’ | eas 50 mM Ca” 10( min) Fic. 3. Several features of PD by Ca’*. concentration. (a) 50mM Ca’*; (b) 100mM Ca?*. | | | | 20 30 60 70 Frequency of rhythm was noted to rise with increase in Ca*t All time course of rhythmical contraction by 50mM Ca?’* is shown in (c). The rhythm continued for 70 min. from which it came spawned in response to over 50mM Ca**. The frequency of the rhythm increased with Ca** concentration. The initiation of rhythm occurred more quickly at 100mM than at 50mM. Rhythm was greater at 50mM than at 100mM, since, owing to its high frequency at 100mM Ca?t, it became superimposed on itself. All the time courses of rhythmical contraction induced by 50mM Ca** are shown in Figure 3c. The highest frequency was noted within 6-8 min. The contraction continued for 70min following immersion of the gonad in the Ca**-rich ASW and then ceased. Generally, the rhythm in young gonad did not occur easily. K* effects Dose-response experiment The experiment was carried out in March. The average weight of one lobule was 0.34 g and the gonads appeared to be either in the resting or young stage. At 100mM, gonad RR was maximium (Fig. 4). The threshold concentration ranged from 9mM_ to 15 mM, with the 50% effective dose being 30mM. Response difference according to sex could not be , RR (PDxy/PDs00K) Concentration of K*, mM Fic. 4. Dose-response curve of potential difference (PD) by K*. Points and vertical bars are means + SEM. The number of preparations assayed appears in parentheses. Abscissa, concentration of K* (mM); ordinate, relative response (RR). detected. Contraction mode Although, when a few drops of 0.5 KCI were added to coelomic fluid, the sea urchin gonad induced rhythmical contraction [4], Kt-rich ASW prepared at various concentra- tions of K* generally failed to produce any rhythm. Contraction due to K* at concentration exceeding 100 mM was noted to be quite active but 444 N. TAKAHASHI AND M. TAKAHASHI to be followed by a state of rigor. That is, gonad relaxation was rendered impossible by K* treat- 10 (6) (G5)) (5) - & a SO (5) : f a a (5 yee (5) , o ar mi RONG eed IE r 10” 10” 10 10 10 10 Concentration of ACh, M Fic. 5. Dose-response curve of potential difference (PD) by ACh. Points and vertical bars are means +SEM. The number in parentheses is number of preparations assayed. Abscissa, concentration of ACh (M); ordinate, relative response (RR). (a) 50mMK’ ee 10(min) (b ) 2 mM ACh 1 0(min) (c ) 2 mM GABA ment. The contraction produced by K* at 50mM is shown in Figure6a. To bring about the relaxation of the gonad, one hour was required. No rhythm could be observed. ACh effects Dose-response experiment This experiment was performed in March. The average weight of one lobule was 0.27 g. The gonads appeared to be in the resting stage. The threshold contraction- concentration was 107° to 10~’M (Fig. 5). RR at concentrations exceeding 10~*M ACh was satu- rated at a value of 0.8. The 50% effective dose was about 10°°M. Sex difference could not be detected. Contraction mode No rhythmical contrac- tion occurred at any ACh concentrations used. Relaxation of contraction by high ACh concentra- ZO MSO MBO 1 0(min) 20 Fic. 6. Potential difference (PD) for each of three stimuli. (a) 50mM K*; (b) 2mM ACh; (c)2mM GABA. Time course of PD in 50mM K* was about one hour and that of ACh and GABA (2mM) was about 20 min. Ca**, K*, ACh and GABA on Sea Urchin Gonad 445 tion (2mM) was possible, as evident from Figure 6b. The gonad was a testis and sperm continued to be released for 15 min. ACh contractions general- ly terminated within 20 min. GABA effects Dose-response experiment The data _pre- sented here have already been presented in the preceding paper [2]. Dose-response is shown in Figure 7. The threshold, 50% effective, and maximal doses were 0.01mM, 0.1mM and 10mM GABA, respectively. 1.0 RR (PDst./ PDs500x) ° stimuli, M Concentration of various Fic. 7. Dose-response curves of potential difference (PD) by Ca**+, K*, ACh and GABA, on the same co-ordinate. Dotted lines indicated rhythmical con- traction. Abscissa, concentration of stimuli; ordi- nate, relative response (RR). Contraction mode The type of GABA con- traction has already been presented in Figure 4 of the preceding paper, but in the present research as well, the contraction was found to be rhythmical (Fig. 6c). This was noted in July. Response to GABA ceased in 20 min. DISCUSSION Ca’* response Palmer [1] found that, on placing a drop of isotonic calcium chloride on the surface of a gonad of Arbacia punctulata, its wall appeared to contract and relax rhythmically. The data of the present research confirm this observation. The rhythmical contraction of a gonad of Strongylocentrotus inter- medius was recorded with an oscillograph. Also, in his investigation, following an injection of CaCl, a definite relation between Ca** concen- tration and both the time prior to visible shedding and duration of the shedding reaction could be clearly discerned. In the present study, rhythm frequency was observed to increase with Ca?+ concentration. The rate of increase in PD was also found to do the same. In regard to gamete shedding in response to Ca**, Palmer found the average time interval between an injection of CaCl, and gamete shedding to depend on sex type. The time of sperm shedding for a 1 ml injection of 0.3 M CaCl was 18 sec and that of egg shedding, 95 sec. In the present in vitro experiments, such variation according to sex could not be detected. Threshold concentration and rate of increase in calcium contraction were the same for both male and female. Rhythmical contraction also occurred at the same concentration (SOmM) in both sexes. The absence of any variation according to sex may possibly have been due to the conditions of the in vitro experiment and/or individual species differ- ences. Comparison of response to Ca’*, K*, ACh and GABA Dose-response curves for Ca2*, K*, ACh and GABA appear on the same co-ordinate in Figure 7. The dotted line indicates rhythmical contrac- tion. The threshold concentration of ACh is lower than that of the others. RR in the case of GABA and K* was as much as 1.0 but that in Ca?* and ACh, 0.8. The duration of response to K* and Ca** was in proportion to their concentration and more than an hour. But for GABA and ACh, the duration was about 20min. GABA and Ca’* induced both rhythmical and phasic contraction but K* and ACh, only slow phasic or long lasting contraction. The gonad displayed at least two kinds of contraction with or without rhythm. Consequent- ly, it may be considered that contractions are produced by at least two different types of gener- ators, one inducing smooth phasic and the other, rhythmical contraction. The present data show the first type of generator to be influenced by K”, ACh and GABA and the second, by Ca** and GABA. Generation in one case appears to involve stable reactions with various stimuli regardless of 446 N. TAKAHASHI AND M. TAKAHASHI the season but the latter is apparently influenced by season since the number of such reactions was noted to increase with gonad maturation. This confirms the possibility of at least different types of generators. Certain muscles of echinoderm are known to be responsive to ACh. In the lantern retractor muscle of the sea urchin, ACh at a concentration of 10-°>M has been found almost invariably to produce powerful contraction which persists with hardly any change for at least 80 min [5]. Moreo- ver, the sea urchin oesophagus has been shown to contract through the action of ACh. Thus, although neuro-muscular transmission in the sea urchin is generally considered cholinergic, the gonad wall responds to GABA as well as ACh [2, 7|. The wall is composed of three layers, outer coelomic epithelium (visceral peritoneum), middle connective tissue with muscle and nerve cells, and an inner germinal layer [8]. The middle layer is further divided into five sub-layers, outer connec- tive tissue, a muscle layer, central connective tissue, a nerve plexus, and inner connective tissue. Davis [9] showed nerve cell processes are present between the visceral peritoneum and the outer connective tissue. Thus, the muscles constitute one layer but the nerves, two layers in the gonad wall of the sea urchin [2]. The one muscle layer may possibly be controlled by various types of nerves whose determination should be made so as to gain an understanding of the spawning mecha- nism in the sea urchin. ACKNOWLEDGMENTS The authors are grateful to Professor K. Kikuchi, President of Sapporo Medical College, and Professor K. Takahashi, Sapporo Medical College, for their en- couragement throughout this work. This work was supported by a grant from the Hokkaido Newspaper Office for Social and Natural Scientific Research (N.T). REFERENCES 1 Palmer, L. (1937) The shedding reaction in Arbacia punctulata. Physiol. Zo6l., 10: 352-367. 2 Takahashi,N. (1987) Gonad response to j- aminobutyric acid in the sea urchin. Zool. Sci., 4: 433-439. 3. Takahashi, N. (1986) The spawning of the sea- urchin, Strongylocentrotus intermedius, by y- aminobutyric acid. Bull. Japan. Soc. Sci. Fish., 52: 2041. 4 Okada, Y., Iwata, K.S. and Yanagihara, M. (1984) Synchronized rhythmic contractions among five gonadal lobes in the shedding sea urchins: coordina- tive function of the aboral nerve ring. Biol. Bull., 166: 228-236. 5 Boltt, R.E. and Ewer, D. W. (1963) Studies on the myoneural physiology of Echinodermata. V. The lantern retractor muscle of Parechinus: responses to drugs. J. Exp. Biol., 40: 727-733. 6 Florey, E. and McLennan, H. (1959) The effects of factor I and of gamma-aminobutyric acid on smooth muscle preparations. J. Physiol., 145: 66-76. 7 Nogi,H. and Yoshida, M. (1984) Inhibitory sub- stance of rhythmic contraction in sea urchin gonad. Zool; Scr, 177873: 8 Kawaguchi, S. (1965) Electron microscopy on the ovarian wall of the echinoid with special references to its muscles and nerve plexus. Biol. J. Okayama Univ., 11: 66-74. 9 Davis, H. (1971) The gonad wall of Echinodermata: a comparative study based on electron microscopy. MSc Thesis, Univ. of California, San Diego, 90 pp. ZOOLOGICAL SCIENCE 4: 447-450 (1987) The Mediation of Cyclic AMP in Octopaminergic Modulation at Neuromuscular Junctions of the Mealworm, Tenebrio molitor Osamu Hipou! and Jun-Icur FuKamr'’? Institute of Agriculture and Forestry, University of Tsukuba, Sakura-mura, Ibaraki 305, and *Laboratory of Insect Toxicology, Institute of Physical and Chemical Research, Wako, Saitama 351, Japan ABSTRACT— The possibility that there is mediation of cyclic AMP in the octopaminergic modulation of neuromuscular transmission was examined. Octopamine (1M) potentiated the amplitude of neurally-evoked excitatory junctional potentials (EJPs) at the neuromuscular junctions on the ventral longitudinal muscle of the mealworm, Tenebrio molitor. Although lower doses of octopamine (0.01 ~M) or cyclic AMP (100 “M) had no effects on EJPs, the amplitude of EJPs was potentiated by these compounds when the phosphodiesterase activity was inhibited with simultaneous application of phosphodiesterase inhibitor, isobutyl-1-methylxanthine (IBMX, 104M). No effects of octopamine, cyclic AMP or IBMX on the time constant of the decay phase of the EJPs and the resting membrane potentials were observed. The results support the presynaptic mediation of cyclic AMP in octopa- © 1987 Zoological Society of Japan minergic modulation at neuromuscular junctions. INTRODUCTION Octopamine is a multifunctional biogenic amine, believed to be a neurotransmitter, a neurohor- mone and a neuromodulator in the insect nervous system [1, 2]. Glutaminergic neuromuscular trans- mission is well modulated by this amine. Pre- synaptic action of octopamine enhances the neuro- muscular transmission in the locust [3], in Man- duca [4], resulting in the potentiation of the amplitude of excitatory junctional potentials (EJPs) and contributing to the potentiation of twitch tension [3, 5]. There is growing evidence that adenosine 3, 5-cyclic monophosphate (cyclic AMP) plays a role as cellular mediator in the nervous system. Octo- pamine is widely distributed in insect nervous system [1, 6] and specific increase in cyclic AMP induced by octopamine has been reported in various neural tissues [7-13]. These observations suggest that a broad variety of octopaminergic actions is mediated via a second messenger system Accepted December 25, 1986 Received November 18, 1986 1 Present address: Laboratory of Biology, Meiji College of Pharmacy, Setagaya, Tokyo 154, Japan. [14, 15]. It seems, therefore, that there is mediation of cyclic AMP in the modulatory action of octopa- mine at neuromuscular junctions in insects. The present work was undertaken to examine the mediation of cyclic AMP by using phosphodiester- ase inhibitor, isobutyl-1-methylxanthine (IBMX) [16]. MATERIALS AND METHODS Larvae of the mealworm (Tenebrio molitor) were used. The neuromuscular preparation em- ployed was similar to that described by Yamamoto and Washio [17]. The ventral longitudinal muscle fibers of an abdominal segment were exposed by slitting open the larvae along the dorsal midline and removing the alimentary canal and fat bodies. The dissected animal was mounted on a perfusion chamber made of Sylgard Resin (Dow-Corning Co.) and was continuously perfused with Tenebrio saline containing 70mM NaCl, 30mM KCI, 14.4 mM MgCh, 0.6mM CaCl,, 445mM glucose and 5mM HEPES. The pH of this saline was adjusted to 7.2 with NaOH. Octopamine HCI and adeno- sine 3’,5-cyclic monophosphate (cyclic AMP) 448 O. HIDOH AND J. FUKAMI were obtained from Sigma Chemical Co. Isobutyl- 1-methylxanthine (IBMX) was obtained from Aldrich Chemical Co. These chemicals were dissolved in the saline. Intracellular recordings from a ventral muscle fiber were made with a glass microelectrode filled with 3M KCl (5-10MQ). To elicit EJPs, the motor nerve innervating the ventral muscle was stimulated at a point close to the segmental ganglion with square pulses of 0.1 msec duration using a pair of fine Ag-hook electrodes. Sixteen EJPs evoked at 2Hz were averaged to estimate their amplitude, the time constant of single ex- ponential decay phases and resting membrane potentials. These averaging treatments were per- formed every one minute. All experiments were carried out at room temperature (24-26 °C). RESULTS The effects of treatments were examined by three parameters: the amplitude of EJPs, the time constant of their single exponential decay phase and resting membrane potentials. Neurally- evoked EJPs showed no facilitation properties when they were evoked at 2Hz. Results obtained are summarized in Table 1. Time constant of single exponential decay phase and resting mem- brane potentials were unchanged with all treat- ments. control treated , | i \ OA +1BMX oom | ae \ Hae N fee N\ cAMP +IBMX a ‘ ner Fic. 1. Action of octopamine, cyclic AMP and IBMX on neurally-evoked EJPs. Sixteen EJPs evoked at 2 Hz were averaged before (control) and 5 min after (treated) the onset of the treatments. Upper two traces were treated with 0.01 ~M octopamine and 10 uM IBMX. Lower two traces were treated with 100 uM cyclic AMP and 10 uM IBMX. Calibration: SmV, 20 msec. Phosphodiesterase inhibitor, IBMX, added to perfusate at a concentration of 10 uM (designated as 10 uM IBMX treatment) had no effect on EJP amplitude. Lower doses of octopamine (0.01 u.M octopamine treatment) also had no effect. In contrast, these two compounds applied at the same time (0.01 4M octopamine plus 104M IBMX treatment) modulated neuromuscular transmis- sion. The amplitude of EJPs was significantly potentiated; the upper two traces in Figure 1 represent this action. The time course comparing 0.01 uM octopamine treatment with 0.01 uM octo- pamine plus 10 .M IBMX treatment is drawn in Figure 2 A. Treatment by octopamine alone did TABLE 1. The effects of octopamine, cyclic AMP and IBMX on the neurally-evoked EJPs Treatment EJP amplitude (mV) Time constant (ms) Resting potential (mV) N control/treated control/treated control/treated OA (1 uM) S24 9E 23 17.8+4.0/17.5+3.8 —4A820s=2 3) — 46n lit Onn OA (1 2M) : +IBMX (10 pM) S 2a lRO/NSES e2,.3, 16.2+2.4/17.1+2.8 =50.01.9/ =49:1--2:379%6 OA (0.01 uM) iSjateysie I(/ al)q [SEN 3) 12.0+0.4/12.8+0.2 te WaeSWPSCVaeil( 2 OA (0.01 uM) x + IBMX (10M) Pellarilall Oe 3 2DaeOI/3 Oae0.© —48.2+1.5/—48.541.9 5 cAMP (100 uM) Use Ite se (0).5) NOQse 223 tS)2ae 22 —49.2+2.0/—48.842.2 5 cAMP (100 uM) if +IBMX (10M) (eae esi sae A Wessel Sse 2.3) —49.8+1.4/—48.5+1.3 6 IBMX (10 «M) ae ZI esoyae le 7/ 13.0+0.4/12.6+0.5 =—5122.5/—48:9 2.1, 5 Each value is the mean and S.E. just before (control) and Smin after (treated) the onset of the treatments. octopamine. cAMP: cyclic AMP. *indicates the mean is significantly increased at P<0.01 by a paired-sample f-test. IBMX: isobutyl-1-methylxanthine. OA: Cyclic AMP in Octopaminergic Modulation 449 (mv ) AEJP amplitude time Fic. 2. Time courses of the effect on the EJP ampli- tude by octopamine, cyclic AMP and IBMX. A: 0.01 ~M octopamine (©) and 0.01 “4M octopamine plus 10 ~M IBMX (@) were added to the perfusate for 5 min. B: 100 4M cyclic AMP (0) and 100 uM cyclic AMP plus 104M IBMX (@) were added to the perfusate for 5 min. Each point represents the mean of difference of the EJP amplitude obtained from 5 or 6 determinations; vertical bars represent S2E: not potentiate EJP amplitude, whereas octopa- mine plus IBMX resulted in gradual potentiation. The results were different in the case of octopa- mine (1u#M) and IBMX(10“M). EJPs were potentiated both by 14M octopamine treatment and 14M octopamine plus 104M IBMxX treat- ment. Potencies inducing potentiation between these two conditions exhibited the same strength. Amplitude of EJPs increased by 6.7+1.6mV (n= 5) with treatment of 1 ~M octopamine alone and by 5.6+0.9mV (n=6) with 1 ~M octopamine plus 10 uM IBMX treatment (means and S.E.) (Table 1). These values were not different by two sample t-test. The action of cyclic AMP had similarities with the potentiating action of octopamine (0.01 “M). One hundred uM cyclic AMP treatment was not effective, whereas 100 uM cyclic AMP plus 10 uM IBMX treatment potentiated the amplitude of EJPs; the lower two traces in Figure 1 represent potentiating action by this treatment. As in the case of 0.01 4M octopamine plus 104M IBMX treatment, the time course of potentiating action is drawn in Figure 2B, and also shows the gradual increase by 100 uM cyclic AMP plus 10 ~«M IBMX treatment. DISCUSSION Presynaptic action of octopamine has been revealed by an increase in the frequency of miniature EJPs (MEJPs) at neuromuscular junc- tions in locust extensor tibiae muscle [3]. Although major changes in cyclic AMP levels occur postsynaptically, octopamine increases cyclic AMP levels in this neuromuscular preparation [12]. Elevation of cyclic AMP levels by phospho- diesterase inhibitors [18, 19] and by a series of cyclic AMP analogues [19] causes an increase in the frequency of MEJPs, showing similarities of action and time course of octopamine on presynap- tic terminals. The elevation of cyclic AMP levels by octopamine in the presynaptic terminals is likely to affect transmitter release by the cyclic AMP-dependent protein kinase, changing the cal- cium levels [20]. At Tenebrio neuromuscular junctions, octopa- mine-induced potentiation of EJP amplitude is contributed to the increase of quantal contents which are estimated by extracellularly-recorded EJP failures, whereas the frequency of MEJPs is not affected by octopamine [21]. These actions suggest the enhancement of calcium accumulation that is associated with the impulse invasion into the presynaptic terminals. In the present study, the amplitude of EJPs was potentiated by 0.01 4M octopamine plus 104M IBMX treatment but not by treatment with 0.01 uM octopamine alone, suggesting that octopamine elevates cyclic AMP levels in the presynaptic terminals. Even 0.01 4M octopamine may elevate cyclic AMP levels; the elevation, however, is so slight that the natural phosphodiesterase inacti- 450 vates cyclic AMP. If this inactivation is inhibited by IBMX, the elevation is sufficient that cyclic AMP exerts potentiating action. In the case of cyclic AMP treatment, the same event related to phosphodiesterase may occur. The elevation of cyclic AMP by 14M octopamine treatment may exceed an optimum level, so that 1 ~M octopamine plus 10 «M IBMX treatment potentiates the ampli- tude of EJPs in the same way as 1 uM octopamine alone. The mediation of cyclic AMP is also suggested by the similar time courses of octopamine and of cyclic AMP treatments shown in Figure 2, and by similar effects on the amplitude of EJPs but not on the time constant of decay phase or the resting membrane potentials. Our results support the presynaptic mediation of cyclic AMP in octopaminergic modulation at insect neuromuscular junctions. It is important to point out that neurally-evoked transmitter release is enhanced by the cyclic AMP-mediated octopa- minergic action, because this release is closely concerned with muscle contraction. REFERENCES 1 Evans, P.D. (1980) Biogenic amines in the insect nervous system. Adv. Insect Physiol., 15: 317-473. 2 Orchard, I. (1982) Octopamine in insects: neuro- transmitter, neurohormone, and neuromodulator. Can. J. Zool., 60: 659-669. 3. O’Shea, M. and Evans, P. D. (1979) Potentiation of neuromuscular transmission by an octopaminergic neurone in the locust. J. Exp. Biol., 79: 169-190. 4 Klaassen, L.W. and Kammer, A.E. (1985) Octo- pamine enhances neuromuscular transmission in developing and adult moths, Manduca sexta. J. Neurobiol., 16: 227-243. 5 Evans, P.D. and O’Shea, M. (1978) The identifica- tion of an octopaminergic neurone and modulation of a myogenic rhythm in the locust. J. Exp. Biol., 73: 235-260. 6 Evans, P. D. (1978) Octopamine distribution in the insect nervous system: distribution, synthesis and metabolism. J. Neurochem., 41: 562-568. 7 Nathanson, J. A. and Greengard, P. (1973) Octopa- mine-sensitive adenylate cyclase: evidence for a biological role of octopamine in nervous tissue. Science, 180: 308-310. 8 Harmer, A.J. and Horn, A.S. (1977) Octopamine sensitive adenylate cyclase in cockroach brain: 10 iit 2 13 14 tS 16 17 18 1S 20 21 O. HIDOH AND J. FUKAMI effects of agonists, antagonists and guanylyl nu- cleotides. Mol. Pharmacol., 13: 512-520. Bodnaryk, R.P. (1979) Characterization of an octopamine-sensitive adenylate cyclase from insect brain (Mamestra configurata Wlk). Can. J. Biochem., 57: 226-2372. Bodnaryk, R.P. (1979) Identification of specific dopamine- and octopamine-sensitive adenylate cy- clases in the brain of Mamestra configurata WIlk. Insect Biochem., 9: 155-162. Bodnaryk, R. P. (1979) Basal, dopamine- and octo- pamine-stimulated adenylate cyclase activity in the brain of the moth, Mamestra configurata, during its metamorphosis. J. Neurochem., 33: 275-282. Evans, P. D. (1984) A modulatory octopaminergic neurone increases cyclic nucleotide levels in locust skeletal muscle. J. Physiol., 348: 307-324. Orchard, I. and Lange, A.B. (1986) Pharmaco- logical profile of octopamine receptors on the lateral oviduct of the locust, Locusta migratoria. J. Insect Physiol., 32: 741-745. Bodnaryk, R.P. (1982) Biogenic amine-sensitive adenylate cyclases in insects. Insect Biochem., 12: 1-6. Evans, P. D. (1985) Biogenic amines and messenger systems in insects. In “Approaches to New Leads for Insecticides”. Ed. by H.C. von Keyserlingk, A. Jager, and C. von Szczepanski, Springer-Verlag, Berlin & Heidelberg, pp. 117-131. Beavo, J. A., Rogers, N. L., Crofford, O. B., Hard- man, J.G., Sutherland, E.W. and Newman, E. V. (1970) Effects of xanthine derivative on lipolysis and on adenosine 3’,5-monophosphate phospho- diesterase activity. Mol. Pharmacol., 6: 597-603. Yamamoto, D. and Washio, H. (1979) The inhibi- tory action of L-glutamic acid esters on the insect neuromuscular junction. Comp. Biochem. Physiol., 63C : 75-80. Fahim,M.A. and Usherwood,P.N.R. (1983) Effects of c-AMP, caffeine, theophyline, and vinb- lastine on spontaneous transmittrer release at locust nerve-muscle junctions. J. Neurobiol., 14: 391-397. Evans, P.D. (1984) The role of cyclic nucleotides and calcium in the mediation of the modulatory effects of octopamine on locust skeletal muscle. J. Physiol., 348: 325-340. Greengard, P. (1976) Possible role for cyclic nu- cleotides and phosphorylated membrane proteins in post-synaptic actions of neurotransmitters. Nature, 260: 101-108. Hidoh, O. and Fukami, J. (1987) Presynaptic mod- ulation by octopamine at a single neuromuscular junction in the mealworm (Tenebrio molitor) J. Neurobiol., (In press). ZOOLOGICAL SCIENCE 4: 451-454 (1987) © 1987 Zoological Society of Japan Diamine Oxidase Activities in Catfish Tissues TAKESHI KUMAZAWA and OSAMU SUZUKI Department of Legal Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan ABSTRACT— Diamine oxidase (DAO) activity was measured in the brain, intestine, kidney, liver and skin of the siluroid catfish, Parasilurus asotus, with putrescine and cadaverine as substrates. The activity was found in the intestine and skin, but no detectable activities were found in other tissues. The apparent Km values for putrescine in the intestine and skin were 85.5~M and 89.1/M, respectively; those for cadaverine in the intestine and skin were 152.1 ~M and 123.0 uM, respectively. Substrate specificity was tested for the intestinal enzyme, and the highest activity was found with putrescine, cadaverine and 1,3-propanediamine, followed by 1,7-heptanediamine and 1,6- hexanediamine. INTRODUCTION Diamine oxidase (DAO; EC 1.4.3.6.) catalyzes the oxidative deamination of both histamine and diamines, such as putrescine and cadaverine [1]. Its distribution is wide among plants, microorgan- isms and vertebrates [2]. In the present study, we report the distribution of DAO in various tissues of the catfish and its characterization. Although the characteristics of DAO have been reported for a few teleostean species [3, 4], no report on DAO appeared for the catfish, and this is also the first demonstration of DAO existence in animal skins. MATERIALS AND METHODS Chemicals Acetylcadaverine-2HCl, acetylputrescine-HCl, agmatine-H,SO,, cadaverine-2HCl, 1, 2- ethylenediamine-2HCl, 1, 7-heptanediamine, 1, 6- hexanediamine, histamine-2HCl, homovanillic acid, horseradish peroxidase (type 1), pargyline- HCl, 1, 3-propanediamine-2HCl, putrescine- 2HC1, spermidine-3HCI and spermine-4HCI were obtained from Sigma Chemical (St. Louis, MO); ornithine from Kanto Chemical (Tokyo); argi- Accepted February 10, 1987 Received January 14, 1987 nine-HCl and histidine-HCl from Nippon Rikaga- kuyakuhin (Tokyo); ethylamine from Tokyo Kasei Kogyo (Tokyo); lysine-HCl from Yoneyama Chemical (Osaka); hydrogen peroxide from Mitsubishi-Gasukagaku (Tokyo). Other common chemicals used were of the highest purity commercially available. Animals Both sexes of the siluroid catfish, Parasilurus asotus, were used as experimental materials. Fishes with body lengths of about 25cm were selected for experiments. After decapitation, the brain, liver, kidney, intestine and skin were rapidly removed and frozen at —80°C until assayed. Assays The tissues were homogenized, and the crude homogenates were used as enzyme source. DAO activities were assayed fluorometrically by the method of Suzuki and Matsumoto [5]. The assay mixture (total 0.6ml) contained 0.1 ml of potas- sium phosphate buffer solution (final concentra- tion 0.08M), 0.1ml of horseradish peroxidase solution (0.5mg/ml), 0.1 ml of homovanillic acid solution (1.0mg/ml), 0.1ml of the homogenate, 0.1 ml of substrate solution and 0.1 ml of distilled water. The pH of the buffer solution was ad- justed to 7.4 in view of the physiological condi- tions. After incubation at 30°C for 30min, the 452 T. KUMAZAWA AND O. SuZzuKI enzyme activities were stopped by adding 2.0 ml of 0.1 N NaOH. The fluorescence intensity was measured with excitation at 323 nm and emission at 426 nm. As blank tests, assay mixtures without substrates are incubated; the substrates are mixed after adding the NaOH solution. Internal stan- dards should be taken by adding appropriate amounts of hydrogen peroxide to the mixtures prior to incubation. The effect of temperature on TABLE 1. DAO reaction was examined with the catfish intestinal enzyme with putrescine and cadaverine as substrates. Assay mixture with 0.08M potas- sium phosphate buffer solutions (pH 7.4) covering 0-60°C were used. The highest activity was found at 30°C with both substrates. From this results, we carried out DAO assay at 30°C. When experiments for substrate specificity were made, the enzyme was incubated in the presence of Diamine oxidase activities in catfish tissues DAO activity (n mole H,O, formed/g wet tissue/30 min) Tissue Putrescine Cadaverine Intestine 33 Se 3) SSE Skin 124.3+18.4 84.2+14.1 Liver N.D. N.D. Kidney N.D. N.D. Brain N.D. N.D. DAO activities were measured with 0.5mM putrescine and cadaverine as substrates. Values are expressed as means of 7 experiments+S.E. N.D., Not detected. 0.1mM pargyline to suppress the contamination of mitochondrial monoamine oxidase. RESULTS The effect of pH was examined with the intes- tinal enzyme with putrescine and cadaverine as substrates. The optimal pH with putrescine was 6.8 and that with cadaverine was 6.5. Table 1 shows DAO activities in the brain, liver, kidney, intestine and skin of the catfish with putrescine and cadaverine as substrates. DAO TaBLE 2. Apparent Km values of DAO in catfish intestine and skin Km (4M) Substrate Intestine Skin Putrescine 85.5 89.1 Cadaverine 1521 123.0 Km values were determined graphically from Lineweaver-Burk plots with 5-6 substrate con- centrations assayed in duplicate upon a single enzyme source prepared from the pooled tissues of 7 catfishes. TABLE 3. Substrate catfish intestine specificity of DAO in Substrate (final 0.5mM) Relative activity (%) Putrescine 100 Cadaverine 100 Spermidine 1.2 Spermine 0 1, 2-Ethylenediamine Dh 1, 3-Propanediamine 99.0 1, 6-Hexanediamine 55.6 1, 7-Heptanediamine 97.9 Agmatine eI Histamine 10.3 Acetylputrescine Sail Acetylcadaverine 31.4 Ethylamine 0 Ornithine 0 Lysine 0 Arginine 0 Histidine 0 The relative activities with each substrate repre- sent the means obtained from duplicate deter- minations upon a single enzyme source prepared from the pooled tissues of 7 catfishes. The incubation mixtures contained 0.1 mM pargyline. Diamine Oxidase Activities in Catfish 453 activities were found only in the intestine and skin. The activities in the intestine were about 9- and 13-fold higher than that in the skin when putrescine and cadaverine were used as sub- strates, respectively. Apparent Km values for putrescine and cadaverine were measured in catfish intestine and skin (Table 2). The values for putrescine and cadaverine were similar between the intestine and skin. Substrate specificity was tested for their oxida- tion by DAO in catfish intestine (Table 3). DAO showed the highest activity toward putrescine, cadaverine and 1, 3-propanediamine, followed by 1, 7-heptanediamine and 1, 6-hexanediamine. DISCUSSION In the present study, the highest DAO activity was found in the intestine of the catfish. The highest activity in the intestine is commonly observed for many vertebrates, such as man, rabbit, guinea-pig, rat, mouse and hen [6-10]. In other organs, however, there are obvious differ- ences in its distribution among different species. The kidney contains a relatively high DAO activ- ity in mammalian species, such as man, pig, dog and cat [7, 9]; while renal activities are low or not detectable in rodents and birds [7-10]. The DAO activity in the liver can be detected only in a few animal species, such as dog, guinea-pig and hen [7,10]. In the present study, DAO activity of catfish brain could not be detected by the fluorometric method. By use of much more sensitive radioisotopic methods, however, the presence of DAO was demonstrated in the central nervous system of a few fish species [3]. Some acetylpolyamines have been reported to be present in human urine, blood and rat tissues [11-13], and not to be end products but intermediates for further reactions [14,15]. In the present study, both acetylputrescine and ace- tylcadaverine could be oxidized with appreciable activities by catfish intestinal DAO. The acety- Ipolyamines probably present in fish tissues and their metabolic regulation by DAO are subjects to be explored in the future. The most probable function of DAO in the intestine seems to be detoxification of toxic di- amines present in the diet or produced by bacte- rial flora. The second function of the intestinal DAO was proposed by Luk et al. [16]; DAO is related to maturation and turnover of mucosal cells. An appreciable DAO activity was also found in catfish skin (Table 1). This is the first demonstra- tion of existence of DAO in animal skins. The nature of fish epidermis is different from those of other vertebrates; they are composed of mucinous tissues [17-19], and similar to intestinal epithelial tissues [19-21] in morphology and function, though they do not function as the site of active absorption. The catfish epidermal cells seem very active in renewal, differentiation and secretion of mucosubstances like intestinal mucosal cells. Thus the presence of an appreciable DAO activity in catfish skin seems to be very reasonable. REFERENCES 1 Gorkin, V. Z. (1983) Substrate specificity of amine oxidases. In “Amine Oxidase in Clinical Research”. Pergamon Press, Oxford, pp. 95-107. 2 Zeller, E.A. (1963) Diamine oxidases. In “The Enzymes”. Ed. by P. D. Boyed, H. Lardy and K. Myrback, Vol. 8, Academic Press, New York, pp. 313-335. 3 Burkard, W. P., Gey, K. F. and Pletscher, A. (1963) Diamine oxidase in the brain of vertebrates. J. Neurochem., 10: 183-186. 4 Matsumiya, M. and Otake,S. (1983) On the di- amine oxidase in the pyloric caeca and the intestine of common mackerel Scomber japonicus . Bull. Jpn. Soc. Sci. Fish., 49: 1695-1699. 5 Suzuki, O. and Matsumoto, T. (1986) Purification and properties of diamine oxidase from human kidney. Biogenic Amines, in press. 6 Bieganski, T., Kusche, J., Lorenz, W., Hesterberg, R., Stahlknecht, C-D. and Feussner, K-D. (1983) Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism. Biochim. Biophys. Acta, 756: 196-203. 7 Waton, N.G. (1956) Studies on mammalian histi- dine decarboxylase. Br. J. Pharmacol., 11: 119-127. 8 Shaff,R.E. and Beaven, M.A. (1976) Turnover and synthesis of diamine oxidase (DAO) in rat tissues. Studies with heparin and cycloheximide. Biochim. Biophys. Acta, 25: 1057-1062. 9 Cotzias,G.C. and Dole, V.P. (1952) The activity of histaminase in tissues. J. Biol. Chem., 196: 235- 242. 10 11 12 13 14 15 454 Bieganski, T. and Ulatowska,M.A. (1983) Di- amine oxidase in the hen. Agents and Actions, 13: 257-262. Seiler, N. and Knédgen, B. (1979) Determination of the naturally occurring monoacetyl derivatives of di- and polyamines. J. Chromatogr., 164: 155-168. Dolezalova, H., Sepita-Klauco, M., Kucera, J., Uchimura, H. and Hirano, M. (1978) Monoacylca- daverines in the blood of schizophrenic patients. J. Chromatogr., 146: 67-76. Seiler, N., Al-Therib, M. J. and Knédgen, B. (1973) Occurrence of monoacetylputrescine in vertebrate tissue. Hoppe-Seyler’s Z. Physiol. Chem., 354: 589- 590. Bolkenius, F. N. and Seiler, N. (1981) Acetylderiv- atives as intermediates in polyamine catabolism. Int. J. Biochem., 13: 287-292. Seiler, N., Bolkenius,F.N., Knédgen,B. and Mamont, D. (1980) Polyamine oxidase in rat tis- sues. Biochim. Biophys. Acta, 615: 480-488. 16 18 19 20 21 T. KUMAZAWA AND O. SUZUKI Luk, G. D., Bayless, T. M. and Baylin, S. B. (1980) Diamine oxidase (Histaminase). A _ circulating marker for rat intestinal mucosal maturation and in- tegrity. J. Clin. Invest., 66: 66-70. Whitear, M. (1970) The skin surface of bony fishes. J. Zool., Lond., 160: 437-454. Harris, J.E., Watson, A. and Hunt, S. (1973) His- tochemical analysis of mucous cells in the epidermis of brown trout Salmo trutta L. J. Fish Biol., 5: 345-351. Yamada, K. (1975) Morphochemical analysis of mucosubstances in some epithelial tissues of the eel (Anguilla japonica ). Histochemistry, 43: 161-172. Curry, E. (1939) The histology of the digestive tube of the carp (Cyprinus carpio communis). J. Mor- phol., 65: 53-78. Weinreb, E. L. and Bilstad, N. M. (1955) Histology of the digestive tract and adjacent structures of the rainbow trout, Salmo gairdneri irideus . Copeia, 3: 194-204. ZOOLOGICAL SCIENCE 4: 455-464 (1987) © 1987 Zoological Society of Japan Experimental Myopia and Glaucoma in Chicks TADASHI OlsHI, JEAN K. LAuBER! and Jerry VRIEND~ Department of Biology, Nara Women’s University, Nara 630, Japan, ‘Department of Zoology, University of Alberta, Edmonton, Canada, and *Department of Anatomy, University of Manitoba, Winnipeg, Canada ABSTRACT—The ocular response of the developing chick to several parameters of the light environment constitutes a useful model system for studies of experimental myopia and glaucoma. We explored the roles of long photoperiod and light intensity extremes in setting a pathological course for the developing eye. Chicks reared under continuous light developed increased eye weight, globe enlargement, decreased corneal diameter, reduced anterior chamber depth, and flat cornea, all effects associated with light-induced avian glaucoma (LIAG). Exposure to very dim light also caused eye enlargement (dim light buphthalmos, DLB), but with less pronounced anterior segment lesions. Under very bright light, globe enlargement was less severe. The LIAG and DLB syndromes appear to be additive, and are probably distinct from one another. INTRODUCTION Domestic chicks reared under continuous light (24L/0D) develop eye enlargement, but with retarded anterior segment growth, and eventual blindness, a condition we have called _light- induced avian glaucoma (LIAG) [1-3]. Several aspects of LIAG have been explored, and a number of lesions associated with the syndrome have been recorded [4], but the primary lesion remains obscure. Very long photoperiods, e.g., 22L/2D or 23L/1D, cause LIAG changes as severe, Or almost as severe, as those occurring under 24L/0D [5, 6]. One of the aspects of LIAG is eventual elevation of intraocular pressure (IOP), the diagnostic finding which permits use of the designation “glaucoma”. When refraction has been monitored during development of LIAG, these eyes show early hypermetropia (presumably due to reduced corneal curvature), then develop the myopia which would be expected with axial lengthening of the eyeball [2, 7]. Even when rearing has not been under con- tinuous, or almost continuous light, myopia may Accepted February 9, 1987 Received December 25, 1986 ensue under several other environmental condi- tions. Thus, very dim light [8], or spectrally restricted light (also dim), even diurnal [9], also causes buphthalmos. The pattern of eye lesions differs somewhat from those of LIAG, and in particular, high IOP has not been recorded at any age so far tested. Thus, we have not used the term glaucoma for this condition, but instead refer to it as dim light buphthalmos (DLB). Corneal flattening is minimal or absent in DLB (when the lighting schedule has been diurnal) [10], and eye enlargement, including axial lengthening, may be even more extreme than in LIAG, Thus, there is every reason to expect myopia, and this has been confirmed in at least ond study [11]. Nevertheless, in both their etiology and in several physical and physiological findings, LIAG and DLB can be shown to be separate and distinct phenomena. When chicks are reared under continuous light which is also very dim, as in the present study, the LIAG and DLB effects are superimposed, and eye enlarge- ment is extreme. A third eye enlargement syndrome has been described when chicks are reared in complete darkness (0L/24D), or almost complete darkness (2L/22D or 1L/23D) [6, 10, 12]. Some caution is 456 T. Otsu1, J. K. LAUBER AND J. VRIEND necessary in interpreting the results of such ex- periments, because what may seem to a human observer to be a “total darkness” protocol may still involve sufficient light to precipitate DLB in chicks. Thus, a seemingly trivial light leak, a pilot light, a photographer’s “safelight”, or a glowing brooder element could flaw an intended 0L/24D protocol. However, it does appear that there is a separate OL/24D-induced eye enlargement phenomenon, although it has not been so well documented as DLB and LIAG, and we have, as yet, only the most subjective guess as to the threshold between “light” and “no light”, in the chick’s preception. Whether or not the OL/24D chicks would be myopic is a moot point, since vision is probably severely impaired during a prolonged stay in complete darkness. Yet another class of eye-affecting environmen- tal factors is exemplified by “lid closure myopia” and related phenomena. We found, in early experiments, that covering one eye, with or without suturing the lids, caused specific re- sponses, including eye enlargement, in that eye only [13]. Meanwhile the environmental lighting condition (e.g., 24L/0D, or dim) had its systemic effect on both eyes, the lid closure effect being additive to the photoenvironment effect [10]. There have recently been a number of studies on the lid suture myopia model, both in chicks and on several mammals in which a similar syndrome has been described [14, and references therein]. In addition, some variations on the theme have been employed, involving, for instance, “altered visual experience”, or the use of vision-restricting goggles or lenses [15-17]. Our exploration of the lid suture myopia phe- nomenon in chicks, against the background of other myopia-inducing, glaucoma-inducing and/ or buphthalmos-inducing factors, will be the sub- ject of a subsequent paper. Here we attempt to distinguish between light intensity effects and photoperiod effects on the size and dimensional parameters of the chick eye. Elsewhere we have dealt with the possible involvement of melatonin in the same system(s) [18], and with the roles of adrenocortical and/or gonadal hormones in rela- tion to LIAG and DLB syndrome [19, 20]. METERIALS AND METHODS Domestic chicks of the Hubbard strain (fast growing, broiler type) were obtained on day of hatch from a commercial hatchery, and reared in floor pens at the University of Alberta Buosci- ences Animal Services Farm. During the early weeks, temperature was kept at recommended levels for young chicks by electric brooder heaters which emitted no visible light. Other environ- mental conditions, especially with respect to light intensity and photoperiod, were strictly control- led. Before the experiments began, chicks were kept under control lighting conditions for the first four days after hatching, so that they could learn the position of the food and water supply, which were thereafter always put in the same locations. This is a necessary precaution if chicks are to be reared later in complete darkness or very dim light. Food (chick starter crumbles) and water were available ad libitum. Five experimental treatments (P, Q, R, S, T) were used in the first experiment, as outlined in Table 1. In the second experiment, four of these treatments (PP-SS) were retained unchanged (ex- cept for numbers of chicks), but room T was replaced by treatment UU. In all lighted rooms, light was supplied by incandescent bulbs in over- head fixtures, so placed in the four quadrants of the room that the entire 3.7m 3.7m floor space was more or less evenly illuminated at bird height. Light intensity was measured before the beginning of Expt. I with an ISCO spectroradio- meter (Instrumentation Specialties Co., Lincoln, Nebr.), wavelength being set sequentially at 50 nm intervals, from 400 to 650nm. The area under the light intensity curve so generated was deter- mined for 50nm bandwidths, and multiplied by the luminosity weighting factor at mid-bandwidth, to arrive at an approximate value for the total light (in lux) to which the chicks were exposed. Of the photoperiods chosen, 12L/12D (lights on 0800-2000), as used in Expts. I/P and II/PP, was considered to be the control treatment, while 23L/1D (dark 0700-0800), as used in rooms Q, QO, R, RR, S and SS, was considered to be “almost-continuous light”, an environmental rear- ing condition expected to produce LIAG [6]. The Experimental Myopia and Glaucoma 457 TABLE 1. Summary of Experimental Treatments Expt/Group Photoperiod ge Light intensity uW/cm?* n(N)' (MST) I/P (control) 12L/12D 0800-2000 10 lux 0.025 10 (50) I/Q 2 LED 0800-0700 10 ‘lux 0.025 10 (50) I/R (dim) 231, Ab} 0800-0700 1.0 lux 0.003 10 (50) I/S (bright) 23L/ 1D 0800-0700 5000 lux 0.86 10 (50) I/T 1L/23D 0800-0900 0.25 lux 0.001 10 (50) II/PP (cont.) ips SAW D) 0800-2000 10. lux (as above) 12 (38) II/QQ 23L/ 1D 0800-0700 10 lux z 7‘ 14 II/RR (dim) 231/ 1D 0800-0700 1.0 lux ‘4 * 11 (31) II/SS (bright) 235) tl 0800-0700 5000 lux ”, % 14 (14) II/UU 0OL/24D (not at all) a — 10 *(N)=additional chicks used in a larger experiment. *=peak at 550nm. one hour of darkness was introduced in these experiments to provide a daily time signal, in case any cyclic phenomena should otherwise “free run” under constant conditions. Of the light intensities chosen for the present experiments, 10lux at bird height was designated the control value; this amount of light, compara- ble to what we have used in previous LIAG experiments [3], was provided by four 60W frosted bulbs hung at approximately 2m, in the four quadrants of a 3.7mX3.7m room. Treat- ment R and RR, at 1.0lux, are here described as dim light [8], provided by four 7.5 W bulbs, lightly coated with black spray paint, and hung 2m from the floor in a similar 3.7mX3.7m room. The bright light treatments S and SS, at over 5000 lux [6], were achieved with 16 reflector floodlamps, 150W each, hung at approximately 1.2m from the floor, and evenly spaced throughout a 3.7m Xx 3.7m room. All light bulbs were replaced with new ones once a month, to forestall burnouts which might affect the results. In treatment UU of Expt. II, we aimed to achieve total darkness, a condition not quite accomplished in our treatment T of Expt. I. Whereas in room T, one hour of very dim light (<0.25lux) was scheduled each morning (0800- 0900) to provide an opportunity for personnel to replenish food and water, in treatment UU we eliminated even this amount of light, by use of several light-tight environment chambers, placed in turn in a light-controlled room. Each chamber measured 61cmx6lcmxX4lcm high, and was made of sheet aluminum, with light-baffled ven- tilation ports. Four to five chicks were started in each chamber, but these numbers were reduced (to forestall overcrowding) to two per chamber by four weeks of age. After an initial four-day “learning period”, these chicks were serviced in complete darkness. Brooder heat was supplied by passing the positive pressure airflow to the cham- bers first through a coil of copper tubing, which was in turn wrapped with electric heating tape and an insulating layer of asbestos. During the seventh week after hatching, chicks of Expt. I were bled (5—6ml, by cardiac punc- ture), then sacrificed by sudden decapitation. All of the Expt.I chicks on which we report here were sacrificed at midday, from approximately 1200-1400 hrs. These Expt. I chicks were part of a much larger series (60 birds per room) from which we obtained blood samples on a “round- the-clock” schedule: the corticosterone and mela- tonin radioimmunoassay for which this plasma was saved have been reported separately [18, 19]. Both eyes were enucleated immediately postmor- tem, trimmed of fat and extraorbital muscles, weighed, and photographed in front and side view (Fig. 1), for later measurement of dimensional parameters. Figure 2 shows the parameters mea- sured on these photographs of freshly enucleated eyes. Each eye was then bisected equatorially 458 T. Otsu, J. K. LAUBER AND J. VRIEND | _=Pr \ Fic. 1. Front and side views of enucleated right eyes. Above, diurnal control chick (Group PP); below, chick reared under continuous light (Group QQ). Light intensity was 10lux for both. Note greatly enlarged globe and flat cornea in the LIAG eye. AXL Fic. 2. Diagrammatic representation of front and side view of right eye photographs, showing the para- meters measured. To maintain orientation, the optic nerve (ON) stump was positioned at 8 o’clock. D-—V=dorsoventral equatorial diameter, N-T=nasotemporal equatorial diameter, AxL= axial length (meridional diameter), GAX=globe axis, AcD=anterior chamber depth, CD=corneal diameter, CRC=corneal radius of curvature. with a sharp razor blade, the retinal half frozen over dry ice for later biochemical studies, and the anterior segment fixed in Bouin’s fluid for later histology. Adrenals and testes were also weighed and saved to Bouin’s. Expt. II chicks were bled then sacrificed, this time by overdose of Nembutal, during their sixth weeks of age, half at 1000-1200 hrs, and half at 2200-2400 hrs (lights on 0800-2000 hrs for diur- nal birds). Thereafter, the same tissues were taken, and the same procedures followed, as for Expt. I. For statistical analysis, we used the MINITAB software program [21], via a terminal interacting with the University of Alberta Amdahl 5860 mainframe computer. Analysis of variance, Stu- dent’s t-test (independent samples), paired t-test and product-moment correlation analysis were employed. RESULTS Eye enlargement, measured as increased eye weight (above and beyond that which could be ascribed to normal growth), occurred under each of the several experimental lighting regimes em- ployed (Figs. 1 and 3, Table 2). Overall, there was an 18.5% increase (Expt. II) in eye weight which could be attributed to continuous light (the LIAG effect). When the continuous light was also of low intensity, the increase in eye size was 30.4% over controls (Expt.1I). The dim light effect was significantly greater than the LIAG effect alone (e.g., p<0.001 for R vs. Q). Eye enlargement also occurred under continuous bright light, although it was less dramatic: 16.0% PP [| ee |] RRG ss 4 24 Ree Pee Se] psososes etatees POLOS 30 I) On Nsasr Sass’ i fe) VGN SNE Seay SVQ EYE WT.-GM. YVR NAN TITS AVBVVH BVRVQ DS WAKY Y x WALA NU RS Fic. 3. Bar graph showing eye weight at 6 weeks of age, in chicks reared under control conditions (PP), or one of four different environmental lighting treatments in Expt. II. For further explanation of the environmental conditions set for this experi- ment, see text and Table 1. Relative eye weight (g/kg B.W.) for the same birds is shown in Table 2. Error bars indicate +SEM. Experimental Myopia and Glaucoma 459 TABLE 2. Eye parameters in chicks reared under several light regimes Group(n) Rel. EWt' D-v"™ N-T AxL AcD CD CRC Experiment I—7 weeks old Bee 9) 112 16.48 16.59 12.64 2.02 7.42 4.35 +0.035 + 0.24 ao (IP?) a (0.23 +0.08 +0.29 +0.07 Q (10) 1.07 /e2i= 17.48* 13.06 Liste 6.45* 4s +0.025 ae WAS se) \)RILT/ ae ONL ae EiT +0.20 +0.17 Reet 8) 14655" L924 "> 18:69%** 14.63*** leona 6.91 S287t +0.053 ste OE Si nee WEIS) ae Ne) a OLS +0.34 +0.20 Sia(7) £3055 17. 4le 17.68* 12.66 O:89m" G2330 a) Sigh e +0.036 ae Osis ae W225) ae W222 +0.05 new) .29 +0.24 Bet t0) 1:367=* S905 = = Lo-olea Sh UO ae 6.59 5,405 °* 20.052 sta) 22 ae WAIN) + 0.18 ae 0)ibI +0.30 +0.14 Experiment II—6weeks old PP (10) £735 16.41 16.71 13.03 aN 7.67 8) +0.042 + 0.06 + 0.09 te OID, +0.06 +0.09 +0.09 QQ (11) 1.60* ol LSelOe as 1322 255 os Tele 6.44*** +0.076 ae WAG ae Ws: + 0.26 +0.07 +0.12 OP tel RR( 8) rol SES0Rs 18.34*** POA ane LBs Uo33) 6.64*** +0.064 ae LAY, 0.35 a, Ov17 +0.05 +0.11 0k ss: (8) 1.45 16.62 L285 12.60 25r8G 6:96" GullGaan +0.052 tas0316 ae) JLi/ se OEY) +0.04 +0.05 ste Oe UU( 9) oni PAE 750i 12.68 3 an TASES SO +0.068 =O hs ae (17 tae OE IW +0.08 +0.09 stay + g/Kg body wt+SEM. *¥ all D-Vs, N-Ts etc. in mm+SEM. Dorsoventral (D-V) diameter, nasotemporal (N-T) diameter, axial length (AxL), anterior chamber depth (AcD), corneal diameter (CD) and corneal radius of curvature (CRC), all in mm+SEM. * significantly different from control, p<0.05. Se KAU aap < 0-001. over controls in Expt.I and 7.4% in Expt. Il (Table 2). In both experiment there was a signi- ficant positive correlation (p<0.05) between body weight and eye weight, but eye weight differences could not be explained by body growth alone: relative eye weight showed a similar pattern of response under the several lighting regimes - (Table 2). The size of the globe increased with eye weight, as shown by both dorsoventral (D-V) and nasotemporal (N-T) equatorial diameters (Table 2). The third eye diameter, axial length (AxL), showed a less pronounced increase, but when AxL minus anterior chamber depth was calcu- lated, (here called GAX, the globe axis), a highly significant degree of correlation (p<0.01) with eye weight was seen (Fig. 4). In comparisons of GAX across groups (Fig. 5) the greatest increase was in 23L/1D dim light (p<0.001 for R vs. P, R=+0.905 1.5 20 25 3.0 35) 40 EYE WT-gm Fic. 4. Correlation of globe axis (see Fig. 2) with eye weight in 7 week-old chicks of Expt. I. All photo- period and light intensity groups are included. 460 T. O1sHi, J. K. LAUBER AND J. VRIEND PL] at] RG iJ ; : 2 S o TH x l| 2 D \o | =| G) iS) Fic. 5. Globe axis diameter (GAX, see Fig. 2) in 7 week-old chicks of Expt.I, reared under several different lighting regimes. For explanation of P, Q, R, S, T, see text and Table 1. Re) 2.0 24 28 S12 EYE WT-gm Fic. 6. Correlation of anterior chamber depth with eye weight in 6 week-old chicks of Expt. II. Shallow anterior chamber is characteristic of light-induced avian glaucoma (LIAG). 1.6 20 24 28 BZ 3.6 EYE WT. -gm Fic. 7. Correlation of corneal radius of curvature with eye weight in 6 week-old chicks of Expt. II. The cornea is flattened in the enlarged eyes of chicks with LIAG. and also for RR vs. PP). For the other groups as well, GAX was significantly above control values, pp(_| t aa] RG ss [A Retacees | XX] roneste : vetenee KROOO SSSI AROS STTRBA GSS SSSA N DPSS FTV OV a gg Gausyggg yyy SHE SSANASSNTVGG one SoHo Wsss AY Ly . Vs CORNEA-RADIUS CURV.- mm NAN SVE SVT 4 Fic. 8. Bar group showing the degree of corneal flattening in Exp. II chicks reared to 6 weeks under several different lighting schedules, as outlined in the text and Table 1. but at the same time, significantly less than under dim light (e.g., p<0.05 for S vs. R; p<0.001 for SS vs. RR) (see also Table 2). With respect to the corneal parameters anterior chamber depth (AcD), corneal diameter (CD), and corneal radius of curvature (CRC) (ref. Fig. 2), the differential responses to continuous light, and to light intensity extremes, further distin- guished chicks in the several lighting regimes from one another, as well as from diurnal controls (Table 2). Overall, there was a modest, but significant (p<0.01), negative correlation be- tween AcD and eye weight (Fig. 6), and a strong positive correlation of CRC with eye weight (p< 0.001) (Fig. 7). Comparison of the CRC re- sponses across the several groups (Fig. 8) reveals that all showed some degree of corneal flattening, but this was most pronounced in LIAG+DLB subjects (groups R and RR); (p<0.001 for each experimental group vs. controls, but not signi- ficant for comparisons of experimental groups with one another). DISCUSSION While the correlations between globe dimen- sions and eye weight were all positive, as might be predicted in a growing system, the relationship between eye weight and the several corneal para- Experimental Myopia and Glaucoma 461 meters was more complex. Because the front and the back of the eye responded differently, and possibly independently, to several permutations of the light environment employed here, the responses of the anterior and posterior segments of the eye are discussed separately below. The posterior segment Influence of photoperiod: The pronounced eye enlargement which occurred here in chicks reared under continuous light is to be expected in LIAG, and confirms several previous studies [1, 3]. Also as expected, body weight was increased in 23L/1D vs. 12L/12D chicks. The eye enlarge- ment was, however, not simply a reflection of this accelerated growth rate: comparison of eye weight relative to body weight showed the same trends as for absolute eye weights. The enlarged eyes of 23L/1D chicks displayed highly significant differences from controls in both D-V and N-T equatorial diameters; at this age, the N-T value was usually the larger of the two. The third diameter we measured, on side view photos of enucleated eyes, was AxL: this dimen- sion was slightly, though not dramatically, in- creased in LIAG vs. control eyes (both 101ux). However, the cornea was also changing shape in 23L/1D eyes (see below), and this tended to negate the overall axial increase. When globe axis (ref. Fig. 2) was plotted, the difference be- tween LIAG and control eyes was highly signi- ficant. Nevertheless, in both LIAG and control eyes, this dimension remains the smallest of the three diameters; i.e., the chick’s eye is not so nearly spherical as is the human eye, the globe being considerably flattened meridionally. Influence of light intensity: Treatments R and S were designed to test the effects of several _ extremes of light intensity on the photosensitivity of the developing eye. In comparing our groups R and S with treatment Q, and all with the diurnal control P, it is important to remember that chicks in all three of these experimental groups (Q, R, S) were reared under 23L/1D, or almost-continuous light: the LIAG symptoms would thus be expected, and did indeed develop (Q and QQ). In dim light of such long photo- period (R and RR), as compared to Q or QQ, body weight was almost the same (although much greater than in P or PP controls), while eye weight of dim-lighted subjects was even greater than in “normal” intensity (10lux) light of the same photoperiod. The low level of light intensity set for room R (1.0lux) was not unlike that we have employed previously in “dim light” experi- ments [8,10], which led us to first use the designation “dim light buphthalmos” (DLB). It is also similar to “dim light” intensity levels used by Harrison et el. [9,11], who also reported eye enlargement even in diurnal chicks. The R vs. Q comparison, of eye weight and/or dimensional parameters, suggests an additive response, the DLB eye enlargement superimposed on the LIAG effects. This supports a suggested explana- tion we proffered based on several previous smaller experiments, although in the earlier cases we had included also a diurnal dim light treat- ment, to better separate the two effects [8, 10]. In every instance, the resulting eye enlargement pattern was consistent with the view that con- tinuous light, leading to LIAG, and dim light, producing DLB, call forth separate and distinct responses in the eye, differing not only in their etiology, but also thier pathological course. The response of chicks to the bright light treatment S or SS was somewhat enigmatic. In body weight and in eye weight, S chicks seemed to be intermediate between diurnal controls and LIAG birds. Thus under 5000 lux, eye weight was significantly greater than for control (p<0.001 for SS vs. PP), but significantly Jess than at 10lux (p<0.05 for SS vs. QQ), and also significantly less than at 1.0lux (p<0.01 for SS vs. RR), even though all three experimental groups were reared under continuous light. Likewise, in the dimen- sional parameters D-V, N-T and GAX, values recorded for S chicks were somewhat higher than for controls (p<0.01 for most parameters). At the same time, these globe dimensions were significantly /ess than those recorded for chicks in dim light. In a previous experiment which closely resembled the present one, one of us also found that under 24L/0D at S5000lux, the expected LIAG eye enlargement was suppressed [6]. 462 T. Otsui, J. K. LAUBER AND J. VRIEND The anterior segment Influence of photoperiod: Corneal diameter (CD) was significantly smaller in LIAG eye, as compared with diurnal controls. This was also as expected, and confirms previous findings in LIAG. In this connection, we have also previous- ly shown that the cornea in LIAG is damaged, leaking lactic dehydrogenase into the aqueous [4], and that growth of the cornea (as evidenced by mitotic counts in the corneal epithelium) is im- paired in LIAG [22]. In the present Expt. I, mean number of dividing cells per standard field was reduced by almost half in Q compared with P; whereas there was a _photoperiod-driven rhythm in normal eyes, with a scotophase peak, this rhythm was damped in all 23L/1D birds (Q, R, S). Our report on corneal mitotic rates has been published separately [18], but the above preliminary finding is included here as well, because it confirms that growth of the cornea is suppressed in the LIAG syndrome. Decreased corneal curvature (i.e., high CRC) is an early finding in LIAG, as evidenced either by measured radius of curvature of the corneal surface, or by +D refractive error [2]. In the present series, we confirmed a reduction in AcD and a dramatic increase of CRC, in LIAG chicks at 6-7 weeks of age. Comparison of AxL and GAX measurements confirms that, while the cornea is becoming flatter, the globe is, in fact, enlarging axially [1, 8]. Reduced corneal curvature would tend toward hyperopia (which was indeed found at 3-4 weeks of age in several earlier studies where refractions were done) [2,7]. However, the overall eye enlargement of LIAG, and especially the increase in GAX, finally overrides the effect on refraction expected from the early anterior segment changes, and this soon results in a net myopia, of increasing severity with age (up to —9D at 6 weeks in one of our previous series) [2]. Influence of light intensity: Whereas light in- tensity differences appeared to have a less drama- tic effect than photoperiod on the globe of the eye, consideration of the corneal parameters re- veals a different picture of the effects of light intensity extremes. The corneal flattening as characteristic of LIAG also occurred in both bright and dim light of 23L/1D. However, the CD and AcD effects, as compared with controls, were most pronounced (i.e., the values were smallest) in bright light, and least pronounced (though still most significant) in dim light. The CD reduction, in fact, fell just short of signi- ficance in groups R and RR (DLB) chicks. CRC was also significantly different in bright light as compared to dim light, in both experiments (p< 0.05 for R vs. Q, and also for S vs. Q). Likewise, in an earlier study, CD and AcD were even more reduced under 5000lux than in 150lux (both continuous light) [6]. Because we did not have a diurnal dim light treatment in the present Expt.I or II, we must look elsewhere to see what might have been expected: earlier experiments showed that dim light of a diurnal photoperiod induces globe enlargement but not corneal flattening [10]. This suggests that whatever increase in CRC occurred here can be attributed to LIAG, not DLB. Rearing in constant darkness: As noted ear- lier, Treatment T was intended as a “complete darkness” protocol, although we felt obliged to allow 1 hr/day of very dim light, to facilitate servicing. There was no question but that these birds found access to food and water: their mean body weight was above average at the time of sacrifice. Eye weight was 28.8% greater in these chicks (p<0.001 for T vs. P), a similar and only slightly less pronounced response than that seen in 23L/1D dim light. In the dimensional para- meters as well, the responses of group T and group R birds were quite similar, and of approx- imately the same magnitude, when both were compared to controls. The CRC constitutes a possible exception: corneal flattening was less pronounced in T vs. P than in R vs. P. These results of dark-rearing echo the findings in several other studies involving a complete darkness pro- tocol [6, 12]. Nevertheless it is tempting to view our T treatment as a kind of “super dim light” treatment but different in one key respect from Treatment R, in that the photoperiod was not long, but in fact, very short (and possibly per- ceived by the Group T chicks as diurnal?). Comparison of the T chick response in Expt. I Experimental Myopia and Glaucoma 463 will the UU response under 0L/24D in Expt. Il suggests, however, that these two treatments cannot be equated. Let it be assumed for the moment that in the UU environment chambers we did indeed achieve total darkness, in the chick’s perception. The UU vs. control compari- son reveals that the eyes were significantly en- larged in total darkness (p< 0.001 for UU vs. PP), and the dimensional parameters correspondingly increased; however, none of these globe dimen- sions were as great as in QQ or RR. The reductions in AcD and in CD were not remark- able in treatment UU, either. From the CRC, however, one sees that the flattening of the cornea was less pronounced in UU than in T, each compared to its own control (P or PP), as well as to Q, R and S responses. Because of the persistent question as to whether either of the “total darkness” environ- ments we provided was indeed so perceived by the subjects, we do not yet feel able to draw conclusions about the effects of constant darkness on chick eye parameters. In summary, in this study we document, and distinguish between, several effects of light on the developing avian eye. Eye enlargement, and increases in the several globe diameters, are characteristic of both light-induced avian glauco- ma (LIAG), brought on by rearing in continuous light, and dim light buphthalmos (DLB). When the environmental light is both continuous and dim, the effects on the posterior segment of the eye are additive. When the continuous light is instead very bright (5000 lux), globe enlargement is less extreme than under light of “normal” intensity (10lux). Of the three globe diameters measured, globe axis (GAX) shows the closest correlation with eye weight, and would be the best predictor of eye enlargement. In the front of the eye, the small flat cornea and shallow anterior chamber of LIAG are less pro- nounced in DLB, even when the dim light is continuous. Some differences, especially of the anterior segment parameters, in responsiveness to light intensity extremes support the conclusion that light intensity effects and photoperiod effects constitute separate and distinct influences on the growing eye. We also investigated eye develop- ment under conditions of complete darkness, but interpretation of these results is uncertain be- cause, we believe, of the difficulty in achieving absolute darkness, which will be so perceived by the avian subjects. The domestic chick used in these studies consti- tutes a particularly useful research subject, for several reasons. Relative to body size, the eyes of birds tend to be large, and are of considerable importance to their owners (i.e., the chick is highly vision-dependent). The LIAG system, especially, provides the investigator with a valu- able model system for glaucoma and myopia research: this condition can be easily brought on at the will of the investigator, it follows a predict- able course, it permits manipulations which would be impossible or unacceptable with human sub- jects, and it presents eyes available for detailed study during a long preglaucoma period. All of these are luxuries not afforded the clinical resear- cher working with human subjects. Since both glaucoma and myopia are clinical entities of some importance for human sufferers, the availability to the investigator of animal models for these vision-threatening conditions presents unique opportunities for both basic and pragmatic research. ACKNOWLEDGMENTS This research was supported by a grant (to JKL) from the Natural Sciences and Engineering Research Council of Canada, and by a Visiting Scientist grant (to TO) from the Alberta Heritage Foundation for Medical Research. REFERENCES 1 Lauber, J.K. and McGinnis, J. (1965) Eye lesions in domestic fowl reared under continuous light. Vision Res., 6: 619-626. 2 Lauber, J.K., Boyd, J. E. and Boyd, T. A. S. (1970) Intraocular pressure and aqueous outflow facility in light-induced avian buphthalmos. Exp. Eye Res., 9: 181-187. 3. Lauber, J. K. and Kivett, V.K.(1981) Environmen- tal control of the rearing conditions and early preglaucomatous lesions in chicks. Exp. Eye Res., 32: 501-509. 4 Kinnear,A., Lauber,J.K. and Boyd, T.A.S. (1974) Genesis of light-induced avian glaucoma. 10 il 12 ie) 464 Invest. Ophthalmol., 13: 872-875. Lauber, J. K., McLaughlin, M. A. and Chiou, G. C. Y. (1985) Timolol and pilocarpine are hypotensive in light-induced avian glaucoma. Can. J. Ophthal- mol., 20: 147-152. Oishi, T. and Murakami,N. (1985) Effects of duration and intensity of illumination on several parameters of the chick eye. Comp. Biochem. Physiol., 81A: 319-323. Shiraki, K., Sotani, T., Oishi, T. and Okuzawa, I. (1981) Effects of continuous light and continuous darkness on the eyes of chicks (in Japanese). Fol. Ophthalmol. Japonica, 32: 1157-1163. Lauber, J. K. and Kinnear, A. (1979) Eye enlarge- ment in birds induced by dim light. Can. J. Ophthalmol., 14: 265-269. Harrison, P.C., Bercovitz, A.B. and Leary, G. A. (1968) Development of eye enlargement of domes- tic fowl subjected to low intensity light. J. Biometeorol., 12: 351-358. Chiu, P.S.L., Lauber, J. K. and Kinnear, A. (1975) Dimensional and physiological lesions in the chick eye as influenced by the light environment. Proc. Soc. Exp. Biol. Med., 148: 1223-1228. Bercovitz, A. B., Harrison, P.C. and Leary, G. A. (1972) Light-induced alterations in growth pattern of the avian eye. Vision. Res., 12: 1253-1259. Osol, G., Schwartz, B. and Foss, D.C. (1986) The effects of photoperiod and lid suture on eye growth in chickens. Invest. Ophthalmol. Vis. Sci., 27: 255- 260. Lauber, J.K., McGinnis, J. and Boyd, J. (1965) 14 15 16 17 18 19 20 743 22 T. Otsu, J. K. LAUBER AND J. VRIEND Influence of miotics, Diamox and vision occluders on light-induced buphthalmos in domestic fowl. Proc. Soc. Exp. Biol. Med., 120: 572-575. Yinon, U. (1984) Myopia induction in animals following alteration of the visual input during development: a review. Curr. Eye Res., 3: 677-690. Wallman.J., Turkel, J., and Trachtman, J. (1978) Extreme myopia produced by modest change in early visual experience. Science, 201: 1249-1251. Hodos, W. and Kuenzel, W. J. (1984) Retinal im- age degradation produces ocular enlargement in chicks. Invest. Ophthalmol. Vis. Sci., 25: 652-659. Hayes, B.P., Fitzke, F.W., Hodos, W. and Hol- den, A.L. (1986) A morphological analysis of experimental myopia in young chickens. Invest. Ophthalmol. Vis. Sci., 27: 981-991. Lauber, J. K., Oishi, T. and Vriend, J. (1986) Plas- ma melatonin rhythm lost in preglaucomatous chicks. J. Ocular Pharmacol., 2: 205-213. Lauber, J.K., Vriend, J. and Oishi, T. (1986) Plas- ma corticosterone in chicks reared under several lighting schedules. Comp. Biochem. Physiol., 86A : 73-78. Oishi, T. and Lauber,J.K. (1986) Light, ex- perimental avian myopia and the role of the sup- rarenals. J. Ocular Pharmacol., 2: 139-146. Ryan, T.A., Joiner, B.L. and Ryan, B.F. (1976) MINITAB Student Handbook, Duxbury Press, Boston, Mass. Oishi, T. (1984) Circadian mitotic rhythm in chick corneal epithelium. J. Interdisc. Cycle Res., 15: 281-288. ZOOLOGICAL SCIENCE 4: 465-474 (1987) © 1987 Zoological Society of Japan Cytochalasin B Affects Selectively the Marginal Cells of the Epithelial Sheet in Culture SHIGEO TAKEUCHI Zoological Institute, Faculty of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT—The epithelial spreading is a fundamental one among morphogenetic movements. To know the mechanism generating force for spreading, the effect of cytochalasin B (cytB) on the corneal epithelium in culture was investigated. Four ug/ml of cytB inhibited completely the epithelial spreading and, at the same time, altered selectively the shapes of marginal cells: they shrinked at leading lamella and retracted quickly leaving fine tails behind, in which the bundles of F-actin were disintegrated to be numbers of fragments, while most of the other cells kept their shapes unchanged, along the cell borders of which F-actin distributed as before the treatment of cytB. The high sensitivity of the marginal cells to cytB led us to consider that F-actin was incessantly polymerized and taken into the intracellular organs generating force which enabled the marginal cells to locomote outwards, and that the outward locomotion, as a main force, spread entire epithelium. INTRODUCTION When the body surface of higher animals was injured, the epithelial cells started to migrate and spread quickly over the denuded area as a co- herent cell sheet, and ceased the movement after the closure of wound. The locomotion of epithe- lial cells, as one of fundamental cellular move- ments in morphogenesis, has attracted the interest of many authors [see 1, 2]. The marginal cells of epithelial sheets in culture were flattened and formed leading lamella at the free end, where they attached to the substratum with both focal and close contacts [1, 3-5]. Lamellipodia, filopodia or microspikes were pro- truded from the leading edges, accompanying ruffling or blebbing [6], as like as in the fibro- blastic cells [7-9]. In the leading lamella of epithelial cells, bundles of microfilaments or of actomyosin were reported to be aligned in a way quite similar with those in fibroblasts [1, 10]. We could therefore assume that the contraction of actomyosin systems in the marginal cells was a main force for the epithelial spreading as in the locomotion of fibroblasts [11]. In order to know Accepted February 10, 1987 Received December 10, 1986 more precisely the mechanism of epithelial spreading, cytochalasin B (cytB), the drug known as an inhibitor of actin filament formation since Schroeder [12], was applied to the cultured epithelia. Both shapes of cells and alignment of bundels of F-actin, as well as an inhibition of spreading of epithelial sheets, were altered re- markably. Above all, the marginal cells were affected most conspicuously. The results lead us to accept the hypothesis that the marginal cells are mainly responsible for epithelial spreading. The entire epithelium was pulled outwards with the marginal cells in which the bundles of F-actin were structuralized incessantly for generating force for the outward locomotion. The details of the observation and the discussion on the subject will be presented in this communication. MATERIALS AND METHODS The eggs of white leghorn obtained commer- cially were incubated for 8 days at 38°C. Millipore filters (MF), PH or RA (Millipore Co.), pore sizes of which are 0.3 4m and 1.2 um respectively, were used for substrata. They will be referred as 0.3MF and 1.2 MF, characterized by numerals expressing pore size. The cover slip (2424 mm) cleaned for culture 466 S. TAKEUCHI use (Matunami Glass Inc.) was used as a substra- tum for an epithelium. 1) Isolation of the epithelium More than 200 sheets of corneal epithelium were isolated in the ordinary procedures using 1lmM_ ethylene- diaminetetraacetic acid (EDTA) in calcium and magnesium free Tyrode solution. The details were presented in a previous paper [13]. 2) Culture methods Each isolated epithelium was combined with a piece of 0.3MF or 1.2 MF, explanted onto the surface of normal Wolff- Haffen’s medium [14] or that containing cytocha- lasin B (Sigma Co., 4, 0.4, or 0.04 ug/ml in a final concentration, cyt:B-W-H), gelled in a hollow in a glass block. After covered with glass slide and sealed with melted paraffin, the explant was incubated for 24, 48, or 96hr at 38°C. CytB-W- H was prepared as follows: 1% agar in Gey’s solution, donor horse serum (Flow Lab.), 50% embryonic extract of 9-day chick embryos, which contained cytB in a concentration of 19, 1.9, or 0.19mg/ml, and Penicillin G-K (Meiji Pharm. Co., 20,000 units/ml) were mixed in a ratio, 7:3:3:1 in volume. CytB was primarily dissolved in dimethylsulfoxide (DMSO) and diluted in embryonic extract. As a result, cytB-W-H con- tained inevitably 2%, at most, of DMSO in a final concentration. The effect of DMSO was checked in the epithelia cultured on W-H containing 2% DMSO alone. Fifty or more sheets of corneal epithelia were explanted onto the surface of cover glass with a small drop of L-15 medium (Leibowitz, L15, Gibco Co.) supplemented with 20% of donor horse serum (Flow Lab.), covered with hollow slide, sealed with melted paraffin and incubated at 38°C. After 24hr of primary culture, only the epithelium started to spread along the glass sur- face was transferred into filming slide with a L-15 medium contained 4 ug/ml of cytB and 20% of horse serum (cytB-L-15) and observed under a phase-contrast microscope, the stage of which was kept at 38°C. The rest of epithelium attached not firmly enough to the glass surface was cultured for another 24hr at 38°C in Petri dish with a large amount of L-15 medium. The epithelium which began to spread was served for cytB treatment as described above. 3) Quantitative assessment The epithelia cul- tured on MF for 48 or 96 hr with cytB-W-H were fixed with Bouin’s fluid for 2hr. After a thorough elimination of picric acid with 70% ethanol, they were stained in toto with Meyer’s hematoxylin and eosin, dehydrated through a series of graded alcohol, cleared with xylene and mounted on slide glass with balsam and cover glass. A ratio between the area of epithelium before and after the culture was used as the index of migratory activity. The details were presented in the previous report [13]. 4) Scanning electron microscopy (SEM) More or less than 10 samples were selected at random from each of 8 groups of culture (two types of MF, and four kinds of cytB-W-H in concentra- tion of cytB) at 24, 48, or 96hr of culture and fixed with 2.5% glutaraldehyde (Taab. Co.) in 0.1M cacodylate buffer (pH=7.2, c-buffer) for 2hr at O°C. Rinsed three times and overnighted in 0.1M c-buffer at 4°C, they were postfixed with 1% OsO, in 0.1M c-buffer for 2hr at O°C, dehydrated through a series of graded ethanol at 0°C and, immediately or after stocked for several days in the refrigerator, dried in a critical point dryer (Hitachi HII) using liquid CO, as a medium. After coated with gold in an ion coater (Eiko Co.), the epithelium was observed with Hitachi $430 scanning electron microscope. 5) NBD-Phallacidin staining The epithelia cultured on MF for 24, 48, or 96hr and those on the glass surface, treated or not treated with cytB, were fixed with 3.5% of neutral formalin in phosphate buffered saline (pH=7.2, PBS) for 30 min at 20°C, rinsed several times in PBS, placed on the bottom of Petri dish, and immersed in a drop of NBD-Phallacidin (NBD-Ph, Wako Pure Chem. Indust.) solution, in which 2 units of NBD-Ph were solved in PBS, for 2hr at 20°C in the moist and dark chamber. After the treat- ment, they were rinsed several times in PBS to eliminate free NBD-Ph, mounted on slide glass with PBS-glycerine (1:1 in volume) and covered with cover slip. 6) Optics A phase-contrast microscope and a microscope equipped with epifluorescent appar- atus and with objective lenses free from auto- fluorescence (UHF X20, UHF X40, and UHF The Effect of CytB on Epithelial Cells 467 100, the last two, for oil immersion, Olympus Co.) were used. For microphotography with the fluorescent microscope, Tri-X films (ISO=400, Kodak) were adopted, which were developed with Super prodol (Fuji Co.) for 7 min at 20°C to enhance the sensitivity from ISO 400 to 800. With the phase-contrast microscope, Neopan F (ISO=32, Fuji Co.) was used without a special treatment. RESULTS I. Quantitative assessment of the effect of cytB on epithelial spreading CytB suppressed completely epithelial spread- ing. In a concentration more than 10 4g/ml, the epithelial cells hardly survived (data not shown). In 4 ug/ml of cytB, in spite of healthy appearance of cells in histological survey, the migratory index (MI) was 1.2+0.2 (an average of 15 cases+a standard deviation) in the epithelia cultured for 96hr in combination with 0.3MF, which was one-sixth of that in the epithelia cultured on normal W-H (7.9+2.3, 10 samples). The value did not differ significantly from that in the epithe- lia cultured on normal W-H in combination with 1.2MF (1.1+0.2, 15), showing complete stop of spreading. Even in a low concectration (0.04 ug/ ml), cytB still worked inhibitory: the MI was 3.3 +1.4 [14], approximately as half as that in those cultured on normal W-H. On W-H containing 2% DMSO alone, the MI (6.0+1.2, 10) differed not significantly from that in those on normal W— H in combination with 0.3 MF at 96hr of culture. TABLE 1. These results were summarized in Table 1. As the cytB in the concentration of 4g/ml sup- pressed the epithelial spreading most effectively, and without giving severe damage to cells, the concentration was used in this series of experi- ments. 2. The early changes of the marginal cells when transferred into the medium containing cytB CytB was observed to affect at first in the marginal cells within 5 min after the epithelium was transferred into cytB—L-15. They swelled up slightly and their leading lamella were narrowed at their width. Then the cells began to retract but some parts of the distal end still attached firmly to the glass surface. As the result, fine thread-like cytoplasmic bridges were left behind, and the more the cell body retracted, the longer the bridges became (Figs. 1 and 3a). It was notewor- thy that the leading lamella never took “arbo- rized” forms as in the fibroblastic cells treated with cytB [15-18]. The retraction of the margin attained to a maximum about 40 min after the application of cytB, and kept the state for several hours (Fig. 1). The retracted marginal cells in cytB-L-15 for 2hr could restart to develop the leading lamella and to migrate within 30 min after brought back to a normal L-15 medium. 3. SEM observation on the epithelia cultured for more than 24hr on cytB-W-H Almost all of the epithelial marginal cells cul- tured on cytB—W-H in combination with 0.3 MF lost the leading lamella to be a hemisphere in shapes (Fig. 2d). Some had poor leading lamella The effect of cytochalasin B on the epithelial spreading Culture medium W-H W-H+DMSO W-H+2% DMSO-+0.04 ug/ml cytB W-H+2% DMSO-+0.4 ug/ml cytB W-H+2% DMSO+4 zvg/ml cytB W-H Substratum ae Hee d.) 0.3 MF 10 USES: 0.3 MF 10 6.0+1.2 0.3 MF 14 3a 14 0.3 MF 12 1.9+0.6 0.3 MF 15 20:2 1.2 MF 15 10E 031 eee SEE EnEEEUE REESE Sap W-H: Wolff-Haffen’s medium. DMSO: dimethylsulfoxide. Av. +s.d.: Average + standard deviation. 468 S. TAKEUCHI with many blebs at the edges (Fig. 2a) as in the aggregated fibroblasts in a medium containing cytB [19], suggesting no sufficient expansion occurred in the cells [20]. Those on W-H without cytB, in contrast, were flat and had well de- veloped leading lamella, from which filopodia, lamellipodia, micro-filopodial processes, blebs or ruffles were protruded as reported before [21] (Fig. 2c). The submarginal cells elongated along the direction in parallel with the marginal line and had microvilli on top as in those cultured on normal W-H, suggesting weak or no effect of cytB on them (Fig. 2a). The inner area differed from place to place in the effect of cytB: Most of places were kept intact. Some still kept regular polygonal shapes, but swelled up slightly, or tended to break con- tacts with the neighbours to be a fibroblast-like cell on the surface of epithelium (Fig. 2b). In the epithelium combined with 1.2 MF, the effect of cytB was not clear. Neither the margin- al, submarginal nor the inner cells changed con- spicuously in their figures on cytB-W-H: The marginal cells scarcely attached to MF and not spread at all, quite similar with those cultured in combination with 1.2 MF on normal W-H (Fig. 2d). 4. The distribution pattern of F-actin in the epithelial cells in the presence of cytB In the cytoplasmic bridges left behind the retracted marginal cells (Figs. 1 and 3a), there were numbers of small fragments bound with NBD-Ph scattered (Fig. 3b). Similar fragments of Pres Be fluorescence were recognized also at perinuclear We ee oe: region in the inner cells (Fig. 3d). Most of eA Ee oS 8 . 4 « Re - a \t.s marginal cells cultured for more than 24hr on Nee | ee cytB-W-H in combination with 0.3MF had hemispherical edges, in which the fluorescence of NBD-Ph was recognized faintly only along cell membrane. By contrast, on normal W-H, the Fic. 1. The process of marginal retraction in the epithelium in cytB-L-15. The numerals at right end in each figure indicate the time after the epithe- lium was transferred into cytB-L-15. The thick black bar on the top figure, 50 um. The thin bars in each mark the fixed points on the glass surface. The Effect of CytB on Epithelial Cells 469 Fic. 2. The epithelial cells observed with an SEM. a. The marginal cells in the epithelium combined with 0.3MF and cultured for 12 hr on cytB—-W-H. In comparison with Fig. 2c below, the retraction of margin and bleds are marked. The white bar, 10 um. b. The submarginal cells and the inner of the epithelium combined with 0.3 MF and cultured for 48 hr on cytB-W-H. The marginal cells retracted completely. Some of inner cells were swelled and loosened connection among them. The white bar, 10 um. c. The marginal cells combined with 0.3MF and cultured for 24 hr on normal W-H. Note the leading lamella spread in flat. The white bar, 5 um. d. The marginal cells in the epithelium cultured for 24 hr, in association with 1.2 MF, on cytB-W-H. The white bar, 5 um. 470 S. TAKEUCHI Fic. 3. The epithelial cells cultured on the glass surface. a. The fine thread-like leading lamella in the epithelial marginal cells after 4 hr of culture in cytB-L-15. The white bar, 50 um. b. The small aggregates of F-actin in the thread-like leading lamella in the epithelial cells cultured for 4 hr in cytB-L-15. Compare with the alignment of F-actin bundles in the well developed leading lamella in Fig. 3c below. The black bar, 50 um. c. The alignment of bundles of F-actin fibers in the marginal cells cultured for 24 hr in association with 0.3 MF on normal W-H. The white bar, 50 um. d. F-actin fibers in the inner cells of the epithelium cultured for 4 hr in cytB—L-15. Note F-actin fibers are aligned along cell borders. The white bar, 20 um. bundles of NBD-Ph were observed to run through cell body from a cytocenter to the leading edges. The distribution pattern was similar with that of microfilaments observed with the aid of a trans- mission electron microscope [1] and also with that of actin filaments observed with the indirect immunofluorescence method using anti-actin anti- body ({10] and Takeuchi, unpublished). In the leading lamella once lost from the marginal cell in the presence of cytB and re- covered in a normal medium, the NBD-Ph posi- tive filaments were radially aligned, running through the leading lamella as in the cells not treated with cytB (Fig.3c). The recovery of NBD-Ph positive filaments was confirmed in both the epithelia cultured on MF on cytB—W-H for 72hr and those on the glass surface in cytB—L-15 for 2hr. In the inner cells, the bundles of NBD-Ph positive filaments were aligned along the polygon- al borders as in the intact epithelium [22] without noticeable changes after the application of cytB, suggesting no effect of cytB on them (Fig. 3d). Apart from the border, however, many flakes of NBD-Ph were recognized to be scattered at perinuclear region (Fig. 3d). DISCUSSION The effect of cytB on the epithelial spreading The Effect of CytB on Epithelial Cells 471 was evident (Table 1). This agreed with the results that the movement of corneal epithelium of newt was arrested with ctyB or cytD [23]. In the epithelia, spreading of which was inhi- bited, the marginal cells were most severely deformed with cytB, even cultured on a substra- tum smooth enough to permit spreading: they lost the leading lamella to be hemisphere in shapes as those cultured on the rough surface without cytB [16]. The deformation began soon after the epithelium was transferred into cytB—L-15 (Figs. 2 and 3a). It could, therefore, be said that the inhibition of epithelial spreading was mainly due to the loss of locomotory activity in the marginal cells accomapnied with the deformation. How did cytB act on the cells to bring about deformation or to disrupt the locomotory activity? The action of cytB on actin filaments There is a variety of elucidation reporting about the mode of action of cytB: CytB bound to one end of an actin filament to block further associa- tion of actin monomers [24-29]. CytB inhibited nucleation of F-actin, so that the formation of actin filaments was delayed [30]. CytB inhibited annealing of F-actin to suppress further elonga- tion [31]. Besides, ctyB was reported to inhibit gelation of actin [26, 30,32-34]. MacLean- Fletcher and Pollard [30] insisted the inhibition of actin-network formation might be more important in living cells than that of actin elongation. Schliwa [35] pursued the changes in the cyto- skeletal system under the presence of cytB. With- in minutes, cytB disrupted the cytoskeletal net- works by severing the filaments into small frag- ments. At the next, cytoplasmic contraction, though disorganized and uncontrolled, occurred to form dense foci (aggregates of filamentous materials). A series of changes ended at a state, where the cytoskeletal system was disintegrated completely. NBD-Ph was reported to associate specifically with F-actin [36]. According to this, the flakes observed in the marginal cells in cytB—L-15, were considered as aggregates of disintegrated F-actin derived from the bundles in the leading lamella as reported by Schliwa [35] (Fig. 3c), or considered as myoid bundles which were assumed to be contracted microfilamentous apparatus at the cell cortex reported by Miranda et al. [37]. Whichever the nature of the flakes might be, it was sure that the bundles of F-actin in the cells were disinte- grated completely. The problems are how, speci- fically in the marginal cells, the F-actin bundles were disorganized, especially at early phase, and how the F-actin bundles were reformed and aligned in accopany with restoration of the lead- ing lamella. The early changes in the marginal cells, when the epithelial sheet was immersed in cytB-L-15 The marginal cells were narrowed very quickly at the leading lamella, when cytB was applied (Fig. 2). This suggested the structure or the machanism keeping the leading lamella flat was most sensitive to cytB. Heath [38] reported that there were networks called as dorsal cortical microfilament sheath (DCMS) in the leading lamella of fibroblastic cells under locomotion. The DCMS consisting of actin and other contractile proteins were recycled from disintegration to reassociation to spread the lead- ing lamella. The DCMS was moved backwards along dorsal surface of the leading lamella and decomposed to be cytoplasmic sol, which were pushed forwards to the leading edges to be reformed as DCMS. Considering similarity of the epithelial cells in locomotory activities to the fibroblastic cells [1, 6], the DCMS was assumed to work also on the leading lamella of epithelial cells. In the presence of cytB, the DCMS could not be structuralized because F-actin polymeriza- tion was inhibited. This might be a reason why the leading lamella was sensitive to cytB and narrowed themselves at first. There might be another explanation for the mode of action of cytB to the leading lamella. According to Godman et al. [39, 40], F-actin was dissociated with actin-binding proteins with cytB and aggregated with myosin as in superprecipita- tion. This might bring about a general contrac- tion of the leading lamella. The mode of action of cytB in living cells will be elucidated more comprehensively when the behavior of actin in situ is fully understood. 472 S. TAKEUCHI The late changes in the marginal cells, the elonga- tion of cytoplasmic bridges The initial shrinkage of width in the leading lamella was followed by elongation of it. It was observed to be elongated straight, not so “arbo- rized” as in the fibroblastic cells treated with cytB [15-18]. This might be explained as follows: after the bundles of F-actin were disintegrated with cytB, the marginal cells could not pull any more the rest of epithelial cells outwards but were, in reverse, pulled inwards, too strongly to permit the slack in the cytoplasmic bridge, by elastic force of themselves and/or of neighbouring cells which had counterbalanced tension arisen inevitably in them. Some parts in the leading edge stuck firmly to the substratum worked as supporting points against the recoiling force. Thus, the more the cells retracted, the longer the cytoplasmic bridges became. This hypothesis was agreed with the reports presented previously: there was strong tension at the marginal area of epithelial sheet in culture. The margin retracted to a considerable extent, when the contacts of marginal cells with the glass surface were disrupted mechanically [41]. Furthermore, the tension accelerated the epithelial spreading seemingly by enhancing the transition of the epithelial cells from a stationary state to a motile [42]. The focal contacts [3] were candidates for the supporting points which stuck firmly to the substratum. Indeed, the focal contacts were recognized in the epithelial margin- al cells [1, 10]. Some of these contacts remained even in the presence of cytB to work as support- ing points against the recoiling force. Stability of F-actin filaments It was quite queer that the bundles of F-actin along borders of the inner cells could not be disintegrated with cytB, while those at perinuclear region were broken down to be small flakes (Fig. 3d). Based on the action of cytB discussed above, the intact bundles were considered to be in a stationary state in which neither polymerization nor disintegration occurred. At the perinuclear region of the inner cells or at the leading lamella of marginal cells, in contrast, F-actin filaments were maintained in an equilibrium between inces- sant association of actin monomers with one end and decomposition at another end as discussed by Brenner and Korn [25]. Fuchtbauer et al. [43] confirmed this in both the fibroblastic cells and the epithelial cells. Capping proteins for F-actin, when injected into the cells in culture, disinte- grated the microfilaments at the site where the focal contacts were formed, causing shrinkage of the cells. According to them, actin monomers were always taken into filaments at the focal contacts. Summerizing the points, the bundles of F-actin were newly aligned only at such active parts in cells as the leading lamella which generated force for works. In the marginal cells which were responsible for generating force for epithelial spreading, F-actin might be always polymerized and the bundles of them might be aligned along tension in order to work most effectively in accordance with formation of firm contacts be- tween cell and substratum. "These results supported our “two step hypoth- esis” that tried to elucidate how the stationary epithelial cells acquired motility (Takeuchi, unpublished): the stationary cells were pulled outwards and under tension, at the first step, the bundles of microfilaments along cell border were discomposed completely and, at the second step, the components were reconstructed to be aligned along the direction of tension through cell body to the leading edge which generated force for locomotion. Only cytB-sensitive microfilaments were, therefore, observed in the marginal cells that made the transition from a stationary state to motile. REFERENCES 1 Heath, J.P. (1982) Adhesions to substratum and locomotory behaviour of fibroblastic and epithelial cells in culture. In “Cell Behaviour”, Ed. by R. Bellair, A. Curtis and G. Dunn, Cambridge Univ. Press, Cambrige, pp. 77-108. 2 Trinkaus, J. P. (1984) Cells into Organs. The Forces That Shape the Embryo, Prentice-Hall, Englewood Cliffs, New Jersey, 543 pp. 3 Izzard,C.S. and Lochner, L.R. (1976) Cell-to- substrata contacts in living fibroblasts: An interfer- ence reflexion study with and evaluation of the technique. J. Cell Sci., 21: 129-160. 10 11 Le 13 14 15 16 17 18 The Effect of CytB on Epithelial Cells Radice, G. P. (1980) The spreading of epithelial cells during wound closure in Xenopus larvae. Dev. Biol., 76: 26-46. Radice, G.P. (1980) Locomotion and _ cell- substratum contacts of Xenopus epidermal cells in vitro and in situ. J. Cell Sci., 44: 201-223. DiPasquale, A. (1975) Locomotory activity of epithelial cells in culture. Exp. Cell Res., 94: 191- 215: Abercrombie, M., Heaysman,J.M. and Pegrum, S.M. (1970) The locomotion of fibroblasts in culture. I. Movements of leading edge. Exp. Cell Res., 59: 393-398. Abercrombie, M., Heaysman,J.M. and Pegrum, S.M. (1970) The locomotion of fibroblasts in culture. II. Ruffling. Exp. Cell Res., 60: 437-444. Abercrombie, M., Heaysman,J.E. and Pegrum, S.M. (1970) The locomotion of fibroblasts in culture. III. Movements of particles on the dorsal surface of the leading lamella. Exp. Cell Res., 62: 389-398. Billig, D., Nicol, A., McGinty, R., Cowin, P., Mor- gan, J. and Garrod, D. (1982) The cytoskeleton and substratum adhesion in chick embryonic corneal epithelial cells. J. Cell Sci., 57: 51-71. Iscenbere, G.; Rathke, P-.C., Hulsmann, N., Franke, W.W. and Wohlfarth-Bottermann, K. E. (1976) Cytoplasmic actomyosin fibrils in tissue culture cells. Direct proof of contractility by visualization of ATP-induced contraction in fibrils isolated by laser microbeam dissection. Cell Tissue Res., 166: 427-443. Schroeder, T. E. (1970) The contractile ring. Fine structure of dividing mammalian (HeLa) cells and the effects of cytochalasin B. Z. Zellforsch. Mik- rosk. Anat., 109: 431-449. Takeuchi, S. (1976) Wound healing in the cornea of the chick embryo. III. The influence of pore size of Millipore filters on the migration of isolated epithe- lial sheets in culture. Dev. Biol., 51: 49-62. Wolff, Et. and Haffen, K. (1952) Sur une méthode de culture d’organes embyronnaires in vitro. Texas Rep. Biol. Med., 10: 463-472. Sanger, J. W. (1974) The use of cytochalasin B to distinguish myoblasts from fibroblasts in cultures of developing chick striated muscle. Proc. Natl. Acad. Sci. USA., 71: 3621-3625. Miranda, A. F., Godman, G.C., Deitch, A. D. and Tanenbaum, S. W. (1974) Action of cytochalasin D on cells of established lines. J. Cell Biol., 61: 481- 500. Atlas, S.J. and Lin, S. (1978) Dihydrocytochalasin B. Biological effects and binding to 3T3 cells. J. Cell Biol., 76: 360-370. Bliokh, Z.L., Domnina, L. V., A., Vasiliev, J.M. and Gelfand, I. M. Smolyaninov, V. (1980) 20 21 22 23 24 26 pif 28 29 30 31 32 473 Spreading of fibroblasts in medium containing cytochalasin B: Formation of lamellar cytoplasm as a combination of several functionally different pro- cesses. Proc. Natl. Acad. Sci. USA., 77: 5919-5922. Van der Schueren, B., Cassiman, J.J. and van der Berghe, H. (1978) Modulation of the effect of colchicine and cytochalasin B on fibroblast aggre- gates by the substratum. J. Cell Sci., 31: 353-367. Erickson, C. A. and Trinkaus, J.P. (1976) Micro- villi and blebs as sources of reserve surface mem- brane during cell spreading. Exp. Cell Res., 99: 375-384. Takeuchi, S. (1983) Would healing in the cornea of the chick embryo. V. An observation and quantita- tive assessment of the cell shapes in the isolated corneal epithelium during spreading in vitro. Cell Tissue Res., 229: 109-127. Owaribe, K., Araki, M., Hatano, S. and Eguchi, G. (1979) Cell shape and actin filaments. In “Cell Motility: Molecules and Organization”. Ed. by S. Hatano, H. Ishikawa and H. Sato, Univ. Tokyo Press, Tokyo, pp. 491-500. Yamanaka, H and Eguchi, G. (1981) Regeneration of the cornea in adult newt: Overall process and behavior of epithelial cells. Differ., 19: 84-92. Brown, S.S. and Spudich, J. A. (1979) Cytochalasin inhibits the rate of elongation of actin filament reagents. J. Cell Biol., 83: 657-662. Brenner, S..L. and) “Kor; B2D-"((1979) ~~ Sub- stichiometric concentrations of cytochalasin D in- hibit actin polymerization. Additional evidence for an F-actin treadmill. J. Biol. Chem., 254: 9982- 9985. Lin. D.C. and Lin, S. (1980) Cytochalasins inhibit actin gelation by binding to filament ends. J. Cell Biol. , 87(2-2): 213a. Flanagan, M.D. and Lin, S. (1980) Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J. Biol. Chem., 255: 835-838. Lin, D.C. Tobin, K.D., Grumet, M. and Lin, S. (1980) Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation. J. Cell Biol., 84: 455-460. Brown, S.S. and Spudich, J. A. (1981) Mechanism of action of cytochalasin: Evidence that it binds to actin filament ends. J. Cell Biol., 88: 487-491. MacLean-Fletcher,S. and Pollard, T.D. (1980) Mechanism of action of cytochalasin B on actin. Cell, 20: 329-341. Maruyama, K., Hartwig,J.H. and Stossel, T. P. (1986) Cytochalasin B and the structure of actin gels. I]. Further evidence for the splitting of F-actin by cytochalasin B. Biochim. Biophys. Acta, 626: 449-450. Weihing, R. R. (1976) Cytochalasin B inhibits actin- 313) 34 35 36 Si 38 474 related gelation of HeLa cell extract. J. Cell Biol., 71: 303-307. Pollard, T.D. (1976) The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba. J. Cell Biol., 68: 579- 601. Hartwig, J. H. and Stossel, T. P. (1979) Cytochala- sin B and structure of actin gels. J. Mol. Biol., 134: 539-553. Schliwa, M. (1982) Action of cytochalasin D on cytoskeletal networks. J. Cell Biol., 92: 79-91. Barak, L.S., Yocum, R.R., Nothnagel, E. A. and Webb, W. W. (1980) Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2- oxa-1, 3-diazole-phallacidin. Proc. Natl. Acad. Sci. USA., 77: 980-984. Miranda, A. F., Godman, G. C. and Tanenbaum, S. (1974) Action of cytochalasin D on cells of estab- lished lines. II. Cortex and microfilaments. J. Cell Biol., 62: 406-423. Heath, J.P. (1983) Direct evidence for microfila- ment-mediated capping of surface receptors on crawling fibroblasts. Nature, 302: 532-534. S. TAKEUCHI 39 40 41 42 43 Godman, G., Woda,B. and Kolberg, R. (1980) Redistribution of contractile and cytoskeletal com- ponents induced by cytochalasin. I. In Hmf cells, a nontransformed fibroblastoid line. Eur, J. Cell Biol., 22: 733-744. Godman, G. Woda, B. and Kolberg, R. (1980) Re- distribution of contractile and cytoskeletal compo- nents induced by cytochalasin. II. In HeLa and HEp2? cells. Eur. J. Cell Biol., 22: 745-754. DiPasquale, A. (1975) Locomotion of epithelial cells. Factors involved in extension of the leading edge. Exp. Cell Res., 95: 425-439. Takeuchi, S. (1979) Wound healing in the cornea of chick embryo. IV. Promotion of the migratory activity of isolated corneal epithelium in culture by the application of tension. Dev. Biol., 70: 232-240. Fuchtbauer, A., Jockusch, B. M., Maruta, H., Kili- mann, M. W. and Isenberg, G. (1983) Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells. Nature, 304: 361-364. ZOOLOGICAL SCIENCE 4: 475-481 (1987) Retinal and 3-Dehydroretinal in the Egg of the Clawed Toad, Xenopus laevis TAKAHARU SEKI, SHIGECHIKA FUJISHITA, MASAMI Azuma! and Tatsuo SuzuKI* Department of Health Science, Osaka Kyoiku University, Hirano Campus, 1-6-7 Nagaremachi, Hiranoku, Osaka 547, ‘Department of Health Science, Osaka Kyoiku University, 4-88 Minamikawahoricho, Tennojiku, Osaka 543, and *Department of Pharmacology, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya, Hyogo 663, Japan ABSTRACT—Retinals in the egg of the clawed toad, Xenopus laevis, were extracted and the composition of the geometric isomers was analysed by using high-performance liquid chromatography. Nearly equal amounts of retinal (all-trans and 13-cis) and 3-dehydroretinal (all-trans and 13-cis) were present at the total quantity about 50 pmol/egg. In the eggs of other frogs, Rhacophorus schlegelii and Rhacophorus arboreus, only retinal was contained at the quantity about 50 pmol/egg. Retinyl ester and carotenoids in the Xenopus eggs were scarce, and retinols, 3-hydroxyretinals or 11-cis isomers of retinals were undetectable in them. Retinals in the Xenopus eggs were suggested to be present in a protein-bound form through the Schiff base linkage, for after reduction by NaBH, neither retinol nor retinyl-product could be found in the organic solvent layer. The metabolic pathways forming the egg retinals are discussed on the basis of retinyl ester and © 1987 Zoological Society of Japan carotenoids found in the Xenopus ovary. INTRODUCTION In animal kingdom, diversity of visual pigments has been ensured by both the variety of the protein and the prosthetic moieties. On the basis of the prosthetic moiety, three kinds of visual pigments can be classified: rhodopsin (and iodop- sin), porphyropsin (and cyanopsin) and xantho- psin [1], containing 11-cis retinal, 11-cis 3- dehydroretinal and 11-cis 3-hydroxyretinal [2-4], respectively. How an animal obtained the capac- ity to use one or a few of the multiformular visual pigments is an interesting problem to be clarified in relation to an adaptation of the animal to the environmental conditions. For this purpose, a clawed toad, Xenopus laevis, is one of the excel- lent materials because this toad contains porphy- ropsin as a principal visual pigment [5] and can be Accepted December 26, 1986 Received October 20, 1986 reared easily in a laboratory under controlled conditions, e.g. on food of the known retinoid composition. Furthermore, it can be induced to lay fertile eggs by administration of gonado- trophic hormone at any time of year [6], and the change of retinoid composition during the de- velopment of eggs into tadpoles can be examined precisely by using high-performance liquid chro- matography (HPLC). At the beginning of the study, we analysed retinoids and carotenoids in the frog eggs and ovary. Retinoids in the egg of frogs in another suborder (Rhacophorus arboreus and R. schlegelii) were also analysed. MATERIALS AND METHODS Pairs of X. laevis were kindly supplied by Dr. Takasaki (Osaka Kyoiku Univ.) or purchased from Hamamatsu Seibutsu Kyozai Co., Ltd. (Shizuoka, Japan). They were reared at 25°C and fed with porcine liver twice a week. The eggs of 476 T. SEKI, S. FUJISHITA et al. X. laevis were induced to breed by injection of chorionic gonadotrophic hormone (Gonatropin; Teikoku Zoki Ltd., Tokyo) to a pair [6], 300 IU to female and 150 IU to male. In some cases, unfertilized eggs were bred, but no difference was found between fertilized and unfertilized eggs in the results reported here. In order to facilitate handling, the jelly layer was removed by immers- ing the eggs in 1% sodium thioglycolate, pH 9-10 [7]; the presence of the jelly layer did not affect the results. A lump of eggs of R. schlegelii was collected at a rice field and that of R. arboreus at a local pond both in Fukui Prefecture on 22 June, 1986. Eggs of R. arboreus hatched on the next day, and so the abdominal parts, where the yolk was retained, of the tadpoles were collected and analysed. Two methods were applied to extract retinoids from eggs. For the extraction of retinal as oxime [8, 9], the procedure described previously [4] was used. For the extraction of nonderivertized reti- nal, the formaldehyde method [10] was used with minor modification. Two hundreds of eggs were homogenized, in a Potter-Elvehjem homogenizer with a motor-driven teflon pestle, in 5ml of 90% aqueous methanol containing 1.2M _ formalde- hyde. After standing at room temperature for 5min, 10 ml of 6M formaldehyde was added and the retinoids were extracted with 10 ml of dichlo- romethane and 20ml of n-hexane by centrifuga- tion at 3500rpm for 5min. The extraction with dichloromethane/n-hexane was repeated three times. Reduction of retinal into retinol by sodium borohydride(NaBH,), formation of retinaloximes with hydroxylamine (NH,OH) and saponification of retinyl ester were performed by the described methods [11]. Retinoids and carotenoids were analysed on a Hitachi 655 HPLC system equipped with a 6x 150mm column of YMC-PACK A-012-3 S-3 SIL (particle size 34m; Yamamura Chemical Laboratories Co., Ltd., Kyoto) with an eluent, 8% diethylether-0.08% ethanol in n-hexane, or with a 4X250mm column of LiChrosorb SI-60 (S «m; Merck, Darmstadt, FRG) with the eluents, 15% diethylether-0.15% ethanol in n-hexane followed by 50% diethylether-0.5% ethanol in n-hexane. The flow rate was at 2ml/min. Absorbance at 350nm was monitored with a Hitachi 638-41 UV-detector and at 450nm with a 875—-UV detector (Japan Spectroscopic Co., Ltd., Tokyo). Quantities of retinaloxime and 3- dehydroretinaloxime were estimated from the peak height of the syn isomers of the equimolar mixture of standard oximes [12]. Absorption spectra were measured with a Hitachi 200-20 spectrophotometer equipped with a X—Y recorder (RY-101; Rikadenkikogyo Co., Ltd., Tokyo). All operations were carried out under red light exceeding 610nm. RESULTS AND DISCUSSION The elution profile of the standard oximes (Fig. 1A) shows the peaks of syn and anti 11-cis retinaloximes (1,1), 11-cis 3-dehydroretinal- oximes (2, 2’) all-trans retinaloximes (3, 3’) and all-trans 3-dehydroretinaloximes (4, 4’). Figure 1B shows the elution profile of retinoids extracted from eggs of R. schlegelii, indicating the peaks corresponding to all-trans retinaloximes (3, 3’) and 13-cis retinaloximes (5, 5’). In Figure 1C, a typical chromatogram of retinoids extracted from the eggs of X. laevis is shown. The several peaks in Figure 1C were assigned to all-trans retinal- oximes (3,3), all-trans 3-dehydroretinaloximes (4, 4°), 13-cis retinaloximes (5,5) and 13-cis 3- dehydroretinaloximes (6,6). Peaks correspond- ing to l1l-cis isomers of retinaloximes and 3- dehydroretinaloximes, or retinol and 3-dehydro- retinol were not detected in the egg extracts. The existence of 3-hydroxyretinaloximes was _ ex- amined by changing the eluent from 8% ethylether-0.08% ethanol in n-hexane to 50% ethylether-0.5% ethanol in n-hexane [4], but any peaks due to 3-hydroxyretinaloximes were unde- tectable. For further analysis of retinals in the Xenopus eggs, the formaldehyde method was applied to the extraction of retinals. Figure 2 shows an elution profile of retinal isomers on HPLC; the peaks attributable to 13-cis and all-trans isomers of retinal (1,5), and those of 3-dehydroretinal (2, 6) are observed, accompanied with small peaks (3, 4) probably due to 9-cis isomers of retinal and Retinals in the Xenopus Egg 477 Xenopus laevis Tt Lim eS (Ghiten) Fic. 1. High-performance liquid chromatograms of (A) standard oximes of retinal and 3-dehydroretinal, (B) retionids extracted from eggs of R. schlegelii and (C) those of X. laevis by the oxime method. Standard oximes contain syn and anti 11-cis retinaloximes (1, 1’), 11-cis 3-dehydroretinaloximes (2, 2’), all-trans retinaloximes (3, 3’) and all-trans 3-dehydroretinaloximes (4, 4’) at 50pmol each. (B) shows the peaks with the retention times similar to those of all-trans retinaloximes (3, 3’) and 13-cis retinaloximes (5,5), and (C) exhibits the peaks corresponding to all-trans retinaloximes (3, 3’), all-trans 3-dehydroretinaloximes (4, 4°), 13-cis retinaloximes (5, 5’)and 13-cis 3-dehydroretinaloximes (6,6). HPLC conditions; column: YMC-PAC A-012-3 S-3 SIL, 4x150min, mobile phase: 8% diethylether-0.08% ethanol in n-hexane, flow rate: 2ml/min, and detection: absorbance at 350 nm. 3-dehydroretinal. Identification of the peak sub- reactivities. Three fractions corresponding to stances was further carried out with the measure- peaks 1 and 2 (fraction 1), peak 5 (fraction 2) and ments of absorption spectra and the chemical peak6 (fraction3) in Figure 2 were collected. 478 T. SEKI, S. FusIsHita et al. sk im © ( imaliow ) Fic. 2. The elution profile of retinoids extracted from 200 eggs of X. laevis by the formaldehyde method and chromatographed on HPLC. The peaks with the retention times similar to those of 13-cis retinal and 13-cis 3-dehydroretinal (1, 2), 9-cis retinal and 9-cis 3-dehydroretinal (3,4) and all-trans retinal and all-trans 3-dehydroretinal (5,6) are obvious. HPLC conditions are same as those of Fig. 1. Absorbance Wavelength ( nm ) Fic. 3. Absorption spectra of the peak substances shown in Fig. 2. The fractions corresponding to peaks 1 and 2 (curve 1), peak 5 (curve 2), peak 6 (curve 3) and the substances eluting out around solvent front (curve 4) in Fig. 2 were collected, redissolved in 1ml of ethanol and the absorption spectra of the fractions were measured. Curve 4 was diluted 5 times before spectrophotometry. Figure 3 shows the absorption spectra of these fractions in ethanol. Fraction 2 shows the absorp- tion maximum at 380nm (curve 2) and fraction 3 at 400nm (curve 3), indicating that the former is all-trans retinal and the latter all-trans 3-dehydro- retinal. Fraction 1 shows the absorption spectrum (curve 1) due to the mixture of 13-cis retinal, 13-cis 3-dehydroretinal and some contaminating material with the absorption maximum around 280nm. The fractions used for spectral analysis were assayed by HPLC after treatment with NH,OH or NaBHy. By reaction with NH,OH, the fraction 1 was converted to a mixture of 13-cis isomers of retinaloxime and 3-dehydroretinal- oxime, the fraction 2 to all-trans retinaloxime and the fraction 3 to all-trans 3-dehydroretinaloxime. The treatment with NaBH, converted the retinal isomers to corresponding retinol isomers. From these results it is concluded that all-trans and 13-cis isomers of retinal and 3-dehydroretinal are included in the egg of X. laevis. An existing state of the retinals in the Xenopus egg was examined by using NaBH, [cf. 13]. Toa homogenate of eggs in 67mM phosphate buffer, pH 6.7, were added NaBH, grains and then the retinoids in the homogenate were extracted by the oxime method or formaldehyde method. The organic solvent layer (dichloromethane/n-hexane) was collected and analysed by HPLC, spectropho- tometry and fluorometry. On HPLC analysis, no peaks corresponding to retinol or 3-dehydro- retinol were detected, suggesting that the retinals are not present in free forms that could be reduced to retinols by NaBH, [13]. The measure- ment of absorption and fluorescence spectra did not show any retinol or N-retinyl-product in the organic solvent layer. These results suggest strongly that the retinal and 3-dehydroretinal in eggs exist as a complex with some protein by forming the Schiff base. The Schiff base com- pound of retinal and protein is converted to N-retinyl-protein by NaBH,, which can not be extracted into the organic solvent layer. Plack and Kon [14] have reported that the retinals in fish eggs [14] and in hen’s eggs [15] are present in such a way that the characteristic properties are masked, probably because they are bound to a lipid and protein. In order to examine the presence of retinyl ester, the materials eluting at the solvent front on HPLC (Fig. 2) were collected and analysed by measurement of absorption spectrum (curve 4 in Retinals in the Xenopus Egg 479 TABLE 1. Amounts and composition of geometrical isomers of retinals in frog eggs Retinal 3-Dehydroretinal He Frogs (pmol/egg) (pmol/egg) ( si all-trans 13-cis all-trans 13-cis EmaviT See) Xenopus laevis 23.6 2.6 25.0) 3.1 54.3 Rhacophorus schlegelii 44.2 6.3 n.d. n.d. 50.5 Rhacophorus_ arboreus 53.4 n.d. n.d. n.d. 53.4 n.d.: not detectable. Fig. 3). The main absorption maxima were pre- sent at 275 and 285nm, and the absorbance around 330nm was about 20% that at 285nm. Saponification of the substance caused only small decrease in HPLC peaks at the solvent front and induced very small peaks due to retinol isomers. The amount of retinyl esters, if any, must be scarce in the egg of X. Jaevis. Curve 4 in Figure 3 also shows that little carotene is present in the egg. As was shown in Figure 1B, the egg of R. schlegelii contains all-trans and 13-cis isomers of retinal as the main stock of retinoids; retinyl ester (at the solvent front) seems to be present but the amount might be far less than that of retinal. The yolk of R. arboreus also contains all-trans retinal as the main stock of retinoids. In any case, Rhacophorus eggs contain no 3-dehydroretinal. Table 1 shows the composition and amount of retinal isomers contained in an egg of the three B species of frogs. All the frogs contain about 50 pmol retinal(s) per egg, and in the case of X. laevis neary equal amounts of retinal and 3- Fic. 4. Retinoids and carotenoids in a ovary of X. laevis. A, elution profiles of an extract obtained from the ovary by the oxime method and analysed tEtaae on HPLC equipped with two detectors monitoring absorbances at 350nm (upper) and at 450 nm (low- 1 er). Under this HPLC conditions (column: LiChro- San . sorb SI-60, 4X250mm, eluent: 15% diethylether- 0.15% ethanol in n-hexane for 15 min followed by 50% diethylether-0.5% ethanol in n-hexane, and Absorbance ; arbitrary unit 3 4 4 flow rate: 2ml/min), the peaks of syn all-trans retinaloxime and 3-dehydroretinaloxime are 5 : cochromatographed (peak 2). B, absorption spec- Pt tra of the original extract from the ovary and the peak substances separated by HPLC (peaks 1, 2, 3, Wavelength ( nm ) 4 and 5 a A). 480 T. SEKI, S. Fusisuita et al. dehydroretinal are present. Plack and Kon [16] have reported that the egg of Rana temporalia contains retinal (1.72mg/g dry matter) and that the proportion of 3-dehydroretinal is 11%, using the Carr-Price method improved by Plack [17]. An interesting problem of the egg retinals is their metabolic origin. To investigate the precur- sor of the egg retinals, retinoids and carotenoids in the ovary of X. laevis were extracted by the oxime method and analysed on HPLC with two detectors monitoring absorbances at 350 and 450 nm (Fig.4A). The elution profile detected at 350nm (upper in Fig. 4A) shows a peak at the solvent front (peak 1) besides the peaks of retinal- oximes (peak 2); under the eluent conditions, syn all-trans retinaloxime and syn all-trans 3-dehydro- retinaloxime are not separated. Figure 4A also shows the absence of 3-hydroxyretinal in the ovary [cf.4]. The elution profile detected at 450nm (lower in Fig.4A) shows some peaks suggesting the presence of carotenoids including xanthophyll (peaks 4 and 5) which have higher polarity than carotene. Figure 4B shows the absorption spectra of the peak substances shown in Figure 4A (peaks 1,2,3, 4 and 5). Curve 1 shows the absorption bands around 330 nm and in the visible region suggesting the presence of retinyl ester and carotene, respectively. Curve 2 shows the absorption spectrum of retinaloximes, and curves 3, 4 and 5 show the characteristic spectra of carotenoids [cf. 18]. Now, two possible metabolic pathways forming egg retinals can be considered. One is the oxidation of retinol and 3-dehydroretinol, which should have been trans- ported from the liver to the ovary, producing retinal and 3-dehydroretinal, respectively. Another is the formation of retinals from carotenoids. Gawienowski et al. [19] have re- ported that f-carotene is converted to retinal by slices of bovine corpus luteum. In the frog ovary, where both retinals and carotenoids are found, such an enzyme as carotene cleavage enzyme [20, 21] may function to produce retinals from carotenoids. The change of the retinoid composition in the Xenopus egg during developement is now under investigation; the amount and the composition of retinals in the abdomen of the tadpoles remain almost constant till about stage 38. Bridges et al. [5] have shown that the visual pigment of Xeno- pus tadpole becomes detectable from stage 39. The retinals in the egg must be a reservoir for the precursor of visual pigment chromophores. The transport and the metabolic change of egg retinals into visual pigment chromophores in the eye is a subject of further investigation. ACKNOWLEDGMENTS The authors thank Dr. H. Takasaki (Osaka Kyoiku Univ.) for her kind supply of pairs of Xenopus laevis and for advices on rearing them. We thank also Drs. M. Ito, K. Tsukida (Kobe Women’s College of Pharma- cy) and Y. Kito (Osaka Univ.) for their discussions. This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 61740420 to T. Seki). REFERENCES 1 Vogt, K. (1983) Is the fly visual pigment a rhod- opsin? Z. Naturforsch., 38¢: 329-333. 2 Vogt, K. and Kirschfeld, K. (1984) Chemical identi- ty of the chromophore of fly visual pigment. Naturwissenschaften, 71: 211-213. 3 Tanimura, T., Isono, K. and Tsukahara, Y. (1986) 3-Hydroxyretinal as a chromophore of Drosophila melanogaster visual pigment analyzed by high- pressure liquid chromatography. Photochem. Photobiol., 43: 225-228. 4 Seki,T., Fujishita,S., Ito,M., Matsuoka, N., Kobayashi, C. and Tsukida, K. (1986) A fly, Dro- sophila melanogaster, forms 11-cis 3-hydroxyretinal in the dark. Vision Res., 26: 255-258. 5 Bridges,C.D.B., Hollyfield, J.G., Witkovsky, P. and Gallin, E. (1977) The visual pigment and vitamin A of Xenopus laevis embryos, larvae and adults. Exp. Eye Res., 24: 7-13. 6 Nieukoop, P. D. and Faber, J. (1956) Normal Table of Xenopus laevis (Daudin). North-Holland, Am- sterdam. 7 Sakai, M. and Kubota, H. Y. (1981) Cyclic surface changes in the non-nucleate egg fragment of Xeno- pus laevis. Dev. Growth Differ., 23: 41-49. 8 Groenendijk,G.W.T., De Grip, W.J. and Dae- men, F.J.M. (1980) Quantitative determination of retinals with complete retention of their geometric configuration. Biochim. Biophys. Acta, 617: 430- 438. 9 Suzuki, T. and Makino-Tasaka, M. (1983) Analysis of retinal and 3-dehydroretinal in the retina by high-pressure liquid chromatography. Anal. 10 11 12 13 14 Retinals in the Xenopus Egg Biochem., 129: 111-119. Suzuki, T., Fujita, Y., Noda, Y. and Miyata, S. (1986) A simple procedure for the extraction of the native chromophore of visual pigments: the formal- dehyde method. Vision Res., 26: 425-429. Bridges,C.D.B. and Alvarez, R.A. (1982) Measurement of the vitamin A cycle. In “Methods in Enzymology”, Vol. 81, Biomembranes. Ed. by L. Packer, Academic Press, New York, pp. 463-485. Makino-Tasaka, M. and Suzuki, T. (1986) Quan- titative analysis of retinal and 3-dehydroretinal by high-pressure liquid chromatography. In “Methods in Enzymology”, Vol. 123, Vitamins and coen- zymes. Ed. by F. Chytil and D.B. McCormick, Academic Press, New York, pp. 53-61. Seki, T. (1984) Metaretinochrome in membranes as an effective donor of 11-cis retinal for the synthesis of squid rhodopsin. J. Gen. Physiol., 84: 49-62. Plack, P.A., Kon,S.K. and Thompson, S. Y. (1959) Vitamin A, aldehyde in the eggs of the herring (Clupea harengus L.) and other marine teleosts. Biochem. J., 71: 467-476. 15 16 17 18 19 20 asl 481 Plack, P. A. (1960) Vitamin A, aldehyde in hen’s eggs. Nature, 186: 234-235. Plack, P.A. and Kon, S.K. (1961) A comparative survey of the distribution of vitamin A aldehyde in eggs. Biochem. J., 81: 561-570. Plack, P. A. (1961) The colorimetric reaction be- tween vitamin A, aldehyde and antimony trichlo- ride. Biochem. J., 81: 556-561. Zechmeister,L. (1962) Cis-trans someric Carotenoids, Vitamins A and Arylpolyenes, Sprin- ger, Wien. Gawienowski, A. M., Stacewicz-Sapuncakis, M. and Longley, R. (1974) Biosynthesis of retinal in bovine corpus luteum. J. Lipid Res., 15: 375-379. Goodman, D.S. and Huang, H.S. (1965) Biosyn- thesis of vitamin A with rat intestinal enzymes. Science, 149: 879-880. Olson, J. A. and Hayaishi, O. (1965) The enzymatic cleavage of f-carotene into vitamin A by soluble enzymes of rat liver and intestine. Proc. Natl. Acad. Sci., USA., 54: 1364-1370. ZOOLOGICAL SCIENCE 4: 483-487 (1987) © 1987 Zoological Society of Japan Chromosome Numbers in 8 Japanese Species of Sea Urchins Kyoko SAOTOME Yokohama City Institute of Health, Takigashira, 1-2-17, Isogo, Yokohama 235, Japan ABSTRACT—Chromosome numbers were examined in 8 Japanese species of sea urchins. In Regularia, Glyptocidaris crenularis belonging to the order Arbacioida had chromosome number of 2n =44 and Temnopleurus hardwickti, Pseudocentrotus depressus, Strongylocentrotus nudus, Hemicentro- tus pulcherrimus and Anthocidaris crassispina belonging to the order Echinoida all showed chromo- some numbers of 2n=42. On the other hand, in Irregularia, both Scaphechinus mirabilis and Astriclypeus manni belonging to the order Clypeasteroida had chromosome number of 2n=46. The chromosome numbers were found to be the same in the species belonging to the same order but differ among three different orders. Arbacioida, Echinoida and Clypeasteroida had chromosome numbers of 2n=44, 2n=42 and 2n=46, respectively. INTRODUCTION The echinoid fauna is especially abundant in Japan [1-4] and about 160 species have been ascertained by Shigei [4]. Systematic study on the echinoids has been carried out from the morpho- logical viewpoint, whereas unsolved systematic problems still remain [3]. In the last ten years, biochemical approach has been tried in echinoid phylogeny [5-8] and the biochemical data have been compared with the phylogenetic relationship based on morphological criteria. On the other hand, cytogenetical technique is also considered to provide valuable information for echinoid tax- onomy [9]. However, chromosomal reports about Japanese echinoids are too few to discuss systema- tic problems [10-12]. The reason for inactivity in chromosome study may be that sea urchins have many chromosomes of small size and that clear metaphase plates are not constantly obtained by sectioning method [10] or squash method [11, 12] used so far. Therefore, an air-drying method has been developed to obtain well-spread chromosome preparation from the morula or the early blastula [13]. In this paper, chromosome numbers in 8 species determined by the air-drying method [13] will be Accepted January 29, 1987 Received December 16, 1986 discussed concerning echinoid taxonomy. MATERIALS AND METHODS Materials Glyptocidaris crenularis, Temno- pleurus hardwickii, and Strongylocentrotus nudus were provided by the Asamushi Marine Biological Station, Tohoku University. Pseudocentrotus de- pressus was obtained from Tsushima, Nagasaki Prefecture and Scaphechinus mirabilis from the Ushimado Marine Laboratory, Okayama Uni- versity. Hemicentrotus pulcherrimus, Anthocidaris crassispina and Astriclypeus manni were supplied by the Misaki Marine Biological Station, Universi- ty of Tokyo. Eggs and sperm were obtained by injecting 0.3 ml of 0.01M acetylcholine chloride into body cavity of adult sea urchins. More than five pairs of males and females were used for preparation. Methods Chromosome preparations were made using the embryos at the morula or the early blastula stage according to the air-drying method previously described [13] with the following mod- ification: 1) Ca-Mg-free sea water [13] or 1mM 3-amino-1, 2, 4-triazole [14] was used to remove fertilization membrane, 2) division was inhibited by colcemid [13] or colchicine, and 3) concentra- tion of colchicine (0.1-1.0mg/ml) and hypotonic KCI solution (0-0.075 M) was changed according to species. 484 RESULTS Figure 1 shows metaphase plates of 8 species of sea urchins and distribution of the diploid chromo- some numbers is summarized in Table 1. Glyptocidaris crenularis (Fig.1A) showed chromosome number of 2n=44 in 76.7% of the 73 cells examined (Table 1). Chromosome numbers of Temnopleurus hardwickii (Fig. 1B), Pseudocen- trotus depressus (Fig.1C), Strongylocentrotus nudus (Fig. 1D), Hemicentrotus pulcherrimus (Fig. 1E) and Anthocidaris crassispina (Fig. 1F) were found to be all 2n=42, and the percentage of cells with 2n=42 was 74.0% in 100 cells, 87.1% in 70 cells, 81.2% in 101 cells, 91.7% in 192 cells and 77.2% in 92 cells examined, respectively (Table 1). Both Scaphechinus mirabilis (Fig. 1H) and Astric- lypeus manni (Fig. 1G) showed the chromosome number of 2n=46 and percentage of cells with 2n =46 was 94.0% in 100 cells and 81.1% in 90 cells K. SAOTOME examined, respectively (Table 1). DISCUSSION In this paper, the clear metaphase plates which are beyond comparison with the metaphase plates already reported about Japanese echinoids [10, 12] have been obtained in many species and chromo- some numbers have been exactly counted (Fig. 1). The chromosome size of sea urchins ranged from about 0.7 um of the smallest size in Temno- pleurus hardwickii (Fig. 1B) to about 5 um of the largest one in Glyptocidaris crenularis (Fig. 1A) of the present paper, with a reservation that the chromosomes tend to contract in colchicine or colcemid. In the distribution of the diploid chromosome numbers (Table 1), the percentage of cells showing the mode was more than 80% in Pseudocentrotus depressus, Strongylocentrotus nudus, Hemicentro- Distribution of the diploid chromosome numbers in 8 species of sea urchins Chromosome numbers TABLE 1. Total No. Species of cells (%) <40 40 41 Glyptocidaris 1B I 1 0 crenularis 100% 1.4 1.4 0 Temnopleurus 100 3 1 IZ hardwickii 100% 3.0 1.0 12.0 Pseudocentrotus 70 2 2 4 depressus 100% 72,8) eS) Dol) Strongylocentrotus 101 2 4 10 nudus 100% 2.0 4.0 9.9 Hemicentrotus 192 3 yy 7 pulcherrimus 100% 1.6 1.0 3.6 Anthocidaris 92 3 4 12 crassispina 100% 3)58) 4.3 13.0 Scaphechinus 100 0 0 0 mirabilis 100% 0 0 0 Astriclypeus 90 0 0 1 manni 100% 0 0 1.1 [2n] 42 43 44 45 46 47 47< Sy mine’ 56 Z 1 Ore 2 4A AO) 16.1 2a ea Omang 74. 5 3 0 OO eZ 42 74.0 5.0 3.00 Oo 0) e220 61 1 0 0 ew © 42 87.1 1.4 0 0 OV ar OnteO 8210 0 0 1 1 if 42 81.2 0 0 0 LO ILO» 10 le Oe 2 0 OF Ole 42 Med WO AD =O) OP OW 71 2 0 0 0 LOp nino 42 Whi, 27! 0 0 OM + OM EO Olan 2 2 94 1 1 46 YO 2208 (220) 94.0" Or 30 a 4 Bi 78 1 1 46 eps, |) Ana 89) Sled lia Fic. 1. ii, C) Pseudocentrotus depressus, Metaphase plates of 8 species of sea urchins. A) Glyptocidaris crenularis, B) Temnopleurus hardwick- D) Strongylocentrotus nudus, Anthocidaris crassispina, G) Astriclypeus manni, and H) Scaphechinus mirabilis. E) Hemicentrotus pulcherrimus, F) Bar, 5 um. 485 Chromosome Numbers of Sea Urchins 486 K. SAOTOME tus pulcherrimus, Scaphechinus mirabilis and Astriclypeus manni but was in the level of 70% in G. crenularis, T. hardwickiti and Anthocidaris crassispina. The reason for a relatively low percentage in the latter species may be that chromosomes flow out on air-drying owing to weakness of cell membrane after hypotonic treat- ment. Chromosome numbers (2n=42) of H. pulcherri- mus and A. crassispina by the air-drying method of this paper were consistent with those reported by Nishikawa [12] using squash method in testis. Nine species, including Clypeaster japonicus described previously [13] and 8 species in this paper (Table 1), can be divided into three groups on the basis of the chromosome numbers; the first group has 2n=44, consisting of G. crenularis, the second one has 2n=42, including T. hardwickii, P. depressus, S. nudus, H. pulcherrimus and A. cras- sispina, and the third one has 2n=46, containing S. mirabilis, A. manni and C. japonicus. When the relationship among the three groups in the light of classification from morphological standpoint was examined, the difference in chromosome number well corresponded to the difference in systematic order of Shigei [3]; the first, the second and the third groups corresponded to the order Arbacioida of Regularia, the order Echinoida of Regularia and the order Clypeasteroida of Irregularia, re- spectively. The chromosome numbers, therefore, were found to be the same in the species belonging to the same order but differ among three different orders; Arbacioida, Echinoida and Clypeaster- oida. Concerning foreign echinoids, Arbacia punc- tulata [15, 16] and Arbacia lixula |9] belonging to the order Arbacioida have chromosome number of 2n=44, and Sphaerechinus granularis [9], Psam- mechinus microtuberculatus [9] and Paracentrotus lividus {17| belonging to the order Echinoida have 2n=42. These data coincided with the present results. In the comparison, the previous data obtained by sectioning method were excluded for low reliability [18]. Concerning chromosome numbers, Arbacioida, Echinoida and Clypeasteroida have respectively 2n=44, 2n=42 and 2n=46, as already noted. Colombera et al. [19] has reported chromosome number of Cidaris cidaris belonging to Cidaroida as 2n=44, with reserve, because of insufficient numbers examined (20 metaphase plates) and the chromosome number of Diadema setosum belong- ing to Diadematoida has been reported as 2n=44 by Shingaki and Uehara [20]. On the other hand, concerning echinoid phy- logeny based on _ morphological viewpoint, although there are various disagreements in detail [3, 21-23], it is generally considered that Cidar- oida is a primitive group, from which Diadema- toida and Arbacioida have evolved and _ that Echinoida and Clypeasteroida are newer groups, the former being descended from Arbacioida [3]. Further, Shigei [3] has proposed from the morpho- logical and paleontological standpoints that Irregu- laria separated from Regularia on the way of evolution from Cidaroida to Arbacioida after Diadematoida branched off (Fig. 2). If chromo- some numbers of Cidaroida and Diadematoida are taken as 2n=44 in spite of knowledge of a single species, diagram of phylogeny based on chromo- some numbers may be drawn like Figure 2. In the diagram, there may be at least three chromosome evolutions; 1) Diadematoida (2n=44) and Arba- cioida (2n=44) have evolved from Cidaroida (2n Echinoida ; 2n=42 Clypeasteroida 2n=46 B Arbacioida 2n=44 Diadematoida A *2n=44 (Cidaridae) * 2n=44 Cidaroida Fic. 2. Phylogeny of echinoids based on chromosome numbers. The scheme was drawn according to the phylogenetic diagram of Shigei [3] with simplifica- tion. *Diploid chromosome number from the knowledge of a single species. Chromosome Numbers of Sea Urchins =44) without change in chromosome number (A of Fig. 2), 2) chromosome number has decreased from 2n=44 to 2n=42 in the evolution from Arbacioida (2n=44) to Echinoida (2n=42) (B of Fig. 2) and 3) chromosome number has increased from 2n=44 to 2n=46 in the evolution from Regularia to Clypeasteroida (2n=46) of Irregular- ia (C of Fig. 2). The mechanism about decrease and increase in chromosome number is still un- known. Though Robertsonian rearrangement may be considered concerning the mechanism, com- parison of arm number, banding pattern and karyotype will be necessary to solve it. Karyotype analysis is now in progress. ACKNOWLEDGMENTS The author expresses her gratitude to Prof. Emer. K. Dan of Tokyo Metropolitan University and to Dr. M. Shigei, University of Tokyo, for valuable discussion. Thanks are also due to the staff of the Marine Biological Stations of the University of Tokyo, Tohoku University and Okayama University and to Waseda University for their kind supply of materials. REFERENCES 1 Nisiyama, S. (1966) The echinoid fauna from Japan and adjacent regions. Part I. Palaeontol. Soc. Japan, spec) Pap... No. I1. 2 Nisiyama, S. (1968) The echinoid fauna from Japan and adjacent regions. Part II. Palaeontol. Soc. Japan, Spec. Pap., No. 13. 3 Shigei, M. (1974) Echinoids. In “The Systematic Zoology 8 (2)—Echinoderms”. Nakayama Book Company, Tokyo, pp. 208-332. (In Japanese) 4 Shigei, M. (1986) The Sea Urchins of Sagami Bay. Maruzen Co., Ltd., Tokyo. 5 Nagaoki, S and Isono, N. (1981) Free amino acids in unfertilized sea urchin eggs. Hiyoshi Sci. Rev. (Keio Univ.), 16: 93-110. (In Japanese) 6 Suzuki, N., Hoshi, M., Nomura, K. and Isaka, S. (1982) Respiratory stimulation of sea urchin sper- matozoa by egg extracts, egg jelly extracts and egg jelly peptides from various species of sea urchins: Taxonomical significance. Comp. Biochem. Phys- iol., 722A: 489-495. 7 Matsuoka, N. (1985) Biochemical phylogeny of the 10 i 12 3 14 15 16 17 18 19 20 21 DD. 1p) 487 sea-urchins of the family Toxopneustidae. Comp. Biochem. Physiol., 80B: 767-771. Matsuoka, N. (1986) Further immunological study on the phylogenetic relationships among sea-urchins of the order Echinoida. Comp. Biochem. Physiol., 84B : 465-468. Colombera, D. (1974) Chromosome evolution in the phylum Echinodermata. Z. Zool. Syst. Evol.- forsch., 12: 299-308. Makino, S. and Niiyama, H. (1947) A study of chromosomes in echinoderms. J. Fac. Hokkaido Univ. Ser. VI, Zool., 9: 225-232. Makino, S. and Alfert, M. (1954) To the question of somatic reduction divisions in sea urchin micro- meres. Experientia, X/12: 489-490. Nishikawa, S. (1961) Notes on the chromosomes of two species of echinoderms, Hemicentrotus pulcher- rimus and Anthocidaris crassispina. Zool. Mag., 70: 425-428. (In Japanese) Saotome, K. (1982) A method for chromosome preparation of sea urchin embryos. Stain Technol., 57: 103-105. Showman, R.M. and Foerder,C.A. (1979) Re- moval of the fertilization membrane of sea urchin embryos employing aminotriazole. Exp. Cell Res., 120: 253-255. Auclair, W. (1965) The chromosomes of sea urchins, especially Arbacia punctulata, A method for studying unsectioned eggs at first cleavage. Biol. Bull., 128: 169-176. German, J. (1966) The chromosomal complement of blastomeres in Arbacia punctulata. Chromosoma (Berl.), 20: 195-201. Colombera, D., Venier, G. and Vitturi, R. (1977) Chromosome and DNA in the evolution of echi- noderms. Biol. Zbl., 96: 43-49. Makino, S. (1950) A Review of the Chromosome Numbers in Animals. Hokuryukan, Tokyo, pp. 31- 33. (In Japanese) Colombera, D., Vitturi, R. and Zanirato, L. (1977) Chromosome number of Cidaris cidaris . Acta Zool. (Stockh.), 58: 185-186. Shingaki, M. and Uehara, T. (1984) Chromosome studies in the several species of sea urchin from Okinawa. Zool. Sci., 1: 1008. (Abstract, In Japanese) Mortensen, T. (1928-1952) A Monograph of the Echinoidea. Vol. 1-15. C. A. Reitzel, Copenhagen. Durham, J. W. and Melville, R. V. (1957) A clas- sification of echinoids. J. Paleontol., 31: 242-272. Philip, G.M. (1965) Classification of echinoids. J. Paleontol., 39: 45-62. j eye i aR a gee 0 Hi a NE RS hea Mire Tete pry ial , % oly , ie eie: ateekie Ww hi Te a er Wy SEE We, AMIE, CK? Pe ees RaW f Phere. op an ges4 , erie | 3 ete t (EES BCLS e eas hee ry é 5 “ he Mt ? . ‘ j yet, ie 3 ge ’ . 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Be ‘ : m tg tie ae p 4 IR "4 ? BME 2 Sh P| t Che ae Vf. Sd ‘ i & a | o : oi a i heed 3 am d ee ; y a um i a gh p Rut x Eee 4 ' pi te ri ‘ Rel i ° 4 ' A ¥ ee. ‘ ie ny oe, ‘ rv - 3 + A y } 4 balae? ' i Way H . BLD 4 : uy ai oa oy ny . ne fl : * (ry TMT Ao. rh tag ant. ¥ to Catered ZOOLOGICAL SCIENCE 4: 489-496 (1987) Twin Formation in Xenopus laevis Eggs Centrifuged before First Cleavage MuTsuKI KUNIEDA and MASAMI WAKAHARA! Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan ABSTRACT—Twin formation was studied in the fertilized Xenopus eggs by means of low speed centrifugation (15-30 Xg) for a short period (20 sec to 5 min) before first cleavage. Dejellied eggs were embedded in gelatin with a vertical orientation (animal pole up), but randomly oriented with reference to the sperm entrance site. They were then spun with the centrifugal force vector at right angles to the animal-vegetal axis. When the eggs were centrifuged at T=0.3-0.6 (30-60% of the time interval to first cleavage), a large number of twin embryos developed, whereas when centrifuged after T=0.7 twin embryos were almost never obtained. Morphological features of the twin embryos were briefly described. They showed a rather continuous spectrum from almost normal to completely double-axes embryos. The twin embryos were tentatively grouped into 6 categories based on the degree of twinning. Twin embryos with clear double-axes were divided into 3 groups in regard to the spatial relationships between the two axes. All double-axes embryos developed from the gastrulae which had shown two blastoporal lips. Possible mechanisms of twinning are discussed in terms of a postcentrifugal modification of the localization of the “dorsal determinants” which specify the future © 1987 Zoological Society of Japan dorsal side of the embryos. INTRODUCTION It is well known that an amphibian egg can produce two complete embryos after separation of the blastomeres at the 2-cell stage [1]. This leads us to conclude that an amphibian egg has an ability to produce two complete sets of axial structures, including such dorsal axial structures as notochord, neural tube and somites. In fact, several ex- perimental treatments of amphibian eggs are known to cause twin formation: Delayed fertiliza- tion (overripeness of eggs) in Rana pipiens [2); rotation of eggs in R.nigromaculata [3] and Xenopus laevis [4-6]; and centrifugation of eggs in Bufo vulgaris [7] and X. laevis [5,8,9]. If an amphibian egg has the ability to produce two complete sets of axial strucutres, a regulation must naturally occur to insure that a single axial structure rather than two axes normally develops. Thus, analysing the twin by means of centrifuga- Accepted January 21, 1987 Received November 8, 1986 * To whom reprints should be requested. tion or egg rotation will aid in understanding the development of dorsal-ventral polarity and also will provide information concerning the regulation of pattern formation in normal development. We have recently succeeded to demonstrate possible existence of the “dorsal determinants”, which specify the future dorsal side of the embryos, in fertilized Xenopus eggs by means of cytoplasmic withdrawal experiments [10]. At present, it seems much important to know the behavior of the “determinants” during the first cell cycle to elucidate the mechanisms of the establish- ment of the dorsal-ventral polarity in amphibian eggs. Twin formation by centrifugation is expected to be a biological tool for analysing the localization of the “dorsal determinants”. In this paper, we first describe the morphologi- cal features of twin embryos developed from the centrifuged Xenopus eggs, and then describe the conditions of centrifugation for obtaining twin embryos. The results obtained support the view that the postcentrifugal modification of the loca- lization of the “dorsal determinants” which specify the future dorsal side of the embryos [10] causes 490 M. KUNIEDA AND M. WAKAHARA the twin formation. MATERIALS AND METHODS Source of embryos The two colonies of Xenopus laevis which are maintained in our laboratory, J and HD groups [11] were used. Eggs from hormone-stimulated female were rinsed briefly with Steinberg’s solu- tion and then inseminated with a testis homogen- ate. Eggs and embryos were usually maintained at 15°C unless mentioned otherwise [12]. Eggs before first cleavage were staged according to the fraction of the interval between fertilization (T=0) and first cleavage (T=1.0) at the time of the relevant manipulation. Embryos were staged according to Nieuwkoop and Faber [13]. Embedment of eggs in gelatin Twenty minutes after fertilization eggs were dejellied in 2.5% thioglycolic acid (pH 8.2) and washed four times in 10% Steinberg’s solution. Nine percent gelatin was prepared according to Black and Gerhart [14], with some modifications: penicillin G potassium (100 IU/ml) and streptomy- cin sulfate (0.1mg/ml) were added instead of gentamycin. Approximately 40 eggs were pipetted at once into molten 9% gelatin (25°C) and then tranferred to a plastic dish (Falcon 1008) contain- ing 2.5ml molten gelatin. Almost all eggs main- tained their normal, vertical orientation (animal pole up) in the molten gelatin. The fewer eggs which had rotated or inverted when tranferred were quickly oriented with forceps to orient the animal pole up. Eggs were aligned approximately along the center line of the dish so that all the eggs could be exposed to the similar centrifugal forces. After the eggs were arranged the dish was placed on an ice-chilled copper plate for 2 min and then placed in a 15°C water bath for 10 min to complete the solidification of the gelatin. Centrifugation of embedded eggs Dishes with eggs were mounted on a holder made from a plastic centrifuge tube. The holders were placed on the swing rotor of a clinical centrifuge and spun at 15°C. The duration of centrifugation indicated below included only the time while the given centrifugal forces were Operating (i.e. acceleration and deceleration periods of approximately 1 min were not included in the “duration”). The centrifugal force vector was at right angles to the animal-vegetal axis of the eggs. Since the eggs were not oriented to bring the sperm entrance site (SES) to the same direction when embedded in the gelatin, the centrifugal force vector varied from egg to egg with respect to the SES. Observation of embryos After centrifugation eggs were incubated at 15°C in the normal, vertical orientation (animal pole up) in gelatin. When the embryos developed to stage 4 (8-cell stage) they were freed from the gelatin by incubating the dish in 35°C water bath for a short period (1-2 min). Embryos freed from gelatin were washed twice with 10% Steinberg’s solution. Developing embryos were periodically observed until they developed to stage 29. Dead and abnormal (except for twinning) embryos were discarded. Eggs and embryos were fixed with a modified Bouin-Holland or Smith’s fixative. Serial sections of 8 um thick were stained with Delafield’s hematoxylin and eosin. RESULTS Brief description of morphological features of twin embryos which developed from centrifuged eggs As a Starting point for experimental analysis, a general description of the morphological features of twin embryos which developed from centrifuged Xenopus eggs was given. Although Black and Gerhart [9] described in detail the conditions of centrifugation for obtaining twin embryos, there has been no systematic description on twin embryos developed from the centrifuged eggs. Approximately 400 “twin embryos” (which in- cluded complete and incomplete double-axes embryos and single-axis embryos with a “wide” sucker; cf. “imbalanced embryos” of Cooke [6]) were obtained. They showed a continuous spec- trum in external and internal morphology from Twin Formation in Xenopus 49] Fig. 1. Typical external views of the “twin embryos” developed from the eggs centrifuged before first cleavage, showing 6 types of twinning, which were divided tentatively based mainly upon the external morphological features. (A) Normal control embryo. Lateral view. (B), Type 1, almost normal but displaying a small protuberance. Lateral view. (C), Type 2, single-axis embryo but containing an apparent wide sucker (an arrowhead). Type 3, single-axis embryo but containing two distinct suckers (two arrowheads). Compare with normal control embryo (inset). Frontal view. (D) Type 4, double-axes embryo with no distinct sucker. Lateral view. (E), Type 5, double-axes embryo one of which contains a distinct sucker, but the other lacks a sucker. Dorsolateral view. (F), Type 6, double-axes embryo both of which show distinct suckers respectively. Lateral view. Bars: 1mm. almost normal to complete double-axes. We divided these embryos into the following 6 catego- ries, based mainly upon the external morphologi- cal features (Fig. 1): Type 1: almost normal (single-axis) embryos, but displaying a small protuberance similar to a secondary embryo (Fig. 1B). Type 2: almost normal (single-axis) embryos, but containing a wide sucker (Fig. 1C). Type 3: single-axis embryos with two distinct suckers (Fig. 1C). Type 4: double-axes embryos with no distinct sucker (Fig. 1D). Type 5: double-axes embryos, one of which contains a distinct sucker but the other lacks a sucker (Fig. 1E). Type 6: double-axes embryos both of which show distinct suckers (Fig. 1F). Of these 6 types, only Types 4, 5 and 6 showed complete duplication of external dorsal axial struc- 492 M. KUNIEDA AND M. WAKAHARA tures. Internal inspections revealed, however, similar indication of duplication of dorsal axial structures (such as somites) even in Types 1, 2 and 3 embryos. Thus, all embryos that showed the morphological features described above (Type 1- Type 6) were tentatively designated as “twin Ci embryos”. Figure 2 shows typical internal structures of several twin embryos. In general, Type 6 twins (two axes—two suckers) contained two complete sets of dorsal axial structures in the half with the sucker and one incomplete set of the axial struc- nt Fic. 2. Internal structures of twin embryos developed from the centrifuged eggs. The level of the sections was indicated in the inserted figures. (A), Type 6 twin (two axes—two suckers), showing two distinct neural tubes (nt) and notochords (nc). (B), Type 5 twin (two axes—one sucker), showing two neural tubes but one distinct notochord. (C), Type 4 twin (two axes—no sucker), showing two neural tubes but no distinct notochord. ev, ear vesicle. Bar: 100m. Twin Formation in Xenopus 493 tures in the other half. The notochord was underdeveloped in this half and so only one intact notochord was observed in the histological sections (Fig. 2B). Similarly, Type 4 twins (two axes-no sucker) showed two incomplete sets of dorsal axial structures: notochordal structures were underde- veloped, but neural tube-like structures were usually developed in both halves of the embryo (igs 2€). Spatial relationships between the two axes were different among embryos regardless of the type of the twinning. Typically, twin embryos were divided into three groups; face-to-face (Fig. 2B), side-by-side (Fig. 2A) and in between (Fig. 2C). The spatial relationship depended upon the origi- nal location of the two dorsal lips of the blasto- pores, or the direction of the invaginations (Fig. 3). Face-to-face twins originated from the embryos which had shown the two dorsal lips just opposite to each other (Fig.3C). Side-by-side twins were derived from the embryos which had had two distinct dorsal lips on one side of the embryo (Fig. 3B). Relationship between centrifugation conditions and frequency of twin formation Eggs were centrifuged at T=0.4—0.6 with two centrifugal forces (15 and 30g) for different periods to establish optimal conditions of centri- fugation for obtaining twin embryos (Table 1). The centrifugal force vector was at right angles to the animal-vegetal axis. The data in Table 1 represent 6 experiments using 3 different females. As controls, 384 eggs were embedded in gelatin but not centrifuged. They were freed from the gelatin after stage 4 and maintained in 10% Steinberg’s solution thereafter in the same fashion as the centrifuged embryos. Approximately half of them were alive at stage 29. Only one embryo from 384 eggs (0.2% of eggs used or 0.5% of embryos alive at stage 29) twinned. In contrast, many twinned embryos developed from centri- fuged eggs: 46 twins (13.6% of eggs centrifuged or 31.1% of the embryos alive at stage 29) developed from 339 eggs centrifuged at 30g for-1 min. Although variations in survival and frequency of twin formation were considerably large from one experiment to another, it was clear that low speed centrifugation caused twin formation. When the eggs were spun at 15 Xg for 1 min or 5 min, a high frequency twinning occurred. At a higher centri- fugal force (30g) shorter periods of centrifuga- tion (20 sec or 1 min) was sufficient for high frequency twinning. | Fic. 3. the blastopore (upper, arrowheads) and the resulting tailbud embryos (bottom). (A), Normal control. (B), Typical “side-by-side” type twin. (C), Typical “face-to-face” type twin. Bottom views of the early gastrula embryos, showing the spatial relationship of two dorsal lips of Bars: 1mm. 494 M. KUNIEDA AND M. WAKAHARA Frequency of twin formation under various centrifugation conditions* TABLE 1. Conditions of No. of eggs centrifugation centrifuged 1Xg 0 min 384 20 sec 331 15Xg 1 min 257 5 min 153 20 sec 335 30 Xg 1 min 339 5 min 161 No. of embryos at stage 29 alive twinnned 187 1 (48.7%) ( 0.5%) ** 64 4 (19.3%) ( 6.3%) 108 25 (42.0%) (23.1%) 34 7 (22.2%) (19.3%) 88 17 (26.3%) (19.3%) 148 46 (43.7%) (31.1%) 9 1 ( 5.6%) (11.1%) * Fertilized Xenopus eggs from 6 different females were centrifuged at T=0.4—0.6, with a centrifugal force vector at right angles to the animal-vegetal axis. ** Percent live embryos at stage 29 which displayed twinning. Stage sensitivity of the egg to centrifugation In a previous study [14], the exact stage sensitiv- ity of the egg to centrifugal effects on modification of the orientation of Xenopus embryonic axis was determined during first cell cycle, showing three different phases of the centrifugal effects; prior to T=0.4, during T=0.4-0.7 and after T=0.7. Thus, we attempted to determine the stage sensi- tivity of the egg to centrifugal effects on the twin formation. Eggs were centrifuged at 15 xg for 5 min at various periods in the first cell cycle. The centrifugal force vector was at right angles to the animal-vegetal axis of the eggs. Figure 4 shows data summarized from 6 experiments using 6 different females. Although there were wide variations in the rate of twin formation among the experiments, it was clear that twin embryos de- veloped largely from eggs centrifuged in the period of T=0.3-0.6. No twin embryos were observed from eggs centrifuged after T=0.7. DISCUSSION It is clear that low speed centrifugation (15 or 30g) at right angles to the egg’s animal-vegetal axis causes twin formation in Xenopus laevis embryos. The results reported here are consistent with the recent studies by Black and Gerhart [9]. Thus, a fertilized Xenopus egg has an ability and probably materials to produce two complete sets of the body plan. Since every twin embryo was always developed 50 C9) jarfoyj| — S{0y/ x2 ©——® Exp:2) 455 = (0) o——© Exp3 895 5 m=—§as Exp4 1144 = 30 o--———© Exp.5 70] iS #——+ Exp.6 218 Ae) © 20 3 1 10 aS) a O —>4#— -——_8 OZ) 04) SOG 08: Op eeyZamemeles Time, normalized to first cleage Fic. 4. Stage sensitivity of Xenopus eggs to centrifuga- tion on the twin formation. Fertilized eggs from 6 different females were centrifuged (15xg for 5 min) at various periods in the first cleavage, with a centrifugal force vector at right angles to the ani- mal-vegetal axis of the eggs. Number on right-top indicates the numbers of eggs centrifuged in each experiment. No twin embryos were observed from eggs centrifuged after T=0.7. Twin Formation in Xenopus 495 from the gastrula which showed two dorsal lips and furthermore the spatial relationship between the two axes of twin embryos depended upon the original location of the two blastoporal lips (Fig. 3), twin formation relates inevitably to the prob- lems how and where the dorsal lip is specified, or more generally, of the establishment of the dorsal- ventral polarity during the early development of embryos. Because the place where cells of the blastopore start to invaginate is known to make the future dorsal side without exceptions [10], the location of the dorsal lip can serve as a manifesta- tion of the ultimate dorsal-ventral polarity. In this respect, the dorsal-most vegetal cells of the 32- or 64-cell Xenopus embryo are reported to contain a set of determinants which enable them to induce neighboring cells to undertake dorsal axis forma- tion [15, 16]. Furthermore, the dorsal-vegetal cells of the 8-cell embryo are known to receive already an ability to form dorsal axial structures [17, 18]. Provided that the set of “dorsal determinants”, which are recently demonstrated to exist in fertil- ized Xenopus eggs by means of cytoplasmic with- drawal experiments [10], specifies the location of the dorsal lip, twin formation by centrifugation is considered to be involved in altered localization of the “dorsal determinants” after centrifugation of egg. That is, normal arrangements of the egg cytoplasm are modified by centrifugation or post- centrifugal rearrangements or the egg contents [14]. Presumably, a bifurcation of the “dorsal determinants” occurs. However, the mechanisms involved in the modification of the localization or partitioning of the “dorsal determinants” by centri- fugation is complex and still remained to be clarified. Recent studies have clearly demonstrated that centrifugations cause a stage-specific modification of the dorsal-ventral polarity: When centrifuged ~ before T=0.4, the future dorsal side is specified in the centrifugal side of the eggs but in the centripe- tal side of the eggs after T=0.4 centrifugation, irrespectively of the sperm entry site [14]. Centri- fugation between T=0.4—-0.45 causes twin forma- tion [9]. This stage-specific effect of centrifugation for obtaining twin embryos was confirmed in this work (Fig.3). Presumably, the stage-specific effects of centrifugation on the modification of the future dorsal-ventral polarity are involved in the drastic changes in the cytoplasmic consistency during the first cell cycle of the fertilized eggs [19]. This is the first description of the morphological diversity of twin embryos developed from the centrifuged eggs. As illustrated in Figures 1 and 2 they showed a rather continuous spectrum in external and internal morphology from an almost normal to a complete double-axes embryos. This variability in twinning is probably due to the degree and localization to which bifurcation of the dorsal determinants after the centrifugation occurs: When the “dorsal determinants” are di- vided evenly into two groups of the vegetal blastomeres at 32- or 64-cell embryo [15, 16], complete double-axes embryos will result, whereas uneven partitioning of the “determinants” results in incomplete twin embryos. Spatial relationship between two axes was also shown to be continuous from face-to-face to side-by-side twin (Fig. 3). When the “determinants” are divided into just opposite to each other face-to-face twin will result, whereas partitioned into two distinct sites on one side of the egg results in side-by-side twin. Twin embryos developed from the centrifuged eggs were divided into 6 categories to allow further quantita- tive analyses of the centrifugal effects on twinning and the modification of the “dorsal determinants”. ACKNOWLEDGMENTS We are grateful to Prof. G.M. Malacinski (Indiana University, Bloomington, Indiana) for his critical reading of the manuscript and valuable discussions. Supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (60440100, 60540452). REFERENCES 1 Kageura, H. and Yamana, K. (1983) Pattern reg- ulation in isolated halves and blastomeres of early Xenopus laevis. J. Embryol. Exp. Morphol., 76: 221-234. 2 Witschi, E. (1952) Overripeness of the egg as a cause of twinning and teratogenesis: A review. Cancer Res., 12: 763-786. 3 Kubota, T. (1967) A regional change in the rigidity of the cortex of the egg of Rana nigromaculata following extrusion of the second polar body. J. 10 fal 496 Embryol. Exp. Morphol., 17: 331-340. Kirschner, M., Gerhart, J.C., Hara, K. and Ubbels, G.A. (1980) Initiation of the cell cycle. and establishment of bilateral symmetry in Xenopus eggs. Symp. Soc. Dev. Biol., 38: 187-215. Gerhart, J., Ubbels, G. A., Black, S., Hara, K. and Kirschner, M. (1981) A reinvestigation of the role of the grey crescent in axis formation in Xenopus laevis. Nature, 292: 511-516. Cooke, J. (1986) Permanent distortion of positional system of Xenopus embryo by brief perturbation in gravity. Nature, 319: 60-63. Motomura, I. (1935) Determination of the embryonic axis in eggs of amphibia and echi- noderms. Sci. Rep. Tohoku Univ. (Ser. 4), 10: 211- 245. Young, R.S., Deal,P.H., Souza,K.A. and Whitfield, O. (1970) Altered gravitational field effects on the fertilized frog egg. Exp. Cell Res., 59 :267 -271. . Black, S.D. and Gerhart, J.C. (1986) High- frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage. Dev. Biol., 116: 228-240. Wakahara, M. (1986) Modification of dorsal-ventral polarity in Xenopus laevis embryos following with- drawal of egg contents before first cleavage. Dev. Growth Differ., 28: 543-554. Katagiri, Ch. (1978) Xenopus laevis as a model for 12 13 14 15 16 17 18 19 M. KUNIEDA AND M. WAKAHARA the study of immunology. Dev. Comp. Immunol., 2: 5-14. Neff, A. W., Wakahara, M., Jurand, A. and Mala- cinski, G. M. (1984) Experimental analyses of cyto- plasmic rearrangements which follow fertilization and accompany symmetrization of inverted Xenopus eggs. J. Embryol. Exp. Morphol., 80: 197-224. Nieuwkoop, P.D. and Faber, J. (1956) Normal Table of Xenopus laevis (Daudin), North-Holland Publ., Amsterdam. Black, S.D. and Gerhart, J.C. (1985) Ex- perimental control of the site of embryonic axis formation in Xenopus laevis eggs centrifuged before first cleavage. Dev. Biol., 108: 310-324. Gimlich, R. L. and Gerhart, J.C. (1984) Early cellular interactions promote embryonic axis forma- tion in Xenopus laevis. Dev. Biol., 104: 117-130. Gimlich, R. L. (1985) Cytoplasmic localization and chordamesoderm induction in the frog embryo. J. Embryol. Exp. Morphol., 89: Suppl., 98-111. Kageura, H. and Yamana, K. (1984) Pattern reg- ulation in defect embryos of Xenopus laevis. Dev. Biol., 101: 410-415. Kageura, H. and Yamana, K. (1986) Pattern forma- tion in 8-cell composite embryos of Xenopus laevis. J. Embryol. Exp. Morphol., 91: 79-100. Elinson, R. P. (1983) Cytoplasmic phases in the first cell cycle of the activated frog egg. Dev. Biol., 100: 440-451. ZOOLOGICAL SCIENCE 4: 497-521 (1987) A Histological Survey on the Development of Circumventricular Organs in Various Vertebrates KAZUHIKO TSUNEKI Department of Biology, Shimane University, Matsue, Shimane 690, Japan ABSTRACT—The development of various circumventricular organs was studied histologically in the lamprey, smelt, catfish, guppy, salamander, frog, lizard, chick, and mouse. The circumventricular organs that appear first in ontogeny are usually the pineal and subcommissural organ. The neurohypophysis (median eminence and neural lobe) also differentiates relatively early in ontogeny. These epithalamic and hypothalamic organs may be fundamental circumventricular organs of verte- brates. Roughly speaking, the choroid plexuses, paraphysis, and paraventricular organ differentiate at © 1987 Zoological Society of Japan about the same time as the neurohypophysis does. subfornical organ, and area postrema differentiate much later in ontogeny. The organum vasculosum laminae terminalis, The developmental sequence of the circumventricular organs roughly corresponds to the phylogenetic development of these organs in a series of vertebrates. INTRODUCTION The ventricular surface of the vertebrate brain is specialized at several places as circumventricular organs (CVOs) [1]. In previous papers [2, 3], I surveyed the occurrence of various CVOs in a series of vertebrates, paying special attention to lower groups, and discussed the phylogeny of these organs. In these papers, however, I did not refer to the ontogeny of the CVOs. Nevertheless, the pertinent description and discussion on the de- velopment of these organs in various vertebrates are obviously indispensable for understanding the overall morphological and evolutionary character- istics of the CVO system in vertebrates. This study is an attempt to describe concisely the develop- ment of various CVOs in several representative vertebrates and to discuss the characteristics of the development of these organs. In this paper, all CVOs in the brains of various vertebrates are together dealt with. This treatment is apparently necessary for relevant comparisons among organs and among animals. There are a large number of papers dealing with the develop- ment of the CVOs, especially in higher verte- brates. However, most papers deal with a single Accepted December 29, 1986 Received October 30, 1986 organ in a single species (e.g. the development of the hypophysis in the mouse, the development of the pineal in the chick, and so on). This kind of detailed work focusing on a particular aspect is of course important to characterize each organ in each animal, but does not necessarily allow ade- quate comparisons among organs and among animals. For appropriate comparisons, all organs should be examined in various vertebrate groups chronologically under the same viewpoint. This kind of comparison is aimed in this paper. The result may serve as a bird’s-eye view on the developmental patterns of the vertebrate CVOs. The review of the literature is kept in minimum, because it is obviously not the purpose of this study and is not indispensable either for the present purpose. The numerous references on the topic may be found in extensive reviews such as Stud- nicka [4], Bargmann [5], Wingstrand [6], Leonhardt [1], and Vollrath [7]. MATERIALS AND METHODS The development of the following 10 species was examined: the brook lamprey, Lampetra reissneri; the smooth dogfish, Mustelus manazo; the Japanese smelt, Hypomesus nipponensis ; the Far Eastern catfish, Silurus asotus ; the guppy, Poecilia reticulata; the hynobiid salamander, Hynobius 498 K. TSUNEKI nebulosus; the ranid frog, Rana japonica; the lacertid lizard, Takydromus tachydromoides ; the domestic chick, Gallus gallus; and the albino mouse, Mus musculus. In Lampetra reissneri, fertilized eggs were obtained through natural spawning of mature adults in a laboratory aquarium. Fractions of embryos and larvae were fixed at appropriate intervals (Table 1). Developmental stages were determined according to the criteria made for Petromyzon marinus [8]. In addition, larvae (ammocoetes) of about 10 cm in total length were collected from a river and their heads were fixed. In Mustelus manazo, intra-uterine embryos were obtained from a pregnant female of 88 cm in total length. These embryos were about 7cm in total length and possessed well developed external gills. These were only dogfish embryos that were avail- able for this study (Table 2). In Hypomesus nipponensis, fertilized eggs were obtained by artificial insemination (dry method). They were attached to the nylon mesh and kept in a laboratory tank. Eight developmental stages of embryos and larvae were studied (Table 3). In Silurus asotus, fertilized eggs were obtained through natural spawning of mature adults in the laboratory aquarium. Ten developmental stages of embryos and larvae were examined (Table 4). In addition to these materials reared in the labora- tory, juveniles of about 15 mm in total length were captured from a river and their heads were fixed. In Poecilia reticulata (flamingo variety), preg- nant females of varying degrees of bulging and darkening of the belly were sacrificed and the embryos were taken out and fixed. In addition to the embryos, swimming larvae of two different stages were examined (Table 5). In Hynobius nebulosus, egg sacs were collected from a pool and were kept in a laboratory tank. Developmental stages were determined according to the illustrations of embryos and larvae of Hynobius_ nigrescens by Usui and Hamsaki (sic, probably Hamasaki) [9] (Table 6). However, accurate determination of stages was often difficult, because the illustrations made by Usui and Hamsaki are not accompanied by the literal description. The present determination is urged to be somewhat tentative. In Rana japonica, egg masses were collected from the rice field and were kept in a laboratory tank. Developmental stages were determined according to Tahara [10, 11] (Table 7). In addition to these materials reared in a laboratory, small frogs shortly after metamorphosis (about 15 mm in head-trunk length) were collected from the field and their heads were fixed. In Takydromus tachydromoides, naturally de- posited eggs were obtained from a laboratory terrarium. These eggs were carefully transferred from the terrarium to the Petri dish of which floor was covered with moist cotton and left there for further development. Developmental stages were determined according to the criteria made for Lacerta vivipara [12] (Table 8). In Gallus gallus (White Leghorn strain), ferti- lized eggs were incubated at about 38°C. Develop- mental stages were determined according to Ham- burger and Hamilton [13] (Table 9). In addition, the brains of chicks of eight days after hatching and adult hens were fixed. In Mus musculus (ICR strain), newly pregnant females were sacrificed at appropriate intervals and the intra-uterine embryos were taken out. Developmental stages were determined according to Theiler [14] (Table 10). In addition, the brains of postnatal mice of three and seven days old and adult mice were fixed. All materials were fixed in Bouin’s solution, embedded in paraffin, and serially sectioned at 6 or 7 ym. Both sagittal and transverse serial sections were prepared at all stages of all species except for Takydromus tachydromoides in which the number of materials was not sufficient. In this species, transverse sections were prepared at every stage, but sagittal sections were made only in a few stages. The sections were stained with paralde- hyde fuchsin (AF) and Masson-Goldner’s method. RESULTS The results are shown in Tables 1 to 10. In the following sections, the developmental characteris- tics of the CVOs are described briefly in each species. The detailed cytological description of the development of the CVOs is not attempted. The aspects which are obvious from the tables are not Ontogeny of Circumventricular Organs repeated in the text. The CVOs of adult indi- viduals of Lampetra, Mustelus, Hypomesus, Silur- us, Poecilia, Hynobius, Rana, and Takydromus are described in a previous paper [2]. Abbrevia- tions are given in the legend following Table 1. Busy readers may skip Results, but see Tables and go to Discussion and Summary. The brook lamprey, Lampetra reissneri (Table 1) Hypothalamic structures In early stages, the 499 ventrocaudal floor of the III ventricle (infundibu- lum) consists of a single layer of ependymal cells. At stage 16°, small nerve fiber bundles appear under the ependymal layer. The fiber layer becomes thicker and is stained with AF at stages 17 and 18, thus constituting the neural lobe (NL) (Fig. 1). At these stages, the ventral surface of the NL is supplied with sinusoid vessels, but is not underlain by the pars intermedia-component of adenohypophysial tissue. The median eminence TABLE 1. Development of circumventricular organs in the brook lamprey, Lampetra reissneri Days after Stage” fertilization Bae ae (ME) NL (OVLT) P PP SCO SD MeCP MCP (22-25°C) 12 6 — _ _ = = = a = =a 13 7 — — _ = = = is hee _ 14 8 2.0-2.8” = = — Sh es Ss = es ce 5 10 3.5-5.0 = = _ See eet ek oe = ft 16~ 11 5.0-6.0 = — _ So oO oa a 16* iS 6.0-6.5 = BE = qe aR ae - = ae 17 21 7.5-8.2 te = = ate tate aay = = + 18 Sit 7.8-8.2 aE =e = ste eat iach = ats + Ammocoetes ca. 100 Be sp se + + 44% + 4. ae Adult ca. 170 + +* fo ELS ate ae 1) Stages were determined according to Piavis [8]. Stage 12=head stage, stage 13=prehatching stage, stage 14=hatching stage, stage 15=pigmentation stage, stage 16=gill-cleft stage, stage 17 =burrowing stage, stage 18=larva stage. *) Crown-rump length (mm). Notes to Tables1 to 10. A mark + means that the organ is differentiated. A mark — means that the organ is not differentiated. A mark + means that the organ is differentiating. An asterisk means that the organ is distinctly stained with AF. An asterisk in parentheses means that the organ is faintly stained with AF. An abbreviation in parentheses such as (OVLT) means that the organ does not show a typical mammalian pattern of differentiation even in adults (see [2]). Data for adults from Tables 1 to 8 were taken from Tsuneki [2]. A clearly separated from each other. horizontal bar in Table 8 indicates the boundary between embryos and adults. Brackets include organs that cannot be Horizontal bars in the other tables indicate the boundary between embryos and larvae (juveniles) and the boundary between larvae and adults. are within a certain range in poikilothermal animals. As shown in each table, temperatures under which embryos and larvae were reared Since the development of Lampetra, Hypomesus, Silurus, Hynobius, Rana, and Takydromus was studied in the earlier half of the year (mainly in spring), lower temperatures in the range indicate the temperatures for the earlier development (in earlier weeks or months) and higher temperatures in the range indicate the temperatures for the later development (in later weeks or months) in each species. Abbreviations are as follows: AP, area postrema; DCP, diencephalic choroid plexus; FO, frontal organ; MCP, myelencephalic choroid plexus; ME, median eminence; MeCP, mesencephalic choroid plexus; NL, neural lobe; OVLT, organum vasculosum laminae terminalis; PA, paraphysis; PE, parietal eye; P, pineal; PP, parapineal; PVO, paraventricular organ; SCO, subcommissural organ; SD, saccus dorsalis; SFO, subfornical organ; SV, saccus vasculosus; TCP, telencephalic choroid plexus; VT, velum transversum. 500 K. TSUNEKI (ME) is not developed in the larvae even at stage 18. At stages 17 and 18, the hypothalamic floor above the adenohypophysial cell cord consists of a single layer of ependymal cells. The rostral part of this floor is also provided with small subependymal nerve fiber bundles and is slightly protruding into the III ventricle. The floor is separated from the underlying adenohypophysial cell cord only by an AF-positive connective tissue sheet. Epithalamic structures The first anlage of the pineal appears at stage 14 as a shallow epithalamic evagination of ependymal cells. However, the ependymal cells of the evagination still contain yolk granules. At stage 15, the evagination takes a OC reissneri. ah, adenohypophysis; n, notochord; oc, optic chiasma. clear omega-shaped structure. The pineal lumen becomes smaller as the development proceeds. At stage 167, the pineal is located above the habenu- lar and posterior commissures. The dorsal wall of the pineal is much thinner than the ventral wall. In later stages (stages 17 and 18), the pineal moves slightly rostrally, but it still does not differentiate a stalk (Fig. 2). At these stages, AF-positive intralu- minal processes are distinct. The parapineal appears at stage 16” as a small aggregation of cells around the rostroventral cor- ner of the pineal. At stages 16°, 17, and 18, the parapineal is a flattened sac formed by a single layer of cells (Fig. 2). The lumen is indistinct. In eee 4 Fic. 1. Sagittal section of the hypothalamo-hypophysial region of a stage 17 larva of Lampetra 500. The left is rostral in this and all following figures of sagittal sections. Fic. 2. Sagittal section of the pineal complex of a stage 17 larva of Lampetra reissneri. Note AF-positive structures in the pineal (P). h, habenula; PP, parapineal. 500. Ontogeny of Circumventricular Organs young larvae, the parapineal is very small com- pared to its relative size in adults. The subcommis- sural organ (SCO) is stained with AF earlier than the NL. (The earlier differentiation of the SCO than the NL is corroborated by the pseudoisocy- anin-fluorescence study in Lampetra planeri |15}].) The dorsal ependymal roof of the cerebral ventricle consists of cuboidal ependymal cells and does not show any histological specialization and thus the paraphysis and the saccus dorsalis (SD) are virtually absent in larvae even at stage 18. 501 Choroid plexuses The mesencephalic and myelencephalic ventricular roofs consist of a single layer of ependymal cells. At stage 15, they still contain yolk granules. In all stages up to 18, the roofs are not folded and thus they are not considered as the typical choroid plexus (CP). The ependymal cells are consistently cuboidal or flat. Blood vessels investing the roofs increase gradual- ly. It is thus hard to say exactly when the cells differentiate into the CP cells. In ammocoetes larvae (ca. 10cm), both the mesencephalic and TABLE 2. The occurrence of circumventricular organs in 7cm-embryos of the smooth dogfish, Mustelus manazo ME eNE. SV -OVLT PVO P JL + + SCO PA + SDF “[Vil7 DEPs TCP|~ SEO MCR> -AP + + + + - Fic. 3. Sagittal section of the hypothalamo-hypophysial region of a 7cm-long embryo of Mustelus manazo. ‘The dorsocaudal wall of the third ventricle represents the incipient SV. The ventral lobe (vl) is still clearly connected to the main part of adenohypophysis (ah). Fic. 4. Sagittal section of the epithalamus of a 7cm-long embryo of Mustelus manazo. The ependymal! sheet of velum transversum (VT) form some folding in other sections of this series. h, habenula; PA, paraphysis; P, pineal. 50. x50. 502 myelencephalic ependymal roofs are folded and thus are considered as the CP. The smooth dogfish, Mustelus manazo (Table 2) Hypothalamic structures The ME is thick and possesses a well developed fiber layer (Fig. 3). The ventral surface of the ME is straight and is apposed to the sac-like adenohypophysis. The ME and adenohypophysis are separated from each other only by an AF-positive connective tissue sheet and sporadic capillaries. The NL is small and possesses a thin fiber layer. It is not stained with AF, but its ventral surface is well vascularized. The caudal and caudolateral walls of the infun- dibular recess consist of several layers of cells (Fig. 3). They are well covered with sinus-like capilla- ries and may represent the anlage of the saccus vasculosus (SV). However, typical coronet cells are not yet detected and the walls are too thick to TABLE 3. Days after Stage” _ fertilization ae [ME NL] (12-14°C) A 5 = = B 7 _— — C 9 = = D 11 = = IE 13 Sl) a ae F US) 5.0-5.5 = ae G 19 6.1-6.3 a ae H 26 6.5-7.0 = ac Adult ca. 110 ae ap K. TSUNEKI be designated as the SV. Both the lateral and posterior recesses of the III ventricle are partly contoured with columnar ependymal cells and thus the paraventricular organ (PVO) is already differentiated. Epithalamic structures The pineal is a long straight tube directed dorsorostrally (Fig. 4). The end-vesicle is slightly dilated. The velum transver- sum (VT) is developed and projects several small ependymal processes into the ventricle. There- fore, it approaches the condition of the diencepha- lic choroid plexus (DCP). In front of the VT, there is a thick ependymal sheet. It consists of a few dense layers of cells and may represent the incipient paraphysis (Fig. 4). Choroid plexuses In the lateral ventricle, the telencephalic choroid plexus (TCP) is well de- veloped. The TCP caudally transforms into the DCP-like VT. These two structures are distin- Development of circumventricular organs in the smelt, Hypomesus nipponensis SV PVO 'P SCO SD VI GCE) (eR) — — + = = = — — Sn ~ Sn ~ SG ot ee — sty ee Na a + + + + + = = = = Te ee a ee + a ee ee. + ee - 1) Stage A=the eye not conspicuously pigmented, the body making a complete turn around the yolk mass, stage B=the eye distinctly pigmented, the body surrounding the yolk in less than one and half turn, stage C=the body surrounding the yolk in more than one and half turn, stage D=prehatching stage, stage E=shorty after hatching. Fic. 5. developed SV. The adenohypophysis (ah) is partly embedded in the hypothalamus. Sagittal section of the pituitary region of a stage C embryo of Hypomesus nipponensis. Note the well x 500. Fic. 6. Sagittal section of the pineal (P) region of a stage C embryo of Hypomesus nipponensis. pc, posterior commissure. 500. Fic. 7. Sagittal section of the pituitary region of a stage F larva of Silurus asotus. ah, adenohypophysis. x 380. Fic. 8. Sagittal section of the pineal (P) region of a stage F larva of Silurus asotus. Note AF-positive structures in the pineal lumen. X380. Fic. 9. Transverse section of the OVLT of a stage E embryo of Poecilia reticulata. An arrow indicates a blood vessel. 440. Fic. differentiated even in adults. 10. Sagittal section of the epithalamus of a stage G larva of Poecilia reticulata. An asterisk indicates the habenular commissure. The DCP is not fully x 500. Ontogeny of Circumventricular Organs 503 guished topologically, but they actually representa the IV ventricle. It is already well folded. The continuous ependymal structure. most caudal roof of the IV ventricle represents a The myelencephalic choroid plexus (MCP) is a small pouch-like posterior tela. large dorsal evagination of the ependymal wall of 504 K. TSUNEKI The smelt, Hypomesus nipponensis (Table 3) Although eight stages from embryos to larvae were studied during 22 days, histological differ- entiation of the CVOs was slow, probably because of low water temperatures. Hypothalamic structures In larvae, the ade- nohypophysis, except for the ventral surface, is embedded in the hypothalamus just as encoun- tered in some adult gobiid teleosts. The neural tissue overlying the adenohypophysis may repre- sent the neurohypophysis, but it does not differ- entiate as such in the larvae studied. However, a thin sheet of tissue above the caudal part of the adenohypophysis consists of nerve fibers and may represent an initial stage of differentiation of the NL. The adenohypophysis and the surrounding neural tissue (the future neurohypophysis) are consistently separated by a connective tissue sheet. The caudal end of the infundibular recess is covered with columnar cells in stage A embryos. It appears like a cup which opens rostrally. In stage B and C embryos, the cells of the cup possess a light, abundant, apical cytoplasm and the cup itself differentiates as the SV (Fig. 5). In larvae, the SV takes the form of a flattened sac extending laterally. The lumen of the sac is occupied by flocculent material. The vascularization is not prominent. In larvae, the SV is even larger than the adenohypophysis in size, especially in its width. (In adult Hypomesus, the SV is also a large organ.) Epithalamic structures The pineal is present as early as in stage A embryos. Throughout the embryonic and larval periods, the pineal lumen is not distinctly open, but is occluded by AF-positive droplet-like structures (incipient outer segment?). In embryos, the pineal is located above the intercommissural area (Fig. 6), but in larvae the pineal is located mainly above the SCO. (In adults, the pineal is extended far rostrally.) The differentiation of the SCO is so gradual that it is difficult to determine the exact time of definite appearance. Choroid plexuses The roof of the ventriculus impar telencephali is formed by a simple ependy- mal sheet. In front of the habenular commissure, the ependymal cells are low columnar in later larvae and may represent the tela chorioidea telencephali or a combination of this structure either with the VT or the SD. The VT and SD themselves are not differentiated in the larvae studied. The dorsal wall of the IV ventricle consists of flat ependymal cells in embryos, but consists of cuboidal or low columnar ependymal cells in larvae. In later larvae, it may represent a tela chorioidea myelencephali. See Tsuneki [2] for terminology on the choroid plexus and its related structures. (An ependymal sheet consisting of flat cells is called a tela membranosa, a straight or slightly undulating ependymal sheet consisting of cuboidal or colum- nar cells is called a tela chorioidea, and a tufted or highly folded ependymal sheet consisting of cuboidal or columnar cells is called a choroid plexus.) The catfish, Silurus asotus (Table 4) Hypothalamic structures In embryos and early larvae, the ventral hypothalamus and ade- nohypophysis are apposed, but are clearly sepa- rated by an AF-positive connective tissue sheet. In stage E embryos, a small mass of cells appears at the ventrocaudal part of the infundibulum where the NL differentiates later. The NL is stained with AF already at its initial stage of differentiation (stage F) (Fig. 7). The ME appears to differentiate slightly later than the NL, although these two components of the neurohypophysis cannot be clearly separable even in adults. At stage F, the SV differentiates as a round sac. During the larval period, the SV is almost as large as the adenohypophysis. (It stands in high contrast to adults where the SV is much smaller than the adenohypophysis.) The organum vasculosum lami- nae terminalis (OVLT) is not clearly differentiated in larvae, although the ventromedial wall of the preoptic recess is slightly bulging into the recess in later larvae. Epithalamic structures At stage C, the dis- tinct pineal appears as an evagination of the dorsal wall of the III ventricle. Although the pineal lumen is patent in some larvae (Fig. 8), it is not distinct in the other larvae and the center of the pineal is irregularly occupied by the AF-positive Ontogeny of Circumventricular Organs 505 TaBLE 4. Development of circumventricular organs in the catfish, Silurus asotus Days after Total Stage’? fertilization length [ME NL] SV OVLT PVO P SCO (DCP) (TCP) (MCP) (17-24C) (mm) i A 1 = = = = = == = = = B 2 aa = = oa = id a = = = C 3 = es ae oF a ye = aa D + 525-60) — = — = ae Se ie tt E 5 6.2 = aE rm = = St ee ise _ ah x F Ui Osi) SE +* 4 _ + Jo dL a dl, ae G 9 8:05-8:53) se Se os a ap ae” = ae + H 11 8.5-8.8 + spe = te ap ARS = AF + I 5) 8.0-8.5 + ape ay AP “PSP” = ar + J 20 8.5 Smal tae ee = ee esta = He + Juvenile 15 + +* ate + AL. aii a a a Adult ca.520 + +* + + + +* o + + ) Stage A=the body not surrounding the yolk mass completely, stage B=the body completely sur- rounding the yolk, stage C=immediately after hatching, stage D=the eye barely visible, barbel rudiments, stage E=the eye distinct, many melanophores on the body, stage F=barbels elongated. TABLE 5. Development of circumventricular organs in the guppy, Poecilia reticulata Days after Total Stage’ fertilization length [(ME) NL] (26-27 C) (mm) A a iv B ui af (G a ale (*) D + +* E 7.1-8.2 + +* F 1 VS=1 7) + +* G 5 8.1-8.7 + +* Adult ca. 45 + +* OVI =PVO" TP SCO (DEP) (MEP) = = + — = — at RE _ = =f $e OE™ aE at 4; fo eS a aE = feo ES = aE + a + 3 + a ae + + ) Stage A=the body surrounding the half of the yolk mass, the eye slightly pigmented, stage B=rudiments of pectoral and anal fins visible, stage C=a membrane completely covering the head, stage D=a small round window in the membrane at the top of the head, the body completely surrounding the yolk, fin rays in the pectoral, anal, and caudal distinct, stage E=a small yolk mass visible externally, fin rays appearing in the dorsal, stage F=immediately after hatching, the yolk mass not visible externally, fin rays developed in all fins including the pelvic. droplet-like structures. In younger larvae, the pineal is located on the intercommissural area, without a distinct stalk. In later larvae, the pineal is gradually extended rostrally and it differentiates a Short stalk. Choroid plexuses The tela chorioidea telen- cephali which consists of cuboidal ependymal cells is distinguished as such in the larvae later than stage F. The tela chorioidea myelencephali is differentiated in the rostral part of the dorsal wall of the IV ventricle at stage G. In earlier stages, the roof of the IV ventricle entirely consists of fiat ependymal cells. 506 K. TSUNEKI The guppy, Poecilia reticulata (Table 5) Hypothalamic structures The neurohypoph- ysis differentiates in stage C embryos. In stage D embryos, the caudal part of the neurohypophysis, that may correspond to the NL, is already distinct- ly stained with AF. The entire neurohypophysis is well vascularized before birth. (Ichikawa et al. [16] also noted that the neurohypophysis of Poeci- lia (= Lebistes) reticulata is stained with AF before birth.) The SV is not discernible in embryos and larvae. In stage B embryos, the ventral wall of the preoptic recess (future OVLT) consists of a single layer of ependymal cells, but is vascularized meningeally. In stage C embryos, thin bundles of nerve fibers appear under the ependymal layer. In stage E embryos, the nerve fiber layer is vascula- rized as well as the ventral surface (meninx) (Fig. No Epithalamic structures The pineal develops as an evagination of the dorsal wall of the III ventricle in stage A embryos. It gradually in- creases in size. The lumen is indistinct throughout the stages studied, and it is partially occluded by AF-positive droplet-like structures. The pineal stalk is short even in stage G larvae. (See Omura and Oguri [17] for the ultrastructural development of the pineal photoreceptor cells in Poecilia.) Choroid plexuses The ependymal cells in front of the habenular commissure is cuboidal in stage B embryos and are columnar in stage C embryos. The ependymal sheet invaginates as a sac into the ventricle in stage D embryos. As the development proceeds, the sac is dilated and may represent the incipient DCP (Fig. 10). The rostrodorsal wall of the IV ventricle consists of columnar ependymal cells in stage C embryos, and it is slightly invaginates into the ventricle in stage D embryos. The invagination is incomplete and does not form an intraventricular sac even in stage G larvae. The salamander, Hynobius nebulosus (Table 6) Hypothalamic structures At stage 46, the in- fundibulum (future NL) is composed of a single layer of ependymal cells. At stage 49, small bundles of nerve fibers appear in the future NL. In the ME, small bundles of nerve fibers are recogniz- able at stages 56 and 62. At stage 46, the ependymal cells located around the sulcus hypothalamicus possess a relatively ample cytoplasm and irregular ventricular protru- sions. These cells may form the incipient PVO. The differentiation of this organ is slow and gradual. TABLE 6. Development of circumventricular organs in the salamander, Hynobius nebulosus Stage” Boras ME NL PVO P SCO PA DCP TCP N ShOmuMer 24 3.3 bye ath S itt = = ee = a 29 3.9 EB Seb a i tla hy S ia i, i 33 6.2 SASS: i AS ie fil aie is ty ms 36 9.5 — = = a + aE fel ad as 39 ill = = - oY eee ae = a os ae 43 12 es Seam ete) fla ee Ee ua ot 46 15 Shee GE Se = — ~ 49 17 ER \ Wee = ai auteeh ne amet + = se 56 19 ke a + = te 62 25 eae ff Se he 4 — + Juvenile” Qi + +* + 4 + * + ao + = + Adult 89 Le a ee ee Ce 4. + + ") Stages were determined according to Usui and Hamsaki [9]. Embryos were kept in water of about 9°C and larvae were kept in water of about. 10 to 24°C. *) Salamanders shortly after metamorphosis (4.5 months after hatching). Ontogeny of Circumventricular Organs 507 . OE gn : . ‘ eres = i ee hag. —— a Ye - * ees Fic. 11. Transverse section of the brain of a stage 39 embryo of Hynobius nebulosus. In this section, the lateral ventricles appear below the third ventricle because of the cranial flexure of the embryo. PA, paraphysis; P, pineal. X120. Fic. 12. Transverse section of the brain of a stage 46 larva of Hynobius nebulosus. PA, paraphysis. 120. Fic. 13. Sagittal section of the epithalamus of a stage 24 larva of Rana japonica. An arrow indicates the pineal anlage. The incipient DCP is seen as a ventricular protrusion below the frontal organ (FO). 190. Fic. 14. Sagittal section of the epithalamus of a stage 26 larva of Rana japonica. The frontal organ moves rostrally outside the scope of this figure. P, pineal. 190. 508 K. TSUNEKI Epithalamic structures At stage 33, a shallow omega-shaped evagination appears in the dorsal wall of the III ventricle. This structure represents the initial differentiation of the pineal. Although the pineal anlage appears in the intercommissural area, the pineal is slightly caudally dislocated during the development in high contrast to the rostrally extended pineal of piscine vertebrates. Thus, in the older larvae of Hynobius it is located mainly on the rostral part of the SCO. At stage 36, the anlage of the paraphysis appears as a shallow evagination in front of a VT-like structure. At stage 39, the anlage be- comes a rostrally directed, large, simple sac (Fig. 11). At stage 43, it is elongated and tube-like in appearance. At stage 56, the tip of the tube is divided into saccules and thus the structure indeed appears paraphysis-like. At stages 56 and 62, the ependymal cells of the paraphyseal stalk are more densely arranged than those of the paraphyseal saccules. Choroid _ plexuses The DCP appears as paired ventricular protrusions at stage 46 (Fig. 12). The cuboidal ependymal cells cover the vascula- rized core of the protrusion. At stage 56, the DCP becomes a median structure which is probably due to a fusion of the pair. At this stage, the ependymal cells are rather flat. (Even in adults, the ependymal cells of the CP are flat. In most vertebrates, however, the ependymal cells of the CP are cuboidal. The flat ependymal cells in Hynobius may be related to the unusually large size of nuclei in this species as well as in other urodeles [18].) The anlage of the TCP appears at stage 43. It is a median ventricular protrusion. In the following stages, it becomes a glomerulus-like in appearance (Fig. 12). At stage 56, the TCP protrudes into the lateral ventricle and its epen- dymal cells become lower in height. The ependymal sheet covering the IV ventricle becomes undulant at stage 49. At stage 56, the MCP differentiates at the rostrolateral region. At stage 62, the entire ependymal roof of the IV ventricle well differentiates as the MCP except for the most caudal part, which represents the simple posterior tela. In Hynobius nebulosus as well as the other species, developmental stages were selected at some intervals. Therefore, even if a certain structure is described to appear at a certain stage, it is quite possible that such a structure actually appears between this stage and a preceding stage TABLE 7. Development of circumventricular organs in the frog, Rana japonica Total Stage” length= "MEY NEv (OVER) PVO> Pa EO SCO PA DCP SFO MCP (mm) 17 4 a = = _ = = _ = = = = 19 5 a = = = = = = = = = 20 6 = = = = te — = = ae 21 7 — — = = _ ~— = = = Di 9 = = = -F sta’ ate ae ae om = 24 10 = = — + + +0) - + — 25 12 —- + = to Gee ote +(*) — + - + 26 16 Se yy Bee = te) ats a a +f = at 28 26 + ap = ae ates ate =p ar a = alr 32 37 de se a =f rahe gat a ala = = + OR amie y samataiis ~ ee eh ee + + + + frog peut Bor ta ey bs + aie ee 3) to udiioee lnnighe an frog 1) Stages were determined according to Tahara [10, 11]. temperatures of 13 to 17°C. Embryos and larvae were kept in water of Ontogeny of Circumventricular Organs examined. The frog, Rana japonica (Table 7) Hypothalamic structures The ME starts to differentiate around stage 26. It consists of a single layer of cuboidal ependymal cells and a thin nerve fiber layer. The NL starts to differentiate at about stage 25 to 26. At these stages, the nerve fiber layer is very thin. In the ventral wall of the preoptic recess, the subependymal fiber layer is scarcely found in tadpoles and thus the OVLT is not differentiated histologically in larvae. In frogs shortly after metamorphosis, a thin nerve fiber layer appears but the organ is still barely specialized. Epithalamic structures The common anlage of the pineal and frontal organ appears as a shallow omega-shaped evagination at stage 19. At stages 20 and 21, it is a rather parenchymatous structure. The anlage is elongated rostrocaudally at stage 22~. The rostral swelling differentiates into the frontal organ and the caudal stalk into the pineal (Fig. 13). The anlage is clearly separated into the frontal organ and the pineal at stage 26, 509 although they are connected by a slender nerve. The pineal bears a distinct lumen after stage 26. At stage 28, the frontal organ is a balloon-like structure with a distinct lumen. In later tadpoles, the frontal organ becomes nearly parenchymatous and moves rostrally. In frogs shortly after meta- morphosis, it is located in the dermis right above the olfactory bulb. In stage 26 tadpoles, a small ependymal sac develops rostrodorsally to the DCP. It may be an anlage of the paraphysis. In stage 32 tadpoles, it is divided into saccules. In tadpoles, the paraphysis is a more or less evaginated structure. In frogs shortly after metamorphosis, the caudal part of the paraphysis is situated in the III ventricle (invagi- nated), being covered by the DCP. Choroid plexuses The TCP is not found at any stages of development. In stage 22* and 24 tadpoles, the anlage of the DCP forms a plate-like ventricular invagination. The anlage becomes an irregularly shaped invagination at stage 25, but its ependymal cells are not yet differentiated as typical ependymal cells of the CP. The ependymal cells gradually increase in amount of the cytoplasm TABLE 8. Development of circumventricular organs in the lacertid lizard, Takydromus tachydro- moides Days after Stage” Guposition? ME NL (OVLT) PVO P- PE SCO PA DCP TCP (SFO) MCP (AP) 29° 4 - = = _ + i ia ze = et 307 74 %) = = + es ka z i -AIl [14] and Asn'-Val°-AII (Okawara, unpublished data), both native AIlIs of the Japanese eel [15]. A small amount of blood was used for microhematocrit determinations. In some cases, the AII values were corrected for changes in the hematocrit as described previously [16]. Experiment I. Observation for a period of 1 to 2hr following transfer of FW eels to SW Thirty FW-adapted eels were transferred into PR-SW, while hiding in the polyvinyl tubes. Groups of seven or eight eels each were killed 15, 30, 60 and 120 min following the transfer. Another ten eels kept in FW were transferred to PR-FW and killed 60 min later. These eels served as controls. Water intake, hematocrit and plasma AII concentration were measured in all fish. The experiments were repeated twice using thirty-eight and eighteen eels. : In order to examine the possible effects of the handling stress, one more experiment was con- ducted using twelve eels. Six eels were kept in an aquarium [30(D) x45(W) Mean+standard error. * p<0.05, compared with control values. 526 Y. OKAWaRA, T. KARAKIDA et al. Water intake (yl /hr /100g) ~ i 40 2 j 30 = = g 5 < Y ° 5 Z 20 @ E Y E B Y) o 8 y) e ao Y} 10 g B O 10hr 1day 2 days 3days Time after tansfer to SW Fic. 1. Changes in drinking rate, plasma AII and hematocrit following transfer of FW eels to SW (Experiment II). Values at 0 time (control) are those obtained in Experiment I. Numbers of eels are indicated in parentheses. Each column repre- sents the mean+SE. The amount of water in- gested was measured for the last hour of 10 hr and 1, 2 and 3 days after immersion in SW. *, p<0.05 and **, p<0.01, as compared with the control. third day (116.9+ 25.1 pg/ml, n=5) (Fig. 1). The hematocrit decreased significantly at day 1 (29.6+ 2.1%, n=8) and at day 3 (29.9+3.1%, n=5) after the transfer. Recalculation of the plasma AII levels at these time points, taking hematocrit values into consideration, according to the method of Okawara et al. [16] yielded values of 104.7+ 18.0 and 95.8+11.3pg/ml, respectively. These values were also significantly different from control values. Experiment III. Effects of SQ14225 on the water ingestion of SW-adapted eels Water intake of the control eels adapted to SW for 10 days was 57.34 14.3 1/100 g (n=7) within 30 min following the vehicle injections and that of eels injected with SQ14225 (1 mg/ fish) was 1.7+ 1.4 4/100 g (n=8). The difference was significant (p<0.01). DISCUSSION In the present experiments, the Japanese eels did not ingest water while in FW. This is in agreement with the results of Oide and Utida [9] and Hirano [6]. We observed only marginal water ingestion (less than 20.7 w1/100 g), during the first one hour after transfer to SW. However, there are several reports indicating that eels ingest water copiously immediately after transfer from FW to SW [7, 8, 17]. Hirano [7] and Takei et al. [8] used oesophagus-cannulated eels and measured water intake with a drop counter. Kirsch and Mayer- Gostan [17] used intact eels and measured water intake using an isotope. The present experiments were conducted on intact eels, measuring water intake by the phenol red method. The difference between our data and those of other investigators may be due to difference in technique, species and source of eels. However, it may be interesting to measure plasma AII concentration at the time when copious drinking is observed, although it is said that the copious drinking is induced by Cl~ reflex [7]. In the present experiment, eels started drinking water soon after being transferred to SW, but the amounts of water intake were small in two of the experiments, even after 120min. During this period, plasma AII concentrations did not change. Thus, plasma AII may not be involved in inducing water intake immediately after the transfer to SW. This hypothesis is consistent with the observation that renin activity in the eels transferred from FW to SW increased between 0.5 and 8hr after the transfer [18]. : Takei et al. [8] observed that a vigorous drinking immediately after the transfer was inhibited by preinjection of SQ14225. Their data suggest the involvement of plasma AII in the vigorous drink- ing induced by Cl” reflex, but there is the possibility that SQ14225 inhibited such drinking through some unidentified mechanisms other than the inhibition of converting enzyme. Water intake and plasma AII both significantly increased 10hr after transfer. After this, water intake increased in parallel with that of plasma All concentration. These findings suggest the possible involvement of plasma AII in water intake Angiotensin II and Water Intake in the Eel observed 10hr after transfer to SW. Our results show that SQ14225 curtailed water intake of eels maintained in SW for 10 days. It may be hypothesized, therefore, that AII is in- volved in the physiological mechanism regulating natural water intake in SW adapted eels. This hypothesis is in agreement with the observations that infusion of SQ14225 inhibited water intake of SW-adapted euryhaline flounder [19] and that administration of SQ20881, another converting enzyme inhibitor, reduced water intake of the SW-adapted euryhaline killifish [20]. Further- more, $Q14225 also reduced natural water intake in mammals [see 5 for a review] and in Japanese quail [1]. Nevertheless, Beasley et al. [21] reported that SQ14225 did not attenuate basal water intake in certain obligatory marine fish such as the winter flounder and the longhorn sculpin. It is possible that stenohaline SW fish such as the winter flounder and the longhorn sculpin may regulate basal drinking through mechanisms independent of the renin-angiotensin system. Further studies using freshwater, marine and euryhaline fishes are needed to clarify this point. ACKNOWLEDGMENTS We are grateful to Professor Tetsuya Hirano, Ocean Research Institute, University of Tokyo, Professor Zvi Yaron, Department of Zoology, Tel Aviv University and Dr. Yoshio Takei, Department of Physiology, Kitasato University for their valuable suggestion and discussion during the preparation of this manuscript. This investiga- tion was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 Kobayashi, H. and Takei, Y. (1982) Mechanisms for induction of drinking with special reference to angiotensin II. Comp. Biochem. Physiol., 71A: 485-494. 2 Kobayashi, H., Uemura, H., Wada, M. and Takei, Y. (1979) Ecological adaptation of angiotensin- induced thirst mechanism in tetrapods. Gen. Comp. Endocrinol., 38: 93-104. 3 Kobayashi, H., Okawara, Y., Maitra, S., Sinhahkim, A. and Ghosh A. (1982) Further studies on angiotensin []-induced drinking in birds. J. Yamashina Inst. Ornithol., 14: 137-142. 4 Nn 10 11 12 13 14 1 16 527 Kobayashi, H., Uemura, H., Takei, Y., Itatsu, N., Ozawa, M. and Ichinohe, K. (1983) Drinking in- duced by angiotensin II in fishes. Gen. Comp. Endocrinol., 49: 295-306. Fitzsimons, J. T. (1979) The Physiology of Thirst and Sodium Appetite, Cambridge Univ. Press, Cambridge/London/New York/Melbourne, pp. 285-288. Hirano, T., Takei, Y. and Kobayashi, H. (1978) Angiotensin and drinking in the eel and the frog. In “Osmotic and Volume Regulation”. Ed. by C.B. Jorgensen and E. Skadhauge, Munksgaard, Copenhagen, pp. 123-128. Hirano, T. (1974) Some factors regulating water intake by eel, Anguilla japonica. J. Exp. Biol., 61: 737-747. Takei, Y., Uemura, H. and Kobayashi, H. (1985) Angiotensin and hydromineral balance: with special reference to induction of drinking behavior. In “Current Trends in Comparative Endocrinology”. Ed. by B. Loft and W. N. Holmes, Hong Kong Univ. Press, Hong Kong, pp. 933-936. Oide,H. and Utida,S. (1968) Absorption and excretion in the Japanese eel. Marine Biol., 1: 172- RET: Takei, Y., Hirano,T. and Kobayashi, H. (1979) Angiotensin and water intake in the Japanese eel, Anguilla japonica. Gen. Comp. Endocrinol., 38: 466-475. Smith, H. W. (1931) The absorption and excretion of water and salts by the elasmobranch fishes. II. Marine elasmobranchs. Am. J. Physiol., 98: 296- 310. Diisterdieck, G. and McElwee, G. (1976) Estima- tion of angiotensin II concentration in human plasma by radioimmunoassay. Some applications to physiological and clinical states. Eur. J. Invest., 2: 32-38. Yamaguchi, K. (1981) Effect of water deprivation on immunoreactive angiotensin II levels in plasma, cerebroventricular perfusate and hypothalamus of the rat. Acta Endocrinol., 97: 137-144. Takei, Y., Okawara, Y. and Kobayashi, H. (1987) Mechanisms regulating drinking in the Japanese quail, Coturnix coturnix japonica. In “Progress in Avian Osmoregulation”. Ed. by M. R. Hughes and A.C. Chadwick, Leeds Philosophical and Literary Society Ltd., Leeds. (in press) Hasegawa, Y., Nakajima, T. and Sokabe, H. (1983) Chemical structure of angiotensin formed with kidney renin in the Japanese eel, Anguilla japonica. Biomed. Res., 4: 417-420. Okawara, Y., Karakida,T., Yamaguchi, K. and Kobayashi, H. (1985) Diurnal rhythm of water intake and plasma angiotensin II in the Japanese quail (Coturnix coturnix japonica). Gen. Comp. 17 18 19 528 Endocrinol., 58: 89-92. Kirsch, R. and Mayer-Gostan, N. (1973) Kinetics of water and chloride exchanges during adaptation of the European eel to sea water. J. Exp. Biol., 58: 105-121. Sokabe, H., Oide,H., Ogawa, M. and Utida, S. (1973) Plasma renin activity in Japanese eels (Anguilla japonica) adapted to SW or in dehydra- tion. Gen. Comp. Endocrinol., 38: 466-475. Carrick, A. and Balment, R.J. (1983) The renin- 20 21 Y. OKAWARA, T. KARAKIDA et al. angiotensin system and drinking in the euryhaline flounder, Platichthys flesus. Gen, Comp. Endocri- nol., 51: 423-433. Malvin, R.L., Schiff,D. and Eiger,S. (1980) Angiotensin and drinking rates in the euryhaline killifish. Am. J. Physiol., 239: R31—-R34. Beasley, D., Shier, D.N., Malvin, R. L. and Smith, G. (1986) Angiotensin-stimulated drinking in marine fish. Am. J. Physiol., 250: R1034-R1038. ZOOLOGICAL SCIENCE 4: 529-534 (1987) Cyclic CMP Alters Squirrel Monkey (Saimiri sciureus) Luteal Cell Structure via Cyclic AMP-Dependent Mechanisms Puitie J. CHAN! Department of Obstetrics and Gynecology, University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine, 401 Haddon Ave., Camden, New Jersey 08103, U.S.A. ABSTRACT—Cyclic cytidine monophosphate (cCMP) is a pyrimidine nucleotide that is not as well-known as the other intracellular messengers such as cyclic adenosine monophosphate (cAMP). The present study was undertaken to determine if cCMP functions to alter cultured luteal cell morphology and if the mechanism involves cAMP. Adult female squirrel monkeys of Guyanan origin were administered daily single i.m. injections of FSH (follicle-stimulating hormone) for 4 days followed by hCG (human chorionic gonadotropin) on the fourth day. Luteal cells were aspirated from ovarian corpora lutea 64 hr after hCG by laparoscopy. The cells were dispersed, washed and cultured in individual center-well petri dishes for 48 hr at 37°C in 5% CO, in air. The test compounds (10 «M each), dbcCMP, dbcAMP, dbcGMP, dbcCMP and 6mM imidazole, or control media were added at the start of culture. At the end of the incubation period, the cells were fixed in methanol and stained with Giemsa stain. The data indicated that dbcCMP restructured the polygonal luteal cells into small narrow-shaped cells with minimal cytoplasm in a manner similar to dbcAMP. However, when imidazole, a phosphodiesterase stimulator, was also present, the cells retained the polygonal shape. Dibutyryl cGMP at the concentration tested did not affect luteal cell morphology. The results suggest that dbcCMP alters the squirrel monkey luteal cell morphology in a manner similar to dbcAMP and © 1987 Zoological Society of Japan that the process requires intracellular cAMP. INTRODUCTION Cyclic nucleotides are a class of intracellular compounds [1-3] which are involved in mediating hormone action and regulating cellular events such as the assembly of microtubules [4,5] and mi- crofilament cell processes [6]. The role of cAMP and cGMP as intracellular second messengers is well-documented [2, 3, 7, 8]. At the present time, there have been no reports on the function of cCMP in the regulation of cell morphology and progesterone synthesis. Cyclic CMP is a pyrimidine compound, in contrast to cAMP and cGMP which are purine compounds. Studies in other cell types indicate that cCMP activates protein kinases [9], hemoglobin synthesis Accepted January 19, 1987 Received November 19, 1986 ' Present address: Department of Obstetrics and Gynecology, Oral Roberts University School of Medi- cine, City of Faith, 8181 S. Lewis, Tulsa, Oklahoma 74104, U.S.A. [10], modulates cell proliferation and increases phosphodiesterase activity in fast growing hepato- ma cells [11]. The present study was carried out to define the action of cCMP, in relation to cAMP and cGMP, on squirrel monkey luteal cell mor- phology. The objective is to determine if cCMP alters cell morphology and to examine the mecha- nism involved in the transformation. MATERIALS AND METHODS Adult female squirrel monkeys (Saimiri sciureus) of Guyanan origin (Buckshire Corp., Perkasie, PA) weighing between 600g and 750g were housed individually in stainless steel flush- type cages. The animals were kept indoors on a 12L:12D cycle and fed a commercial monkey feed and water ad libitum. The hormonal-stimulating regimen [12] consisted of administering the female monkeys with 4 days of follicle-stimulating hor- mone (FSH-P, Burns-Biotec Laboratories Inc., Omaha, NE) at a daily dosage of 1 mg intramuscu- 530 P. J. CHAN larly followed by a single i.m. injection of 250 IU human chorionic gonadotropin (hCG, APL Ayerst Laboratories, New York, NY) on the final day of FSH treatment. It has been shown that there is no seasonal effect on squirrel monkey oocyte de- velopment and that follicle cells such as luteal cells may be harvested at any time of the year [13]. At 64 hr after hCG treatment, the squirrel monkeys were anesthetized (15mg ketamine/animal i.m.) and laparoscoped. The luteal cells were aspirated from the corpora lutea in the ovaries using a 1 ml tuberculin syringe fitted with a 25 gauge needle. The cells were dispersed into the culture medium, gently minced, washed and pipetted into the center well of individual Falcon #3037 petri dishes and incubated as low-density cultures at 37 C in a moist atmosphere of 5% CO, in air. The test com- pounds, 104M each of dbcCMP, dbcAMP, dbcGMP, dbcCMP plus 6 mM imidazole or control media were added at the start of culture. After 48 hr of incubation, the cells were fixed in methanol and stained with Giemsa stain [14]. Photomicro- graphs of the stained cells in the different treat- ments were taken and analyzed using the Zeiss Videoplan computerized image analyzer as de- scribed below. Culture medium The culture medium consisted of Medium 199 with 25mM HEPES buffer, Earle’s salts and L-glutamine (GIBCO, Grand Island, New York) and 75mg/1 penicillin and 75 mg/I streptomycin. The medium was filtered through a 0.22 micron syringe filter (Nalgene Co., Rochester, NY) and supplemented with 20% filtered and _heat- inactivated fetal bovine serum (HyClone, Logan, (U/10)). Videoplan computerized image analysis Morphometric measurements of the photo- micrographs of the cells were quantitated using the Zeiss Videoplan computerized image analyzer equipped with statistical software. The image analyzer operated by translating the movements of the tracing of the outline of selected two- dimensional objects using an electronic input pen placed on a magnetized tabloid. The area and perimeter of the nucleus and individual cell outline were measured. The data collected were express- ed as mean+S.E. For convenience, the units of measurement were in arbitrary units. The actual perimeter in microns may be calculated by multi- plying each value by 0.2545. In the case of the area value, multiplying each area value by 0.0648 should provide the actual value in square microns. Statistical analysis Differences in the perimeter and area measure- ments were tested for significance using the Dun- nett’s t-test for comparisons with the control with heterogeneous variance and unequal treatment sample sizes. A P<0.05 value was considered significant. RESULTS The presence of 104M dbcCMP in the media resulted in a significant reduction (P<0.01) in the cross-sectional area of the squirrel monkey luteal cell (Table 1). The cross-sectional area of the nucleus of the dbcCMP-treated cell was also significantly smaller in comparison with the control cell nucleus. When the cAMP-specific phospho- diesterase stimulator, imidazole, was present along with dbcCMP, the cells did not show the character- istic reduction in cell shape. Light microscopic observations of the cells revealed differences in shape of dbcCMP-treated and control luteal cells (Fig. 1). The control cells were polygonal-shaped while the dbcCMP-treated cells were small and narrow-shaped with less cytoplasm. The dbcCMP cells also appeared to have less pseudopod forma- tion and less granular cytoplasm compared with the control cells. Some of the dbcCMP-treated cells had a wide spindle-shaped appearance. We did not observe any other cell types along with the cultured luteal cells. When imidazole was present along with dbcCMP (Fig. 2), the luteal cell mor- phology was similar to the control cell morphology and did not have the small narrow-shaped appear- ance characteristic of dbcCMP-treated cells. The luteal cells became smaller and narrow- shaped when 104M dbcAMP was added to the culture media (Fig. 3). The decrease in cell shape and structure was significant (P<0.001) in com- parison with the control (Table 1). The cross- Cyclic CMP and Squirrel Monkey Luteal Cell 331 TaBLE 1. The effect of cyclic nucleotides on the morphology of squirrel monkey (Saimiri sciureus) luteal cells in vitro airéatmatt Mean area of cell nucleus Mean area of entire cell (sq. units) (sq. units) Control 73.7+4.6 (27) 628.5 +36.2 (22) 10 uM dbcCMP 58.6+4.0 (28)? 451.0+34.6 (33)? 10 uM dbcCMP+ FT ee PSLOE=2.3 (32) 603.3 +50.7 (22) 10 uM dbcAMP 52.0+4.0 (33)? 396.6+25.1 (26)° 10 uM dbcGMP 73.0+3.7 (26) 701.6+42.4 (23) * Significant difference from respective control (P<0.05). > Significant difference from respective control (P<0.01). Values are expressed as mean+S.E. (sq. arbitrary units). Values in parentheses indicate number of cells. Fic. 1. The morphology of squirrel monkey luteal cells Fic.2. Two views of squirrel monkey luteal cells in growing in low-density cultures after 48 hr of in- low-density cultures after 48 hr of incubation in the cubation in either control media (A) or in the presence of 104M dbcCMP and 6mM imidazole; presence of 104M dbcCMP (B). Note the de- light microscopy (A) and phase contrast (B). Mag- creased cell size and reduced cytoplasm in the —_ nification 400. dbcCMP-treated cells. Magnification x 400, phase contrast. 532 P. J. CHAN Fic. 3. The effect of 104M dbcAMP (A) or 104M dbcGMP (B) on the morphology of squirrel mon- key luteal cells in low-density cultures after 48 hr of incubation. Magnification <400, phase contrast. sectional area of the cell nucleus was also de- creased by the dbcAMP treatment. Some of the cells showed a pronounced spindling effect. In contrast, the addition of 10 uM dbcGMP to the cultures did not have an effect on cell morphology (Fig. 3). An analysis of the cross-sectional area of the dbcGMP-treated luteal cells also did not reveal any significant differences from control cells (Table 1). DISCUSSION This report is the first demonstration of in vitro cultured squirrel monkey (Saimiri sciureus) lutei- nized granulosa cells or luteal cells. In the present study, we have demonstrated that the action of dbcCMP on the alteration of the morphology of cultured squirrel monkey luteal cells is similar to the action of dbcAMP on these cells. This suggests that the function of cCMP may be similar to cAMP. The size of the luteal cell soma and nucleus are both decreased by dbcCMP and dbcAMP treatment. Our observation that dbcAMP alters the luteal cell morphology is supported by studies showing the same effect in other cell types such as fibroblasts [6, 15, 16] and granulosa cells [17, 18]. The alteration in cell size by cAMP and by other compounds that increase intracellular cAMP such as luteinizing hormone (LH) has been correlated to active steroidogenesis in the cell [17] and may involve reorganization of the tubulin and intermediate filaments [18, 19] through calmodulin-mediated mechanisms [20]. We surmise that cCMP may be involved in the control of steroidogenesis in luteal cells because of the similarity of dbcCMP-treated cells and dbcAMP-treated cells. The synthesis of progester- one by the squirrel monkey luteal cells will be examined in a future experiment. There is very little information on the role of cCMP in the cell. Cyclic CMP is a pyrimidine compound, unlike cAMP and cGMP which are purine compounds. A related compund, cytidine- 3’-monophosphate is a ribonuclease inhibitor and it is possible that CCMP may also function as such. The role of cCMP in other cell activities such as stimulating cancer cell proliferation has been reported [21,22]. A study done on pigeon crop-sac mucosal epithelial cells suggests a role of cCMP as a mediator in the proliferation of cells [23]. The activity of cCMP-phosphodiesterase (PDE) has been shown to be high in slow growing hepatoma cells and low in rapidly-dividing cells [24] and the cCMP-PDE appears to be a separate and different PDE from the cAMP-specific PDE [25]. Another study suggested a role of cCMP in stimulating hemoglobin synthesis [10] probably via protein kinases [9]. In this study we postulate a role of cCMP in the control of steroidogenesis in luteal cells. The action of dbcCMP on squirrel monkey luteal cells can be inhibited by imidazole, a cAMP-specific PDE stimulator implying that the mechanism of action of dbcCMP requires in- tracellular cAMP. We cannot exclude the possibil- Cyclic CMP and Squirrel Monkey Luteal Cell ity that the imidazole may be stimulating PDE to inactivate both intracellular cAMP and exogenous dbcCMP, although this argument is weak because it has been shown that dibutyryl cyclic nucleotide analogues are resistant to degradation by PDE [26]. It is possible that the role of cCMP is to be a mediator or co-factor for cAMP-induced cell shape changes, calcium-mediated events and _ steroid- ogenesis. In this study, we also tested dbcGMP because certain concentrations of cGMP appear to affect intracellular cAMP production [27]. Our data indicated that dbcGMP was not involved in the alteration of luteal cell morphology. It appears that cGMP does not play a role in the physiological processes in the squirrel monkey luteal cell. The results of the present study suggest that cCMP alters the squirrel monkey luteal cell mor- phology in a manner similar to cAMP and that the process requires intracellular cAMP. ACKNOWLEDGMENTS This research was supported in part by the Foundation of the University of Medicine and Dentistry of New Jersey. REFERENCES 1 Michelson, A.M. (1983) The Chemistry of Nu- cleosides and Nucleotides, Academic Press, New York, NY, pp. 1-40. 2 Robison,G.A., Butcher,R.W. and Sutherland, E.W. (1971) Cyclic AMP, Academic Press, New York, NY, pp. 1-51. 3 Sutherland, E. W. (1972) Studies on the mechanism of hormone action. Science, 177: 401-407. 4 Garland, D.L. (1979) CAMP inhibits the in vitro assembly of microtubules. Arch. Biochem. Bio- phys., 198: 335-337. 5 Means,A.R., Tash, J.S., Chafouleas, J. G., Lagace, L. and Guerriero, V. (1981) Regulation of the cytoskeleton by Ca‘ *-calmodulin and cAMP. Ann. N. Y. Acad. Sci., 383: 69-84. 6 Willingham,M.C. and Pastan,I. (1975) Cyclic AMP and cell morphology in cultured fibroblasts. J. Cell Biol., 67: 146-159. 7 Goldberg, D.L., Haddox, M.K., Hartle, D. K. and Hadden, J. W. (1972) The biological role of cyclic 3’, 5’-guanosine monophosphate. Proc. 5th Int. Congr. Pharmacol, Krager, Basel, pp. 149-169. 8 10 11 12 13 14 15 16 17 18 19 20 7a 553 Goldberg, D.L. and Haddox, M. K. (1977) Cyclic GMP metabolism and involvement in biological regulation. Ann. Rev. Biochem., 46: 823-896. Vardanis, A. (1980) A unique cyclic nucleotide- dependent protein kinase. J. Biol. Chem., 255: 7238-7243. Canas, P.E. and Congote, L. F. (1982) Effects of cyclic nucleotides on hemoglobin synthesis in fetal calf liver cells in culture. Can. J. Biochem., 60: 1-7. Wei, J.W. and Hickie,R.A. (1983) Decreased activities of cyclic CMP phosphodiesterase in Morris hepatomas having varying growth rates. Int. J. Biochem., 15: 789-796. Dukelow, W.R. (1970) Induction and timing of single and multiple ovulations in the squirrel mon- key (Saimiri sciureus). J. Reprod. Fertil., 22: 303- 309. Chan, P.J. and Dukelow, W.R. (1984) Variations in squirrel monkey responses with seasonal and environmental conditions. Zool. Sci., 1: 471-476. Freshney, R.I. (1983) Characterization. In “Cul- ture of Animal Cells”. Ed. by R.I. Freshney, Alan R. Liss, Inc., New York, pp. 159-161. isies Aww elones iC. and) Puck ain (1971) Further changes in differentiation state accompany- ing the conversion of CHO cells to fibroblastic form by dibutyryl adenosine 3’,5’-monophosphate and hormones. Proc. Natl. Acad. Sci. USA, 68: 1648- 1652. Johnson, G.S., Friedman, R.M. and Pastan, I. (1971) Restoration of several morphological charac- teristics of normal fibroblasts in sarcoma cells treated with adenosine-3’: 5’-cyclic monophosphate and its derivatives. Proc. Natl. Acad. Sci. USA, 68: 425-429. Lawrence, T.S., Ginzberz, R. D., Gilula, N. B. and Beers, W. H. (1979) Hormonally induced cell shape changes in cultured rat ovarian granulosa cells. J. Cell Biol., 80: 21-36. Soto, E. A., Kliman, H.J., Strauss, J. F. II, Paavo- la, L. G. (1986) Gonadotropins and cyclic adenosine 3’, 5’-monophosphate (cAMP) alter the morphology of cultured human granulosa cells. Biol. Reprod., 34: 559-569. Herman, B. and Albertini, D.F. (1984) A time- lapse videoimage intensification analysis of cytoplas- mic organelle movement during endosome trans- location. J. Cell Biol., 98: 565-576. Chan, P. J. and Dukelow, W. R. (1985) Calmodulin level changes associated with cyclic AMP treatment in cultured squirrel monkey oocytes and sperm. Zool. Sci., 2: 219-223. Bloch, A. (1975) Isolation of cytidine 3’, 5- monophosphate from mammalian tissues and body fluids and its effects on leukemia L1210 cell growth in culture. Adv. Cyclic Nucleotide Res. , 5: 331-338. ip 23 24 M5) 534 Bloch, A., Dutschman,G. and Maue,R. (1974) Cytidine 3’,5’-monophosphate. II. Initiation of leukemia L1210 cell growth in vitro. Biochem. Biophys. Res. Commun., 59: 955-959. Anderson, T.R., Mayer,G.L. and Nicoll, C.S. (1982) Cyclic nucleotides and the control of epithe- lial cell proliferation: Cyclic CMP may be a partial mediator of the response of the pigeon crop-sac to prolactin. J. Cyclic Nucleotide Res., 7: 225-234. Wei, J.W. and Hickie, R.A. (1983) Decreased activities of cyclic CMP phosphodiesterase in Morris hepatomas having varying growth rates. Int. J. Biochem., 15: 789-796. Cheng, Y.C. and Bloch, A. (1978) Demonstration P. J. CHAN 26 Zi) in leukemia L-1210 cells of a phosphodiesterase acting on 3’: 5’ cyclic CMP but not on 3’: 5’-cyclic AMP or 3°: 5’-cyclic GMP. J. Biol. Chem., 253: 2522-2524. Kaukel, E., Mundhenk, K. and Hilz, H. (1972) N6-monobutyryl adenosine 3’: 5’ monophosphate as the biologically active derivative of dibutyryl adeno- sine 3’: 5’' monophosphate in Hela 53 cells. Eur. J. Biochem., 27: 197-200. Whitfield, J.F., Boynton, A.L., Macmanus, J. P., Sikorska, M. and Tsang, B.K. (1979) The reg- ulaiton of cell proliferation by calcium and cyclic AMP. Mol. Cell. Biochem., 27: 155-179. ZOOLOGICAL SCIENCE 4: 535-542 (1987) Entrainment of Cricket Circadian Activity Rhythm after 6-Hour Phase-Shifts of Light-Dark Cycle KENJI TOMIOKA and YOSHIHIKO CHIBA Environmental Biology Laboratory, Biological Institute, Yamaguchi University, Yamaguchi 753, Japan ABSTRACT— Adult crickets Gryllus bimaculatus show a circadian locomotor rhythm peaking early in the dark fraction of 12-h light to 12-h dark cycle (LD). Reentrainability of the locomotor rhythm to 6-h delayed or advanced LD was investigated in 68 crickets under a constant temperature of 26°C with three different illumination levels, i.e. 10, 100 and 400lux. The LD was shifted by either lengthening or shortening of the light fraction. More cycles were needed for reentrainment in phase-advance than in phase-delay. Decrements of light intensity increased the cycles needed in both phase-delay and phase-advance. When the LD was shifted by 6-h in either direction, the LD masked the rhythm dependently on the phase of the rhythm, either inducing or inhibiting the activity; the strongest masking was observed in the subjective night. Interestingly, a second peak was developed to form a double peaked activity pattern after phase-shifting in 31 animals, more frequently when the illumina- tion level was 10lux. The results are discussed mainly in relation to effects of LD on the circadian © 1987 Zoological Society of Japan rhythm. INTRODUCTION Circadian rhythms, freerunning with a period of about 24-h under constant conditions, synchronize to certain environmental 24-h cycles (zeitgeber). The most powerful zeitgeber is light-dark cycles; reentrainment of the rhythm by its phase shifts revealed its efficacy in entrainment [1]. In addi- tion, photic informations related to the LD, bypassing the circadian pacemaker, modulate waveforms of the overt rhythms. The modula- tion, termed masking effect [2], has been pro- foundly studied in mammals such as rats [3] and squirrel monkeys [4]. Thus, behavioral rhythms under LD may reflect pacemaker-dependent effects of light as well as masking effects. The cricket G. bimaculatus shows clear circa- dian activity rhythm peaking in the light fraction as nymph but in the dark fraction as adult [5]. A series of surgical and electrophysiological experi- ments [6-10] has revealed that the rhythm is controlled by a circadian system composed of bilateral optic lobe pacemakers, a secondary Accepted December 22, 1986 Received August 4, 1986 oscillatory system outside of the lobes, and the compound eye as the necessary source of photore- ceptive information for entrainment. The activity rhythm of crickets with optic nerves severed, therefore, freeruns because of a lack of photic information. However, we found that the rhythm of the optic nerve severed animals eventually resynchronized to the LD without any sign of forced phase-shifting [7]. Our explanation for this was that the reentrainment was attributable to the nerve regeneration which was not so sufficiently completed that the zeitgeber information con- veyed was too weak to force the rhythm to resynchronize by abrupt phase-shifting; in other words, light was felt subjectively too weak. The present experiment was carried out to investigate the reentrainment of the cricket activ- ity rhythm to 6-h phase-shifted LDs with three different illumination levels (10,100, and 400 lux). The results revealed that the entraining ability of LD depends on light intensity, support- ing Our previous explanation. In addition, it was shown that the phase-shifting of LD often induced a double peaked activity pattern as well as circad- ian phase-dependent maskings. 536 K. TOMIOKA AND Y. CHIBA MATERIALS AND METHODS All experiments were carried out with male crickets, Gryllus bimaculatus which were taken from our laboratory colonization kept under 12-h light to 12-h dark cycle (LD 12:12, L: 06°°-18) and a constant temperature of 26+0.5°C, hereaf- ter called the standard conditions. Locomotor activities were recorded individually with an acto- graph with a rocking substratum whose movement caused by a moving animal was sensed by a microswitch connected to an _ event-recorder (Yokogawa, XP-490) and a digital printer (Chi- no, Procos-V). Phase-shift experiments included studies with three illumination intensities, bright-LD (L: about 400 lux), medium-LD (L: about 100lux) and dim- TIME LD (L: about 10lux), and with 22, 23 and 23 animals, respectively. Lighting was manipulated with the use of 20 W cool white fluorescent lamps which were programed to produce an LD of 12-h of light and 12-h of darkness per day. Light intensity was adjusted either by changing the number of lamps or by shading them. Tempera- ture was 26+0.5°C. Six-hour phase-shifts of LD were carried out by either lengthening or shorten- ing the light fraction. RESULTS In the cricket Gryllus bimaculatus, diurnally peaked activity rhythm is reversed to peak noctur- nally 3 to 5 days after the imaginal molt [5]. The endogenous rhythm reversal [5] could be ieqt dl seals ils a= fe eed that) if ry i he ee t 4 il 5 i i t 43 SU RNLee aii t spo Fania if . il ali c, OF DAY Fic. 1. Double plotted activity records of 4 crickets showing a reentrainment to the shifted LD 12:12 with 10lux. Lighting conditions are indicated in the left half of the figure: brackets show a dark fraction. IM, imaginal molt. The animals were diurnal in nymphal stage, but became nocturnal a few days after the imaginal molt. At the early stage of the rhythm reversal, an intense activity occurred to form a double peaked activity in A. In response to a 6-h delayed LD, a slowly delaying (open arrow heads) and an immediately synchronized component occurred. The latter seems to be a positive masking effect of lights-off. After phase-shifts of LD, a second component either being restricted in the light fraction or spreading widely from late night to midday appeared (arrow heads). The second component either disappeared with the completion of reentrainment (A, B) or persisted for weeks (G)D): Reentrainment of Cricket Rhythm Soy observed under the three LDs of different illu- mination levels; daily locomotor activity was initi- ated at lights-on in nymphal stage and at lights-off in adults (Figs. 1-3). At the early stage of the reversal, an intense activity often occurred at lights-on to make a double peaked activity together with the newly developed nocturnal peak, but, in most cases, this lights-on activity became to terminate earlier day after day and ew ee ha _— ~ TIME OF DAY Fic. 2. Double plotted activity records of 3 crickets under phase-shifting of LD 12:12 of 100lux. Brackets show a dark phase. IM, imaginal molt. The animals were reentrained by shifted LDs. An animal shown in A consistently exhibited a single peaked nocturnal activity, while other two animals shown in B and C exhibited a second activity com- ponent (arrow heads) which extended from late at night to about midday. TIME OF DAY Fic. 3. Double plotted activity records of two crickets under various lighting conditions with illumination level of 400lux. Brackets show a dark phase. IM, imaginal molt. A single nocturnal component con- sistently occurred in A, whereas a second compo- nent (arrow) was developed to produce a double peaked pattern in B. eventually disappeared (example: Fig. 1A). The LD was first phase-shifted, at the earliest, 4 days after the establishment of the nocturnal rhythm and was done repeatedly. Figures 1-3 exemplify the activity recording of fully entrained animals. In dim-LD, two kinds of activity peaks were clearly observed after a 6-h delay of the LD (Fig. 1); one occurred in synchrony with the new LD from the very beginning and the other (open arrow heads in Figure 1) moved backward slowly to attain resynchronization in several days. The latter was less prominent in most cases, and almost disappeared in higher illumination levels (Figs. 2 and 3). In view of the masking effect of LD, the slowly moving peak may be endogenous under circadian control and receives sometimes inhibitory effect (negative masking) of light, whereas the other peak may be regarded as exogenous, being induced by lights-off (positive masking). Thus, we adopted this slowly moving 538 K. TOMIOKA AND Y. CHIBA TABLE 1. of illumination Transient cycles after 6-h delay- and advance-shift under different intensities Illumination Transient cycles (days) level (lux) advance delay 400 Delo ae Wet (Us) 100 Dj Oet=sletela(2 1) 10 6.00 + 2.33 (17) 3.50+0.76 (18) 4.26+0.71 (19) 10.70 +3.20 (10) Numerals in brackets indicate the number of animals used for estimating the values. TABLE 2. Activity patterns during phase shifting under different intensities of illumination No. of animals with Illumination tevel Cu), = Ne eee Peace double peaked nocturnal rhythm rhythm 400 Dap) I) Ta) 100 13} 15 8 ( 6) 10 Nps 6 16 (14) Totals 67 36 31 (27) Numerals in brackets indicate the number of animals showing a second component broadened from late at night to midday. activity onset for estimating the transient cycles (number of cycles required for reentrainment). Animals showing obscure onsets were excluded from data. The number of transient cycles varied with the direction of shifts and with the light intensity: it was shorter in delay-shifts (ANOVA, P<0.01) and in higher light intensity (ANOVA, P<0.01) (Table 1). The asymmetry-effects of delay- and advance-shifts and the light intensity dependency of the transient cycles conform to results obtained from other nocturnal animals [cf. 1]. In addition to these activity peaks related with lights-off, a peak (second component) was some- times developed at lights-on, as the LD was shifted, to make a double peaked activity in all illumination levels (arrow head in Figs. 1, 2B, C and 3B). This second component often broad- ened from late at night to midday and sometimes merged with the nocturnal peak (Figs. 1C, D, 2B, C and 3B). Table 2 summarizes the relationship between the occurrence of double peaked activity and the light intensity. The occurrence of the second component depended on the light intensity (chi-square test, P<0.01): the lower the light intensity was, the greater the number of animals with the second component was. In 4 out of 31 animals which developed the double peaked activ- ity, the second component occurred only during the 6-h phase-advance and was restricted in the light fraction. It’s offset advanced keeping pace with the nocturnal peak, but it’s onset never entered the dark fraction. The component thus disappeared on the completion of reentrainment of the rhythm (Fig. 1A, B). The remaining 27 animals showed the broadened second component which persisted for weeks and resynchronized to the shifted-LD with several transient cycles (Figs. 1C, D, 2B, C and 3C). The component, however, sometimes later became to be restricted in the light fraction and was often observed to be synchronized with the lights-on (Fig. 1C, D). On the basis of these facts, the broadened second component seems to be regulated by a combina- tion of an endogenous pacemaker causing an activity preceding lights-on and the LD as an exogenous zeitgeber inducing lights-on activity (i.e. positive masking effect of light). In some animals the nocturnal component became very faint or even disappeared to form an almost diurnal pattern by the second component (Figs. 1D and 2C). Of the 68 animals examined, one animal from the dim-LD series failed to synchronize to the LD, showing a freerunning rhythm consistently (Fig. 4). The LD, however, modulated the rhythm in two ways: (1) the relative coordination, which is a phenomenon that the rhythm changes the freerunning period systematically dependently on the phase-angle relationship between the LD Reentrainment of Cricket Rhythm 539 the thins Ne = | eft fia thes ‘| i UR eLALSL| ae i! rH i “alll — Mh TIME OF DAY Fic. 4. Double plotted activity record of a cricket showing a freerunning rhythm under shifting LD 12:12 with 10lux. A bracket indicates a dark phase. IM, imaginal molt. The freerunning activ- ity started with two peaks in the dark phase, which later fused in a single component. The period of the rhythm was shortened when the onset of activ- ity occurred early in the dark phase. The positive masking occurred at lights-on after 6-h advance of LD on day 33. and it, being explained to occur when zeitgeber is below but near the threshold for entrainment and (2) the positive masking effect. The freerunning rhythm started with two activity components in the night (around day 10), and the components merged around day 28 as the second component reached the lights-on. The relative coordination was evident after first and second phase-shifting; the rhythm freeran with shorter period when the onset of the activity occurred early in the dark onset. The masking effect was seen after 6-h advance of LD on day 33. Thereafter, an activity component appeared at lights-off, being inten- sified with the passage of day while the freerun- ning component approached the lights-off. These results suggest that the LD of 10 lux might be below but near the threshold for entrainment in this particular animal. DISCUSSION 1. Entrainment We proposed previously a view that, in the crickets with regenerated-optic nerves, the LD being felt subjectively very weak could not force the rhythm to phase-shift abruptly, but entrain it [7]. One of the purposes of this study was to see whether the view was effective for intact crickets. The number of transient cycles was shown to depend on both the light intensity and the direc- tion of the shift: it was longer in low light intensity and in advance-shifts (Table 1). In dim-LD with a illumination level of 10lux, the most animals showed the adult nocturnal peak gradually advancing or delaying to synchronize the newly phased LD without abrupt phase shift- ing (Fig. 1A, B). In addition, some of them needed extremely long transient cycles for resyn- chronization (Fig. 1C) and one failed to entrain (Fig. 4). The evidence implies the illumination level of 10 lux is near threshold level for entrain- ment of the rhythm. These results are consistent with our previous view [7], suggesting that the entraining ability of LD depends on the amount of information about it being conveyed to the circadian pacemaker. The asymmetry-effect of delay and advance shifts observed in the cricket, where the delay- shifts completes faster than advance, is commonly seen in nocturnal animals, such as rats [11] and flour beetles [12]. The opposite occurs in diurnal animals: chaffinches [13] and lizards [14]. Wever [15] proposed a mathematical model simulating various properties of circadian rhythms to explain a relation between the freerunning period and the transient cycles. According to his model, there is a “natural period” of about 24.5-h at which advance- and delay-shifts last equally long. With longer period advances last longer than delays; with shorter period the opposite occurs. The model seems to be effective for G. bimaculatus, where the freerunning period in constant dim light (LL) of 10lux as adult is 25.49+0.49 (mean +§.D.)-h [5]. 2. Double peaked activity rhythm About 45% of the cricket developed the double peaked activity with the adult nocturnal and the second components after phase-shifting. The occurrence of the pattern was low in LD with higher light intensity (Table 2), suggesting that the LD with higher illumination level entrains the circadian system more stably. Involvement of endogenous control in the double peaked activity 540 K. ToMIOKA AND Y. CHIBA is evident by the fact that both of the two activity components showed transients before completion of the reentrainment to the newly phased LD no matter whether either of the components dis- appeared (Figs. 1 and 2B). A double peaked pattern was also observed at the early stage of the rhythm reversal. This pattern is similar to that after the 6-h phase- advance in a following respect: an intense lights- on activity, which was restricted in the light fraction, became to terminate earlier day after day and eventually disappeared (example: Fig. 1A). The similarity may suggest that these two double peaked activities share a common phys- iological mechanism. That the activity peak occurs twice in one circadian cycle has been reported for some ani- mals. Whether the peaks are underlain by sepa- rate oscillations has called much attention of re- searchers, because the kinetic analyses sometimes suggest that they are of physiologically different nature (the mosquitoes Aedes aegypti [16], Culex pipiens molestus [17], Culiseta incidens [18], the silkmoth Hyalophora cecropia [19], the New Zea- land weta Hemideina thoracica [20], the cricket Teleogryllus commodus [21], the hamster Meso- cricetus auratus {22]). Recently results of lesion experiments appeared in both vertebrates and invertebrates, strengthening the view of ‘separate oscillations’; unilateral ablation of the candidate pacemaker tissue eliminates the double peaked activity to make a single peaked rhythm in the hamster [23] and in the cricket (T. commodus) [24]. The other view is that one circadian pacemaker controls the two peaks, being adopted where no physiological difference can be detected between the peaks (the cockroach Periplaneta americana [25}). The circadian system of G. bimaculatus is sug- gested to be composed of three constituents, i.e. the circadian pacemakers in the bilateral optic lobes, a driven system outside the optic lobe regulating locomotor activity under control from the pacemaker, and the compound eye as a photoreceptor for entrainment [10]. The two peaks of the double peaked activity here seem to be driven by a common pacemaker. The facts arguing this statement are that the two peaks resynchronized to the newly phased-LD in similar time courses and that the double peaked activity survived unilateral removal of the lobe [26]. However, to clarify the entire physiological mechanism underlying the cricket’s double peaked activity, further critical study is deserved. 3. Masking effect of LD The activity pattern is controlled essentially by endogenous mechanism but receives exogenous modification from LD cycle. This effect of LD which operates independently of its effects on the endogenous circadian system was classically termed “masking effect” [2]. The present study revealed that the LD induced “positive” or “nega- tive” masking dependently on the phase of the circadian rhythm. Negative masking occurred as more or less potent inhibition of the delaying nocturnal component when the first half of the subjective night phase (the night phase for the circadian pacemaker) was exposed to light after 6-h phase-delay of the LD (Figs. 1 and 2). Posi- tive masking, as lights-on activity, however, was induced by exposure of the second half of the subjective night phase to the light by 6-h advance of the LD (Fig. 1A, B). The positive masking action of light decreased cycle by cycle, dis- appearing with completion of re-entrainment. Lights-off also induces a positive masking at the subjective night when the first half of the subjec- tive night was exposed to light (cf. Figs. 1A—C and 4). In summary, the most effective phase for the masking of the LD is the subjective night phase of the circadian activity rhythm. The circadian phase dependence of masking action of light has been investigated profoundly for some vertebrate species: such as the nocturnal prosimian Galago crassicaudatus [27], the rat [3], the diurnal squirrel monkey Saimiri sciureus [4] and the chicken [28]. The most effective circadian phase for masking action of light seems to be variable among species. In Galago and Saimiri light is most effective in the subjective day [4, 27], whereas the opposite is effective in nocturnal rats [3]. The masking of chicken brain temperature rhythm is maximal on both the ascending and descending slopes of the rhythm [28]. The charac- teristic circadian phase dependency of masking in Reentrainment of Cricket Rhythm each species may have an adaptive value in that animals can respond properly to light given at unexpected time. ACKNOWLEDGMENTS This work was supported by grants from Ministry of Education, Science and Culture of Japan. 10 11 WA REFERENCES Aschoff, J., Hoffmann, K., Pohl, H. and Wever, R. (1975) Reentrainment of circadian rhythms after phase shifts of the zeitgeber. Chronobiologia, 2 : 23— 78. Aschoff, J. (1960) Exogenous and endogenous components in circadian rhythms. Cold Spring Harbor Symp. Quant. Biol., 25: 11-27. Borbely, A. A. and Huston, J.P. (1974) Effects of two-hour light-dark cycles on feeding, drinking and motor activity of the rat. Physiol. Behav., 13: 795- 802. Gander, P.H. and Moore-Ede, M. C. (1983) Light- dark masking of circadian temperature and activity rhythms in squirrel monkeys. Am. J. Physiol., 245: R927-R934. Tomioka, K. and Chiba, Y. (1982) Post-embryonic development of circadian rhythm in the cricket, Gryllus bimaculatus: a rhythm reversal. J. Comp. Physiol., 147: 299-304. Tomioka, K. and Chiba, Y. (1982) Persistence of circadian ERG rhythm in the cricket with optic tract severed. Naturwissenschaften, 69: 395-396. Tomioka, K. and Chiba, Y. (1984) Effects of nymphal stage optic nerve severance or optic lobe removal on the circadian locomotor rhythm of the cricket, Gryllus bimaculatus. Zool. Sci., 1: 385-394. Tomioka, K. and Chiba, Y. (1985) Optic lobe- compound eye system in cricket: a complete circad- ian system. J. Interdiscipl. Cycle Res., 16: 73-76. Tomioka, K. and Chiba, Y. (1986) Circadian rhythm in the neurally isolated lamina-medulla complex of the cricket, Gryllus bimaculatus. J. Insect Physiol., 32: 745-755. Tomioka, K. (1985) Residual circadian rhythmicity after bilateral lamina-medulla removal or optic stalk transection in the cricket, Gryllus bimaculatus. J. Insect Physiol., 31: 653-657. Halberg, F., Nelson, W., Runge, W.J., Schmitt, O.H., Pitts, G. and Reynolds, O. E. (1971) Plans for orbital study of rat biorhythms. Results of interest beyond the biosatellite program. Space Life Sci., 2: 437-471. Chiba, Y., Cutkomp, L. K. and Halberg, F. (1973) 13 14 iS 16 7) 18 WY) 20 21 22 23 24 25 26 541 Circadian oxygen consumption rhythm of the flour beetle, Tribolium confusum. J. Insect Physiol., 19: 2163-2172. Aschoff, J. and Wever, R. (1963) Resynchroniza- tion der Tagesperiodik und Zeitgeberperiodik. Z. vergl. Physiol., 46: 321-335. Fischer, K. (1961) Untersuchungen zur Sonnen- kompassorientierung und Laufaktivitat von Sarag- deidechsen, (Lacerta viridis Laur). Z. Tierpsychol., 18: 450-470. Wever, R. (1966) The duration of re-entrainment of circadian rhythms after phase-shifts of the Zeitge- ber. J. Theor. Biol., 13: 187-201. Taylor, B. and Jones, M.D.R. (1969) The circad- ian rhythm of flight activity in the mosquito Aedes aegypti (L.): The phase-shifting effects of light-on and light-off. J. Exp. Biol., 51: 59-70. Chiba, Y., Kubota, M. and Nakamura, Y. (1982) Differential effects of temperature upon evening and morning peaks in the circadian activity of mosquitoes, Culex pipiens pallens and C. pipiens molestus. J. Interdiscipl. Cycle Res., 13: 55-60. Clopton, J. R. (1984) Mosquito circadian and circa- bi-dian flight rhythms: a two-oscillator model. J. Comp. Physiol., 155: 1-12. Truman, J. W. (1974) Physiology of insect rhythms. IV. Role of the brain in the regulation of the flight rhythm of the giant silkmoth. J. Comp. Physiol., 95: 281-296. Christensen, N.D. and Lewis, R.D. (1982) The circadian locomotor rhythm of Hemideina thoracica (Orthoptera; Stenopelmatidae): the circadian clock as a population of interacting oscillators. Physiol. Entomol., 7: 1-13. Wiedenmann, G. and Loher, W. (1984) Circadian control of singing in crickets: two different pace- makers for early-evening and before-dawn activity. J. Insect Physiol., 30: 145-151. Pittendrigh, C.S. and Daan, S. (1976) A functional analysis of circadian pacemaker in nocturnal ro- dents. V. Pacemaker structure: A clock for all seasons. J. Comp. Physiol., 106: 333-353. Pickard, G. E. and Turek, F. W. (1982) Splitting of the circadian rhythm of activity is abolished by unilateral lesions of the suprachiasmatic nuclei. Science, 215: 1119-1121. Wiedenmann, G. (1983) Splitting in a circadian activity rhythm: the expression of bilaterally paired oscillators. J. Comp. Physiol., 150: 51-60. Wiedenmann, G. (1980) Two peaks in the activity rhythm of cockroaches controlled by one circadian pacemaker. J. Comp. Physiol., 137: 249-254. Tomioka, K. and Chiba, Y. (1985) Modification of circadian waveform by 6-hr phase shifts of LD cycle in the cricket, Gryllus bimaculatus. Zool. Sci., 2: 1000. 542 K. TOMIOKA AND Y. CHIBA 27 Randorph, M. (1971) Role of light and circadian temperature in the unanesthetized chicken: its cir- rhythms in the nocturnal behavior of Galago crassi- cadian rhythm of responsiveness to light. Brain caudatus. J. Comp. Physiol. Psychol., 1: 115-122. Res., 101: 1-9. 28 Aschoff,J. and Paul,U. von S. (1976) Brain ZOOLOGICAL SCIENCE 4: 543-549 (1987) Relationship Between Daily Variation of Locomotor Activity and That of Plasma Corticosterone Levels in the Newt, Cynops pyrrhogaster pyrrhogaster ATSUHIKO CHIBA and KtyosHi AOKI Life Science Institute, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102, Japan ABSTRACT—The daily variation of locomotor activity and that of plasma corticosterone levels were investigated in the newt, Cynops pyrrhogaster pyrrhogaster. Under LD 12:12 (Light on 07:00, off 19:00), the newts were active chiefly during the light period, and the daily variation of locomotor activity clearly showed a biphasic pattern. Plasma corticosterone levels increased during the dark period and reached a maximum value just prior to the light period just as is observed in diurnal mammals. The daily variation of plasma corticosterone levels also showed a biphasic pattern and virtually paralleled that of locomotor activity. If the light-dark cycle was reversed (Light on 19:00, off 07:00) by doubling the first light period, the daily patterns of both locomotor activity and of plasma corticosterone levels were also reversed within 7 days. These results suggest that, in the newt, the daily variation of plasma corticosterone levels is closely associated with that of locomotor activity at © 1987 Zoological Society of Japan least under the light-dark cycles of LD 12:12. INTRODUCTION A number of studies have established that in mammals, the circadian rhythms of plasma gluco- corticoid levels are closely linked to their rest- activity cycles including their sleep-wake and fasting-eating cycles [1-10]. In non-mammalian species, however, several studies have been car- ried out to investigate the daily variations of plasma glucocorticoid levels [11-14]. The rela- tionship between the levels of these hormones and the rest-activity cycles, however, was not precisely examined in non-mammalian studies. Corticosterone is the major glucocorticoid se- creted by the adrenal gland of amphibians [15, 16]. The existence of a daily rhythm of plasma corticosterone has already been demonstrated in frogs living freely in their natural habitat [14]. Recently, the present authors have succeeded in a long-term recording of the locomotor activity in the newt, Cynops pyrrhogaster pyrrhogaster, under experimental conditions [17]. In the present study, the writers recorded the Accepted February 4, 1987 Received January 7, 1987 locomotor activity and measured the plasma corti- costerone levels in Cynops to clarify the rela- tionship between the daily variation of locomotor activity and that of plasma corticosterone levels under experimental conditions. MATERIALS AND METHODS Animals Adult female newts, Cynops pyrrhogaster pyr- rhogaster, over 50mm in snout-vent length were purchased from commercial suppliers in Tokyo from March to October. Prior to the following investigations, all animals were maintained for at least a month in aquaria at 22°C under a control- led light-dark cycle of LD 12:12 (Light on 07:00, off 19:00). Food was scattered in the aquaria once a week. Locomotor activity recordings Four newts were housed per small plastic recording box (17cm long, 8.5cm wide) which was placed separately in a light-tight container. Water was maintained in the recording box at 1.5cm depth and it was continuously circulated 544 A. CHIBA AND K. AOKI through a gravel filter. The container had on its ceiling a 4W fluorescent lamp, which was con- nected to an automatic timing device to control the photoperiod. The intensity of the light was adjusted to 50lux at the surface of the water by inserting pieces of thin paper beneath the lamp. The temperature of both water and air in the container was kept at 22°C. No food was given throughout the experiment. The newts were initially kept under a normal light-dark cycle of LD 12:12 (Light on 07:00, off 19:00) and subse- quently exposed to a reversed light-dark cycle (Light on 19:00, off 07:00) achieved by doubling the first light period. Locomotor activity (counts/5 min) of the newts in each recording box was recorded by an infrared photoswitch (NT-10, Takenaka Electronic In- dustrial Co., Ltd.) connected to a micro-com- puter. The beam of the photoswitch was adjusted so that it would be located just below the surface of the water and also be directed along the longitudinal axis of the recording box. The signals were generated when the newts moved and intercepted the beam. The number of the signals was recorded as an index of locomotor activity. For visual display, time series of locomotor data were computer-plotted to simulate Esterline Angus recorder actogram. The authors have already recorded previously the locomotor activ- ity from the recording box containing a single newt each and have obtained the actogram in the same manner as the present investigation. These former actogram clearly showed locomotor rhythms entrained to LD 12:12 and free-running in constant darkness (Fig. 1). In an experimental series to investigate the relation to plasma glucocorticoid level, the daily variation of locomotor activity was determined under the normal light-dark cycle two days before the light-dark reversal, and on the Ist, 3rd, 5th and 7th day under the reversed lighting regimen; the counts of locomotor activity were summed up every two hours over a 24hr-period, beginning at 10:00 a.m., and expressed as a relative locomotor activity (percentage) to the mean 24 hr-activity of the last 7 days under LD 12:12 (Fig. 3). Measurement of plasma corticosterone For measurement of plasma corticosterone, the newts were divided into two groups. In order to keep approximately the same density as in loco- motor activity recording, each group consisting of 20 to 25 newts was housed per plastic tank (37 cm long, 21cm wide) which was placed separately in a light-tight container. Water for the tank and illumination in the container were also supplied in the same manner as in the locomotor activity recording. The temperature of both water and air in the container was maintained at 22°C. No food was given throughout the experiment. The first group was kept under the normal light-dark cycle Time of Day Fic. 1. A sample of the locomotor activity record of one recording box containing a single newt. To aid in visual inspection the record has been duplicated and arranged 48 hr-time base. The newt was initially entrained to a light-dark cycle of LD 12:12 (Light on 07:00, off 19:00) diagrammed at the top of the figure. On the indicated day, the newt was placed in constant darkness. Locomotor Activity and Corticosterone in Newt 545 for at least two weeks and then sacrificed. The 2nd group was exposed to the reversed light-dark cycle after being kept under the normal light-dark cycle for at least two weeks, and then sacrificed on the 7th day of the reversed lighting regimen. An average of 3 newts in each group were sacrificed every 4 hr over a 24hr-period, begin- ning at 10:00 a.m., by decapitation within 30 sec after removal from the tank. The effluent blood from each newt was collected in heparinized tubes and was then immediately centrifuged to obtain the plasma sample. The amount of corticosterone was determined by a radioimmunoassay employing an antibody for corticosterone (Miles Yada Ltd.) and [1, 2, 6, 7-H] corticosterone (91 Ci/mmol, The Radio- chemical Centre Amersham). Methylene chloride was added to each plasma sample. The mixture was shaken on a Vortex mixer, then centrifuged, and the organic phase was evaporated by a stream of nitrogen gas. The dried material was recon- structed in benzene: methanol (85:15, vol: vol) and subjected to chromatography on Sephadex LH-20 column (0.9X25cm) in a solvent system of benzene: methanol (85:15, vol: vol). The fraction containing corticosterone was collected and evaporated by a stream of nitrogen gas. The residue was dissolved in 0.05M Tris HCl buffer (pH 8.0) containing 0.1M NaCl, 0.1% NaN; and 0.1% bovine serum albumin and incubated with labelled corticosterone and the antibody. De- xtran-coated charcoal was used to separate the bound from the free hormone. The least detect- able amount of the assay was 0.08ng/ml plasma. The inter-assay and intra-assay coefficient of variation was 13.6% and 4.0%, respectively. For statistical analysis, the Student’s ¢ test was em- ployed. The procedure described above was repeated until a sample size of 4 for locomotor recording, and a sample size of more than 9 per each point during the 24hr-period for blood collection were obtained. RESULTS Under the normal light-dark cycle (LD 12:12), the newts were active chiefly during the light period (Fig.2). The daily locomotor activity began before the onset of light and ended at the light to dark transition. After the light-dark reversal, the phase of the daily onset of locomotor activity gradually delayed and finally shifted approximately 12 hr from that seen under the normal light-dark cycle. On the other hand, the locomotor activity ended at the end of the light period from the initiation of the reversed lighting regimen. In all samples of the activity records, the rest-activity cycles were almost completely reversed within 7 days after the light-dark re- Time of Day 12 24 (hr) a 1: — a Fic. 2. A sample of the locomotor activity record of one recording box containing 4 newts. To aid in visual inspection the record has been duplicated and arranged on 48 hr-time base. The newts were initially entrained to a light-dark cycle of LD 12:12 (Light on 07:00, off 19:00) diagrammed at the top of the figure. On the indicated day, light period was continued for 24 hr from 07:00 till 07:00 and the reversed light-dark cycle was started. 546 A. CHIBA AND K. AOKI versal. The daily variation of locomotor activity under the normal light-dark cycle clearly showed a biphasic pattern (Fig. 3A). The amount of loco- motor activity peaked sharply just before the onset of light and then decreased during the first half of the light period. The second peak appeared near the end of the light period. The lowest amount of activity was observed around the middle of the dark period. On the Ist, 3rd and Sth day after the light-dark reversal, the locomotor activity rhythms were still in the transient period leading to the new phase, and the daily patterns of locomotor activity were atypical (Fig. 3B-D). On the 7th day, the same pattern as observed under the normal light-dark cycle was shown with the phase shifting approx- imately 12 hr (Fig. 3E). Under the normal light-dark cycle, the daily variation of plasma corticosterone levels also showed a biphasic pattern (Fig. 3A). The plasma corticosterone increased during the dark period and reached a maximum value at 06:00 just prior to the light period. The value at 06:00 was significantly higher (p<0.02) than the value at 22:00. During the light period, the plasma corticosterone decreased one time but then in- creased again showing a smaller peak (not signi- ficant) at the end of the light period (at 18:00). The minimum value was observed in the early part of the dark period (at 22:00). On the 7th day after the light-dark reversal, the daily pattern of plasma corticosterone levels was similar to that demonstrated under the normal light-dark cycle (Fig. 3E). The plasma corticos- terone increased during the dark period of the reversed light-dark cycle and reached the max- imum value at 18:00 just prior to the light period. The value at 18:00 was significantly higher (p< 0.05) than the value at 10:00. During the light period, the plasma corticosterone had decreased once slightly and then showed a second peak (not significant) as shown under the normal light-dark cycle. This peak, however, appeared around the middle of the light period (at 02:00) instead of at the end. Then the level dropped to the minimum value in the early part of the dark period (at 10:00). } Corticosterone(ng/ml) © Plasma Relative Locomotor Activity Plasma Corticosterone(ng/ml) 10 22 10 Time of Day (hr) Fic. 3. (A) The daily variation of plasma corticoster- one levels and that of locomotor activity in the newts adapted to a normal light-dark cycle of LD 12:12 (Light on 07:00, off 19:00), and (B) in the newts on the Ist, (C) the 3rd, (D) the 5th and (E) the 7th day under a reversed light-dark cycle (Light on 19:00, off 07:00). Each point (A, E) represents the mean+SEM of plasma corticosterone levels, and number in brackets indicates the number of sampled animals. A significant difference is indi- cated in figures. Each column (A-E) represents the mean+SEM of relative locomotor activity of 4 recording boxes. Each box contained 4 newts. The light-dark cycles are diagrammed at the bottom of each figure. Locomotor Activity and Corticosterone in Newt 547 Under the reversed light-dark cycle, the phases of both the maximum and the minimum value of the plasma corticosterone levels on the 7th day had already shifted approximately 12 hr from those recorded under the normal light-dark cycle. DISCUSSION It has been established that in diurnal mammals the peak values of plasma glucocorticoid levels are observed early in the morning [18-24] and in nocturnal species late in the afternoon [1, 25]. In non-mammalian species, however, the relation- ship between the daily variation of plasma gluco- corticoid levels and that of locomotor activity has not been precisely described [11-14]. It has been reported that in amphibians, circa- dian rhythms of locomotor activity are not clearly evident and that a large amount of “noise” is observed in their activity records [26, 27]. However, the authors have recently investigated the circadian rhythms of locomotor activity in the Cynops, and these rhythms were clearly day- active under light-dark cycles of LD 12:12 (Fig. 1). In the present study, the actogram from those recording boxes containing 4 female newts each has also demonstrated that these newts were clearly day-active (Fig. 2). The plasma corticos- terone increased during the dark period and reached the maximum value just prior to the light period just as is observed in diurnal mammals (Fig. 3A, E). This similarity between mammals and the newt may suggest that a similar mecha- nism for glucocorticoids secretion exists in both classes of vertebrates. In the newts, the daily variation of locomotor activity clearly showed a biphasic pattern, while that of plasma corticoster- one levels had only one significant peak (Fig. 3A, E). The daily variation of plasma corticosterone levels, however, virtually paralleled that of loco- motor activity both under the normal light-dark cycle and after the adaptation to the reversed light-dark cycle (Fig. 3A, E). These data indicate that the daily variation of plasma corticosterone levels is closely associated with that of locomotor activity in the newts as well as in mammalian species. It has been reported that in rats the circadian rhythms in both rest-activity and plasma corticos- terone were altered concomitantly under various lighting conditions including constant darkness and constant light [8]. The plasma corticosterone levels tended to increase when they were asleep or during inactive periods, and the levels tended to decrease when they were during active periods regardless of lighting conditions [8]. Further, several authors have suggested that the alteration in the circadian rhythms of locomotor activity, such as that induced by the restriction of feeding, might act as a predominant zeitgeber of the circadian rhythms of the pituitary-adrenal system rather than the light-dark cycle [4-6, 8, 28]. In the newts, the results presented in Figure 3 demonstrated that a change in the phase of the locomotor activity rhythm was accompanied by a change in that of the plasma corticosterone rhythm. The present authors’ results, however, do not answer the question as to whether the daily rhythm of plasma corticosterone levels is synchronized predominantly by that of locomotor activity rather than by the imposed light-dark cycle. In amphibians, it has been reported that in a natural environment during midsummer spawning period, plasma corticosterone levels of European green frogs were higher during the night when the frogs developed an important activity associated with the various components of sexual behavior, yet they were usually active throughout the day in this season [14]. The spontaneous locomotor activity recorded in the present study might be composed of various facets of activity. The newts might be demonstrating sexual behavior under the given experimental conditions. Therefore, the authors did not exclude the possibility of the circadian rhythms in the pituitary-adrenal system in the newts being linked definitely to the rhythms of the activity associated with some components of sexual behavior. In addition, for non-domesti- cated animals such as newts, exposure to ex- perimental conditions may have some effects on both behavior and plasma corticosterone levels. For instance, it was recently shown in male rough-skinned newts that the exposure to stressful stimuli elevated the levels of plasma corticoster- one and inhibited sexual behavior [29]. From this 548 A. CHIBA AND K. AOKI point of view, it would be of great interest to examine, in newts, the relationship between plas- ma corticosterone levels and specific types of locomotor activity. ACKNOWLEDGMENTS We thank Dr. W. Heiligenberg for helpful discussion and criticism of the manuscript and Dr. S. Kikuyama for reviewing it. We are also grateful to Dr. M. Kikuchi for extensive technical assistance with the experiments. This work was supported by 58540473 Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 Halberg, F., Peterson, R.E. and Silber, R. H. (1959) Phase relations of 24-hour periodicities in blood corticosterone, mitoses in cortical adrenal parenchyma, and total body activity. Endocrinolo- gy, 64: 222-230. 2 Perkoff,G.T., Eik-Nes, K., Nugent, C.A., Fred, H.L., Nimer, R.A., Rush, L., Samuels, L.T. and Tyler, F.H. (1959) Studies of the diurnal variation of plasma 17-hydroxy-corticosteroids in man. J. Clin. Endocrinol. Metab., 19: 432-443. 3. Orth, D.N., Island, D. P. and Liddle, G. W. (1967) Experimental alteration of the circadian rhythm in plasma cortisol (17-OHCS) concentration in man. J. Clin. Endocrinol. Metab., 27: 549-555. 4 Krieger, D.T. (1974) Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity. Endocrinology, 95: 1195-1201. 5 Holley, D.C., Beckman, D.A. and Evans, J. W. (1975) Effect of confinement on the circadian rhythm of ovine cortisol. J. Endocrinol., 65: 147- 148. 6 Moberg,G.P., Bellinger, L.L. and Mendel, V.E. (1975) Effect of meal feeding on daily rhythms of plasma corticosterone and growth hormone in the rat. Neuroendocrinology, 19: 160-169 7 Gibbs, F. P. (1976) Correlation of plasma corticos- terone levels with running activity in the blinded rat. Am. J. Physiol., 231: 817-821. 8 Morimoto, Y., Arisue,K. and Yamamura, Y. (1977) Relationship between circadian rhythm of food intake and that of plasma corticosterone and effect of food restriction on circadian adrenocortical rhythm in the rat. Neuroendocrinology, 23: 212- pea, 9 Takahashi, K., Inoue, K. and Takahashi, Y. (1977) Parallel shift in circadian rhythms of adrenocortical activity and food intake in blinded and intact rats 10 11 12 13 14 15 16 7) 18 19 20 21 exposed to continuous illumination. Endocrinology, 100: 1097-1107. ) Honma, K. and Hiroshige, T. (1978) Internal syn- chronization among several circadian rhythms in rats under constant light. Am. J. Physiol., 235: R 243-R 249. Boehlke, K.W., Church, R.L., Tiemeier, O. W. and Eleftheriou, B.E. (1966) Diurnal rhythm in plasma glucocorticoid levels in channel catfish (Icta- lurus punctatus). Gen. Comp. Endocrinol., 7: 18- Ze Singley, J. A. and Chavin, W. (1971) Cortisol levels of normal goldfish, Carassius auratus L., and re- sponse to osmotic change. Am. Zool., 11: 653. Redgate, E.S. (1974) Neural control of pituitary- adrenal activity in Cyprinus carpio. Gen. Comp. Endocrinol., 22: 35-41. Leboulenger, F., Delarue,C., Belanger, A., Per- roteau,I., Netchitailo, P., Leroux, P., Jegou, S., Tonon,M.C. and Vaudry,H. (1982) Direct radioimmunoassays for plasma corticosterone and aldosterone in frog. I. Validation of the methods and evidence for daily rhythms in a natural environ- ment. Gen. Comp. Endocrinol., 46: 521-532. Carstensen. H., Burgers, A.C.J. and Li, C.H. (1961) Demonstration of aldosterone and corticos- terone as the principal steroids formed in incubates of adrenals of the American bullfrog (Rana cates- beiana) and stimulation of their production by mammalian adrenocorticotropin. Gen. Comp. En- docrinol., 1: 37-50. Macchi, I. A. and Phillips, J.G. (1966) In vitro effect of adrenocorticotropin on corticoid secretion in the turtle, snake, and bullfrog. Gen. Comp. Endocrinol., 6: 170-182. - Aoki, K., Kikuchi, M. and Chiba, A. (1984) The role of the pineal organ in circadian rhythm of the newt. In “Animal Behavior: Neurophysiological and Ethological Approaches”. Ed. by K. Aoki, S. Ishii and H. Morita, Japan Scientific Societies Press, Tokyo, pp. 243-251. Bliss, E.L., Sandberg, A.A., Nelson, D.H. and Eik-Nes, K. (1953) The normal levels of 17- hydroxycorticosteroids in the peripheral blood of man. J. Clin. Invest., 32: 818-823. Migeon, C.J. French, A.B., Samuels, L.T. and Bowers, J. Z. (1955) Plasma 17-hydroxy- corticosteroid levels and leucocyte values in the rhesus monkey, including normal variation and the effect of ACTH. Am. J. Physiol., 182: 462-468. Harwood, C.T. and Mason, J. W. (1956) Effects of intravenous infusion of autonomic agents on peripheral blood 17-hydroxycorticosteroid levels in the dog. Am. J. Physiol., 186: 445-452. Peterson, R.E. (1957) Plasma corticosterone and hydrocortisone levels in man. J. Clin. Endocrinol. 22 ps) 24 5) Locomotor Activity and Corticosterone in Newt Metab., 17: 1150-1157. Zolovick, A., Upson, D. W. and Eleftheriou, B. E. (1966) Diurnal variation in plasma glucocorticoster- oid levels in the horse (Equus caballus). J. Endocri- nol., 35: 249-253. McNatty, K.P., Cashmore,M. and Young, A. (1972) Diurnal variation in plasma cortisol levels in sheep. J. Endocrinol., 54: 361-362. Leshner, A.I., Toivola, P.T.K. and Terasawa, E. (1978) Circadian variations in cortisol concentra- tions in the plasma of female rhesus monkeys. J. Endocrinol., 78: 155-156. Guillemin, R., Dear,W.E. and Liebelt, R.A. (1959) Nychthemeral variations in plasma free corticosteroid levels of the rat. Proc. Soc. Exp. Biol. Med., 101: 394-395. 26 Pf] 28 ao 549 Binkley,S., Adler, K. and Taylor,D.H. (1973) Two methods for using period length to study rhythmic phenomena. J. Comp. Physiol., 83: 63-71. Demian, J.J. and Taylor, D. H. (1977) Photorecep- tion and locomotor rhythm entrainment by the pineal body of the newt, Notophthalmus viridescens (amphibia, urodela, salamandridae). J. Herpetol., 11(2): 131-139. Krieger, D.T. and Hauser, H. (1978) Comparison of synchronization of circadian corticosteroid rhythms by photoperiod and food. Proc. Natl. Acad. Sci. USA, 75: 1577-1581. Moore, F. L. and Miller, L. J. (1984) Stress-induced inhibition of sexual behavior: Corticosterone in- hibits courtship behaviors of a male amphibian (Taricha granulosa). Horm. Behav., 18: 400-410. + vat Ke Ait r 5 Trai rae enh i A ZOOLOGICAL SCIENCE 4: 551-555 (1987) © 1987 Zoological Society of Japan The Effects of Preputialectomy on Aggression in Male Mice SUSUMU HAYASHI Department of Biology, Faculty of Education, Kagoshima University, Kagoshima 890, Japan ABSTRACT—To investigate the relationship between preputial glands and aggression, male mice underwent one of the following three surgeries, bilateral (XX), unilateral (PX) or sham (PP) preputialectomy. The mice were paired off and placed for 14 days in cages which were divided by a wire net barrier. The pair was permitted to come into contact with each other periodically. Social behavior during removal of the barrier was observed. PP-PP, PP-PX and PP-XX pairs showed vigorous aggressive behavior than PXK-PX, XX-XX and PX-XX pairs. PP males were more apt to become dominant than PX or XX males. aggressive behavior of its owner. INTRODUCTION The preputial glands of male mice have been frequently implicated in olfactory communication. Male preputial odors attract females [1-4]. It has also been suggested that there is a relationship between preputial odors and aggressive behavior. Preputial sebum that has been applied to a castrated male promotes attacks by a conspecific male [5,6]. These findings, however, are con- cerned with the pheromonal function of preputial odors which act on conspecific recipients other than donors. There are very few reports which deal with the reactions of animals to their own odors [7]. This may be partly because the pheromonal concept implies transmission of olfac- tory information from one animal to a second animal and partly because the effects of an animal’s own odors are likely to be confused with motivational changes. The present investigation intended to study the relationship between aggressive behavior and the preputial glands which may affect conspecifics including its owner. For this purposes the glands were bilaterally or unilaterally segregated in some male mice and sham operations were performed in others. Accepted February 12, 1987 Received November 20, 1986 It was concluded that the preputial gland affects the GENERAL MATERIALS AND METHODS Mice (Mus musculus) of the ICR-JCL strain were used. They were housed in groups of 5 or 6 in plastic cages (23X16X12cm), and received food and water ad /ib. in a light and temperature controlled room. When the males were 110-140 days of age, they were isolated for 11 days. On the 10th day of isolation, they were anesthetized with sodium pentobarbital and underwent one of three surgeries, a bilateral preputialectomy (XX), a unilateral preputialectomy (PX) or a sham operation (PP). Each male was placed together with an assigned male for 14 days from the day after the surgery in a cage divided by a wire net barrier, inhibiting tactile responses between co- habitants. The barrier was removed for the first 24 hr and for 26 min daily from the 3rd to 14th day of cohabitation. Social behavior such as attacks, bites or mounts were recorded while the barrier was removed. These behaviors were counted as one bout when the animals parted from each other regardless of the duration time of the behavior. Attacks and bites were put together as aggressive behaviors. In preliminary experiments, the activities of male mice were observed with ANIMEX before and after the surgery. Although the activities weakened slightly immediately after the surgery, they were restored after 24 hr. There were no Sy S. HAYASHI significant differences among the motor activities of PP and XX males after the surgery. EXPERIMENT I Methods Males used in this experiment were paired with an animal which underwent the same kind of surgery. Males of about half the pairs (7 PP—PP pairs, 7 PX-PX pairs and 8 XX-XX pairs) were familiar with each other because they had been fed in the same cage from weaning (28 days old) up until isolation. The other pairs were compris- ed of males who were unfamiliar with each other (10 PP-PP pairs, 13 PX-PX pairs and 8 XX-XX TABLE 1. cohabitation (26 minXx12 day) pairs). In addition, preputial glands removed from the males of unfamiliar XX-XX and PX- PX pairs were freed from fatty and connective tissues, the fluid content gently sqeezed out, and the wet weight measured. The remaining prepu- tial glands of PX—PX pairs and PP—PP pairs were removed immediately after cohabitation and weighed. Results Results are shown in Table 1. Dominant- subordinate relationships were established in almost every pair by the 3rd day of cohabitation, and there were no changes in social status throughout the remaining observation period. Dominant males of both familiar and unfamiliar Social behaviors exhibited by paired males in the same preputial state during Pair N Dominance Aggression Mount Median (Range) Median (Range) familiar PP-PP 7 Dominant 80 (18-123) 2 ( 0-12) Subordinate 0( 0- 6) 0( 0- 0) PX-PX 7 Dominant 21 ( 3- 26) 0( 0- 5) Subordinate 3( 0- 10) 0( 0- 1) XX-XX 8 Dominant 7( 2- 34) 1( 0- 6) Subordinate 1( 0- 11) 0( 0- 3) unfamiliar PP-PP 10 Dominant 45 (33-127) 8 ( 0-52) Subordinate 0( 0- 10) 0 ( 0-10) PX-PX 13 Dominant 19( 0- 48) 13 ( 2-46) Subordinate 0( 0O- 6) 1 ( 0-25) XX-XX 8 Dominant 32 ( 3- 44) 48 (10-82) Subordinate 0( 0- 6) 2 ( 0-14) TABLE 2. Comparison of preputial weight (Mean+SE) Pair Segregation Dominance Weight (mg) PP-PP after Dominant 62.4+8.3° Cohabitation Subordinate 41.9+3.3 PX-PX before Dominant Misael" Cohabitation Subordinate 559) se 13 after Dominant Denon Cohabitation Subordinate I) se 2.3" XX-XX before Dominant 39.9+6.0 Cohabitation Subordinate SLOse 25) * Weight of a unilateral gland. * Mean -+standard error. Preputial Glands and Aggression in Mice PP-—PP pairs were more aggressive than those of PX-PX or XX-XX pairs (Mann-Whitney U-test; P<0.01). Although the number of aggressive behaviors displayed by PX and XX males was less than that of PP males, there was no qualitative difference among them. Concerning aggression, unfamiliar pairs were more active than familiar pairs only when the preputial glands were re- moved bilaterally. The remarkable difference between familiar and unfamiliar groups was a mounting behavior. The behavior was mainly displayed by unfamiliar pairs (P<0.01), and with dominant XX males showing the highest frequen- cy (P<0.05). In regard to preputial weight, the glands re- moved from dominant males of PP—-PP and PX- PX pairs after cohabitation were heavier than those of subordinate ones (t-test, PP—PP; t=2.33, P<0.05, PX-PX; t=2.22, P<0.05) as shown in Table 2. The glands increased in weight during cohabitation because the preputial weight of a dominant PI male after the cohabitation was heavier than that before cohabitation (Wilcoxon matched-pairs signed-ranks test, T=4, N=13, P <0.01). Although it was not as marked as in the dominant male, the weight of the preputial gland of a subordinate male increased, too (T=11, N= 11, P<0.05). EXPERIMENT II In Exp.1, dominant males of PP—PP pairs were more aggressive than PX or XX males. The difference could be accounted for in two ways. PP males may provoke aggressive behavior in a cohabitant more than PX or XX males. Alterna- tively, PP males were motivationally more aggres- 553 sive than PX or XX males. To clarify this point, Exp. II was performed. Methods Male mice which had undergone one of three surgeries as in Exp.I were placed with an un- familiar male of a different preputial condition. Fourteen PP-XX pairs, 11 PP-PX pairs and 9 PX—XX pairs were used and the other procedures were the same as in Exp. I. Results As shown in Table 3, PP males showed more aggressive behaviors than cohabitants when they were paired with XX males (P<0.01) or PX males (P<0.05), although 3 XX males and 2 PX males dominated the PP cohabitants. In PX-XX pairs, 6 PX and 3 XX males became dominant. DISCUSSION It was reported that preputial odors provoke aggressive behaviors in recipients [5,6]. In this experiment, however, neither XX males of PP- XX pairs nor PX males of PP—PX pairs showed ageressive behaviors toward a_ cohabitant although they were the recipients of preputial odors. They retained a lower status than a PP male. These results suggest that a preputialec- tomy decreases aggressivity in the excised mouse. In other words the preputial gland may maintain the aggressiveness of its owner. The reduction of ageressivity in PX-XX, PX-PX and XX-XX pairs seemed to be attributable to a decline in aggressive motivation rather than a decline of aggression inducing stimuli. McKinney and Christian [8] found that PP-XX TABLE 3. Social behaviors exhibited by paired males in different preputial states Pair N Males Aggression Mount Median (Range) Median (Range) PP-PX 11 PP 54 (0-183) 7 (0-29) PX 0 (0-138) 0 (0-20) PP-XX 14 PP 33 (0-179) 3 (0-41) XX 0 (O0- 50) 0 (0- 3) PX-XX 9 PX 14 (0- 85) 9 (0-27) XX 1(0- 63) 1 (0-16) 554 S. HAYASHI pairs exhibited more fights than PP—PP or XX- XX pairs. Their findings were not consistent with those of the present experiment, due perhaps, to a methodological difference. They observed only 15 min of cohabitation and it was in the early stages of establishing dominance. In the present experiment, however, observation did not com- mence until 48 hr after the start of cohabitation and consequently most social orders were estab- lished by the beginning of observation. Bronson and Marsden reported that preputial glands of dominant males are heavier than those of subordinate ones, and that preputial glands of subordinate males are lighter than isolated males [9]. These individual variations in preputial weight are partly induced by social environment; a mouse with heavier preputial glands does not necessarily dominate one with lighter glands. Cohabitation with a strange male increases the preputial weight. Although it occurs without physical contact (prevented by a wire net barrier), it is much more effective when the barrier is removed periodically [10]. Preputial glands of dominant males increased markedly in the present experiment. This is in accord with previous findings [9,10]. However, preputial glands of subordinate males also increased in weight although the increase was not as marked as in dominant males. Bronson and Marsden housed 2 male mice in a cage for 2 weeks without a barrier, while the mice of the present experiment came into physical contact with each other for only 26 min a day. Preputial growth of subordinate males would probably not occur without a partition. Aggressive behaviors were displayed only by dominant males. Considering that preputial glands seem to maintain aggressivity, the glands may play an important role in social order. Mugford [11] observed that pairs of pre- putialectomized males spent less time on aggres- sion with one another than did pairs composed of sham operated males. He attributed the reduc- tion of aggressive behaviors to operation trauma and lack of pheromonal stimulus. In the present investigation, cohabitation began on the day fol- lowing the operation. It cannot be denied that surgery may have imposed some pain or stress which could indirectly influenced aggression. However, it did not seem to disturb comparative studies because PP males were not intact but underwent sham operations and the frequency of social behaviors (aggression and mounts) exhi- bited by unfamiliar PP-PP pairs was comparable to that of XX-XX pairs. Any other social behaviors as grooming, chasing and so on were rarely observed in this experimental situation. In addition, activities observed with ANIMEX did not reveal any differences among PP, PX and XX males. Recently, Wiesler et al. [12] and Novotny et al. [13] studied the function of substances in urine such as 2-(sec-butyl)-4, 5-dihydrothiazole and de- hydro-exo-brevicomin, both of which are not contained in preputial secretions. They observed that the substances act as aggression-inducers, sex-attractants, and estrous-inducers. On the other hand, it is well known that preputial odors attract female mice, and there are some reports about preputial sebum which promotes the attack of opponents. Those substances contained in urine and preputial secretions may be effective as cues which indicate maleness without particular meaning. Mounting behavior between males, which was very similar to the mounting behavior between a male and a female except intromission, was dis- played mainly by dominant males regardless of their preputial state. Although the meaning of the mounting between males was not determined, the difference between familiar and unfamiliar pairs suggested that individual recognition pre- vented mounting behavior. XX-XX_ males showed the most frequent mounting behavior among unfamiliar pairs. This seems to be caused not only by a reduction in agonistic motivation but also by a decline in the maleness of a cohabitant. Preputial materials stimulate the owner of the glands. It is probably olfactory stimuli that affect the owner, considering the other functions of the preputial glands, although this must be confirmed by impairing olfactory pathways. REFERENCES 1 Bronson, F.H. and Caroom, D. (1971) Preputial Preputial Glands and Aggression in Mice gland of the male mouse: Attractant function. J. Reprod. Fertil., 25: 279-282. Hayashi, S. and Kimura, T. (1973) Responses of normal and neonatally estrogenized female mice to the odor of males. Sci. Pap. Coll. Gen. Educ. Univ. Tokyo, 23: 39-43. Hayashi, S. (1979) A role of female preputial glands in social behavior of mice. Physiol. Behav. , 23: 967- 969. Hayashi, S. (1985) A preputial odor imprinted on female mice. J. Ethol., 3: 89-91. Jones, R.B. and Nowell, N. W. (1973) Effects of preputial and coagulating gland secretions upon aggressive behavior in male mice: a confirmation. J. Endocrinol., 59: 203-204. Homady, M.H. and Brain, P.F. (1982) Effects of marking with preputial gland material on the attack directed towards long-term castrates by isolated males. Aggress. Behav., 8: 137-140. Kimura, T and Hagiwara, Y. (1984) Responses of male and female laboratory mice to the odor of conspecifics of the same sex: Effects of previous 10 11 12 13 555 isolation and gruping. J. Ethol., 2: 121-126. McKinney, E. D. and Christian, J.J. (1970) Effect of preputialectomy on fighting behavior in mice. Proc. Soc. Exp. Biol. Med., 134: 291-293. Bronson, F.H. and Marsden, H.M. (1973) The preputial gland as an indicator of social dominance in male mouse. Behav. Biol., 9: 625-628. Hayashi, S. (1986) Effects of a cohabitant on preputial gland weight of male mice. Physiol. Behav., 36: 299-300. Mugford, R. A. (1973) Intermale fighting affected by home cage odors of male and female mice. J. Comp. Physiol. Psychol., 84: 289-295. Wiesler, D. P., Schwende, F. J., Carmack, M. and Novotny, M. (1983) Structural determination and synthesis of a chemical signal of the male state and a potential multipurpose pheromone of the mouse Mus musculus. J. Org. Chem., 49: 882-884. Novotny, M., Harvey, S., Jemiolo, B. and Alberts, J. (1985) Synthetic pheromones that promote inter-male aggression in mice. Proc. Natl. Acad. Sci. USA, 82: 2059-2061. fs aul ; i sesh rr io ee alae. é tlle netalenery yaa se : ‘, + i ; Aa eye iad ty as ae eth unio si sania Ci ee bias Ee Oe a Cer tai ‘kia E ve ne abet he a fe abesy ey. Hise >| "eT a ". ye a Fi " ie Ve} ARK if i 4 u > ; | rae ts ti uy ' Theat § iy ; uf h fay isc , : } ZOOLOGICAL SCIENCE 4: 557-561 (1987) © 1987 Zoological Society of Japan Annual Ovarian, Fat Body and Liver Cycles of the Grass Lizard Takydromus stejnegeri in Taiwan HsiEN Yu CHENG and Jun Yi Lin! Department of Zoology, National Taiwan University, Taipei, Taiwan 107, and ‘Department of Biology, Tunghai University, Taichung, Taiwan 400, R.O.C. ABSTRACT—The female Takydromus stejnegeri in Taiwan has a definite breeding season from March to August. Most females produced two clutches but a few females may produce one or three clutches a year. The number of eggs per clutch for most clutches was 2 (with range from 1 to 4). The clutch size increased significantly during the breeding season. The smallest and largest mature females were 41 mm and 57mm in snout-vent length, respectively. Most newly hatched individuals (<25mm in snout-vent length) occur from May to September. Fat body size was inversely correlated with reproduction. The mean liver weights peak in June. Both annual mean liver and fat body weights of T. stejnegeri females were similar to those of males during this same period. INTRODUCTION During the past ten years, a wealth of data has accumulated on annual reproductive and lipid storage patterns in both temperate and tropical lizards [1-7]. However, the reproductive and energetic biology of most lizards remains poorly known, particularly for insular populations in the Pacific region [8]. The paucity of reproductive and energetic data on tropical and subtropical Asian lizards has been one of the obstacles to a fuller understanding of evolutionary strategies of saurian reproduction and energetics [9-11]. The present study reported here was made as part of our comparative study on reproductive and energetic biology of lizards in Taiwan, a subtrop- ical Pacific island. MATERIALS AND METHODS Lizards were collected monthly from November 1975 to December 1976. An additional collection was made on April 27, 1977. Individuals for this study were collected from grassy field and culti- vated area of sugarcane and sweet-potato around Accepted February 14, 1987 Received December 23, 1986 the Tunghai University campus, Taichung, Taiwan (24°10'N; elevation 180 meters) during midday (from 11:30 am to 2:00 pm) when their activities were at the peak. Specimens were etherized within two hours after capture, weighed to the nearest 0.01g and their snout-vent length (SV-length) measured to the nearest 0.1mm. Each specimen was then made a small abdominal cut and preserved in 10% formalin for further examinations. Livers and fat bodies were, thereafter, removed and weighed to the nearest 1mg. Yolked follicles, oviducal eggs, and corpora lutea were counted. Yolked follicles and oviducal eggs were then weighed to the nearest 0.1 mg and their greatest diameter was measured to the nearest 0.1mm. Females were classified as reproductive when they contained either oviducal eggs or bright yellow ovarian follicles greater than 1.8mm in diameter. To minimize the influence of SV-length on the results [12], only a relatively narrow range of adult SV-lengths was included for analyses. The metereological data of Taichung City (9 km from Tunghai campus; 83.8m above sea level) were obtained from the Central Weather Bureau, Taipei, and shown in a Cheng and Lin’s article [ASH- 558 : H. Y. CHENG AND J. Y. LIN RESULTS The smallest and largest mature females were 41.0mm and 56.8mm SV-length, respectively. However, samples (n=10) contained only one mature individual with the 41.0-43.5mm _ SV- length class. This mature individual derived from the March sample. Thus, also to minimize the influence of the SV-length on the results, 43.6mm SV-length was chosen as the smallest female adult size used in the analyses. From November 1975 to December 1976, a total of 105 females of T. stejnegeri, larger than 43.6 mm in SV-length, were collected. The overall yearly mean SV-length for adult females was’48.6mm. The monthly SV- length means were ‘similar among all months (ANOVA, p=0.90) and ranged from 44.4mm to 51.6mm. a Reproduction In 1976, almost all (>90%). adult females captured from March through August were clas- sified as reproductive (N=75, Fig. 1). The num- ber of reproductively active females declined sharply in September, and reached the lowest 14 12 100 Ey Gis XN 80 Ao <= 40 20 iol ot D) let ioa) tien] Sart Vata asl Mc) 1976 Fic. 1. level (0%) in late September (the samples of this month were collected on September 28). No yolked follicles or oviducal eggs were observed among the thirty adult females sampled during September to December 1976 and November 1965 to February 1976 (Fig. 1). During 1976, most females produced two clutches. By May and June, the majority (68%) of the adult females contained both yolked folli- cles and oviducal eggs or both yolked follicles and corpora lutea. A few females may produce three clutches or merely one clutch. Because females containing oviducal eggs occurred in mid-March and would have oviposited in late March or early April, these females would have had nearly five months for reproduction, long enough to produce a second and perhaps a third clutch. The mean time necessary to produce a clutch was estimated to be two months. Females would reach maturity in every month during the breeding season but most females achieve their mature size from late breeding season (July-August) to the early period of the next breeding season (March or April). The younger adult (reaching maturity in late breeding season) unlikely could produce more 5 3 Oo, fo) ~ fo) 8. 2 73 EE 0 VERE 0 WENN o WEEN 9 REEEEN (SCE CEN CS ESS | JPA AGS eID) RES ES Cor Mtn 1977 Monthly changes in the reproductive state of adult females (SV-length>43mm) from November 1975 to December 1976 (with additional data on April 1977). Solid line shows the percentage of each sample that contained yolked follicles, oviducal egg or both; dashed line shows the percentage of each sample that contained oviducal eggs. Sample size is shown above each month. Female Reproduction of Takydromus 559 than one clutch during that year. To sum up, many females produced their first clutch in late April and May and a second clutch in July and August. A few females may produced a first clutch in late March-early April, a second clutch in June, and a third clutch in August. Younger females may produce only one clutch in August. The mean number of eggs for all clutches was 2.2 as estimated by the mean number of ovarian yolked follicles (N=43; range: 1-4; mode=2) or by the mean number of oviducal eggs (N=27; range: 1-4; mode=2). Clucth size had no signif- icant correlation with SV-length. However, only females larger than 53mm in SV-length had clutch size of four eggs. The clutch size of three eggs occurred only in the females larger than 46mm SV-length. The number of eggs for the first clutch was 2.0 (N=15) as estimated by the mean number of oviducal eggs of females collected during March, April and May. This was lower than the mean number 2.33 (N=12) for the second or the last clutch of the adults collected during June, July and August. Sizes of oviducal eggs, both weights and max- imum diameters, were very similar in eggs of the same clutch; but between females there was considerable variation. Maximum oviducal egg diameters ranged from 5.8 to 11.9mm. No females with oviducal eggs less than 10.1mm in 200 150 X Liver wt. 100 50 (mg) 1976 Fic. 2. Monthly changes of liver and fat body weights of adult females from November 1975 to December 1976. Solid line indicates mean liver weight changes; dashed line indicates mean fat body weight changes. Vertical lines show plus and minus one standard error about the means; numbers are sample sizes. diameter had yolked follicles at the same time. Inter-uterine migration of eggs was found to have occurred in 14% of gravid females with no apparent bias toward the right or left oviduct. there was a slight difference between the numbers of oviducal eggs in the right and the left oviducts (1.18 vs. 0.93, favoring the right). The mean incubation time of oviducal eggs (n= 3) was 32 days. All eggs were incubated at room temperature (25-28°C). The mean hatchling SV- length was 20.8mm. Maturity was estimated to occur between 9 to 12 months of age. Livers and fat bodies Monthly mean liver weights were at higher level from March through September with a peak in June. During the late fall and winter, the monthly means of liver weights were low (Fig. 2). No considerable fat bodies occurred in the spring and summer months. The marked increase in sizes of fat bodies occurred in September after the end of the breeding activities and reached the peak in October and November. The weight of fat bodies decreased markedly in winter to their lowest size in March (Fig. 2). DISCUSSION The female T. stejnegeri in Taiwan has a def- inite breeding season from March to August (Fig. Co = 720 “1M Apog ie E ¢ 560 H. Y. CHENG AND J. Y. LIN 1). Most females produced two clutches but a few females may have produced three clutches or merely one clutch during the 1976 breeding sea- son. The female T. stejnegeri in Taiwan, as T. tachydromoides in Japan [14], has a multiple- brooded cyclic reproductive pattern. The earliest clutches were laid in March or early April, and the last clutches were laid in early September. Since the incubation time is about one month, the hatchings are expected to occur from May to early October and this is concordant with observations of recently hatched young (<25 mm in SV-length) occurring from May to September. The reproductive cycle of male T. stejnegeri during the same period was reported by Cheng and Lin [13]. The testicular changes in weight showed that T. stejnegeri had a distinct reproduc- tive cycle: mainly reproductively active during the spring and summer, and quiescent in September and October. During the winter, male T. stey- negeri had a potential for breeding; 1.e., accessory organs were hypertrophied and _ spermiation occurred [13]. However, no breeding activities of female T. stejnegeri occurred during the same period of the winter. In T. stejnegeri, females cease their reproductive activities while the males still show reproductive potential, as in some species of lizards such as Leiolopisma rhomboida- lis (Scincidae) [15], Agama agama (Agamidae) [16, 17], and Hemidactylus frenatus (Gekkonidae) in Taiwan [4]. The number of eggs per clutch was found to increase slightly but significantly during the second half of the 1976 reproductive season. Since the second clutch began development dur- ing the middle of the rainy season, it appears likely that an increased food supply during that time would allow for greater investment in repro- duction. Several studies have demonstrated a positive correlation between rainfall and food abundance or lizard growth [18, 19]. The possibil- ity that this increase in egg number was due to a general increase in the size of individual lizards in the population or sampling bias, was eliminated by an examination of the mean SV-length of each month’s samples. There was essentially no differ- ence in the mean SV-length between combined spring (March, April and May) and combined summer (June, July and August) samples. The cyclical lipid storage and utilization system is an important adaptation in lizards to seasonal fluctuation of the environment for reproduction or winter dormancy [9]. All lizards with a cyclical lipid storage and utilization system are expected to be cyclical breeders. Both liver and fat body weights have been used as indices of body lipid content and consequently stored energy [20]. Fat body size in both sexes of T. stejnegeri was essentially inversely correlated with reproduction, though some accumulation of fat occurred while reproductive activities were still taking place. The absence of fat bodies during spring and summer probably did not result from physiological inabil- ity to accumulate energy or from the shortage of food. It appears more likely that reduced size of fat bodies is due to the high expenditure of energy for breeding activities and to the reduction of foraging time during the breeding season. Liver is probably an intermediary organ for both the storage and utilization of lipids, especial- ly for the reproductive purpose [21,22]. The peak of the mean liver weight occurred in June, suggesting that June was the end of intense reproductive effort allowing the energy accumula- tion for lipid storage to begin (Figs. 1 and 2). As to the precise role of the liver functions, further experiments are needed. Although climate at study areas is subtropical, the annual reproductive and energetic patterns of T. stejnegeri and the other two species of lizards studied in Taiwan are more similar to the patterns found in temperate lizards. Even the house gecko, Hemidactylus frenatus, shows a definite breeding season in Taiwan, although it has a continuous reproductive pattern in Java [3, 13, 20, 23]. In the present species (7. stejnegeri) and the other species studied (Japalura swinhonis), vitel- logenesis begins in spring and continues untill mid-summer, followed by a period of the repro- ductive quiescence and rapid build-up of fat reserves prior to winter [4, 20,25]. In general, this pattern fits the reproductive strategy de- scribed by Tinkel et al. [25] of early maturing, relatively small clutch sized, mutiple-brooded lizards. Female Reproduction of Takydromus ACKNOWLEDGMENTS This study was partially suported by a grant from the National Science Council (NSC75-—0201-—B002-29 to HYC) of the Republic of China and a grant from the Taipei Zoological Garden (to JYL). The facilities for the computer word processing and calculation were provided by the Department of Zoology of National Taiwan University. Figures were prepared by Tsai Tsan-Dung. 11 12 REFERENCES Fitch, H.S. (1982) Reproductive cycles in tropical reptiles. Occas. Papers Mus. Nat. Hist. Univ. Kansas, 96: 1-53. Guillette, L.J. and Sullivan, W.P. (1985) The re- productive and fat body cycles of the lizard, Scelo- porus formosus. J. Herpetol., 19: 474-480. Lin, J. Y. and Cheng, H. Y. (1984) Ovarian cycle in the house gecko, Hemidactylus frenatus, in Taiwan with reference to food stress in winter. Bull. Inst. Zool., Academia Sinica, 23: 21-28. Lin, J. Y. and Cheng, H. Y. (1986) Annual repro- ductive and lipid storage patterns of the agamid lizard, Japalura swinhonis mitsukurii in southern Taiwan. Bull. Inst. Zool., Academia Sinica, 25: 13- 23: McKinney, R. B. and Marion, K. R. (1985) Repro- ductive and fat body cycles in the male lizard, Sceloporus undulatus, from Alabama, with compari- sons of geographic variation. J. Herpertol., 19: 208-217. Vitt, L.J. (1983) Reproduction and_ sexual dimorphism in the tropical Tetid lizard Cnemid- ophorus ocellifer. Copeia, 1983: 359-366. Vitt, L. J. and Goldberg, S. R. (1983) Reproductive ecology of two tropical iguanid lizards: Tropidurus torquatus and Platynotus semitaeniatus. Copeia, 1983: 131-141. Schwaner, T.D. (1980) Reproductive biology of lizards on the American Samoan Islands. Occas. Papers Mus. Nat. Hist. Univ. Kansas, 86: 1-53. Derickson, W. K. (1976) Lipid storage and utiliza- tion in reptiles. Am. Zool., 16: 711-723. Pianka, E.R. (1976) Natural selection of optimal reproductive tactics. Am. Zool., 16: 775-748. Stearns, S.C. (1976) Life-history tactics: a review of ideas. Q. Rev. Biol., 51: 3-47. Atchley, W.R.,Gaskins, C.T., and Anderson, D. (1976) Statistical properties of ratios. I. Empirical results. Syst. Zool., 25: 137-148. 13 14 iS) 16 JZ) 18 i 20 Dai 22 23 24 JE) 561 Cheng, H. Y. and Lin, J. Y. (1977) Comparative reproductive biology of the lizards, Japalura swinho- nis formosensis, Takydromus septentrionalis, and Hemidactylus frenatus in Taiwan. I. Male reproduc- tive cycles. Bull. Inst. Zool., Academia Sinica, 16: 107-120. Telford, S.R. (1969) The ovarian cycle, reproduc- tive potential, and structure in a population of Japanese lacertid, Takydromus tachydromoides. Copeia, 1969: 548-567. Wilhoft, D.C. (1963) Gonadal histology and sea- sonal changes in the tropical Australian lizard Leiolopisma rhomboidalis. J. Morphol., 113: 185- 204. Chapman, B.M. and Chapman, R.F. (1964) Observations on the biology of the lizard Agama agama in Ghana. Proc. Zool. Soc. Lond., 143: 121- 132) Marshall, A. L. and Hook, R. (1960) The breeding biology of equatorial verterbrates: reproduction of the lizard Agama agama lionotus Boulenger at Lat. 0°01'N. Proc. Zool. Soc. Lond., 134: 197-205. Ballinger, R.E. (1977) Reproductive strategies: food availability as a source of proximal variation in a lizard. Ecology, 56: 628-635. Stamps, J. A. (1977) Rainfall, moisture and dry season growth rates in Anolis aeneus. Copeia, 1977: 415-419. Cheng, H. Y. and Lin, J. Y. (1978) Comparative reproductive biology of the lizards, Japalura swinho- nis formosensis, Takydromus septentrionalis and Hemidactylus frenatus in Taiwan. II. Fat body and liver cycles of the males. Bull. Inst. Zool., Academia Sinica, 16: 107-120. Hahn, W.E. (1967) Estradiol-induced vitellogene- sis and concomitant fat mobilization in the lizard Uta stansburiana. Comp. Biochem. Physiol., 23: 83-93. Reddy, P.R.K. and Prasad, M.R.N. (1972) Sea- sonal variations in the pattern of lipids in the sexual segment of kidney and liver of the Indian house lizard, Hemidactylus flaviviridis Ruppell. Comp. Biochem. Physiol., 41A: 63-76. Church, G. (1962) The reproductive cycles of the Javanese house geckos, Cosymbotus platyurus, Hemidactylus frenatus, and Peropus miultilatus. Copeia, 1962: 262-269. Lin, J. Y. (1979) Ovarian, fat body and liver cycles in the lizard Japalura swinhonis formosensis in Taiwan (Lacertilia: Agamidae). J. Asian Ecology, 1: 29-38. Tinkle, D., Wilber, H.M., and Tilley, S.G. (1979) Evolutionary strategies in lizard reproduction. Evolution, 24: 55-74. sea Eu Siac aden thay ihe Die, dere is et vole | re ah tua Logit Tae rOgRy Wo YR o 2 fhe Sone wl i TS Wel abet Pay Pyne a ~ , Me ee eg this igri i ioe } 'Dains ye is Ws Ma srt ube er F oe \ Hel ais ) 7 ; t ey t i % Ae i { ay f nwa su ibe ane ma susid wonengage (2 Bi ere aes Mceaaneln elder ela tiie (ROEM) el Sees RRM eRe AEC IRC IED: OO REEDS Re aaaair ty FE ROS AALS ey aR nears is phe rm EO oe am ay PY beuhet | . if? or ‘ vey < ve ANN FN wae, iL et aay Pe ight vn aly i , i eae ae: Re ee He pay said a a i ee ae he al ii } ik om | Wet y betty hee) eagee ol Pant y Pee Mey . oD rey habe Bilt PUM da ak cutee at ca , i bail A) nih ie hare eh 2 eB ok y he’ f aft f yet Bic, ft r] } alt mo et e sa Se OE a) LER W OS e RN A Ame Hy Pulte Le tt ! ‘ : i within iS. aces Ke 20 a pei aL : ‘ / " ha i as Ch rhs PRO 4 ae he Nar Tee % Stubs I ‘ ' ae hi Seg my ¥ A peal a yea renee by LO Um phet Hib vey ay 9 vayy oh bob" % Re Taree Py : nt i CY Bes a hes tage Loe Ni Se tee Beane MY oe Sa ah t ORE SERS pete ve mt lgkuees aN fy Go emg a Tn LE Pitre Oe hPL kel a De Me eee eh Cot On LMM Ch Totes bh ahi oh per a wah ON | sean ‘ : Pirie seedy ee Ve ae ( ; Ae ee ah oC =D, Cas 4 - Oy) 7 pag j ‘ shy) Ree 6 if ¢ 4 er i Carer: Pe Ta Mi Wel? ey) ? oe tr ; " ‘ fT PR Oe ; eeu PESLIe ya Calta oml s Se ree Ect moans | we ers Cay Mee A “Se bil af are epee: $ ; ye ; Hi ay H pt f ; , i : A ‘ } i f > Sam) HP P| \ Dh by SH kaha A My te yt ; ba ie fk i Pity @ DPS ee eh: £8 ¥j sy " We hy iG aa ne Beh ‘> + PT bss f 4 a4 : F belule ey 5 ; m ‘5 ca ri f , ai} i ied a ip renesney y 7 ¥ i % te i. , tj . f ve . i { yall P * , | Oh cE ? 4 R ¥ a | ‘ fy ; ; «be i F j ide ee =) ~ t i faa q 4 or ; y 7 ; , 49 ee OO \ ay" ihe au ZOOLOGICAL SCIENCE 4: 563-567 (1987) © 1987 Zoological Society of Japan Advertisement Calls of Two Taiwan Microhylid Frogs, Microhyla heymonsi and M. ornata MITSURU KURAMOTO Department of Biology, Fukuoka University of Education, Munakata, Fukuoka 811-41, Japan ABSTRACT—The advertisement calls of two Taiwan microhylid frogs, Microhyla heymonsi and M. ornata, were analyzed with sound spectrograph to clarify acoustic characteristics and a possible cause of acoustic divergence. The calls of the two species were composed of a series of pulse groups (notes). Several notes constituted distinct note groups in M. heymonsi, while no such note groups were involved in M. ornata. In M. heymonsi the note lasted longer, pulses in a note were fewer, and pulses were repeated more slowly than in M. ornata, but the energy distribution and wave form were quite similar in both species. These indicate that the calls of the two sympatric species have diverged essentially in temporal features, not in frequency features. Between populations of M. ornata from Taiwan and those of the Ryukyu Islands, considerable differences were found in temperature relationships of temporal acoustic features. INTRODUCTION Acoustic features of anuran amphibians reflect the phylogenetic relationships, that is, closely related species should have similar acoustic fea- tures as a consequence of their common descent. From the function of anuran calls, however, it is expected that the acoustic features have diverged to maximize the efficiency in attracting conspecific females. Thus, sympatric species have distinct specific calls even if they are closely related to each other. Selection will favor for acoustic divergence where two species with similar calls are sympatric [1]. Four frog species of the family Microhylidae occur in Taiwan [2], but no acoustic works have been performed. In this study I analyzed adver- tisement calls of two species, Microhyla heymonsi and M. ornata, to clarify the species-specificity in their acoustic features. The two species occur widely in southeastern Asia and resemble each other in their size, shape, and habitat, but differ in larval morphology [3]. In Taiwan, M. heymon- si is confined to the southern half where it is sympatric with M. ornata, while the latter ranges Accepted December 25, 1986 Received August 8, 1986 to the northern half and to the Ryukyu Islands. By comparing the acoustic features of the two species with each other and with those of Thai- land and the Ryukyu Islands reported previously [4,5], a possible cause of acoustic divergence is suggested. MATERIALS AND METHODS Advertisement calls of M. heymonsi were re- corded at Manchou (Pingtung Co.) and Chiahsien (Kaohsiung Co.) and those of M. ornata at Manchou, Chiahsien, Chi-an (Hualien Co.), Mucha and Nankang (Taipei City) from 1976 through 1984. All calls were recorded in the field with casette tape recorders (Sony TC-1015, TC- 1051 and TCM-100) and the sounds were ana- lyzed with sound spectrographs (Rion SG—09 and Kay Sona-Graph 7800). Air temperature records were taken near the calling males. Male speci- mens obtained from these localities were 20- 23mm in snout-vent length. The calls of the two species were composed of a series of pulse groups. In the following, “note” means a pulse group, “note length” the time from the beginning of the first pulse to the beginning of the last pulse in a note, “note interval” the time from the beginning of a note to the beginning of 564 the next note, and “note gap” the time from the end of a note to the beginning of the next note. Significance tests used were t-test and F-test. RESULTS The mean length of advertisement calls of M. heymonsi from Manchou (25.5°C) was 15.5 sec and consisted of about 18 notes (N=42). The mean values for M. heymonsi from Chiahsien (24.5°C) were 22.1 sec and 17 notes (N=20). In some calls notes were repeated with a regular interval, but in others some of the notes formed A B M. KuRAMOTO distinct note groups consisting of two, three, four and more notes (Figs. 1A and 2A-2C). Accord- ingly, whole call structures can be described as 1-1-1-1-1-1-1-1, 1-1-1-2-2-4-3-4-2 and so on, where each figure indicates the number of notes constituting a note group. Every call began with a series of single notes which may or may not be followed by several multi-note groups. For the sake of convenience, a note which does not form a multi-note group is regarded as constituting a single-note group. Generally the notes in a multi-note group were longer, contained more pulses and showed faster TIME Fic. 1. (SEC) Diagrammatic representation of pulse patterns of a whole call of M. heymonsi (A) and a part of a call of M. ornata (B). Vertical bars represent pulses. Multi-note groups in M. heymonsi are underlined. PERBRRRBGS bhhhits FERRRERED: beERL LL TEREREBEEE bekbhin. EPERRESEDA bbbbhe. FREQUENCY (kHz) WPOPPRDT LL tbh: byppbNDHI Vite MU WONPPERD AN iis ili .@] I T 2 1 ME STE epeuaged ATTEN reikbbtin 2 fe) | (SEC) Fic. 2. Sound spectrograms of advertisement calls of M. heymonsi (A-C) and M. ornata (D-F) analyzed with 300 Hz filter. (A) Two successive single-note groups and (B) a 2-note group recorded at Chiahsien, 24.5°C. (C) A 4-note group recorded at Manchou, 25.5°C. M. ornata calls are from Manchou, 25.5°C (D), Mucha, 22°C (E), and Nankang, 18.5°C (F). Taiwan Microhylid Calls 565 TaBLE 1. Temporal characteristics of pulse groups (notes) in advertisement calls of M. heymonsi and M. ornata (m+SD) Manchou (25.5°C) Chiahsien (24.5°C) Type of Note note group N Note length No. pulse Pulse rate N Note length No. pulse Pulse rate (sec) (pulse/sec) (sec) (pulse/sec) M. heymonsi 1-note te 0074-0105 Glee Age 3028-19 19" 0532-0204 OSES IO 9 27-304. 2-note Ist 11 0.24+0.04 O40 S025 EZ) 862 10°32 20:03 9.8+0.8 27.9+0.4 Pade it 0:27-0:02 OS -E0 oled tal one Ole O37 = O02 SMES OL6n 2828-12054 3-note Ist 10 0.27+0.02 9.040.7 30.0+1.0 2 0.3440.03 10.5+0.7 28.0+0.4 Onde 10770:292-01039) 51020220399 34a) 27703800 12:0-0:0 928-7 --0.6 Sree O29 002e OM O39 oie nO ee ON OOS teleost Own 22853-1013 M. ornata 1457020202019 tel228-- 0167) 87 21-8 16 7 0)25320102" 13 4=20:9" 5021-09 pulse repetition rate than the notes in a single- note group (Table 1). These tendencies were more evident in the latter notes within a note group. Note gaps (0.17—-0.19 sec in Manchou and 0.21—0.22 sec in Chiahsien) did not differ signif- icantly between various kinds of multi-note groups (P<0.05). In the calls from Manchou the gaps between multi-note groups (0.68-0.69 sec) were significantly shorter than those between single-note groups (1.09 sec, P<0.01). The dominant frequencies were at about 3 kHz in Manchou and at about 2.8kHz in Chiahsien calls. They were the second harmonic bands with fundamental frequencies of 1.5 and 1.4kHz, re- spectively. Sound energy was concentrated be- tween 1-4kHz (Fig.3A). The wave form of pulses (Fig. 3C) was regular and symmetric with a sharp rise time. 2a a > See N The advertisement calls of M. ornata were te- corded in several localities at various air tempera- tures. The calls were much longer than those of M. heymonsi, and often lasted for a few minutes and involved more than a hundred notes. As in M. heymonsi, the calls of M. ornata were com- posed of notes, but the notes were repeated at a regular interval without forming note groups (Fig. 1B). More pulses were involved in a note and the pulse rate was much faster in M. ornata than in M. heymonsi when compared at the same temper- ature (Fig. 2D, Table 1). Note intervals and note gaps were short compared with those of M. heymonsi. Dominant frequencies, about 2.7 to 3kHz, and energy distribution (Fig. 3B) and the wave form of pulses (Fig. 3D) were similar to those of M. heymonsi, although the rise time was longer than that of M. heymonsi. | C 0. 02SEC Fic. 3. Power spectrums (45 Hz filter) and wave forms of a pulse in advertisement calls of M. heymonsi recorded at Chiahsien, 24.5°C (A, C), and M. ornata recorded at Nankang, 18.5°C (B, D). Abscissa in A and B is relative intensity. 566 M. KurRAMOTO PULSE RATE (PULSE/SEC) TEMPERATURE (%T) PULSE RATE (PULSE/SEC) & 70 60 50 14 16 18 20 22 24 26 TEMPERATURE (7) Fic. 4. Correlation between air temperature and pulse rate in advertisement calls of M. ornata from Taiwan (A), and from the Ryukyu Islands (B). Mean pulse rates are indicated by hollow circles in M. ornata of Taiwan and solid circles in M. ornata of the Ryukyus. Vertical bars represent one standard deviation on both sides of the mean. Solid squares in A are mean values for M. heymonsi and upper line in B is the regression for Taiwan M. ornata given in A. When two males of M. ornata were calling in close proximity, the notes were given always alternately, never synchronously or overlapping. Note intervals were longer in alternately emitted _ calls than singly emitted ones. At 22°C the note interval was 0.84+0.10 sec (m+SD, N=S) in the former, while 1.07+0.03 sec (N=8) in the latter, the difference being highly significant (P<0.01). Note length and pulse rate did not differ in either situations. Pulse rate was directly, and note interval and note length were inversely, correlated with air temperature (Fig.2D-2F). Linear regression equations were P=4.12T—48.32 (Fig. 4A), I= —0.07T+2.38, and L= —0.02T+0.74 where P is pluse rate (pulse/sec), I is note interval (sec), L is: note length (sec) and T is temperature (°C), differences of the regression coefficients from 0 being highly significant (P<0.01) in the former two and significant (P<0.05) in the last. The number of pulses and dominant frequency were temperature-independent. DISCUSSION Acoustic features as revealed by sound spectro- grams seem very distinct between M. heymonsi and M. ornata at nearly the same temperature (cf. Fig.2A and 2D). heymonsi at high temperatures are quite similar to those of M. ornata at low temperatures (cf. Fig. 2A and 2F). Pulses constitute distinct notes, and However, the calls of M.- the frequency constitution and wave form are essentially identical in both species. These results indicate that M. heymonsi and M. ornata share many acoustic features, and suggest that they have derived from a common ancestral stock, probably rather recently. Obviously, acoustic divergence in the two species has occurred mainly in two temporal features, pulse rate and note group formation. Another microhylid M. inorna- ta from south Taiwan (Yu, personal communica- tion) and from Thailand [4] has quite different vocalization. In Manchou and Chiahsien, M. heymonsi and M. ornata called simultaneously in the same place. It is obvious that the females discriminate the calls of conspecific males from those of the other species in these situations. Among the acoustic parameters examined, pulse rate should be the most important cue for species discrimina- tion because the other parameters differ only slightly. Gerhardt [6], applying synthetic calls to the American treefrog Hyla versicolor, demon- strated that the pulse-repetition rate is critical for species discrimination. Since pulse rate is a function of temperature, the female’s, as well as the male’s auditory system should be adjusted in some way so as to offset the temperature effects. Note groups may be used as a cue for species discrimination, too. Multi-note groups seemed to become predominant over single-note groups as the breeding season of M. heymonsi proceeded, like multi-note calls of Rhacophorus taipeianus Taiwan Microhylid Calls 567 and multi-pulse calls of Polypedates leucomystax [7]. Among the M. ornata populations here ex- amined, only Manchou and Chiahsien populations are sympatric with M. heymonsi. Because no acoustic data were available for M. ornata of Manchou and Chiahsien at below 24.5°C, it could not be confirmed whether regressions of tempera- ture and pulse rate or note interval differed between sympatric and allopatric populations of M. ornata with M. heymonsi. Heyer [4] reported the M. heymonsi calls from Thailand recorded at 28°C. Note length (0.48 sec) and pulse rate (23 pulses/sec) are much longer and much slower, respectively, than those of the Manchou population at 25.5°C. The call of M. ornata from Thailand recorded at 25-28°C [4] has a note length of 0.23-0.31 sec, pulse number 10- 18, and a pulse rate of 53-60 pulses/sec. Com- parisons of these data with those of Taiwan populations indicate that the parameters of M. ornata are similar, while those of M. heymonsi differ considerably between the two areas. The acoustic divergence of the two species seem to be more pronounced in Thailand probably due to character displacement, the phenomenon exem- plified in several other frogs [8, 9]. By contrast, the M. ornata populations of the Ryukyu Islands [5] have different acoustic charac- teristics from those of Taiwan. The mean note length is longer and the mean pulse number in a note is fewer in Ryukyu than in Taiwan popula- tions. The relationship of temperature and pulse rate is on a distinct regression, P=0.74T+3.55 (Fig. 4B), the difference between the regression coefficients of Taiwan and the Ryukyus being highly significant (P<0.01). Apparently the Ryukyu populations of M. ornata have temporal acoustic features distinct from Taiwan and Thai- land populations, although their body sizes are similar. Since M. ornata is the only microhylid in the Ryukyu Islands, there has been no selective pressure which may shift the temperature rela- tionships of temporal acoustic features. ACKNOWLEDGMENTS A part of this study was financed from the National Science Council, Republic of China. Professors K.-H. Chang and F.-J. Lin, Zoological Institute, Academia Sinica, R. O. C., provided help when I was staying in the Institute in 1982 and 1984. Professor Y. Minamide, Department of Special Education, and Professor S. Ikeura, Department of Foreign Languages, Fukuoka University of Education, offered their facilities for sound analysis. REFERENCES 1 Bogert, C.M. (1960) The influence of sound on the behavior of amphibians and reptiles. In “Animal Sounds and Communication”. Ed. by W.E. Lanyon and W.N. Tavolga, AIBS Publ. No.7, Washigton, D.C., pp. 137-320. 2 Lue, K.-Y. and Chen, S.-H. (1985) The Amphibians of Taiwan. C.-H. Chang, Publisher, Taiwan, 190 pp. (In Chinese) 3 Pope, C.H. (1931) Notes on amphibians from Fu- kien, Hainan, and other parts of China. Bull. Am. Mus. Nat. Hist., 61: 397-611. 4 Heyer, W.R. (1971) Mating calls of some frogs from Thailand. Fieldiana: Zool., 58: 61-82. 5 Kuramoto,M. (1977) Mating calls of the frog, Microhyla ornata, from the Ryukyu Islands. Bull. Fukuoka Univ. Educ., Pt. III, 26: 91-93. 6 Gerhardt, H.C. (1978) Temperature coupling in the vocal communication system of the gray tree frog, Hyla versicolor. Science, 199: 992-994. 7 Kuramoto, M. (1986) Call structures of the rhaco- phorid frogs from Taiwan. Sci. Rep. Lab. Amph. Biol., Hiroshima Univ., 8: 45-68. 8 Fouquette,M.J., Jr. (1975) Speciation in chorus frogs. I. Reproductive character displacement in the Pseudacris nigrita complex. Syst. Zool., 24: 16-23. 9 Littlejohn, M.J. and Loftus-Hills, J.J. (1968) An experimental evaluation of premating isolation in the Hyla ewingi complex (Anura: Hylidae). Evolution, 22: 659-663. Pe EN ND eg Br reat bie 4 yard eere rain oa | ig a, ion eatroe elie otk cele Ven: ci viele eee t. oo WAT . oi , ; Le s i ‘ &. Pe ‘pat i i i J & i it Movemeyeyatak MG ao ; it rie OVE: cube aes cabo = and -->: evolutionary pathways; refer to the text for explanation of these solid and broken lines. In this evolutionary flow, the morphological change of the mandibular palp from the in- termediate type between the P2 and P3 to the P3 type spontaneously occurred. This flow in the group with the fused urosome finally reached the most apomorphic A-T-P4-F3 subgroup (C. simile [5,7,8]) through the A-~T-P3 or P4—F2 one. The latter is subdivided into two pathways at the D-S—P1 subgroup (C. arenarium [3, 7] and C. orientale [10, 19]); one pathway had the ordi- narily segmented mandibular palp and the other had various positions of the terminal segment attached to the proximal segment. In the former pathway, we can permit the evolutionary flow Evolutionary System of Corophium 373 from the D-S-—P1 subgroup to the D-S-PIr one (C. sinense [12, 14]) according to the side pathway of the mandibular palp from the P1 type to the Pir type (Fig. 1A), but we cannot hypothesize further pathways in this flow, as we have not found variations of the ordinarily segmented man- dibular palp and this mandibular palp in the group with fused urosome. The latter pathway went to the D-T-P2 or P3 subgroup (C. brevis [9], C. panamense [9] and C. rioplatense [8]) in the group with segmented urosome during the evolutionary change of the mandibular palp from the P1 type to the intermediate one between the P2 and P3 types, and further reached the D-T- P3-F1 subgroup in the group with fused urosome according to evolutionary changes in the urosome and mandibular palp as well as those in the flow with the sexually similar antenna 2. This flow is supported by the fact that the teeth on the dactyl of gnathopod 2 are present in the D-T-P2 or P3 subgroup and all subgroups with fused urosome. Lastly, we will consider the evolutionary path- ways in the group with fused urosome and sexual- ly dimorphic antenna 2. In this group, we can set up 9 subgroups by combining the three types of fused urosome (F1, F2 and F3 ) with the P3, P4 and P5 type in the mandibular palp. Seven of these 9 subgroups and 3 intermediate species have been found to date (Fig. 3). In the evolutionary pathways among these 9 subgroups, two schema can be shown for each of the following assump- tions: 1) that the evolutionary change in the urosome occurred subsequent to the formation of three types in the mandibular palp; 2) that the change in both organs occurred in a reverse order. If we accept the former assumption, we will be able to show the pathways drawn with a solid line in Figure 3. That is, after the formation of three subgroups, D-T-P3-F1 (C. insidiosum [5, 8], C. lacustre [5,7, 8] and C. uenoi [18]), D- T-P4-F1 (C. sextonae [3,7]) and D-T-P5-F1 (C. crassicorne |5, 7,8, 15]), and the intermediate species, C. clarencense [9], between the D-T—P4- F1 and the D-T—P5-F1 subgroups, the D-T-P3- Fl one evolved to the D-T-P3-F3 one (C. louisianum [8]) through two intermediate species, C. acherusicum [3,5,7,8] and C. californianum [9], and the D-T-—P3-F2 one (C. tuberculatum [5, 8]); the D-T-P4-F1 one to the D-T-P4—F3 one (C. acutum [5,7,8] and C. baconi [9]) through the D-T-P4-F2 one; the D-T-P5-F1 one to the D-T-—P5-F3a one (C. lobatum [12]) through the D-T-—P5-F2 one. Reversely, if the latter assump- tion is accepted, we will be able to show the pathways drawn with a broken line in Figure 3. That is, after the formation of three subgroups, D-T-P3-F1, D-T-P3-F2 and D-T-P3-F3, the D-T-P3-F1 one evolved to the D-T-P5-F1 one through the D-T-P4—-F1 one and the in- termediate species, C. clarencense; the D-T-P3- F2 one to the D-T-—P5-F2 one through the D-T- P4—F2 one; the D-T—P3-F3 one to the D~T-P5- F3a one through the D-T-P4—F3 one. As mentioned above, I intended to present a new evolutionary system for the corophiid amphi- pods in this paper based on four morphological features, antenna 2, urosome, mandibular palp and dactyl of gnathopod 2, but this system is incomplete. Difficulties are caused by the insuf- ficient information provided in many poorly de- scribed species. In order to improve the present systematics of the genus Corophium, many known corophiid species must be redescribed, new spe- cies need to be described and figured and new key characteristics must be found to provide more insight into the classification of corophiid amphi- pods. ACKNOWLEDGMENTS I wish to thank Dr. E.L. Bousfield of the National Museum of Canada for his useful comments. Thanks are also due to Dr. Y. Suzuki of the Biological Labora- tory, Asia University, for providing me with facilities to work and for his critical comments on the manuscript. I am grateful to Mr. N. Sawada of the Marine Ecological Research Co. Ltd., Osaka, for his personal communica- tion concerning Corophium lobatum and for allowing me to use a drawing (Fig. 2) in this paper. REFERENCES 1 Barnard, J.L. (1973) Revision of the Corophiidae and related families (Amphipoda). Smithson. Contr. Zool., 151: 1-27. 2 Myers, A.A. (1981) Amphipoda Crustacea. I. Family Aoridae. Mem. Hourglass Cruises, 5(5): 1- TD. 10 11 574 Crawford, G.I. (1937) A review of the amphipod genus Corophium with notes on the British species. J. Mar. Biol. Assoc. U.K., 21: 589-630. Boesch, D. F. and Diaz, R. J. (1974) New records of peracarid crustaceans from oligohaline waters of the Chesapeake Bay. Chesapeake Sci., 15: 56-59. Bousfield, E. L. (1973) Shallow water gammaridean Amphipoda of New England, Cornell Univ. Press, Ithaca, N. Y., 312 pp. Hurly, D.E. (1954) Studies on the New Zealand amphipodean fauna. No.7. Family Corophiidae, including a new species of Paracorophium. Trans. R. Soc. New Zealand, 82: 431-460. Lincoln, R. J. (1979) British Marine Amphipoda: Gammaridea, Chaucer Press, 658 pp. Shoemaker, C.R. (1947) Further notes on the amphipod genus Corophium from the east coast of America. J. Wash. Acad. Sci., 37: 47-63. Shoemaker, C.R. (1949) The amphipod genus Corophium on the west coast of America. J. Wash. Acad. Sci., 39: 66-82. Stock, J. H. (1952) Some notes on the taxonomy, the distribution and the ecology of four species of the amphipod genus Corophium (Crustacea, Mala- costraca). Beafortia, 21: 1-10. Shoemaker, C.R. (1934) Two new species of Corophium from the west coast of America. J. Wash. Acad. Sci., 24: 356-360. 12 13) 14 15 16 iv, 18 19 A. HIRAYAMA Hirayama, A. (1987) Two peculiar species of corophiid amphipods (Crustacea ) from the Seto Inland Sea, Japan. Zool. Sci., 4: 175-181. Heard, R.W. and Sikora, W.B. (1972) A new species of Corophium Latreille, 1806 (Crustacea: Amphipoda) from Georgia brackish waters with some ecological notes. Proc. Biol. Soc. Wash., 84: 467-476. Zhang, W. (1974) A new species of the genus Corophium (Crustacea, Amphipoda, Gammaridea) from the southern coast of Shangtung Peninsula, North China. Stud. Mar. Sinica, 9: 139-146. Hirayama, A. (1984) Taxonomic studies on the shallow water gammaridean Amphipoda of west Kyushu, Japan. II. Corophiidae. Publ. Seto Mar. Biol. Lab., 29: 1-92. Ledoyer, M. (1977) Contribution a Vetude de Pécologie de la faune vagile profonde de la Méditerranée Nord occidentale. 1. Les gammarides (Crustacea, Amphipoda). Bull. Mus. Civ. Stor. Nat. Verona, 4: 321-421. Stebbing, T.R. R. (1906) Amphipoda I: Gammar- idea. Das Tierreich, 21: 1-806. Stephensen, K. (1932) Some new amphipods from Japan. Annot. Zool. Japon., 13: 487-501. Stock, J.H. (1960) Corophium volutator forma orientalis Schellenberg, raised to specific rank. Crustaceana, 3: 188-192. ZOOLOGICAL SCIENCE 4: 575-578 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Number and Size of Eggs in the Three Emerald Damselflies, Lestes sponsa, L. temporalis and L. japonicus (Odonata: Lestidae)! “MAMORU WATANABE and YAsuyo ADACHI Department of Biology, Faculty of Education, Mie University, Tsu-shi, Mie 514, Japan ABSTRACT—Females of the emerald damselflies, Lestes sponsa, L.temporalis and L. japonicus, were captured in the wild and their reproductive organs were observed. The number of mature eggs was rather constant during their reproductive ages at about 200, 100 and 100 in L. sponsa, L. temporalis and L. japoni- cus, respectively. The egg maturation seemed to occur continuously after a particular developmental stage of adult female. The mature egg size of L. temporalis was more than two times of that of other species and decreased with age. The realized fecundity seemed to be the largest in L. sponsa. INTRODUCTION The reproductive biology of the emerald dam- selflies, Lestes spp., has been focussed upon the mating behaviour in relation to sperm precedence [1], the life history [2-4], and the adult move- ments [5]. However, there have been no reports on the fecundity and oviposition pattern of these damselfly species. Since these species deposit eggs in tandem with male, it may be difficult to obtain eggs from females under laboratory condi- tion. By dissection of feral females, Watanabe and Adachi [6] have already clarified the fecundi- ty and estimated the number of eggs actually deposited by a damselfly, Copera annulata, which also shows oviposition behaviour in tandem. In this paper, we attempt to clarify the fecundity of the emerald damselflies, L. sponsa, L. temporalis Accepted January 26, 1987 Received December 13, 1986 " Comparative Ecological Studies of Coenagrionoidea in Woodlands V. and L. japonicus, by counting the number of eggs in ovaries of feral adults by dissection. MATERIALS AND METHODS Three species of the emerald damselflies were collected in Mie and Shizuoka Prefectures both in the warm-temperate zone of Japan. Because of low density of L.sponsa in these prefectures, supplementary females were collected in Fukushi- ma Prefecture. All females were captured during various behaviours, e.g. feeding, roosting, tan- dem flight and ovipositing, from June to October in 1985. Females were put into 50% ethyl alcohol, as soon as they were netted. The condition and the length of their hind wings and abdomen were recorded. They were classified into seven age classes (T, I, II], P, M, MM, and MMM) mainly by the colour of the abdomen [6]. The former four age classes are regarded as immature females. The latter three are at the reproductive stages, in which the females show mating and ovipositing behaviours. All the dissected females were examined for the number of eggs remaining in the ovaries. The height, the width and the length of the bursa copulatrix were measured for calculation of its volume as a spheroid. Eggs in the ovaries were classified into three categories, i.e. mature, sub- mature and immature eggs, as the case of C. annulata [6]. Volume of the mature egg was also caiculated as an oval. The number of mature and submature eggs was 576 counted directly. Since immature eggs were not divided individually under a microscope, we were not able to evaluate the number. Instead, we assessed the abundance of immature eggs in the ovaries by four degrees: r, +, ++ and ++, denoting rare, a few, common and abundant, respectively. All means are reported with stand- ard errors. RESULTS AND DISCUSSION Thirty-six females of L. sponsa, 62 of L. tem- poralis and 73 of L. japonicus were collected in the field and dissected. Since there were no size variations with the ages collected (F-test, P>0.1), all of the data were pooled. The lengths of the abdomen and the hind wings were 31.9+0.1mm and 24.3+0.3mm in L. sponsa, 36.9+0.1mm and 26.9+0.5mm in L. temporalis and 31.6+ 0.1mm and 22.0+0.1mm in L. japonicus, re- spectively. Newly emerged females (age T) had little fat body in the cavity of the abdomen. The fat body Xx 103mm 1000 r 500 L (@) L. sponsa 100 Volume of Bursa Copulatrix ! 1 ! ! | | I ! | ! ! ! ! ! ' yn 10 T I WP M MMMMM T Age Change in the volume of the bursa copulatrix in relation to female age. Sample size was 2, 1, 1, 10, 12, 9 and 1 in L. sponsa (left), 13, 14, 6, 11, 5, 6 and 7 in L. temporalis (middle) and 9, 10, 9, 19, 6, 10 Fic. 1. M. WATANABE AND Y. ADACHI enlarged toward the age M and then decreased. Such a change in quantity of the fat body was also observed in C. annulata [6]. At age T, the bursa copulatrix was of a thin ellipsoidal shape. The volume was about 0.02, 0.05 and 0.02mm°, in L. sponsa, L. temporalis and L. japonicus, respectively (Fig. 1). The sizes were the same during ages I and II in all species. They became thicker in age P (about 0.1-0.2 mm°*), but no sperm was found in them. In mature females (M), the bursa copulatrix became oval shape with filled sperms (Fig. 2). The shape and the size of the bursa copulatrix were similar with each other among these three species. The volume was rather constant after the age M. This fact suggests that male emerald damselflies may remove the sperm of previous males and insemi- nate with a similar volume, because multiple matings are the rule in mature females as re- ported by Waage [1]. About 1mm? was the maximum volume of the bursa copulatrix in all species. The volume was ten more times larger than that of C. annulata [6]. L. temporalis L. japonicus I Po MMMMMMT I II P M MMMMM and 10 in L. japonicus (right), in age classes of T, I, Il, P, M, MM and MMM, respectively. Broken line shows the estimated volume in the age II of L. sponsa, because the bursa copulatrix was damaged during the dissection. Eggs of Three Emerald Damselflies 577 = : common oviduct vagina haa vagina common oviduct vagina common oviduct Fic. 2. Illustration of bursa copulatrix filled by sperm in a mature female of (a): L. sponsa, (b): L. tem- poralis and (c): L. japonicus. The abundance of immature eggs increased with age significantly (r7=0.23, p<0.001 in L. temporalis; r*=0.32, p<0.001 in L. japonicus), but was not significant (r>=0.02, p>0.2) in L. sponsa (Fig. 3). Submature eggs appeared at age P in L.sponsa and L. temporalis, while those were observed at age M in L. japonicus. Except the number of eggs in age MMM of L. sponsa, the submature eggs of all species increased according to the reproductive ages. Mature eggs of L.sponsa and L. temporalis were found after age P, while those of L. japoni- cus appeared after age M. Little difference in the egg number between age M (or P) and MMM was observed in each species. The number was rather constant at about 200, 100 and 100 in L. sponsa, L. temporalis and _ L. japonicus, respectively. Thus, it is expected that the number of mature eggs is maintained throughout the reproductive period (M to MMM). This shows that egg maturation occurred continuously during their reproductive ages in the emerald damselfly. An increase in number of immature eggs with the age also supports that the number of mature eggs is generated after oviposition. Volume of matured eggs in L. temporalis de- creased significantly (t-test, P<0.005) with the age (Table 1). An egg at the age MMM had a volume of about 75% of that at the age P. The egg of L. tempolaris was the largest. The de- crease in egg volume was also observed in L. sponsa (t-test, P<0.005), though the difference between maximum (age P) and minimum (age MM) volume was about 1.13 times. On the other hand, no egg size variation with age was found in L. japonicus, egg volume of which was the smal- lest among three species. Although we have already reported the smaller eggs deposited by older females of C. annulata [6], its adaptive significance still remained unknown. In general, the decrease of egg size is assumed to be due to the decrease in yolk content [7]. In the butterfly such as Pararge aegeria, neither egg survival nor larval survival is correlated with the egg size [8]. However, there was no report on the possible difference in hatchability, length of larval period or survival rate of larvae between larger and smaller eggs in damselfly species. It is likely that females of L. temporalis increase their fecundity by depositing smaller eggs at an older age, at which they probably counteracted their shorter life-expectancy with deterioration of fat body. In L. japonicus smaller egg size may be due to less yolk content. Considering lowest mature egg number in ovaries of each female, the fecundity might be the smallest among these species. On the other hand, either egg size or the size variation of L. sponsa was intermediate. The realized fecundity seemed to be the largest, be- cause the mature egg number in ovaries per female was double of that in other species. Further studies regarding the oviposition pattern of the emerald damselfly are needed to explain the relationship between egg size and female age. 578 M. WATANABE AND Y. ADACHI un ttt (e) t D if x} au ++ e) + ----- %5 wa © <> (0) = DE SE See eeLF v =) = 50 § = 25 nw of (0) Zw 200 L. sponsa L. temporalis L. japonicus 2) a n WwW L lOO ay = (e) z 0) ie eT CT MM MMM Tp Sige DIES eUn MUIR M MM MMM rf i BD aM M MM MMM dt Fic. 3. Change in the quantity of immature eggs (upper) and the number of submature (middle) and mature (lower) eggs in relation to female age of L. sponsa (left), L. temporalis (middle) and L. japonicus (right). See Fig. 1 for sample size in each age class. TaBLE 1. Change in egg volume with age in three emerald damselfly species (X10 *mm°, +SE) P M MM MMM L. sponsa 3.45 +0.04 (80) 3.24+0.03 (110) 3.06+0.04( 90) 3.34+0.09 (10) P<0.005 L.temporalis 6.09+0.13(10) 5.11+0.10( 10) 5.01+0.08( 56) 4.66+0.06 (70) P<0.005 L. japonicus — — 2.40+0.05( 60) 2.61+0.03 (100) 2.66+0.05 (91) N.S. ( ): number of mature eggs counted. 3 Pickup,J., Thompson,D.J. and Lawton, J. H. ACKNOWLEDGMENTS (1984) Odonatologica, 13: 451-459. We wish to express our sincere thanks to Dr. K. Ueda 4 Lutz, P. E. (1968) Ecology, 49: 576-579. for critical reading, and to Mr. N.Ohsawa for a 5 Utzeri, C., Carchimi, G., Falchetti, E. and Belfiore, technical assistance. This study was partly supported by C. (1984) Odonatologica, 13: 573-584. the Grant-in-Aid for Encouragement of Young Scien- 6 Watanabe, M. and Adachi, Y. (1987) Odonatologi- tists, No. 60740356 from the Ministry of Education, ca, 16 (in press). Science rand eure Ob Japan 7 Kimura, K. and Tsubaki, Y. (1985) Appl. Entomol. Zool., 20: 500-501. 8 Wiklund, C. and Persson, A. (1983) Oikos, 40: 53- REFERENCES 63 1 Waage, J.K. (1982) Odonatologica, 11: 201-209. 2 Gower,J.L. and Kormondy, E.J. (1963) Ecology, 44: 398-402. ANNOUNCEMENT CUPT P. RICHTER PRIZE IN PSYCHONEUROENDOCRINOLOGY The International Society of Psychoneuroendocrinology (ISPNE) invites submission of manuscripts for the Curt P. Richter Prize 1988. The award includes an honorarium of US $ 1,000 (sponsored by Pergamon Press) and a travel grant of up to US $1,000 (offered by the Society) to the 19th International Congress of the ISPNE in Groningen, The Netherlands, August 22 to 26, 1988, where the prize will be awarded. The winning paper will automatically be considered for publication in the Journal “Psychoneuroendocrinology”. Manuscripts should be submitted in quadruplicate to: Gerhard Langer, M.D. Associate Professor of Psychiatry Secretary of the ISPNE The deadline for submission is January 31, 1988. All submissions will be screened by a committee of established psychoneuroendocrinologists. Before submission of manuscript, for detailed information please contact the Secretary of the ISPNE, Gerhard Langer, M.D., Associate Professor of Psychiatry, Department of Psychiatry, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. £ i] ip iP if Published by the Japanese Society of Developmental Biologists The journal is devoted to the publication of original papers dealing with any aspects of developmental phenomena in all kinds of organisms, including plants and micro-organisms. Papers in any of the following fields will be considered: developmental genetics, growth, morphogenesis, cellular kinetics, fertilization, cell division, dormancy, germination, metamorphosis, regeneration and pathogenesis, at the biochemical, biophysical and analytically morphological levels ; reports on techniques applicable to the above fields. At times reviews on subjects selected by the editors will be published. Brief complete papers will be accepted, but not preliminary reports. Members of the Society receive the Journal free of charge. Subscription by institutions is also welcome. Papers in DGD, Vol. 29, No. 3. (June 1987) 19. REVIEW: M. Oxapa and S. KopayasuHt: Maternal Messenger RNA as a Determinant for Pole Cell Formation in Drosophila Embryos. 20. K.TAKAMUNE and Cu. Kataairi: The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg. 21. G.A. Buznixov, L.A. MaALcHENKO, G.V.DELONE and V. YA. Bropsky: The Embryos Obtained from the Karyoplasts of Starfish Oocytes: Their Development and Sensitivity to Cytostatic Neurotransmitter Antagonists. 22. R.E. HINKLeEy, R.N. EDELSTEIN and P.I.IvVONNET: Selective Identification of Sperm Fused with the Surface of Echinoderm Eggs by DNA-Specific Bisbenzimide (Hoechst) Fluorochromes. 23. M. Asasuima, H. NAKANO, T. MatsuNAGA, M. SuGcimoTto and H. TAKANO: Purification of Mesodermal-inducing Substances from Carp Swim Bladders I. 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Effects of Increasing CO, upon a Tropical Rainforest Ecosystem Kopama, A.: A Comparative Karyological and Cytophotometric Study of Normal and Wounded Stem Tissues of Lathyrus odoratus L. Kapono, Y. anD E.L. ScuneiDerR: Floral Biology of Trapa natans var. japonica Kamiya, N.: Cytoplasmic Streaming in Giant Algal Cells: A Historical Survey of Experimental Approaches Order form Send to THE BOTANICAL SOCIETY OF JAPAN Toshin Building Hongo 2-27-2, Bunkyo-ku, Tokyo 113, Japan THE BOTANICAL MAGAZINE, TOKYO [] Individuals: ¥ 7,000 p.a. CJ Institutions: ¥ 17,500 p.a. Name (Please print): Address: Date: Signature: NARISHIGE THE ULTIMATE NAME IN MICROMANIPULATION OUR NEW MODELS MO-102 and MO-103 MAKE PRECISION MICROMANIPULATION SO EASY! (Photo: by courtesy of Olympus Optical CO., LTD) SOME FEATURES of MO-102 and MO-103: * The manipulator head is so small that it can be mounted directly on the microscope stage. There is no need for a bulky stand. * Hydraulic remote control ensures totally vibration-free operation. * 3-D movements achieved with a single joystick. Micromanipulators Microelectrode pullers Stereotaxic instruments NARISHIGE SCIENTIFIC INSTRUMENT (eset 7 LABORATORY CO.,LTD. 4-9-28, Kasuya, Setagaya-ku, Tokyo 157 JAPAN Telephone: 03-308-8233 Telex: NARISHG J27781 (Contents continued from back cover) Watanabe, M. and Y. Adachi: Number and size of eggs in the three emerald damselflies, Lestes sponsa, L. temporalis and L. japoni- cus (Odonata: Lestidae) (COMMUNICA- TION). cocoon 575 Taxonomy Kuramoto, M.: Advertisement calls of two Taiwan microhylid frogs, Microhyla heymon- SUMAN eI VIRMOTIGIG 5 or or tee ois Aceh g's sy sieSee 563 Hirayama, A.: Notes on the evolutionary systematics of the genus Corophium ....... 569 ZOOLOGICAL SCIENCE VOLUME 4 NUMBER 3 JUNE 1987 CONTENTS REVIEWS Price, D. A., N. W. Davies, K.E. Doble and M.J. Greenberg: The variety and distribu- tion of the FMRFamide-related peptides in MOM MSCS: ac Reo es ee ee 395 Asashima, M., T. Oinuma and V.B. Meyer- Rochow: Tumors in amphibia ............ 411 ORIGINAL PAPERS Physiology Ohnishi, K.: Proposed tertiary olfactory pathways in a teleost, Carassius auratus ...427 Takahashi, N.: Gonad response to y-amino- butyric acid in the sea urchin .............. 433 Takahashi, N. and M. Takahashi: Gonad response to calcium and a comparison of the effects of calcium, potassium, acetylcholine and y-aminobutyric acid on the sea urchin SOMA See eee A eine ysis mbes mie ee a 441 Hidoh, O. and J. Fukami: The mediation of cyclic AMP in octopaminergic modulation at neuromuscular junctions of the meal-worm, Renebrio MOOT sc bud ska ae ene es 447 Kumazawa, T. and O. Suzuki: Diamine oxidase activities in catfish tissues .......... 451 Oishi, T., J.K. Lauber and _ J. Vriend: Experimental myopia and glaucoma in CHICKS eae. oo snongoga00Gn0Gb0GE0N0C 455 Cell Biology and Biochemistry Takeuchi, S.: Cytochalasin B affects selec- tively the marginal cells of the epithelial sheet im.culture)..co. ace nice saat ra 465 Seki, T., S. Fujishita, M. Azuma and T. Suzuki: Retinal and 3-dehydroretinal in the egg of the clawed toad, Xenopus laevis INDEXED IN: Current Contents/LS and AB & ES, Science Citation Index, ISI Online Database, CABS Database Genetics Saotome, K.: Chromosome numbers in 8 Japanese species of sea urchins ............ 483 Developmental Biology Kunieda, M. and M. Wakahara: Twin forma- tion in Xenopus laevis eggs centrifuged before first cleavage 35.4.5. 489 Tsuneki, K.: A histological survey on the development of circumventricular organs in various vertebrates ..3......,0-e pene ae 497 Endocrinology Okawara, Y., T.Karakida, M. Aihara, K. Yamaguchi and H. Kobayashi: Involve- ment of angiotensin II in water intake in the Japanese eel, Anguilla japonica ............ 523 Chan, P.J.: Cyclic CMP alters squirrel mon- key (Saimiri sciureus) luteal cell structure via cyclic AMP-dependent mechanisms ........ 529 Behavior Biology Tomioka, K. and Y. Chiba: Entrainment of cricket circadian activity rhythm after 6-hour phase-shifts of light-dark cycle ............. 525 Chiba, A. and K. Aoki: Relationship between daily variation of locomotor activity and that of plasma corticosterone levels in the newt, Cynops pyrrhogaster pyr- TROQASUET voces cincadins a icin) pee 543 Hayashi, S.: The effects of preputialectomy on aggression in male mice ................ 551 Ecology Cheng, H. Y. and J.Y. Lin: Annual ovarian, fat body and liver cycles of the grass lizard Takydromus stejnegeri in Taiwan .......... Spi (Contents continued on inside back cover) Issued on June 15 Printed by Daigaku Printing Co., Ltd., Hiroshima, Japan TOOLOGICAL SCIENCE An International Journal ZOOLOGICAL SCIENCE The official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Hideshi Kobayashi (Tokyo) Toshin-building, Hongo 2-27-2, Bunkyo-ku, Managing Editor: Tokyo 113, Japan. Tel. (03) 814-5675 oo oedichiro Kawashima (Hiroshima) @iicers: o VA Sater President: Nobuo Egami (Tsukuba) Takeo ni (Hiroshima) Secretary: Yasuto Tonegawa (Urawa) Sire? eae aaa (Hiroshima) Treasurer: Tadakazu Ohoka (Tokyo) y y Librarian: Shun-Ichi Uéno (Tokyo) baa a SteaagauBoaye” “ProwardA. Bern (Berkeley) Walter Bock (New York) Aubrey Gorbman (Seattle) Horst Grunz (Essen) Robert B. Hill (Kingston) Yukio Hiramoto (Tokyo) Susumu Ishii (Tokyo) Yukiaki Kuroda (Mishima) Koscak Maruyama (Chiba) Roger Milkman (Iowa) Hiromichi Morita (Fukuoka) Kazuo Moriwaki (Mishima) Tokindo S. Okada (Okazaki) | Andreas Oksche (Giessen) Hidemi Sato (Nagoya) Hiroshi Watanabe (Shimoda) | Mayumi Yamada (Sapporo) Ryuzo Yanagimachi (Honolulu) ZOOLOGICAL SCIENCE is devoted to publication of original articles, reviews and communications in the broad field of Zoology. The journal was founded in 1984 as a result of unification of Zoological Magazine (1888-1983) and Annotationes Zoologicae Japonenses (1897-1983), the former official journals of the Zoological Society of Japan. ZOOLOGICAL SCIENCE appears bimonthly. An annual volume consists of six numbers of more than 1000 pages including an issue containing abstracts of papers presented at the annual meeting of the Zoological Society of Japan. 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New subscriptions and renewals begin with the first issue of the current volume. All rights reserved. No part of this publication may be reproduced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1987, The Zoological Society of Japan Publication of Zoological Science has been supported in part by a Grant-in-Aid for Scientific Publication from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 4: 579-591 (1987) © 1987 Zoological Society of Japan REVIEW The Importance of Inducing Factors for Determination, Differentiation and Pattern Formation in Early Amphibian Development — Horst GRUNZ Department of Zoophysiology, FB 9 (Biologie) University GAS Universitatsstr., 4300 Essen 1, FRG INTRODUCTION It is generally accepted that the amphibian embryo is one of the best studied eucaryotic systems in developmental biology. Since now absolutely new methods (fluorescent cell lineage markers, highly purified or homogeneous mor- phogenetic factors, monoclonal antibodies, elec- trofusion of amphibian cells, techniques of genetic engineering) are available, the molecular pro- cesses, including the expression of regional and tissue specific genes, of the fairly well described morphological and morphogenetic events during embryogenesis can be further studied in detail. A growing number of scientists focus their interest on molecular processes during amphibian oogenesis, cleavage, embryonic induction and pattern forma- tion. An increasingly favoured species is the South African clawed frog, Xenopus laevis, since its eggs can be obtained under laboratory conditions throughout the whole year. Of special importance are the mechanisms of gene expression and gene regulation during early embryonic development. Powerful tools are now available to study such events also in the very complex amphibian embryo [1, 2]. This holds true also for the problem of early embryonic induction. Although an overwhelming amount of data from many laboratories is avail- able, the exact molecular mechanisms of embryonic induction are still obscure. Therefore it is highly desirable to get a better understanding of Accepted March 10, 1987 EM\THSONIAN APR 2 1 1988 Essen, LIBRARIES processes responsible for mesoderm formation, midblastula transition [3] and induction of the central nervous system. It should be pointed out that most of those processes are basic topics in cell biology and molecular genetics. Therefore, those results in amphibians can be considered to be closely related to embryonic events in higher vertebrates, which are less suitable for many studies because of technical or ethic (human embryo) reasons. PECULIARITIES IN THE XENOPUS EMBRYO Since Xenopus laevis is now frequently used for embryological and biochemical studies, first I like to mention basic results, which were achieved in our laboratory. In contrast to Triturus species or Ambystoma mexicanum the jelly coat of Xenopus laevis or X. borealis is removed by culture medium containimg cysteine hydrochloride or other di- sulfide cleaving agents [4]. K. Buiting in our lab could show that a prolonged treatment of early gastrulae with cysteine hydrochloride results in malformations of the head area of the swimming larvae. If early gastrulae are treated with cysteine longer than 10 min, we got defects of the olfactory placodes, eyes or the whole brain area (synoph- thalmia, cyclopia, Figs. 1 and 2). Therefore the procedure should be performed under strictly controlled conditions. We recommend the follow- ing standard conditions: 3.5% cysteine hydrochlo- ride in Holtfreter solution, pH 7.35 at 20°C for 10 min; Holtfreter solution: 0.059 M NaCl, 0.00067 580 H. GRuUNZ Fic. 1. Transversal section of the head area of an embryo, which was treated in the early gastrula stage with cysteine hydrochloride for 13min. The larva has formed two fused eyes (synophthalmia). Bar: 0.1mm. Fic. 2. Transversal section of the head area of an embryo, which was treated in the early gastrula stage with cysteine hydrochloride for 16min. The larva has formed a cyclopic eye with two lenses. ta: tapetuum, le: lens. Bar: 0.1mm. M KCl, 0.0009 M CaCl,:2H,O). In addition we emphasize the macroscopical control, since varia- tions in the thickness of the jelly coat of different spawns require a shorter or prolonged treatment. After artificial insemination, however, the jelly coat of 2—4-cell stages can be removed within 3-4 min under the same experimental conditions. Also in biochemical experiments, using cleavage stages up to the early tadpole stage, control embryos should be raised up to stage 46 [5] to find out negative effects of cysteine. Malformations (re- duced size of the eyes) can easily be identified in the stereomicroscope. It must be pointed out that malformations of the head area can be caused by various substances [6-9]. Although it can be speculated that the defects are correlated with impaired morphogenetic movements and abnor- mal formation of the prechordal plate, responsible for the induction of the archencephalic brain area [10], the exact mechanism of action of cysteine at the molecular level is not known. In contrast to Triturus species (urodela) the ectoderm of Xenopus similar to other anura consists of several distinct cell layers [11], which mechanically can be separated by fine very flexible glass needles with a possible tip diameter less than 2 ym. Also in the upper blastopore lip an outer and deep layer can be distinguished [12]. Nieuw- koop and Florschiitz [13] have postulated that only the deep layers develop into mesodermal deriva- tives, while the superficial cell sheet is thought to have epidermal character. Preliminary results of U. Koch in our laboratory indicate that also the outer ectoderm possess a weak capacity to differ- entiate into mesoderm. Another peculiarity in Xenopus should be short- ly mentioned. If inducer substances in pellet form (inducers bound to biologically inactive y- globulin) are implanted into the blastocoel of early gastrulae (implantation method after Spemann and Mangold [14]; Fig. 3a2), the pellet is later found in most larvae within the intestine. In contrast to the situation in Triturus species the pellet does not come into contact with the compe- tent presumptive belly epidermis, which is a prerequisite for inductions. Another effect is the unspecific formation of additional tail structures, probably caused by the mechanical splitting of the inducing archenteron during invagination by the implanted material. This effect can tven be observed with pellets of y-globulin without any neuralizing or mesodermalizing activity. There- fore the implantation technique cannot be recom- mended for Xenopus laevis. In contrast the sandwich-technique [15] is an excellent test- method also for Xenopus (Fig. 3b). Inducing Factors in Amphibian Development 581 METHODS TEST a a) blastopore lip b) inductorpel let Implantation method ectoderm : (~) b iductorpellet Sandwich method ectoderm \ Cc filterpaper Filterpaper method Fic. 3. Diagrammatic representation of 3 important test methods: a) implantation-technique after Man- gold, al) implantation of blastopore lip into the blastocoel of an early gastrula, a2) implantation of an inducer pellet, b) sandwich-technique after Holt- freter, c) test method for soluble agents. The filter-paper prevents the curling up of the ectoderm. The hanging drop culture technique is shown elsewhere (Tiedemann, [23]). THE IMPORTANCE OF MORPHOGENETIC FACTORS FOR THE EARLY EMBRYONIC DEVELOPMENT Spemann and Hilde Mangold could show in their famous implantation experiment in 1924 (Fig. 3a1) that a certain area of the embryo (the upper blastopore lip) is able to induce a secondary axis in a host embryo. Therefore Spemann called this pa.i of the embryo “organizer”. Its role in embryogenesis is described in detail elsewhere [16, 17]. The interest of embryologists focussed on the question, which factors located in the blastopore lip are responsible for the induction process of the central nervous system. Since morphogenetic factors are present in the embryo in very low concentrations, the isolation of those neuralizing factors was not successful for long time. The same holds true for endoderm or mesoderm inducing factors. They will induce in competent ectoderm notochord, somites, pronephros, intestine etc. Crude factors could be isolated from guinea pig bone marrow [18, 19]. However, a vegetalizing factor, protein in nature (molecular weight 13,000 Daltons), was isolated from chicken embryos and was purified to homogeneity [20]. Also from swimmbladder a factor with mesodermalizing activity was partially purified [21]. Also crude neuralizing factors could be obtained from am- phibian embryos [22]. Recently a mesodermaliz- ing factor was found in conditioned medium of a Xenopus laevis cell line (XTC-Cells) by Smith, London. All the just mentioned factors (in solution or bound to biologically inert protein) can be tested by the implantation method, sandwich- technique or hanging-drop-culture [23]. In all three test methods or slightly modified techniques competent ectoderm is the target for the inducing factors (Fig.3). Depending on the different biological activity of the inducing factors, the ectoderm, which in normogenesis forms epidermis [24], differentiates into endodermal, mesodermal or neural differentiations [11, 25, 26]. Of interest was the observation that certain lectins (Concana- valin A (Con A) and Ulex europeus agglutinin) will evoke neural inductions in competent ectoderm [25-31]. It can be speculated that those lectins mimic the action of the “natural” neuralizing factors, which are thought to interact with specific receptors (glycoproteins) on the plasmamembrane of the reacting tissue (ectodermal target cells). There are strong indications that the internaliza- tion is not a prerequisite for the biological function of neuralizing factors [32]. It should be pointed out that the competent ectoderm must be triggered by certain threshold concentrations of the inducer (Con A) to form neural tissues [27]. Apparently not only the quality but also the quantity of receptors on the plasmamembrane of the target cells play a crucial role in the realisation of neural structures [28, 33]. We could show that the superficial layer of the competent ectoderm of Xenopus laevis, which binds much less Con A than the deep layer, is unable to form neural structures. 582 H. Grunz On the other hand, the deep layer of the ectoderm of the late gastrula, which has lost its competence to react to neuralizing stimuli, binds similar amounts of Con A as competent early gastrula ectoderm (deep layer) [33]. These data support the view that the loss of competence is not correlated with a change in the amount of recep- tors, but more likely with intracellular regulatory events at the genome level. POSSIBLE ROLE OF IONS AND CYCLIC AMP IN EMBRYONIC INDUCTION AND THE PROBLEM OF AUTONEURALIZATION By Holtfreter [34] it was shown that amphibian ectoderm can form neural structures under un- physiological conditions without any interaction with a specific inducer. Recently Hildegard Tiede- mann found that HEPES-containing medium also induces neural structures [35]. Also phorbolester is a potent substance to evoke neural structures in competent ectoderm [36]. Normal culture condi- tions, which are without effect in other species (Triturus alpestris or Xenopus laevis), can cause autoneuralization in Ambystoma mexicanum (Ax- olotl). It is well known that neuralization in contrast to mesodermalization can be evoked by unspecific treatment of ectoderm with different substances except lithium chloride. One explana- tion for such effects was that a certain amount of cells of the ectoderm piece was fully or partially impaired by the treatment, which will liberate inducing substances and will induce the remaining unaffected cells [37]. It could be argued that after Con A treatment the tissue is also damaged. However, neither Takata’s group nor ours did find necrotic cell material in the explants. Therefore the effect of Con A could be considered as an activation of processes, which are more closely related to the natural inducers. It could be assumed that the different neural inducing sub- stances have the activity in common that they interact on the same or a succeeding link in the chain of molecular events, leading to neural induction and differentiation. This could hold true also for the neuralizing effect of phorbolester [36]. On the other hand we cannot support the view that ions or cyclic AMP are involved in the inducing processes as primary signals. We could show that cyclic AMP has no neuralizing effect in Tritutus alpestris or Xenopus laevis species, which in contrast to Ambystoma are very resistant against treatments, leading to autoneuralization in Ambystoma. Therefore, data describing neura- lization in Ambystoma after the treatment with cAMP or other substances must be discussed with great caution. By experiments using vegetalizing factor and calciumionophore A 23187 the exclusive role of ions as primary signals for embryonic induction can be ruled out [29, 38]. On the other hand ions Ca** are important cofactors in signal transduction, for example, the activation of pro- teinkinase C [39], which could play part in the process of neural induction and differentiation. INFORMATION TRANSFER AND COMMUNICATION BETWEEN EMBRYONIC CELLS The central nervous system is formed by the interaction between the invaginating chorda- mesoderm and the overlaying neuroectoderm, which forms the neural plate. It could be shown by transfilter experiments that free migrating factors are transmitted from the inducing chorda- mesoderm to the reacting ectoderm [40]. It is likely that those factors are released from the inducing chordamesoderm by exocytosis into the intercellular space between the chordamesoderm and the ectodermal target cells [41]. Those neuralizing factors then could interact with specific receptors on the plasmamembrane of the target cells. That the neuralizing factors in contrast to the vegetalizing factor apparently must not be internalized to become biologically active can be concluded from experiments with factors, which were prevented from internalization by the binding to sepharose beads [27, 32, 42]. The isolation and characterization of neural factors from Xenopus laevis is described in detail elsewhere [22]. On the basis of earlier and recent data obtained in our laboratory it can be postulated that the transmis- sion between inducing and reacting tissue takes place by short distance transport [43]. Together with Dr. Tacke in my laboratory it could be shown that there must be established a close juxtaposition Inducing Factors in Amphibian Development 583 between inducing upper blastopore lip (chorda- mesoderm) and reacting ectoderm for the realiza- tion of neural structures. At least 75% of the ectodermal cells must come into close contact with the chordamesodermal cells. This process is time dependent. If the chordamesoderm of Xenopus laevis is removed from the sandwich within 3 hr (cell contact area below 75%) no or only small amount of neural structures is formed. These data will be published in detail elsewhere. Furthermore we could document that a close association of combined inducing and reacting tissues is realized in various species (Triturus vulgaris, Rana esculenta and Xenopus laevis) after different periods of time. Earlier data (Grunz, unpublished results) could show that chordamesoderm of Ambystoma takes up close cell-to-cell contacts to reacting ectoderm much faster than recombinants of Triturus alpestris. These data may explain why chordamesoderm of Ambystoma induces neural structures after 5 min contact with ectoderm, while in Triturus vulgaris chordamesoderm needs 4 hr to trigger the reacting ectoderm [44, 45]. A long distance diffusion of inducing factors is unlikely because of the following reasons. During the gastrulation process an unlimited transport of neuralizing factors, released from the invaginating chordamesoderm, could induce not only the neuroectoderm but also the presumptive epidermis after migration of the inducers through the blasto- coel. Furthermore already during cleavage mesoderm inducing factors released from the vegetal blastomeres could not only trigger the presumptive marginal zone to form the mesoderm, but also the animal pole material {presumptive ectoderm) after crossing the blastocoel. However, there exists also the possibility that secreted inducers are inactivated by inhibitors present in the blastocoel. The data so far available support the view that inducing factors exert their biological activity after short distance migration in areas of close juxtaposition of inducing and reacting tissue. It is likely that a critical number of cells within a piece of ectoderm must be stimulated with a certain threshold concentration for the realization of neural structures. It is highly probable that the susceptibility for the neuralizing or other inducing factors also depends on critical phases of the cell cycle, which corresponds to the exposition of specific receptors for inducing signals and en- docytotic processes, important for the recycling of receptors [33]. On the basis of our experiments with combined upper blastopore lip and ectoderm it seems unlikely that only one or few cells are triggered first, which in turn will stimulate their neighbours. Presumably in the first step a critical number of cells must be stimulated. However, we don’t exclude the possibility of secondary cell interactions (cell communication within the same germ layer) including the still obscure molecular processes of homoiogenetic induction [46-51]. That every competent ectodermal cell within the same sandwich apparently cannot react on the same inducing signal in the same way, can be concluded from the fact that always different cell types are formed in the mesodermal or neural induced tissue. IMPORTANCE OF THE EXTRACELLULAR MATRIX FOR EARLY EMBRYOGENESIS Boucaut and coworkers and the group of Naka- tsuji in Japan could show that extracellular matrix proteins (fibronectin and laminin), covering the blastocoelic side of the ectoderm, are important as contact guidance for the invaginating chorda- mesoderm [52, 53]. When specific antibodies directed against fibronectin are injected into the blastocoel of blastulae the gastrulation movements are inhibited [54]. In the early neurula stage also glucosaminoglycans are present in the intercellular gap between chordamesoderm and neural plate [41]. However, it can be excluded that those polysaccharides and the fibrillar high molecular weight proteins are themselves responsible for the neural induction. Together with Boucaut’s group we could show that the binding of anti-fibronectin (anti-FN) to ectoderm does not prevent neural induction, when anti-FN-treated ectoderm was combined with upper blastopore lip in the sand- wich-technique [55]. These data indicate that the extracellular matrix proteins in normogenesis are responsible for the close juxtaposition of inducing and reacting tissue only, a prerequisite for the short distance transport of neuralizing factors. Those factors, proteins in nature, have presumably 584 H. GRUNZ a much smaller molecular weight than the fibrillar extracellular matrix proteins. THE ROLE OF VEGETALIZING AND MESODERMALIZING FACTORS IN EARLY EMBRYOGENESIS A vegetalizing factor with endodermal and mesodermal inducing activity has been isolated from chicken embryos and was purified to homogeneity. Its chemical features and its bio- logical activity are described in detail elsewhere [20, 56]. We could show that this factor is able to trigger competent ectoderm to form endodermal and mesodermal derivatives [25]. Also neural structures were formed under certain conditions by the ectoderm, treated with vegetalizing factor. However, those differentiations are thought to be formed by secondary cell interactions [47-49]. A similar factor could be present in the vegetal half of amphibian embryo. We know from our earlier experiments that in the 8-cell-stage-embryo the differentiation potentials of the 4 animal blasto- meres differ significantly from the 4 vegetal blasto- meres [57]. By more detailed studies [58-60] could be shown that all blastomeres of the 8-cell-stage- embryo possess distinct developmental capacities. The results show that very early in amphibian development there exist already specific animal/ vegetal and dorsal/ventral and also lateral gra- dients [61]. Nieuwkoop demonstrated that the mesodermal marginal zone including the upper blastopore lip is formed by the interaction of the vegetal with the animal blastomeres of early cleavage and blastula stages [62]. Asashima [63] found that the vegetal material of the mid blastula stage possesses the highest mesodermal inducing capacity. Gurdon and colleagues demonstrated that within 3 hr after combination of material of the vegetal and the animal hemispheres of early blastulae the activation of actin genes takes place [64]. By transfilter experiments we could show that mesodermal inducing factors are passing through Nucleopore filter pores of 0.4 ~m from isolated vegetal pole material to ectodermal target cells. By electron microscopy we could exclude that cell protrusions have penetrated into the Nucleopore filter pores. Therefore also cell contacts between the inducing and the reacting tissue could be ruled out [65]. Those experiments convincingly show that gap junctions are not relevant for the primary steps in mesodermal induction. Therefore the asymmetric malformations, observed especially in the head area of larvae after injection of antibodies directed against the 26 KDa gap junction protein in blastomeres of 8- to 32-cell stages of Xenopus laevis, must be considered as a result of inhibition of secondary steps (control of cell proliferation, cell cycle etc.) [29, 66, 67]. Since gap junctions are thought to permit the exchange of molecules up to 1 KDa only, it is unlikely that inducing factors (molecular weight over 10,000 Da) are transmitted from cell to cell via gap junctions. As a working hypothesis we suggest that a mesodermalizing factor is exported from the vegetal pole material and will interact with receptors of cells of the presumptive mesodermal zone of the embryo. Of interest was our observation that in the transfilter experiments single animal cap formed ventral mesodermal derivatives (heart structures, coelo- mic epithelium, and blood cells), while aggregates of 4 or 6 animal caps differentiated into dorsal mesodermal structures (notochord, somites and neural tube). Because of the increase of the initial cell mass of the reacting ectoderm the differ- entiation pattern is shifted from ventral to dorsal mesodermal structures [47, 65]. The experiments of Boterenbrood and Nieuwkoop [68] indicate that the ventral and lateral endoderm induces ventral mesodermal structures, while the dorsal endoderm evokes dorsal mesodermal derivatives. They con- cluded from their results that the difference in mesodermal inducing capacity of the dorsal mesoderm as against the lateral and ventral mesoderm is probably purely quantitative in char- acter. From our data it could be concluded that in addition to the inducing capacity of the inducing tissue several other parameters are controlling the realization of a certain differentiation pattern, 1.e. the initial cell mass, inducer concentration, incuba- tion time, size of the contact zones of the recom- binants, and phase of competence. It should be mentioned that we tried to isolate the mesodermalizing factor from supernatant of vegetal pole material of early blastula by binding to Heparin-Sepharose. However, so far we were Fic. Fic. Inducing Factors in Amphibian Development 585 5 4. Confluent layer of XTC-cells of Xenopus laevis. Several days after confluency also aggregates (Ag) and freely floating spheres (Sp) can be observed. The monolayer is out of focus. Inset: Focus on the monolayer of the XTC-cells. Bar: 0.1mm. 5. A small sphere of XTC-cells (compare with Fig. 4) was wrapped by late blastula ectoderm (sandwich- method) and cultured for 5 days at 17°C. The explant has formed blood cells (E) and coelomic epithelium. Similar differentiations were formed by gastrula ectoderm, treated for 1/2 hr with XTC- conditioned medium. Late blastula ectoderm after incubation with 50 “g/ml FGF for 10 min differentiates in similar way. Bar: 0.1mm. : 586 H. Grunz Fic. 6. Differentiations formed by late blastula ectoderm, treated for 20min with 50 ug/ml FGF. Somites (so) and peripheral endoderm (en) can be seen. Bar: 0.1mm. Fic. 7. Differentiations formed by late blastula ectoderm, treated for 40 min with 50 ng/ml FGF. The explant has formed intestine (in), mesenchyme and blood cells (not present in this section). Bar: 0.1mm. Fic. 8. Differentiations formed by late blastula ectoderm, treated for 40 min with 50 ug/ml FGF. The explant has differentiated into somites (so) and neural tube (n). Similar differentiations have formed after the treatment of blastula ectoderm with XTC-conditioned medium (supernatant of XTC-cultures). Bar: 0.1mm. Inducing Factors in Amphibian Development 587 not successful. It can be suggested that the inducer is present in very low concentrations only or/and it is inactivated during the different steps of isola- tion. Dr. Slack, London, has reported that fibroblast growth factor (FGF) induces ventral mesodermal structures in competent ectoderm [69]. However, we could show by the treatment of ectoderm of middle blastulae with very high concentrations of FGF (50 ug/ml) (Boehringer, Mannheim) for 10- 60 min that this growth factor is also able to evoke dorsal mesodermal structures (Fig. 8). When the ectoderm of middle blastulae is incubated for relatively long period (2 hr), the explants develop in yolk-rich spheres. Similar results we obtained with high concentrations of vegetalizing factor [25]. The tissue could be identical with undiffer- entiated endoderm. The histotypic differentiation of endoderm depends on the presence of mesodermal derivatives [70]. This fact is corrobo- rated by the observation that shorter incubation time of ectoderm with FGF (5-10 min) results not only in the differentiation of ventral mesodermal structures (Fig.5), but in few cases also into endodermal derivatives, i.e. intestine and en- dodermal epithelium (Figs. 6 and 7). Another mesodermalizing factor could be iso- lated by Smith, London, from the supernatant (conditioned medium) of a confluent layer of a Xenopus-cell line (XTC-cells) [71]. He could show that ectoderm, treated with the conditioned medium, differentiates into dorsal mesodermal structures (notochord, somites and neural tube) [72]. However, when we used early gastrula ectoderm instead of blastula ectoderm ventral mesodermal and endodermal structures are also formed after short incubation period (Fig. 5) (Tacke and Grunz, unpublished results). It should be mentioned that the XTC-cells after confluency form compact freely floating spheres (Fig. 4). These small aggregates can be wrapped by compe- tent ectoderm (sandwich method). It is of interest that the ectoderm is stimulated by the XTC- aggregates to form ventral mesodermal (blood cells, coelomic epithelium and intestine) and mus- cle structures as well (Figs. 5 and 8). Therefore the factor from XTC-cells or FGF cannot be consi- dered as specific ventral or dorsal mesodermalizing factor, respectively [73]. So far it is not clear whether two different (a dorsal and a ventral mesodermalizing factors) inducers are present in the dorsal and the ventral vegetative material of the intact embryo. We know that the basic FGF-factor, purchased from Boehringer, Man- nheim, which we used for our experiments, still contains other components. So far we cannot exclude that these factors are also biologically active. The HPLC-purification and the test of the different fractions are in progress (joint experi- ments with Dr. Tiedemann, Berlin). It could be speculated that homogeneous vege- talizing factor, XTC-factor and FGF contain simi- lar domains, which are responsible for the bio- logical activity. This could be concluded from the fact that all three factors are able to initiate the formation of endoderm or vegetal and dorsal mesoderm. Besides certain structural differences all three factors could contain specific amino acid sequences. It could be argued that all three factors give a similar primary vegetalizing signal, followed by secondary cell interactions (activation of mesodermalizing factors), which will cause the formation of mesodermal derivatives, which in turn will induce neural structures under certain experimental conditions. It is known that FGF can be bound to Heparin- Sepharose. In preliminary experiments we could show that after addition of Heparin-Sepharose CL-6B to conditioned medium (supernatant of confluent layers) of XTC-cells (1 ml gel to 4 ml supernatant) the inducing activity of the XTC- inducer(s) is not fully abolished. We obtained in 25% of the cases mesodermal inductions. This could mean that in contrast to FGF there is only a weak affinity of XTC-factor(s) to MHeparin- Sepharose. However, we found no inductions at all, when we added Con A-Sepharose to the XTC-conditioned medium and treated competent ectoderm with the supernatant. This could mean that XTC-factor contains a-D-mannoside or/and a-D-glucoside residues. In contrast the fraction with the highest mesodermal inducing capacity isolated from carp swimmbladder does not bind to Con A-Sepharose [21]. 588 CONCLUSIONS AND PERSPECTIVES There are known several vegetalizing (meso- dermalizing) factors, protein in nature, which can be obtained in highly purified form. This holds true for the FGF and the factor(s) from XTC-cells. The amino acid sequence has already been de- scribed for the pituitary basic FGF and the acidic bovine brain FGF [74]. Since large quantities of conditioned medium (supernatant of confluent cultures) of XTC-cells can be obtained, a further purification and _ characterization of XTC- mesodermalizing factor(s) can be expected. Next steps would include the cloning of vegetalizing (mesodermalizing) and neuralizing factors. Since the factors, just mentioned, mimic the biological effects of the genuine factors present in the embryo, it can be suggested that they contain similar domains, which are important for the biological activity. The exact mode of the induc- tion process could also be studied with specific antibodies directed against the inducers. Fur- thermore other modern techniques (for example electro-cellfusion, adapted to amphibian cell mate- rial [75]) will be useful for studies on cytoplasmic/ nuclear interrelationships including gene regula- tion. These homogeneous proteineous inducing fac- tors will be powerful tools for studies of the chain of molecular events, which take place during the determination of cells and pattern formation in early embryogenesis, closely related to differential gene expression. ACKNOWLEDGMENT Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt “Steuerung der Differenzierung von ein- und wenigzelligen eukaryontischen Systemen”) and, in part, by the University GHS Essen. We thank Sabine Effenberger for the preparation of the histological sections. REFERENCES 1 Sargent, T. G. and Dawid, I. B. (1983) Differential gene expression in the gastrula of Xenopus laevis. Science, 222: 135-139. 2 Miyatani,S., Winkles,J.A., Sargent,T.D. and H. Grunz 10 11 WD 13 14 15 16 17 Dawid, I. G. (1986) Stage-specific keratins in Xeno- pus laevis embryos and tadpoles: The XK81 gene family. J. Cell Biol., 103: 1957-1965. Newport, J. and Kirschner, M. (1982) A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell, 30: 675-686. Gusseck, D. J. and Hedrik, J. L. (1971) A molecu- lar approach to fertilization. I. Disulfide bonds in Xenopus laevis jelly coat and a molecular hypothesis for fertilization. Dev. Biol., 25: 337-347. Nieuwkoop, P. D. and Faber, J. (1956) Normal Table of Xenopus laevis (Daudin). North Holland, Amsterdam. Stockard, C. R. (1909) The artificial production of one-eyed monsters and other defects, which occur in nature, by the use of chemicals. Anat. Rec., 3: 167- By Stockard, C. R. (1909) The development of arti- ficially produced cyclopean fish. “The magnesium embryo”. J. Exp. Zool., 6: 285-337. Adelmann, H. (1936) The problem of cyclopia. Part I. Quart. Rev. Biol., 11: 161-183. Adelmann, H. (1936) The problem of cyclopia. Part II. Quart. Rev. Biol., 11: 284-305. Mangold, O. (1931) Das Determinationsproblem. Dritter Teil. Das Wirbeltierauge in der Entwicklung und Regeneration. Ergeb. Biol., 7: 193-403. Asashima, M. and Grunz,H. (1983) Effects of inducers on inner and outer gastrula ectoderm layers of Xenopus laevis. Differentiation, 23: 157-159. Keller, R. E. (1981) An experimental analysis of the role of bottle cells and the deep marginal zone in gastrulation of Xenopus laevis. J. Exp. Zool, 216: 81-101. Nieuwkoop, P. and Florschiitz, P. (1950) Quelques caracteres speciaux de la gastrulation et de la neurulation de l’oeuf de Xenopus laevis, Daud. et de quelques autres Anoures. Arch. Biol. (Liege), 61: 113-150. Spemann, H. and Mangold, H. (1924) Uber Induk- tion von Embryonalanlagen durch Implantation artfremder Organisatoren. Wilhelm Roux’ Arch. Entwicklungsmech. Org., 100: 599-638. Holtfreter, J. (1933) Nachweis der Induktionsfahig- keit abgetdteter Keimteile. Isolations- und Trans- plantationsversuche. Wilhelm Roux’ Arch. Ent- wicklungsmech. Org., 128: 584-633. Nakamura, O. and Toivonen, S. (1978) Organizer- A Milestone of a Half-Century from Spemann. Biomedical Press, Elsevier/North-Holland. Nieuwkoop, P. D., Johnen, A. G. and Albers, B. (1985) The epigenetic nature of early chordate development. Inductive interactions and compe- tence. Cambridge University Press, Cambridge, London. 18 19 20 21 22 23 24 25 26 fad | 28 29 Inducing Factors in Amphibian Development Yamada, T. (1958) Induction of specific differ- entiation by samples of proteins and nucleoproteins in the isolated ectoderm of Triturus gastrulae. Experientia, 14: 81-87. Yamada, T. (1961) A chemical approach to the problem of the organizer. In “Advances in Mor- phogenesis, Vol. 1”. Ed. by M. Abercrombie and J. Brachet, Academic Press, New York, pp. 1-53. Born, J., Hoppe, P., Schwarz, W., Tiedemann, H.., Tiedemann, H. and Wittmann-Liebold, B. (1985) Embryonic inducing factor: Isolation by high per- formance liquid chromatography and chemical prop- erties. Biol. Chem. Hoppe-Seyler’s, 366: 729-735. Asashima, M., Nakano, H., Matsunaga, K., Hashi- mota, K. and Shimada, K. (1986) Purification of mesodermal inducing substance and protein syn- thesis using this material. In “Cellular Endocrinolo- gy: Hormonal Control of Embryonic and Cellular Differentiation”. Ed. by. G. Serrero and J. Hayashi, Alan R. Liss, Inc., New York, pp. 55-66. Janeczek, J., Born, J., Hoppe, P., Schwarz, W., Tiedemann, H. and Tiedemann, H. (1986) Informa- tive molecules and induction in early embryogene- sis. In “Cellular Endocrinology: Hormonal Control of Embryonic and Cellular Differentiation”. Ed. by. G. Serrero and J. Hayashi, Alan R. Liss, Inc., New York, pp. 11-24. Tiedemann, H. (1986) Test of embryonic factors: advantages and disadvantages of different proce- dures. In “Cellular Endocrinology. Hormonal Con- trol of Embryonic and Cellular Differentiation”. Ed. by G. Serrero and J. Hayashi, Alan R. Liss, Inc., New York, pp. 25-34. Grunz, H. (1973) The ultrastructure of amphibian ectoderm treated with an inductor or actinomycin D. Wilhelm Roux’ Arch., 173: 283-293. Grunz, H. (1983) Change in the differentiation pattern of Xenopus laevis ectoderm by variation of the incubation time and concentration of vegetaliz- ing factor. Wilhelm Roux’ Arch., 192: 130-137. Grunz, H. (1983) Early embryonic induction in amphibians, a model system of the information transfer between cells. In “Developmental Biolo- gy”, An Afro-Asian Perspective. Ed. by. S. C. Goel and R. Bellairs, pp. 73-86. Takata, K., Yamamoto, K. Y. and Ozawa, R. (1981) Use of lectins as probes for analyzing embryonic induction. Roux’ Arch. Dev. Biol., 190: 92-96. Grunz, H. (1985) Effect of Concanavalin A and vegetalizing factor on the outer and inner ectoderm layers of early gastrulae of Xenopus laevis after treatment with Cytochalasin B. Cell Differ., 16: 83- 92. Grunz,H. (1985) Information transfer during embryonic induction in amphibians. J. Embryol. 30 Sit 32 33 34 35 36 Si 38 39 40 41 42 43 589 Exp. Morphol., 89: Suppl., 349-363. Gualandris,L., Duprat,A.M. and _ Rouge, P. (1987) Cross-linking of membrane glycoconjugates is not a sufficient condition for neural induction by Con A. Cell Differ., (in press). Takata, K. (1985) A molecular profile of the receptor responsive to the neural-inducing signals in the amphibian competent ectoderm. Zool. Sci., 2: 443-453. Born, J. Hoppe, P., Janeczek, J., Tiedemann, H. and Tiedemann, H. (1986) Covalent coupling of neuralizing factor from Xenopus to Sepharose beads: no decrease of inducing activity. Cell Differ. , 19: 97-101. Tacke, L., and Grunz, H. (1986) Electron micros- copy study of the binding of Con A-gold to super- ficial and inner ectoderm layers of Xenopus laevis in correlation to the neural inducing activity of this lectin. Roux’ Arch. Dev. Biol., 195: 158-167. Holtfreter, J. (1944) Neural differentiation of ectoderm through exposure to saline solution. J. Exp. Zool., 95: 307-340. Tiedemann, H. (1986) The molecular mechanism of neural induction: neural differentiation of Triturus ectoderm exposed to hepes buffer. Roux’ Arch. Dev. Biol., 195: 399-402. Davids, M., Loppnow,B., Tiedemann, H. and Tiedemann, H. (1987) Neural differentiation of amphibian gastrula ectoderm exposed to phorbol- ester. Roux’ Arch. Dev. Biol., 196: 137-140. Chuang, H. H. (1955) Untersuchungen tiber die Reaktionsfahigkeit des Ektoderms mittels sublethal- er Cytolyse. J. Acad. Sinica, 4: 151-186. Siegel, G., Grunz,H., Grundmann, U., Tiede- mann, H. and Tiedemann, H. (1985) Embryonic induction and cation concentrations in amphibian enbryos. Cell Differ., 17: 209-219. Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal transduction and tumor promo- tion. Nature, 308: 693-698. Saxen, L. (1961) Transfilter neural induction of amphibian ectoderm. Dev. Biol., 3: 140-152. John, M., Janeczek, J., Born, J., Hoppe, P., Tiede- mann, H. and Tiedemann, H. (1983) Neural induc- tion in amphibians. Transmission of a neuralizing factor. Roux’s Arch. Dev. Biol., 192: 45-47. Born, J., Grunz,H., Tiedemann, H. and Tiede- mann, H. (1980) Biological activity of the vegetaliz- ing factor: decrease after coupling to polysaccharide matrix and enzymatic recovery of active factor. Roux’s Arch., 189: 47-56. Grunz, H. and Staubach, J. (1979) Cell contacts between chorda-mesoderm and the overlaying neuroectoderm (presumptive central nervous sys- tem) during the period of primary embryonic induc- tion in amphibians. Differentiation, 14: 59-65. 590 44 Johnen, A. G. (1956) Experimental studies about 45 46 47 48 49 50 51 32 53 54 5 56 S/ the temporal relationships in the induction process. I. Experiments on Amblystoma mexicanum. Proc. Acad. Sci. Amst., ser. C, 59: 554-561. Johnen, A. G. (1956) Experimental studies about the temporal relationships in the induction process. II. Experiments on Triturus vulgaris. Proc. Acad. Sci. Amst., ser. C, 59: 652-660. Nishijima, K., Noda, S., Kurihara. K. and Sasaki, N. (1978) Differentiation of partially mesodemal- ized ectoderm; homoiogenetic and heterogenetic induction by primarily induced part of the ectoderm. Dev. Growth Differ., 20: 275-281. Grunz, H. (1979) Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass. Wilhelm Roux’ Arch., 187: 49- ih Asahi, K., Born, J., Tiedemann, H. and Tiede- mann, H. (1979) Formation of mesodermal pattern by secondary inducing interactions. Wilhelm Roux’ Arch., 187: 231-244. Minuth, M. and Grunz, H. (1980) The fromation of mesodermal derivatives after induction with vegeta- lizing factor depends on secondary cell interactions. Cell Differ., 9: 229-238. Albers, B. (1985) Kompetenz als entscheidender Faktor bei der raumlichen Begrenzung der Neural- anlage (Ambystoma mexicanum). Ph. D. Thesis, Koln. Grunz, H., Born,J., Tiedemann, H. and Tiede- mann, H. (1986) The activation of a neuralizing factor in the neural plate is correlated to its homoiogenetic inducing activity. Roux’ Arch. Dev. Biol., 195: 464—466. Boucaut, J. C. and Darribere, T. (1983) Fibronectin in early amphibian embryos. Cell Tissue Res., 234: 135-145. Nakatsuji, N., Hashimoto, K. and Hayashi, M. (1985) Laminin fibrils in newt gastrulae visualized by the immunofluorescent staining. Dev. Growth Differ., 27: 639-643. Boucaut, J. C, Darribere, T., Boulekbache, H. and Thiery, J. P. (1984) Prevention of gastrulation but not neurulation by antibodies to fibronectin in amphibian embryos. Nature, 307: 364-367. Grunz, H., Darribére, T. and Boucaut, J. C. (1987) Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions. Roux’ Arch. Dev. Biol., 196: 203-209. Geithe, H.-P., Asashima, M., Asahi, K., Born, J., Tiedemann, H. and Tiedemann, H. (1981) A vege- talizing inducing factor—Isolation and chemical properties. Biochim. Biophys. Acta, 676: 350-356. Grunz, H. (1977) The differentiation of the four animal and the four vegetal blastomeres of the H. GruNZz 58 59 60 61 62 63 64 65 66 67 68 69 70 71 eight-cell-stage of Triturus alpestris. Wilhelm Roux’ Arch., 181: 267-277. Kageura, H. and Yamana, K. (1983) Pattern reg- ulation in isolated halves and blastomeres of early Xenopus laevis. J. Embryol. Exp. Morphol., 74: 221-234. Kageura, H. and Yamana, K. (1986) Pattern farma- tion in 8-cell composite embryos of Xenopus laevis. J. Embryol. Exp. Morphol., 91: 79-100. Yamana, K. and Kageura, H. (1987) Reexamina- tion of the “regulative development” of amphibian embryos. Cell Differ., 20: 3-10. Scharf, S. R. and Gerhart, J. C. (1980) Determina- tion of the dorsal-ventral axis in eggs of Xenopus laevis: Complete rescue of UV-impaired eggs by oblique orientation before first cleavage. Dev. Biol., 79: 181-198. Nieuwkoop, P. D. (1969) The formation of the mesoderm in urodelean amphibians. I. Induction by the endoderm. Wilhelm Roux’ Arch., 162: 341-373. Asashima, M. (1975) Inducing effects of the pre- sumptive endoderm of successive stages in Triturus alpestris. Wilhelm Roux’ Arch., 177: 301-308. Gurdon, J. B., Mohun, J., Brennan, S. and Cascio, S. (1985) Actin genes in Xenopus and their developmental control. J. Embryol. Exp. Morphol., 89: (Suppl.), 125-136. Grunz,H. and Tacke,L. (1986) The inducing capacity of the presumptive endoderm of Xenopus laevis studied by transfilter experiments. Roux’s Arch. Dev. Biol., 195: 467-473. Warner, A. E., Guthrie,S.C. and Gilula, N. B. (1984) Antibodies to gap-junctional protein selec- tively disrupt junctional communication in the early amphibian embryo. Nature, 311: 127-131. Tseng, M.-P. Chuang (1986) On the cellular com- munication of amphibian embryogenesis. In “Cellu- lar Endocrinology: Hormonal Control of Embryonic and Cellular Differentiation”. Ed. by G. Serrero and J. Hayashi, Alan R. Liss, Inc., New York, pp. 35-44. Boterenbrood, E. C. and Nieuwkoop, P. D. (1973) The formation of the mesoderm in urodelean amphibians. IV. Its regional induction by the en- doderm. Wilhelm Roux’ Arch. Dev. Biol., 173: 319-332. Slack, J. M. W., Darlington, B.G., Heath, J: K. and Godsave, S. F. (1987) Mesoderm induction in early Xenopus embryos by heparin-binding growth factors. Nature, 326: 197-200. Okada, T. S. (1960) Epithelio-mesenchymal rela- tionships in the regional differentiation of the digestive tract in the amphibian embryo. Roux’ Arch. Dev. Biol., 152: 1-21. Pudney,M., Varma,M.G.R. and Leake, C. J. (1973) Establishment of a cell line (XTC-2) from 72 19 74 Inducing Factors in Amphibian Development South African clawed frog, Xenopus laevis. Ex- perientia, 29: 466-467. Smith, J. C. (1987) A mesoderm-inducing factor is produced by a Xenopus cell line. Development, 99: 3-14. Dale, L., Smith, J.C. and Slack, J.M.W. (1985) Mesoderm induction in Xenopus laevis: a quantita- tive study using a cell lineage label and tissue- specific antibodies. J. Embryol. Exp. Morphol., 89: 289-312. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., 75 5911 Denoroy,L., Klepper,R., Gospodarowicz, D., Bohlen, P. and Guillemin, R. (1985) Primary struc- ture of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino- terminal sequence of bovine brain acidic FGF. Proc. Natl. Acad. Sci. USA, 82: 6507-6511. Grunz, H. and Tacke, L. (1986) Embryonic induc- tion in amphibians. In “New Discoveries and Tech- nologies”. Ed. by. H. C. Slavkin, Alan R. Liss Inc., New York, pp. 135-138. ZOOLOGICAL SCIENCE 4: 593-606 (1987) © 1987 Zoological Society of Japan REVIEW The Derivation of Terrestrial Cave Animals SHUN-ICHI UENO Department of Zoology, National Science Museum (Nat. Hist.), Shinjuku, Tokyo 160, Japan INTRODUCTION It was believed for more than a century that terrestrial cave animals were derived from ances- tors which had entered into limestone caves, colonized there and adapted themselves to various environmental factors peculiar to the underground world. All the eminent biospeologists in former times, including Viré [1], Racovitza [2], Jeannel [3, 4] and Vandel [5], did not throw any doubt on this point. The situation did not change even after the Second World War and all the biospeological studies were confined in calcareous areas, although considerable development was achieved in this field of science, not only in taxonomic and zoogeographic studies but also in the ecology and physiology of cave animals. On the other hand, aquatic cave animals were already known from outside calcareous areas near the end of the last century [6]. Various kinds of phreatic animals, mostly eyeless and colourless, were subsequently found, first from wells [7, 8] and then from interstitial waters along rivers and seashores [9]. In Japan, the first phreatic animal, a planarian, was found in November 1889 from a well at Ichigaya in Tokyo and was described in 1916 [10]. Following this lead, subterranean species of freshwater worms, water-mites and isopod and amphipod crustaceans were recorded in the next two decades, mostly from wells dug or driven into alluvial layers distant from karstic terrains [11]. Similar data were rapidly accumu- lated in the 1950’s and 1960’s, and it became a common knowledge that so-called aquatic cave Received February 18, 1987 animals, at least most of small-sized ones, origi- nated in the interstitial zone irrespective of the geological nature of the areas concerned. It is, therefore, incredible that no biospeologists sus- pected the existence of terrestrial cavernicoles in non-calcareous areas. In the past decade or so, however, our concept of terrestrial cave animals has made a drastic change, and now we are fully aware of the fact that they are not confined to limestone caves but widely occur wherever environmental condition is suit- able for their existence. They have not been met so far in pure granitic terrains, alluvial plains and small oceanic islands, but these blanks seem to have been caused either from ecological reasons or from existence of extrinsic barriers to dispersal of their ancestors. The purpose of the present review is to delineate the process of recent development in the study of terrestrial cavernicoles, and to demonstrate what is the original habitat of most terrestrial troglobionts. Since the upper hypogean fauna of the Japanese Islands has not yet been thoroughly studied, the following account is main- ly based on better known groups with relatively numerous species, especially on trechine beetles. An abstract of the present paper was already published in a previous volume of this journal [12], and a concise outline of the recent development of Japanese biospeology was given in the Tenth Anniversary Special Volume of the Speleological Society of Japan [13]. BIOSPEOLOGICAL STUDY OF LAVA CAVES In 1968, the National Parks Association of Japan and the Japanese National Subcommittee for the 594 S.-I. UENo Conservation of Terrestrial Animals in the Inter- national Biological Program organized a joint project for investigating the natural history of Mt. Fuji-san [14]. Since this volcano is famous not only for its beautiful shape but also for the abundance of lava tubes, study of the cave fauna was taken up in the project, and its execution was entrusted to me. I was, however, rather reluctant to accept the responsibility, because it was generally believed that lava caves were not sufficiently old to develop Fic. 1. Map showing the distribution of lava caves (including complex tree-molds) in the Fuji area; young lava flows are indicated by horizontal hatching; old ones are shown by fine dots; middle-aged lavas are omitted. Derivation of Cave Animals 595 specialized cavernicoles. This belief was founded on the data that all but one of the lava caves theretofore examined were sterile and devoid of troglobionts. Nevertheless, I myself had to under- take the task, as I was unable to find anyone willing to study the supposedly meagre fauna of lava caves. Before the new project was drawn up, there had been two biologists who had taken a particular interest in the lava cave fauna of Mt. Fuji-san. They were Hajime S. Torii [15, 16] and Masao Kumano [17], both of whom considered that some clue for clarifying the origin and adaptation of cave animals could be obtained by a comparative study between the fauna of old lava caves and that of young ones. Neither of them succeeded in accom- plishing an expected result, but their working hypothesis seemed to make a good starting point. And thus, I commenced my own investigation along the same line as that of Kumano. With the consistent support of expert cavers, my study progressed systematically and rapidly, and soon I became aware of the fundamental error of our former belief. Excepting frozen ones, most lava caves in the Fuji area were inhabited by certain terrestrial animals, especially by spiders and millipedes. Those found in young caves usually did not show appreciable morphological modification adaptive to subterranean life, where- as those occurring in old ones were often highly modified and similar to species previously known = 2 Fic. 2. A habitat of troglobionts in an old lava cave (Komakado-kaza-ana Cave) at the southeastern foot of Mt. Fuji-san. Photo by Takanori Ogawa. only from limestone caves. The faunal discrepancy was so decisive that the age difference between young and old caves appeared to bear on the differentiation of cave animals. Many young caves in the Fuji area are de- veloped in a lava flow spouted in 864 on the northwestern side of the volcano, that is, they are about 1,100 years old, while older caves lie in southeastern, southern and western lava flows 8,000-13,000 years old (Fig. 1). Therefore, there is a lapse of 7,000-—12,000 years between the birth of old caves and that of young ones. However, 10,000 years or so did not seem sufficiently long for differentiation of troglobiontic animals, and be- sides, all the troglobionts found in Fuji lava caves belong to groups, whose members are widely distributed in the neighbouring areas. If we regard them as morphologically modified independent of their relatives, we cannot explain the close similar- ity between lava cave forms and limestone cave ones, unless very unusual parallel evolution could have taken place between them. Thus, I had to seek for other factors than mere age to account for the faunal difference between old and young caves. Examining the ecological data amassed during the course of cave explorations, I realized that there was a definite difference in environmental conditions between old and young caves [18]. Young caves are mostly composed of bare lava and devoid of soil, and the climate is usually subject to 596 S.-I. UENO diurnal and seasonal fluctuations because of the porous nature of the rock. On the contrary, old caves are more or less covered with layers of soil and frequently have muddy floors; the climate is usually stable throughout the year as in limestone caves (Fig. 2). It is therefore evident that the former is not suited for the habitats of highly specialized cavernicoles, especially of such soil- dependent animals as chthoniid pseudoscorpions and trechine beetles. When it is created, a lava cave is completely bare. As time goes by, its roof becomes gradually eroded, forested and finally covered with thick layers of humus and soil. This coating keeps the underground climate stable and protects the en- vironment from substantial changes. On the other hand, the soil gradually percolates into the cave with rain water, accumulates on the floor, and fills up small voids in the rock. For this reason, old caves are always wet and often have shallow pools of groundwater. The presence of soil is also indispensable for the existence of many specialized cavernicoles, since autotrophic microorganisms living in the clay or silt deposits synthesize certain vitamins in the absence of light and serve as the nutrition of young cavernicoles [5, 19]. After all, habitats suitable for specialized cavernicoles are yielded only in old caves. It was necessary to see whether the existence of troglobionts in environmentally stabilized lava caves is peculiar to the Fuji area or universally observed. Accordingly, we extended our studies to all the areas in which lava caves were known, that is, the Island of Daikon-jima on Lake Naka- umi in western Honshu, Aso Volcano in central Kyushu, the Satsuma Peninsula in southwestern Kyushu, and the Island of Fukué-jima of the Gotds off the western coast of Kyushu. The results of the explorations accorded well with that obtained in the Fuji area, almost all the old caves examined having been inhabited by highly specialized caver- nicoles (Fig. 3) [20, 21]. It was confirmed beyond doubt that the controlling factor in the lava cave fauna was the environmental conditions, not sim- ply the age of the caves concerned. The biospeological importance of lava caves was subsequently recognized by Howarth, who discov- ered many extraordinary troglobionts on the 1.0mm Fic. 3. Gotoblemus ii S. Uéno, a specialized anoph- thalmic trechine beetle endemic to old lava caves on the Island of Fukué-jima of the Gotés off the western coast of Kyushu. Island of Hawaii [22]. All these troglobionts belong to groups autochthonous to the island group and have become differentiated under the tropical climate, so that they cannot be directly compared with temperate forms. Howarth’s study is, however, important in clarifying that a special- ized cave fauna can exist wherever there is a suitable environment for colonization and adapta- tion [23]. Next problem to be cleared up was to determine whether or not the troglobionts extant in lava caves had evolved after the eruption of lava flows bearing the caves concerned. It was difficult to approach the subject directly from the data obtained by the studies of lava cave inhabitants. Fortunately, however, a new light was shed from a different direction and led us to a new field of biospeology. Derivation of Cave Animals 597 BIOSPEOLOGICAL STUDY OF ARTIFICIAL CAVES In the autumn of 1970, when I was still in an inextricable maze of lava cave problems, an anophthalmic trechine beetle was discovered by Masahisa Ohrui in an abandoned adit of a gold mine on the Izu Peninsula [24]. After a careful examination, it became apparent that the beetle was a new species closely related to a lava cave inhabitant endemic to the southeastern foot of Mt. Fuji-san (Fig. 4). Subsequent investigations re- vealed that the adit had an interesting fauna very similar to that of old caves in the Fuji area. Most important was the discovery of an eyeless spider called Falcileptoneta caeca, which was common between the two areas [25]. Fic. 4. Trechiama ohruii S.Uéno, an anophthalmic trechine beetle first found in an old gold mine on the Izu Peninsula. The location of the gold mine was not very far from the nearest lava flow in the Fuji area, but was still more than 20 km distant in a bee-line. Be- sides, the geological feature of the intervening area was very intricate and not comparable to relatively simple calcareous terrains or lava fields. If the troglobiontic spiders extant in the two areas became independently differentiated from a com- mon ancestor, they could not be perfectly identical with each other, even though a striking parallelism could have taken place. Therefore, certain under- ground routes passable for the spider must exist between Fuji and Izu however implausible it seemed to be. This inference was also supported by the fact that the mine adit was only 100-150 years old, or much younger than the youngest known lava cave in the Fuji area. No troglobiont could have undergone an appreciable morpho- logical modification within such a short time. 1.5mm Fic. 5. Trechiama echigonis S. Uéno, an anophthalmic trechine beetle first discovered from the upper hypogean zone of Central Japan. 598 S.-I. UENO Five years prior to the discovery of the mine fauna on the Izu Peninsula, Kintaro Baba discov- ered an anophthalmic trechine beetle, which was in every respect troglobiontic, from the narrow fissures of shale about 2 m below the surface (Fig. 5) [26]. In our present concept, the habitat of this beetle is typically upper hypogean, but at that time I was unable to distinguish it from the endogean domain, although I was much surprised at its unusual depth. The realization that artificial cavities could harbour specialized fauna directed my eyes once again to the importance of Baba’s discovery. Similar environment under the earth might well be the original habitat of terrestrial Sa ay Fic. 6. troglobionts found in the mine adit. It was, however, not easy to dig deep holes into the ground for searching for minute animals of considerable rarity. An easier way to obtain the same result seemed to me to be the faunal investigation of artificial cavities already dug into the ground by someone else, and most abundant of them seemed to be mine adits of various kinds. Besides, there were several records suggestive of a promising future of this project. In 1954, Shigeru Nomura examined an abandoned mercury mine in eastern Kyushu and found that its fauna was very similar to those of nearby limestone caves [27]. Though lying in a calcareous area, the adit itself Ie 5 mn I i Chaetotrechiama procerus S. Uéno, a very remarkable anophthalmic trechine beetle hitherto known only from an abandoned mine in southwestern Shikoku. Derivation of Cave Animals 599 was dug into a small hill mainly composed of chert. In the following year, I myself paid a visit to that area and examined the adit in question and two other adits of another mine, all of which were found rich in specialized fauna [28, 29]. At about the same time, J. Balazuc and J. Demaux explored a hematite mine in southern France, reporting that it harboured various terrestrial troglobionts [30]. However, all these findings were made in either calcareous terrains or their vicinities, so that their importance in biospeology was overlooked, until a rich troglobiontic fauna was found in the gold mine on the Izu Peninsula widely distant from any calcareous areas. Standing on a new solid ground, we started in a renewed study of mine fauna with the hope of pursuing the origin of terrestrial cavernicoles. As in the case of the faunal study of Fuji lava caves, our exploration of artificial cavities pro- gressed systematically and rapidly, but at first the results were not so good as expected. We had to endure repeated disappointment and almost gave up the whole plan. After a few years of painstak- ing efforts, however, we finally located several mines rich in specialized cavernicoles, first in the vicinities of Kyoto and Osaka, and then in the Island of Shikoku (Fig. 6). Once fertile mines were found, subsequent researches became much easier, and a large quantity of new materials were rapidly accumulated. We are now fully aware of the fact that specialized hypogean faunas are found in various kinds of artificial cavities including mines, under- ground shelters, conduits, prospecting adits at dam sites, and so on. - The age of the cavities is not significant with the faunas, since highly specialized troglobionts are found in old adits 100 or more years old as well as in very young ones that were dug only 2 or 3 years before. Moreover, their existence are not directly dependent on the nature of rocks into which the cavities in question are dug; igneous and metamorphic rocks are equally suit- able to sedimentary ones [31, 32]. However, most favourable are such clastic rocks as mudstones, shales, schists and breccias. Tuff mines are also frequently, but not always, good for habitats of specialized cavernicoles. On the other hand, pure granite is always devoid of specialized fauna; this was one of the main reasons why our investigation of mine fauna was not successful at the beginning. It is apparent at present that the sterility of granitic cavities is mainly ascribed to ecological causes. When eroded, pure granite only produces pure sand, so that granitic cavities are usually devoid of clay or silt indispensable for the exist- ence of many terrestrial cavernicoles. The situa- tion is similar to that observed in young lava caves which are almost always bare and very clean. The sterility of granitic terrains was also confirmed later by the study of the upper hypogean fauna, as will be described in the following chapter. RECOGNITION OF THE UPPER HYPOGEAN ZONE Alongside of the faunal investigation of artificial cavities, we tried a more direct method of finding out terrestrial troglobionts from their supposed natural habitats, that is, excavation from the surface. This was a laborious and time-consuming work, so that its progress was slow and expected results were not gained for some time. After 1975, however, we were able to locate several places inhabited by anophthalmic trechine beetles, and realized that they were always met on or near the bedrock beneath thick layers of soil. Though scanty, these data seemed to indicate that terres- trial troglobionts could be regularly found in Narrow spaces just above the bedrock, and if so, we could locate their habitats more easily by looking for such spots as the overlying soil mantle was relatively thin. Keeping this in mind, we continued our searches for anophthalmic trechines mainly in the vicinities of Lake Biwa-ko, because that area was rather easily accessible and looked promising judged from the data obtained by cave explorations. Our expectation was soon realized, especially by the painstaking efforts of Yoshiaki Nishikawa, who alone discovered six new species of anophthalmic trechines from outside caves between the autumn of 1978 and the summer of 1980. They were described in 1980, together with other species, in a revision of the trechine beetles belonging to the group of Trechiama ohshimai [33]. Of the twenty- two species recognized in this paper, six were then 600 S.-I. UENO known only from caves of some kind (two of the six were later found in extra-cave habitats), thir- teen were obtained from the underground habitats we specially looked for, and the remaining three were taken from both inside and outside caves. Thus, it was proved beyond all reasonable doubt that so-called troglobiontic trechines were not confined in caves but widely occurred in narrow spaces between the soil layer and the bedrock. At that time, I regarded this habitat as the lowest zone of the endogean domain, though it was unusually deep and looser than ordinary endogean environ- ment. In the same year, Juberthie and his colleagues published the result of their study on the fauna of a zone below the deepest layer of the soil, conclud- ing that terrestrial cavernicoles were not confined in calcareous terrains but widely distributed in the shale and crystalline rock areas [34, 35]. They considered that the extra-cave environment suit- able for the existence of terrestrial troglobionts was a zone extending just above the bedrock, which was characterized by its high porosity due to the presence of fissures and interconnecting spaces. Because of this feature and of the regular occurrence of troglobionts, they distinguished the zone from the endogean domain and named it “le milieu souterrain superficiel”. Their conclusion perfectly accorded with ours, though the methods of approach were different. While we dug out eyeless animals directly from their habitats, they dug holes into the ground, inserted baited traps and attracted troglobionts. The latter method was not very successful in Japan though we tried it later, probably because of the low population density of Japanese forms. One of the most important achievements made by the French scholars was the discovery of undoubted connections between a limestone cave and the milieu souterrain superficiel. This was made at Moulis on the Pyrénées in southern France, where a subterranean laboratory had been established in a limestone cave. Placing baited traps in a scree on the hillside above the laboratory cave, they obtained four troglobiontic species of bathysciine beetles; two of them were native to the area, but the other two were imported species which had escaped from terraria and settled down in the cave [36, 37]. This was an irrefutable proof that the extent of their habitat was not restricted to the cave itself but spread out all over the milieu souterrain superficiel. At present, it is indisputable that many terres- trial cave animals distributed in temperate regions originated in the milieu souterrain superficiel, or the upper hypogean zone. Specialized upper hypogean fauna exists from near the seashore to the height of at least 2,000 m (Figs. 7 and 8). It has been unknown in certain areas, for instance, pure granitic terrains and alluvial plains. The former is sterile because it is usually sandy and devoid of clay or silt. However, terrestrial troglobionts may survive even on granitic hills, if there are certain oases suitable for their existence. An example is Z0Ozu-san in northeastern Shikoku, which is a small granitic hill largely devoid of upper hypogean fauna. In spite of such an unfavourable condition, an anophthalmic trechine beetle endemic to the hill occurs in the upper hypogean zone in a small andesitic gully on its northeastern slope [38]. The sterility of alluvial plains can be ascribed to the Fic. 7. A schistous gully on Okuminagawa-yama in central Shikoku, which harbours an anophthalmic trechine beetle of the genus [shikawatrechus. Derivation of Cave Animals 601 Fic. 8. Excavation of an upper hypogean habitat of an Ishikawatrechus on Okumina- gawa-yama. Fic. 9. Schematic cross section of a colluvium showing the habitat of troglobionts. UHZ-upper hypogean zone; S-soil layer. 602 S.-I. UENO absence of continuous spaces under the earth. To our present knowledge, upper hypogean habitats of terrestrial troglobionts can be located without much difficulty in small gullies or at the sides of moderate-sized streams (Fig. 9). They are invariably found beneath colluvia emplaced under steep slopes and fed by seepages. The thickness of the soil mantle is usually 50 cm or more, but where the mantle is thinner, terrestrial troglobionts can be met at the depth of only 10-20 cm. Unfortu- nately, however, favourable colluvia are not very common, and once they are excavated, their recovery cannot be expected within a short time. Besides, population density of Japanese troglo- bionts is much lower than that of continental forms. This makes procuration of adequate mate- rials very difficult. We know that specialized upper hypogean fauna exists over most of the Japanese Islands and that it consists of various groups of terrestrial troglobionts, but most of them have not yet been studied even taxonomically. For in- stance, white eyeless polydesmid millipedes have been known from many stations in West Japan, but always from a single female which is useless for taxonomic study. Only the exception is trechine beetles, which are, though also rare, fairly well known through my own efforts and unfailing collaborations of many friends of mine. After the discoveries made in Japan and France, upper hypogean fauna was also found in Romania [39]. It has been unknown in the United States [40], but must exist there, especially on the Appalachian Mountains where a very rich cave fauna has been reported. Recent discovery of an anophthalmic trechine beetle in an abandoned coal mine in Kentucky is an infallible indication of its existence [41]. CONCLUDING REMARKS Terrestrial cavernicoles comprise many groups of animals derived from various sources. Recent investigations of tropical and subtropical cave faunas have shown that there exist relatively few but very interesting terrestrial troglobionts in the tropics [42, 43]. Derivation of those tropical troglobionts may not be the same as that of temperate ones; for instance, herbivorous cixiid homopterans [44] and extremely long-legged re- duviid heteropteran [45], both from Hawaiian lava caves, may have entered underground through the spaces around tree roots, and small eyeless cockroaches of the Ryukyu Islands [46] may have colonized limestone caves through porous coral reefs. However, a great majority of terrestrial troglo- bionts occur in the temperate areas of both the Northern and Southern Hemispheres, and most of them seem to originate in the upper hypogean zone. Since they are invariably hygrophilous, they probably have their roots in ripicolous ancestors, which lived under stones on water edges of narrow streams. Their colonization in the upper hypogean zone may have been initiated by dispersal along seepages, debouchures of which usually attract various hygrophilous animals, especially in drier seasons. Continuous spaces in the upper hypogean zone, or intricate microcaverns, are equivalent to caves in an ordinary sense for such minute crea- tures as insects and other terrestrial arthropods. We have to realize clearly that “caves”, which are defined as “underground cavities large enough to permit human entrance”, are an artificial concept not applicable to small animals. The above conclusion cannot be applied to all terrestrial troglobionts even in the temperate zone. Notable exceptions are those derived from endo- gean ancestors and from pholeophilous ones. Cavernicoles of endogean origin can be recognized by a thickset or parallel-sided body form and relatively short appendages (Fig. 10). Their close relatives are always found in the endogean do- main, not in the upper hypogean zone. Probably descended from humicolous ancestors, these endo- gean animals may have found their way into depths of soil layer. Most of them remain there nowa- days, but there are some that have penetrated deeper and settled down in caves. Contrary to the cave animals of endogean origin, descendants of pholeophiles, mostly represented by guanobionts, usually do not show high specialization adaptive to subterranean life. They have become cavernico- lous only because deposits of bats’ guano are found in caves. There are, however, exceptions like the springtails belonging to the Aphoromma—Coeco- loba group, which are all troglobiontic and have Derivation of Cave Animals 603 0.8mm Fic. 10. Stygiotrechus nishikawai S. Uéno from Gonji- ana Cave/Mine on the Kui Peninsula, an anophthal- mic trechine beetle derived from an endogean ancestor. undergone intensive allopatric speciation [47]. The realization that most terrestrial troglobionts occurring in temperate areas originated in the upper hypogean zone has broadened the scope of biospeology to a considerable extent. We can now fill in wide blanks in our knowledge about the distribution of terrestrial cave animals, which was formerly gained by the investigation of sporadical- ly distributed limestone caves and lava tubes. In the Japanese Islands, about 130 species of anoph- thalmic trechine beetles have been known from natural caves and endogean habitats. Faunal investigation of artificial cavities and the upper hypogean zone has already doubled the number, and new findings are still being accumulated at a considerable speed. Many new species have been found in such areas wholly devoid of natural caves as the eastern part of the Chigoku Hills in western Honshu [48] and coastal area on the Japan Sea side of central Honshu (unpublished data). According- ly, we can analyse phylogeny and distribution of Japanese trechines on a much sounder basis than before (Fig. 11). The discovery of the upper hypogean fauna has also revealed that terrestrial troglobionts occur in such areas as were immersed by Pleistocene transgressions. Endemic species are commonly found in such areas immersed in the early Pleis- tocene as the Hokuriku District, Niigata region and the Mogami-gawa drainage area in northeast- ern Honshu, which indicates that their speciation must have taken place after the subsequent regres- sion. Even in certain areas immersed in the last Interglacial Age, there sometimes occur endemic trechine beetles and other arthropods. These evidences, together with the fact that many troglo- bionts are endemic to lava flows only 8,000—13,000 years old, suggest that the speciation of troglo- bionts must have progressed much more rapidly than it is generally supposed. Barr cited previous authors’ calculations that 100,000-1,000,000 gen- erations would be required for accomplishment of the loss of eyes, considering that troglobionts had colonized caves as late as the Last Interglacial, about 300,000 years ago [49]. Based upon the data on reproductive isolation and genetic distance, Sbordoni has surmised that the time required for speciation in caves is rather long, possibly not less than 5X 10°-10° years [50]. I am not in a position to argue if the calculations presented by these authors are correct or not, as I am not a geneticist, but I am fully satisfied that colonization of caves including microcaverns and subsequent speciation of troglobionts usually took place after the Last Interglacial Age. Preadaptation to subterranean existence may require a longer time, so that the calculations cited above may include it if they are really correct. Z Recently, Juberthie proposed a new definition of the subterranean environment, describing that it consists of all the intercommunicating cavities of various sizes isolated from the surface and char- acterized by the total darkness and relatively stable climatic condition [51]. He then developed his idea and built up a hypothesis on the colonization of caves by terrestrial troglobionts and _ their subsequent isolation and speciation [52]. His discussion seems largely reasonable, although his explanation of the role of glacial—interglacial climatic changes seems to me better interpreted as S.-I. UENO 604 Fic. 11. Maps showing the distribution of anophthalmic trechine beetles in Japan. Map A is based solely on the species known from limestone caves. Map B is drawn from our present knowledge enlarged by the discoveries of mine and upper hypogean faunas; the blank at the southeastern part of the Island of Shikoku was to some extent filled after the manuscript of this paper had been sent to the press. hypogean fauna, but there still remain many other problems not yet cleared up. It is to be hoped that more intensive studies will be made from different that of stadial—interstadial ones in the Last Glacial Age. Anyway, biospeology has made a consider- able progress by the discovery of the upper Derivation of Cave Animals aspects of cave animals so as to develop the biospeology in a harmonious way. 10 11 1 13 14 15 16 REFERENCES Viré, A. (1900) La Faune Souterrain de France. 159 pp. J.-B. Bailliére et fils, Paris. Racovitza, E.G. (1907) Essai sur les problémes biospéologiques. Biospéologica, I; Arch. Zool. exptl. gén., (IV), 6: 371-488. Jeannel, R. (1926) Faune cavernicole de la France, avec une étude des conditions d’existence dans le domaine souterrain. Encycl. ent., 7: 1-334, 15 pls. Paul Lechevalier, Paris. Jeannel, R. (1943) Les fossiles vivants des cavernes. L’Avenir de la Science, (n.s.), (1): 1-321, 12 pls. Gallimard, Paris. Vandel, A. (1964) Biospéologie—La Biologie des Animaux Cavernicoles. XVIII+619 pp. Gauthier- Villars, Paris. Vejdovsky, F. (1882) Thierische Organismen der Brunnenwasser von Prag. 70 pp., 8 pls. Selbstverlag, Prag. Spandl, H. (1926) Die Tierwelt der unterirdischen Gewasser. Spelaologische Monographien, 11: I- XII+ 1-235. Speldologisches Institut, Wien. Chappuis, P. A. (1927) Die Tierwelt der unterirdi- schen Gewasser. Die Binnengewasser, 3: i-v+1- 175, 4 pls. E. Schweizerbart, Stuttgart. Delamare Deboutteville,C. (1960) Biologie des Eaux Souterraines Littorales et Continentales. 740 pp., 2 folders. Hermann, Paris. Ijima, I. and Kaburaki, T. (1916) Preliminary de- scriptions of some Japanese triclads. Annot. zool. japon., 9: 153-171. Uéno, S.-I. (1957) Blind aquatic beetles of Japan, with some accounts of the fauna of Japanese subterranean waters. Arch. Hydrobiol., Stuttgart, 53: 250-296, 3 pls. Uéno, S.-I. (1985) The derivation of terrestrial cave animals. Zool. Sci., Tokyo, 2: 852. Uéno, S.-I. (1986) Recent development of bio- speology in Japan. J. speleol. Soc. Japan, 10th Anniv. spec. Vol., 17-23. (In Japanese, with En- glish summary.) Uéno, S.-I. (1971) The fauna of the lava caves around Mt. Fuji-san. I. Introductory and historical notes. Bull. natn. Sci. Mus., Tokyo, 14: 201-218, pls. 1-4. Torii, H. S. (1962) A consideration of the distribu- tion of some troglobionts of Japanese caves III. Jpn. J. Zool., 13: 423-440. Torii, H. S. (1965) Die Lavahéhlen am Fue des Fudschijama (Japan) und deren Tierwelt. Die Hohle, Wien, 16: 18-24. 7 18 19 20 21 22 28 24 25 26 A 28 29 30 DIL 32 33 34 605 Kumano, M. (1943) The animals of the lava caves around Mt. Fuji. Hakubutsugaku Zasshi, Tokyo, 39 (73): 51-56. (In Japanese.) Uéno, S.-I. (1971) Notes on the lava cave fauna of Mt. Fuji. Fuji-san: Results co-op. sci. Surv. Fuji, 752-759. Fuji Kyuko, Tokyo. (In Japanese, with English summary. ) Caumartin, V. (1959) Quelques aspects nouveaux de la Microflore des cavernes. Annls. Spéléol., 14: 147-157, pl. 5. Uéno, S.-I. and Morimoto, Y. (1970) The fauna of the insular lava caves in West Japan. I. General account. Bull. natn. Sci. Mus., Tokyo, 13: 443-454, 1 folder, 1 pl. Uéno, S.-I. (1977) The fauna of the lava caves in the Far East. Proc. 6th int. Congr. Speleol., Olomouc 1973, 5: 237-242. Howarth, F. G. (1972) Cavernicoles in lava tubes on the Island of Hawaii. Science, Washington D.C., 175: 325-326. Howarth, F. G. (1980) The zoogeography of spe- cialized cave animals: a bioclimatic model. Evolu- tion, Lawrence, 34: 394-406. Uéno, S.-I. (1972) A new anophthalmic Trechiama (Coleoptera, Trechinae) found in an old mine of the Izu Peninsula, Central Japan. Annot. zool. japon., 45: 111-117. Yaginuma, T. (1972) The fauna of the lava caves around Mt. Fuji-san. IX. Araneae (Arachnida). Bull. natn. Sci. Mus., Tokyo, 15: 267-334. Uéno, S.-I. (1972) A new endogean Trechiama (Coleoptera, Trechinae) from the northern side of Central Japan. Annot. zool. japon., 45: 42-48. Nomura, S. (1959) On the fauna of lime-stone caves in Oita Prefecture (9). Collect. & Breed., Tokyo, 21: 174-175, 177. (In Japanese, with English title.) Uéno, S.-I. (1959) A new troglobiontic trechid found in the mines of central Kyushu. Mem. Coll. Sci. Univ. Kyoto, (B), 26: 285-290. Uéno, S.-I. (1960) A new species-group of the genus Rakantrechus (Coleoptera, MHarpalidae). Mem. Coll. Sci. Univ. Kyoto, (B), 27: 37-44. Balazuc, J. (1962) Troglobies des cavités artificiel- les. Spelunca (4° Sér.), Mém., (2): 104-107. Uéno, S.-I. (1977) The biospeleological importance of non-calcareous caves. Proc. 7th int. speleol. Congr., Sheffield 1977, 407-408. Uéno, S.-I. and Kashima, N. (1978) An Introduc- tion to Speleology. Exploring the Underground World in Darkness. Blue Backs, B-361. 233 pp. Kodansha, Tokyo. (In Japanese.) Uéno, S.-I. (1980) The anophthalmic trechine bee- tles of the group of Trechiama ohshimai. Bull. natn. Sci. Mus., Tokyo, (A), 6: 195-274. Juberthie, C., Delay, B. and Bouillon, M. (1980) Sur l’existence d’un milieu souterrain superficiel en 35) 36 Sf 38 39 40 41 42 43 606 zone non calcaire. C. R. Acad. Sci. Paris, (D), 290: 49-52. Juberthie, C., Delay, B. and Bouillon, M. (1980) Extension du milieu souterrain en zone non- calcaire: Description d’un nouveau milieu et de son peuplement par les Coléoptéres troglobies. Mem. Biospéol., 7: 19-52. Juberthie, C. and Delay, B. (1981) Ecological and biological implications of the existence of a “super- ficial underground compartment”. Proc. 8th int. Congr. Speleol., Bowling Green 1981, 1: 203-206. Juberthie, C., Bouillon, M. and Delay, B. (1981) Sur l’existence du milieu souterrain superficiel en zone calcaire. Mém. Biospéol., 8: 77-93. Uéno, S.-I. (1981) New anophthalmic Trechiama (Coleoptera, Trechinae) from northern Shikoku, Japan. J. speleol. Soc. Japan, 6: 11-18. Juberthie, C., Delay, B., Decou, V. and Racovita, Gh. (1981) Premiéres données sur la faune des microespaces du milieu souterrain superficiel de Roumanie. Trav. Inst. Spéol. “Emile Racovitza”, 20: 103-111. Barr, T. C., Jr. and Holsinger, J. R. (1985) Specia- tion in cave faunas. Ann. Rev. Ecol. Syst., 16: 313- S37. Barr, T. C., Jr. (1986) An eyeless subterranean beetle (Pseudanophthalmus) from a Kentucky coal mine (Coleoptera: Carabidae: Trechinae). Psyche, Cambridge, 93: 47-50. Howarth, F. G. (1973) The cavernicolous fauna of Hawaiian lava tubes, 1. Introduction. Pacif. Ins., 15: 139-151. Peck, S. B. (1977) Recent studies on the inverte- S.-I. UENO 44 45 46 47 48 49 50 Sil a2 brate fauna and ecology of sub-tropical and tropical American caves. Proc. 6th int. Congr. Speleol., Olomouc 1973, 5: 185-194. Fennah, R. G. (1973) The cavernicolous fauna of Hawaiian lava tubes, 4. Two new blind Oliarus (Fulgoroidea: Cixiidae). Pacif. Ins., 15: 181-184. Gagné, W.C. and Howarth, F.G. (1975) The cavernicolous fauna of Hawaiian lava tubes, 7. Emesinae or thread-legged bugs (Heteroptera: Re- duviidae). Pacif. Ins., 16: 415-426. Asahina, S. (1974) The cavernicolous cockroaches of the Ryukyu Islands. Mem. natn. Sci. Mus., Tokyo, (7): 145-156, pl. 18. Yosii, R. (1956) Monographie zur Hohlencollem- bolen Japans. Contr. biol. Lab. Kyoto Univ., (3): i+1-109+ 1-22, 50 pls. Uéno, S.-I. (1985) The group of Trechiama oni (Coleoptera, Trechinae) —its distribution and dif- ferentiation—. Mem. natn. Sci. Mus., Tokyo, (18): 163-198. Barr, T. C., Jr. (1968) Cave ecology and the evolu- tion of troglobites. Evol. Biol., New York, 2: 35- 102. Sbordoni, V. (1982) Advances in speciation of cave animals. Mechanisms of Speciation, ed. by C. Barigozzi, 219-240. Alan R. Liss, New York. Juberthie, C. (1983) Le milieu souterrain: étendue et composition. Mém. Biospéol., 10: 17-65. Juberthie, C. (1984) La colonisation du milieu souterrain; théories et modéles, relations avec la spéciation et l’évolution souterraine. Mém. Bio- spéol., 11: 65-102. ZOOLOGICAL SCIENCE 4: 607-611 (1987) © 1987 Zoological Society of Japan Frequency Block in the Giant Axons of a Sabellid Worm, Pseudopotamilla occelata TAKASHIRO Hicucui, Hiroyuki NAKAMURA!, KATSUHIKO SAwaucHE! and HirosH1 OkumurRA!” Department of General Education, Higashi-Nippon-Gakuen University, Ishikari-Tobetsu, Hokkaido 061-02, and 'Akkeshi Marine Biological Station, Hokkaido University, Akkeshi, Hokkaido 088-11, Japan ABSTRACT—In the sabellid worm, Pseudopotamilla occelata, there is a graded decrease in the capacity for the conduction of action potentials along the giant axons. Failure of a faithful conduction of successive action potentials under a constant frequency stimulation, namely, the frequency block, occurs near the mid-body position. All action potentials are eventually blocked in an area posterior to this. These phenomena require neither a special geometry of the axon nor the effects of the accumulation of much preceding axon activity. It is likely that there are gradual changes in the properties of the membrane along the axon. Temporal changes in the properties of the membrane are also involved in the mechanism of the frequency block. INTRODUCTION Body contraction in the sabellid worm, Pseudo- potamilla occelata, during the rapid withdrawal reflex occurs in the anterior half of the body. End-to-end shortening is never observed. This phenomenon is caused by the conduction block of single action potentials in the giant axons, whose activity induces the contractions of segmental longitudinal muscles, at the mid-body [1]. Conduction block of single action potentials in the giant axons of this worm does not require any special geometrical features of the axons. Although isthmus-like structures within the giant axon in the mid-body position (around the 40th body segment) are characteristic, the conduction block occurs in a region anterior to these struc- tures, and does often in a region of uniform geometry [1]. Thus, in this worm, it appears that the low safety factor for conduction is not depend- ent upon geometrical factors such as a sudden Accepted March 26, 1987 Received February 17, 1987 * Present address: Department of Biology, Japan Women’s University, Mejirodai, Bunkyo-ku, Tokyo 112, Japan. increase in the membrane surface area [2-5] or axonal bifurcation [6-10]. Current injection experiments on the giant axons of this worm have revealed that an area of low safety factor for conduction was relatively large around the apparent site of the conduction block of single action potentials [1]. Following this result, we demonstrate in this papaer a frequency block which develops towards the site of the conduction block of single action potentials, and show that there is an inherent gradient in the ability to conduct action potentials along the giant axons of Pseudopotamilla occelata. MATERIALS AND METHODS Sabellid worms, Pseudopotamilla occelata, were obtained near the Marine Biological Station of Hokkaido University (Akkeshi, Hokkaido) and maintained in an aquarium in circulating sea water (at 10-15°C) before use. Experiments were carried out on semi-dissected worms pinned in a chamber filled with natural sea water. Worms having 100-140 body segments (each segment of ca. 1 mm in length) were selected for experiments. The first nine segments form the head and thorax, 608 T. Hicucui, H. NAKAMURA et al. and the remainder form the abdomen. The giant axons in the dorsomedial position within the ventral nerve cord were exposed, by removing the body wall on the dorsal surface and overlying digestive tract, from the posterior half of the thorax to the middle of the abdomen. The axons were stimulated extracellularly in the thorax by twin or bursts of rectangular current pulses (50- 100 psec in duration). Action potentials were recorded intracellularly, with KCl-filled glass mi- croelectrodes (10-20 MQ in resistance), and suc- cessively at several points along the giant axon. After the electrophysiological experiments, the specimens were fixed in Bouin’s fluid. Serial transverse sections (10 um in thickness) were cut and stained with hematoxylin-eosin solution and observed under the light microscope. RESULTS Previous experiments [1] have shown that the region of the conduction block of single action potentials was effectively restricted to an area between the 20th and the 40th body segments. He i This experimental result also indicated that the single action potentials could consistently be con- ducted within the anterior part of the abdomen. A gradual developement of a frequency block along the giant axon was found to occur in the anterior part of the abdomen. In response to electrical stimulation of giant axons, a train of three action potentials with an interspike interval of 10 msec were elicited (Fig.1A). Control responses were recorded in the 13th segment. In most cases the frequency block occurred more posterior to the 15th segment. Faithful conduction of all action potentials was observed in the 18th segment. The amplitude of the last (the third) action potential decreased posteriorly. In the 21st segment, a reduced potential, which was due to the electrotonic spread of the blocked action potential, was observed. The second potential was blocked in the 23rd segment, resulting in successful conduc- tion of only the first action potential beyond this point. The remaining action potential (the first action potential) was then blocked in the 30th segment. The geometrical features of this axon, reconstructed from serial sections around the LU 5OmvV aN oo Bydiogt 10 msec ih St 100pm Pome ae ey eel Imm (2. ty 22 28. eee cleo Fic. 1. (A) Frequency block of the giant axon, and schematic representation of the arrangement of external stimulation (St) and intracellular recordings. Control responses (C) were recorded in the 13th segment. Each pair of control response and the response in the 18th (1), 21st (2), 23rd (3) or 30th (4) segment were recorded simultaneously. (B) Giant axon geometry around the region of the frequency block. Recording sites of 1-3 are indicated by closed circles. Numbers below the axon indicate the body segment. Frequency Block in the Giant Axons 609 100msec = |somv 5msec Fic. 2. Train and twin pulse experiments. (A) Responses to a train of pulses with a 10 msec interpulse interval. Numbers indicate the body segment where recordings were performed. (B) Responses to twin pulses with varying intervals in each segment corresponding to those in A. recording region (Fig. 1B), show only regular repetition of segmental constrictions, which are natural features of the giant axons of sabellid worms [11]. No special geometrical features could be observed which may be responsible for the frequency block. The responses at four different points along the giant axon to both trains of pulses (15 pulses with an interpulse interval of 10 msec) and twin pulses with varying intervals are shown in Figure. 2. In the 15th segment, the train of 15 action potentials could still be recorded, although a gradual de- crease in the amplitude of the action potentials was observed. A refractory period of approximately 2 msec was measured in this segment. In the 18th segment, all of the action potentials in the train were still recorded, but the gradual decrease in the amplitude was enhanced. The twin pulse experi- ment in this segment showed the prolongation of the relative refractory period. The frequency block clearly occurred at some point between the 18th and the 22nd segment. Only the initial four action potentials were conducted to the 22nd segment. The remaining action potentials were blocked possibly near the 22nd segment since the electrotonically spreading potentials were relative- ly large. In this region, the refractory period was prolonged to 7 msec. Electrotonic potentials with a delay of 3-6 msec between the two pulses also indicated that the region of refractoriness was near the recording site. No more than the first two action potentials were conducted to the 32nd segment. In this segment there were no electro- tonic potentials, indicating that the region of refractoriness was far from the recording micro- electrode. Although development of the frequen- cy block increased from the 22nd to the 32nd segment, there was no further prolongation of the refractory period in that region. In all of the preparations used (20 animals), the frequency block was consistently observed in a region anterior to the apparent site of the conduc- tion block of single action potentials. There was some variation in the development of the frequen- cy block along the axon between animals. When the interpulse interval was 10 msec, the number of action potentials which could be conducted along the axon reduced linearly in some preparations and non-linearly in other ones. DISCUSSION On the basis of the results of the present and previous studies [1], the giant axon in the anterior half of Pseudopotamilla has been divided into three areas with reference to the capacity for the conduction of action potentials (Fig.3B). They have been named (1) normal, (2) frequency blocking, and (3) blocking areas. In the normal area, which encompassed the head and thoracic segments to approximately the 15th segment, the giant axons show general characteristics commonly observed in many other axons. On the other hand, all action potentials were completely blocked in 610 T. Hicucui, H. NAKAMURA ef al. A =) : ees Fic. 3. Schematic representation of the giant axons of Pseudopotamilla occelata. (A) Diagram of a pair of giant axons. (B) Enlarged diagram of one of the giant axons in the abdomen. N: normal area. F: frequency blocking area. B: blocking area. I: region of isthmus-like structures. P: giant axon in the posterior half of the body. (C) Schematic representation of a decrease in the capacity for the conduction (solid line) and hypothetical reduction in the density of sodium channels (dotted line) along the giant axon. Note that a gradual decrease in the density of sodium channels occurs from the end of the normal area, and develops continuously over the frequency blocking and the blocking area. the blocking area, around the 30th segment (20th- 40th segment). In the frequency blocking area, namely, the area between the normal and the blocking areas, single action potentials could consistently propagate, but the frequency block of a train of action potentials developed gradually towards the blocking area. Although the bound- aries of these areas were not distinct and varied from preparation to preparation, a gradual change in the capacity for conduction along the axon was commonly observed. One common characteristic of both the conduc- tion block of single action potentials and the frequency block, which has by now been demon- strated in several species, is that the axons have a zone with a special geometry [2, 5-7]. A second common feature is that the conduction block appears following stimulation at high frequency producing an accumulation of extracellular potas- sium ions [5, 9]. However, on the basis of the histological study, the first possibility must be excluded as a possible mechanism for the frequen- cy block since the block occurs in an area of uniform geometry. The second feature must also be excluded since the occurrence of the frequency block was independent of the accumulation of extracellular potassium ions produced by preced- ing axon activity. In the previous paper [1], it was hypothesized that the conduction block of single action poten- tials might be induced by changes in the properties of the membrane, such as a reduction in the density of sodium channels [12, 13], and that the isthmus-like structures were secondary features associated with a change of membrane structure which was in turn responsible for the change in membrane properties. A gradual development of the frequency block may also be explainable if the density of sodium channels gradually decreases along the axon towards the blocking area (Fig. 3C). This assumes that both the frequency block and the conduction block of single action poten- tials are induced by the same factors, and that the frequency blocking area and blocking area are continuous, although there is a gradient in the degree of the change in membrane excitability. It is likely that the mechanism of the frequency block in this animal involves not only a local change in the membrane properties but also a temporal change in membrane excitability. The twin pulse experiment (Fig. 2) showed that the refractory period was 7 msec in both the 22nd and the 32nd segments. However, pulse trains in which the interpulse interval was 10 msec led to the block of action potentials after the first four and the first two action potentials in the 22nd and the 32nd segments respectively. This suggests that there is a cumulative effect of the preceding axon activity on the recovery of the membrane excitability. In a few preparations we observed a restoration of conduction during the spike train, i.e. intermittent conduction block (our unpublished data). Although the data are lacking, it is possible that the effects of the preceding action potentials, other than the accumulation of extracellular potassium ions, may be closely related to the local change of the membrane properties. This worm has a great capacity for regeneration. When the worm is cut at the mid-body position, the head and thorax regenerate from the cut end of Frequency Block in the Giant Axons the posterior half. Giant axons also regenerate to form the giant system in the head and thorax. The responses to current injection across the giant axon membrane during the course of this regeneration are similar to those around the blocking area in the intact animal, namely, rise of the threshold de- polarization and fall of the amplitude of the action potential in both cases [1, 14]. It is therefore conceivable that the phenomena observed during this study may be the result of changes in the membrane properties of the giant axons. ACKNOWLEDGMENT The authors wish to thank Dr. P. L. Newland, Hok- kaido University, for his reading and correcting of the manuscript. REFERENCES 1 Higuchi, T., Nakamura, H., Sawauchi, K. and Oku- mura, H. (1986) Withdrawal reflex and conduction block in the giant axon of a sabellid worm (Pseudo- potamilla occelata). J. Exp. Biol., 126: 433-444. 2 Parnas,I., Spira,M.E., Werman,R. and Berg- mann, F. (1969) Nonhomogeneous conduction in giant axons of the nerve cord of Periplaneta america- na. J. Exp. Biol., 50: 635-649. 3. Mellon, D. and Kaars, C. (1974) Role of regional cellular geometry in conduction of excitation along a sensory neuron. J. Neurophysiol., 37: 1228-1238. 4 Castel, M., Spira, M. E., Parnas, I. and Yarom, Y. (1976) Ultrastructure of region of a low safety factor in inhomogeneous giant axon of the cockroach. J. Neurophysiol., 39: 900-908. 5 10 Uh 12 13 14 611 Spira,M.E., Yarom, Y.and Parnas,I. (1976) Modulation of spike frequency by regions of special geometry and by synaptic inputs. J. Neurophysiol., 39: 882-899. Parnas, I. (1972) Differential block at high frequen- cy of branches of a single axon innervating two muscles. J. Neurophysiol., 39: 909-923. Grossman, Y., Spira, M. E. and Parnas, I. (1973) Differential flow of information into branches of a single axon. Brain Res., 64: 379-386. Hatt, H. and Smith, D. O. (1976) Synaptic depres- sion related to presynaptic conduction block. J. Physiol., 259: 367-393. Smith, D. O. (1980) Mechanisms of action potential propagation failure at site of axon branching. J. Physiol., 301: 243-259. Smith, D. O. (1980) Morphological aspects of the safety factor for action potential propagation at axon branch points in the crayfish. J. Physiol., 301: 261- 269. Mellon, D., Treherne, J. E., Lane, N. J., Harrison, J. B. and Langley, C. K. (1980) Electrical interac- tions between the giant axons of a polychaete worm (Sabella penicillus L.). J. Exp. Biol., 84: 119-136. Smith, D. O. (1977) Ultrastructural basis of impulse propagation failure in a nonbranching axon. J. Comp. Neurol., 176: 659-670. Raymond, S. A. and Lettvin, J. Y. (1978) After- effects of activity in peripheral axons as a clue to nervous coding. In ”Physiology and Pathology of Axons”. Ed. by S. G. Waxman, Raven Press, New York, pp. 203-225. Higuchi, T., Nakamura, H., Okumura, H. and Sawauchi, K. (1983) Propagation of action poten- tials in giant axons of sabellid worms, Pseudopota- milla occelata. Zool. Mag., 92: 526. (In Japanese) ZOOLOGICAL SCIENCE 4: 613-620 (1987) Electron Microscopic Observations of the Alveolar Brush Cell of the Bullfrog TOSHIAKI GOMI, AKIHIKO KIMURA, HIROMASA TSUCHIYA, TAKESHI HASHIMOTO, KAzuyosHt Hicasut' and SHozo Sasa! Department of Anatomy, School of Medicine, Toho University, Tokyo 143, and ‘Department of Histology, Kanagawa Dental College, Yokosuka 238, Japan ABSTRACT—The alveolar brush cells of bullfrogs were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). SEM showed brush cells with long, thick, unique microvilli of ca. 80-90 in number, existing in concave cavities in the capillary network. TEM showed slightly expanded cylindrical (columnar) brush cells. They were constricted in the area directly below the free cell surface of the lateral side of the cell, resulting in a flask or pear shape. Marked junctional complexes were found in the constricted part of the cells. The microvilli were about 0.8-1.0 4m in length and about 0.15-0.25 um in diameter. A large number of fine filaments extended from the top of the microvilli to the supranuclear region. Vesicles, and granules which had moderate electron density and various shapes were found from near the free cell surface to the supranuclear region. The most characteristic feature of brush cells is the presence of companion cells, which have osmiophilic lamellated bodies and poorly developed cell organs. From these morphologi- cal characteristics, our study suggested the possibility that the alveolar brush cells might have sensory or chemoreceptive functions in spite of the fact that it includes no nerve ending in addition to their © 1987 Zoological Society of Japan absorptive or secretory functions. INTRODUCTION Rhodin and Dalhamn [1] were the first to study the rat tracheal epithelium with an electron micro- scope. They were also the first to report the existence of non-ciliated cells with brush borders consisting of well-developed microvilli, and they named this cell the “brush cell”. Thereafter, many researchers reported the existence of brush cells in the respiratory system; these cells have been found in the nasal cavity [2-5], larynx [6], trachea and bronchi [7-14]. However, since Low [15], a large number of authors have studied the ultrastructure of the alveoli [16-21], while there are only a few reports on alveolar brush cells, the third kind of alveolar epithelial cell, other than the epithelial cells of Type I and Type II. Meyrick and Reid [12], Luciano et al. [22], Hijiya et al. [23] and Accepted March 16, 1987 Received November 5, 1986 Scheuermann et al. [24] reported on those of rats, Scheuermann and Timmermans [25] on those of red eared turtles, and Gomi [26] on those of striped snakes. We previously reported briefly that alveolar brush cells exist in bullfrogs [27]. We then made a further study in which a scanning electron microscope (SEM) and a transmission electron microscope (TEM) were used to identify brush cells among the alveolar epithelial cells. We observed the ultrastructure of these cells very carefully, and obtained some interesting findings. Here we compare the ultrastructure of the brush cells we observed with that reported in earlier reports, and we include some discussion on their functions. MATERIALS AND METHODS Commercially available adult male bullfrogs (Rana catesbeiana) were used. Their body weights were about 200-250 g. 614 T. Gomi, A. Kimura et al. After being rendered unconscious by a blow to the head, they were thoractomized, and the lungs were quickly extracted. The extracted organs were sliced into sections of 1-2 mm square (for TEM) and 5—6 mm square (for SEM), and the specimens were fixed in 2% glutaraldehyde buffered with 0.1M sodium cacodylate (pH 7.4, at 4°C) for 2 hr. At the beginning of pre-fixation, deaeration of the specimens was carried out using an aspirator or an injection syringe. Thereafter, the specimens were rinsed with the same buffer, and post-fixation was achieved with a treatment of 1% osmium tetroxide (at 4°C) for 1.5 hr. Specimens for SEM After fixation, de- hydration with ethanol was done. Specimens were dried to the critical point with isoamyl acetate, and after they were dried thoroughly they were attached to a stub with silver conducting paint for double evaporation using carbon and platinum- paradium. They were examined and photo- graphed at an acceleration voltage of 15 kV using a JXA-S0A (JEOL). Specimens for TEM After fixation, de- hydration with ethanol was performed. The specimens were embedded in Epon by ordinary methods, after which they were thinly sectioned with a diamond knife, and this was followed by electron-staining with uranyl acetate and lead citrate. Observation was then carried out with a JEM-100B (JEOL) under an accelerating voltage of 80 kV and with an H-600 (HITACHI) under an accelerating voltage of 75 kV. RESULTS Observation by SEM With SEM we could see that brush cells had long and thick microvilli of a unique form (Figs. 1-3), which clearly distin- guished them from other epithelial cells. Brush cells existed in the concave cavities of the capillary network. The form of their microvilli was clearly different from that of microvilli of other epithelial cells covering the capillary. Brush cells had about 80-90 microvilli of uneven heights. They were protuberant in the center. Observation by TEM The alveolar brush cells were slightly expanded cylinders (columnar), and constricted in the area directly below the free cell surface of the lateral side of the cell resulting in a flask or pear shape (Figs. 4-6). The upper part of the cell cytoplasm slightly expanded to the alveolar lumen. As for the form of connection with the adjoining cells, marked junctional complexes existed in the area where cells were constricted directly below the free cell surface (Figs. 4a, 5a, 7 and 8), while lateral interdigitation was found in the lower area (Fig. 6). The microvilli were about 0.8-1.0 ~m in length and about 0.15-0.25 um in diameter. Inside these unique microvilli, numer- ous fine filaments extended from the top to the supranuclear region of the cytoplasm (Figs. 4a, Sa, 7 and 8). Most bundles of filaments ran, and were markedly developed, along the central long axis of the brush cells. Vesicles existed in apical cytoplasm between filament bundles near the free cell surface, with their orifices found in the cellular surface between microvilli (Figs. Sa and 7). Vacuoles existed in lateral side of the apical cytoplasm (Fig. 4a). Granules, which had moderate electron density and different forms ranging from almost round to comma-shaped beads, were present in the region extending from the area near the free surface to the supranuclear region (Figs. 4a and 8). Oval to long rod-shaped mitochondria, endoplasmic re- ticula and free ribosomes and microtubules existed in the supranuclear region (Figs. 4a, 5a, 7 and 8). Immediately above the nucleus, oval lysosomes and Golgi apparatus existed (Figs.5a and 7). Golgi apparatus was composed of several lamellae, and both edges of the lamellae expanded substan- tially in some Golgi apparatus (Fig. 7). Glycogen granules were scattered throughout the cytoplasm, except for the area where filaments developed in the top of the central long axis of the brush cells (Figs. 4-6). Microtubules were present at the supranuclear region where filament bundles be- came sparse (Fig. 4a). The nucleus occupied about a third to a half of the cell, and was omnipresent in the base of the cytoplasm with a concave, irregular form. The chromatin of the nucleus aggregated in the periphery of the karyoplasm, and usually a nu- cleolus was observed (Figs. 4-6). On one side of the adjacent cells, the very thin cytoplasmic processes of the alveolar epithelial Alveolar Brush Cell of Bullfrog 615 Fics. 1-3. 3 ® $ c ° — * PO) | 7) ," Pai" Pi dag ; ri en ah Alveolar brush cells (arrow) are seen here in concave cavities of the capillary network. Long, thick and unique microvilli, about 80-90 in number, can be seen protruding into the alveolar lumen. (Inset: High magnification of the alveolar brush cell in Fig. 1). (Fig. 1 1400, Inset 12200, Fig.2 x7000, Fig. 3 6000) cells, which continuously covered the capillary, extended further to cover the upper side of the brush cells. The other side neighbored with a cell that was a slightly expanded, cylinder-shaped cell (companion cell) in most cases (Figs. 4-6). Most of the cytoplasm of this companion cell was occupied by an irregularly-shaped nucleus, and contained only a few osmiophilic lamellated bodies (Figs.5 and 6). Cell organs were poorly de- veloped, and mitochondria, rough endoplasmic reticula and free ribosomes were scattered in the cytoplasm. Most of these cells extended very thin cytoplasmic processes onto the neighboring capil- lary (Figs. 4-6). Between companion cells and brush cells, a very small intercellular space existed in most cases (Figs. 4 and 6), but sometimes there was a Slightly expanded intercellular space be- tween the lower parts of their bodies (Fig. 5). DISCUSSION On the surface of the alveolar brush cell, there were long, thick and peculiar microvilli, and fine filaments characteristically extending from the top of the microvilli to the supranuclear region of the cytoplasm, which phenomenally differentiated it from other alveolar epithelial cells. As we previously reported, brush cells are oval 616 T. Gomi, A. Kimura et al. Fics. 4-6. Flask-shaped (pear-shaped) alveolar brush cells (BC) showing marked junctional complexes (arrows) near the free cell surface. Each alveolar brush cell is accompanied by a companion cell (CC). Most of the companion cells contain only a few osmiophilic lamellated bodies (LB) (5, 6). Sometimes there is a slightly expanded intercellular space (+) between the lower parts of companion cell and brush cell (5). (Fig. 4 3100, Fig.5 3500, Fig. 6 5200) (spherical), expanding toward the both sides of the __ brush cells is not fixed, but varies with function, cell [27]. Besides their oval shape, this study found which would account for the different forms of some brush cells that were slightly expanded and __ brush cells that have been found in other organs cylinder-shaped. This suggests that the shape of and tissues. Numerous fine filaments in the long Alveolar Brush Cell of Bullfrog 617 Fics. 4a,5a,7and8. High magnification of the supranuclear regions of the alveolar brush cells. Figs. 4a, 5a in the facing page are high magnification of Figs. 4 and 5. Long, thick and unique microvilli can be seen protruding from the cell surface into the alveolar lumen. Inside these unique microvilli, there are numerous fine filaments (FL) extending from the top to the supranuclear region (4a, 5a, 7). In addition to filaments, there are vesicles (arrows) (5a, 7), vacuoles (4a), granules (GR) (4a, 8), mitochondria (5a, 7, 8), Golgi apparatus (GA) (5a, 7), endoplasmic reticula, free ribosomes, glycogen granules (7, 8), lysosomes (LY) (7), microtubules (MT) (4a) and a centriole (CT) (8) in the cytoplasm. The orifice of a vesicle can be seen at the cell surface (thick arrow) (5a, 7). (Fig. 4a 12200, Fig. 5a 13600, Fig. 7 9800, Fig. 8 x 13600) axis of the cell shrink depending on the function of the cell, which may change the shape of the entire cell. On one side of the adjacent cells, the very thin cytoplasmic processes of the alveolar epithelial cells covered the upper side of the brush cells. The other side neighbored with a companion cell. Meyrick and Reid [12] and Hijiya et al. [23] reported that brush cells neighbor the very thin cytoplasmic processes of the Type I cell (squamous lining type) and the Type II cell including osmiophilic lamellated bodies (cuboidal secretion type). Most companion cells that were found in this study possessed the features of both Type I and Type II alveolar cells, which are usually found in the alveolar epithelial cells of the lungs of higher animals. Luciano and Reale [28] reported that there is a very small intercellular space between brush cells and adjacent cells. A number of lateral interdigita- tions penetrate deep in the adjacent cells to form desmosomes with the adjacent cells, which differs from our result. This study showed that a very small intercellular space existed between most brush cells and companion cells, while there was a somewhat larger intercellular space between the lower parts of some brush cells and their compan- ion cells. Differences in cellular activity may be responsible for the expansion or shrinkage of intercellular space. Various theories have been proposed concern- ing the function of brush cells; including that they are undifferentiated cells [6-8, 29], that they have an absorptive function [1, 12, 30], and that they are receptors [11, 12, 23, 31]. Rhodin [7], Spoendlin [6], Leeson [8], and Johnson and Young [29] stated that brush cells are undifferentiated cells. Howev- er, we do not think this is true for the following reasons: 1) numerous filament bundles do not exist in undifferentiated cells [28], and 2) cell organs develop poorly in undifferentiated cells. Rhodin and Dalhamn [1] and Meyrick and Reid [12] suggested that brush cells are absorption cells, because they resemble absorption celis of the 618 T. Gomi, A. Kimura et al. intestinal epithelium in that they have a brush border. However, Luciano and Reale [28] negated this when they reported that they did not found any trace of horseradish peroxidase in the cyto- plasm of brush cells in the gallbladder to which those substances were applied. On the other hand, Qwarnstr6m and Hand [30] stated that horseradish peroxidase that was applied to the main excretory duct of the submandibular glands of rats was absorbed by brush cells. Our study showed vesicles existing from apical cytoplasm between filament bundles near the free cell surface to the supranuclear region, with their orifices existing on the cellular surface between microvilli. Almost round to comma-shaped bead granules that had moderate electron density existed with these vesi- cles and vacuoles in the supranuclear region. This study did not clearly show whether or not these granules were secretory granules, but the fact that these granules were located only in the supranu- clear region, as well as their form, suggest a possibility that they may have an absorptive function, as mentioned above, or a secretory function. Concerning the function of the brush cell, Luciano et al. [11] reported that the tracheal brush cell in rats is a chemoreceptor, since it forms an afferent synapse with a dendrite, 1.e., the epithelio-neural junctions; they also suggested that the alveolar brush cell might have a chemorecep- tive function, since its shape, though the nerve ending had not found, was the same as that of the tracheal brush cell. Meyrick and Reid [12] also support the chemoreceptive function idea. According to Hijiya et al. [23], administration of Bleomycin increased the number of alveolar brush cells, and SEM pictures revealed that the microvilli were protruding toward the alveolar duct, not from the top of the cell, but from the side, and that a structure considered to be of unmyelinated nerve fibers was found near the basement membrane of this cell, thus possibly having the activities of a kind of chemoreceptor. According to the observa- tion by Higashi et al. [31] of brush cells in the gallbladder, the microvilli and cell organs of the brush cells had a similar shape to the type I taste cell of the taste buds [32], in the base of which basal cells exist. From these results, they sug- gested that the brush cells act as receptors, and the basal cells have some functional relationship with the brush cells. In the alveolar brush cell of the bullfrog, the form of the microvilli and the presence of filaments, microtubules, granules and vesicles in cytoplasm resemble that of the type I taste cell of the taste buds [33]. Moreover, the alveolar brush cell resembles the fifth cell type of the olfactory epithelium in that the upper part of the cell cytoplasm slightly expanded to the alveolar lumen, unique microvilli protruded from the sur- face of the cell, a large number of fine filaments extended from the top of the microvilli to the supranuclear region, and it has marked junctional complexes near the free cell surface at the con- stricted part [34]. Andres [3] reported that sensory nerve fibers entered the olfactory epithelium and synapsed with this fifth cell type. This study suggested that the alveolar brush cells in bullfrogs might have sensory or chemoreceptive functions besides their absorptive and secretory functions already described. Long, thick microvilli of a unique form protruded from the cell surface, and the upper part of the cytoplasm expanded to the alveolar lumen. These observations, combined with the report published by Hijiya et al. [23] in which SEM pictures revealed that the microvilli were protruding toward the alveolar duct, not from the top of the cell but rather from the side, suggest that the brush cells are shaped so as to be convenient to receive any information from a substance that may be present in the alveolar lumen, for example, a stream of air that reached the alveoli. Furthermore, the fact that the shape of the alveolar brush cell is quite similar to that of tracheal brush cell, which possesses epithelio- neural junctions, and that the shape of the alveolar brush cell is entirely similar to the type I cell of taste buds and to the fifth cell type of the olfactory epithelium suggest the possibility that the alveolar brush cells might have sensory or chemoreceptive functions in spite of the fact that it includes no nerve ending; it also suggested that there is a close relationship between the brush cells and their — adjacent cells, which in turn suggests that the brush cells may receive information on the secre- tion of osmiophilic lamellated bodies that acts as a surfactant involved in the essence of gas exchange and the lamination of the blood-air barrier (expan- Alveolar Brush Cell of Bullfrog sion of alveoli). A summary of this paper was presented at the 11th International Congress on Electron Microscopy. 10 11 12 13 14 LS) 16 £7 REFERENCES Rhodin, J. and Dalhamn, T. (1956) Electron mi- croscopy of the tracheal ciliated mucosa in rat. Z. Zellforsch., 44: 345-412. Brettschneider, H. (1958) Elektronenmikroskopis- che Untersuchungen an der Nasenschleimhaut. Anat. Anz., 105: 194-204. Andres, K. H. (1969) Der olfaktorische Saum der Katze. Z. Zellforsch., 96: 250-274. Adams, D.R. and Wiekamp, M.D. (1984) The canine vomeronasal organ. J. Anat., 138: 771-787. Adams, D.R. (1986) The bovine vomeronasal organ. Arch. Histol. Japon., 49: 211-225. Spoendlin, H. (1959) Elektronenmikroskopische Untersuchungen am respiratorischen Epithel der oberen Luftwege. Pract. Oto-rhino-laryngol., 21: 484-498. Rhodin, J. (1959) Ultrastructure of the tracheal ciliated mucosa in rat and man. Ann. Otol. Rhinol. Laryngol., 68: 964-974. Leeson, T.S. (1961) The development of the trachea in the rabbit, with particular reference to its fine structure. Anat. Anz., 110 (Suppl.): 214-223. Watson, J. H. L. and Brinkman, G. L. (1964) Elec- tron microscopy of the epithelial cells of normal and bronchitic human bronchus. Am. Rev. Resp. Dis., 90: 851-866. Konradova, V. (1966) The ultrastructure of the tracheal epithelium in rabbit. Folia Morphol., 14: 210-214. Luciano, L., Reale, E. and Ruska, H. (1968) Uber eine ,,chemorezeptive“ Sinneszelle in der Trachea der Ratte. Z. Zellforsch., 85: 350-375. Meyrick, B. and Reid, L. (1968) The alveolar brush cell in rat lung -—a third pneumonocyte. J. Ultra- struct. Res., 23: 71-80. Jeffery, P. K. and Reid, L. (1975) New observations of rat airway epithelium: a quantitative and electron microscopic study. J. Anat., 120: 295-320. Allan, E. M. (1978) The ultrastructure of the brush cell in bovine lung. Res. Vet. Sci., 25: 314-317. Low, F.N. (1952) Electron microscopy of the rat lung. Anat. Rec., 113: 437-450. Scheuermann, D. W. and De Groodt-Lasseel, M. H. A. (1981) Comparative ultrastructural explora- tion of the alveoli in the normal lung. Verh. Anat. Ges 5..507-341- Gomi, T. (1983) Electron microscopic studies on the alveolar epithelial cells of the striped snake 18 19 20 21 22 23 24 25 26 27 28 29 30 31 619 (Elaphe quadrivirgata). J. Med. Soc. Toho, 29: 577- 593. Hashimoto, T., Gomi, T., Kimura, A. and Tsuchiya, H. (1983) Light and electron microscopic study of the lung of the giant salamander, Megalo- bateracus japonicus davidanus. J. Med. Soc. Toho, 29: 724-733. Bartels, H. and Welsch, U. (1984) Freeze-fracture study of the turtle lung: 2. Rod-shaped particles in the plasma membrane of a_ mitochondria-rich pneumocyte in Pseudemys (Chrysemys) scripta. Cell Tissue Res., 236: 453-457. Kimura, A., Gomi, T., Tsuchiya, H., Fujita, H. and Hashimoto, T. (1984) Comparative anatomical observation on the lung blood-air barrier of verte- brates. Acta Anat. Nippon., 59: 516. Matsumura, H. and Setoguti, T. (1984) Electron microscopic studies of the lung of the salamander, Hynobius nebulosus: 1. A scanning and transmission electron microscopic observation. Okajimas Folia Anat. Jpn., 61: 15-31. Luciano, L., Reale, E. and Ruska, H. (1969) Biir- stenzellen im Alveolarepithel der Rattenlunge. Z. Zellforsch., 95: 198-201. Hijiya, K., Okada, Y. and Tankawa,H. (1977) Ultrastructural study of the alveolar brush cell. J. Electron Microsc., 26: 321-329. Scheuermann, D. W., Van Meir, F. and De Groodt- Lasseel, M. H. A. (1980) Ultrastructural study of alveolar brush cells in normal and _ terbutaline treated rats. Electron Microscopy (Proc.), 2: 138- 139) Scheuermann, D. W. and Timmermans, J. P. (1982) Peculiar observations with the scanning electron microscope in the pulmonary epithelium of the fresh water turtle. Abst. 10th Int. Congr. Electron Mi- crosc., Hamburg, No. 3, 597-598. Gomi, T. (1982) Electron microscopic studies on the alveolar brush cell of the striped snake (Elaphe quadrivirgata). J. Med. Soc. Toho, 29: 481-489. Gomi, T., Murakami, K. and Kitazawa, Y. (1980) The lung epithelium of lower vertebrates. J. Med. Soc. Toho, 27: 674-680. Luciano, L. and Reale, E. (1979) A new morpholo- gical aspect of the brush cells of the mouse gallblad- der epithelium. Cell Tissue Res., 201: 37-44. Johnson, F.R. and Young, B. A. (1968) Undif- ferentiated cells in gastric mucosa. J. Anat., 102: 541-551. Qwarnstrom, E. E. and Hand, A. R. (1982) A light and electron microscopic study of the distribution and effects of water-soluble radiographic contrast medium after retrograde infusion into the rat sub- mandibular gland. Arch. Oral Biol., 27: Pig ee Higashi, K., Takano, K. and Sasa, S. (1982) Ultra- structural aspects of the brush cell of the mouse 82 33} 620 gallbladder. Zool. Mag. (Tokyo), 91: 158-164. Takeda, M. and Hoshino, T. (1975) Fine structure of taste buds in the rat. Arch. Histol. Japon., 37: 395-413. Fujimoto, S. (1984) In “Human Histology, vol. 7. Sense Organs: Gustatory Organ”. Ed. by E. Yama- 34 T. Gomi, A. Kimura et al. da and H. Hashimoto, Asakura Shoten, Tokyo, pp. 43-64. Okano, M. (1984) In “Human Histology, vol. 7. Sense Organs: Olfactory Organ”. Ed. by E. Yamada and H. Hashimoto, Asakura Shoten, Tokyo, pp. 1- Sil, ZOOLOGICAL SCIENCE 4: 621-626 (1987) © 1987 Zoological Society of Japan Karyotype Analysis of Two Japanese Salamanders, Hynobius nebulosus (Schlegel) and Hynobius dunni Tago, by Means of C-banding! CHIKAKO IKEBE, KEN-ICHI AOKI? and SEI-ICHI KoHNO2 Department of Biology, Faculty of General Education, and *Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan ABSTRACT—The karyotypes of Hynobius nebulosus from Nagasaki-shi (Nagasaki Prefecture) and Minabe-cho (Wakayama Pref.), and those of H. dunni from Takamori-cho (Kumamoto Pref.) and Oita-shi (Oita Pref.) were analyzed by the conventional Giemsa-staining and C-banding methods using their embryos. Intraspecific chromosome variations were detected in chromosomes 1, 5, 8, 10 and 13 of H. nebulosus and in chromosome 20 of H. dunni. C-banding patterns of 17 identified chromosome pairs (chromosomes 1-13 and 4 pairs of small-sized chromosomes) of H. nebulosus and H. dunni were similar, except for the variant bands. INTRODUCTION The two pond type hynobiid salamanders, Hynobius nebulosus and Hynobius dunni, are considered to be phylogenetically close because of similar morphological characters in egg-capsules, larvae and adults [1-3]. H. nebulosus is distrib- uted widely in the western part of Japan including the Kinki, Chugoku, Shikoku and Kyushu dis- tricts, while H. dunni is distributed only in the Kyushu district [1-3]. Chromosome analyses of H. nebulosus and H. dunni have been performed by Makino [4, 5] and Sato [6] in the 1930s, using the testis-sectioning method, and the diploid chromosome numbers of both species were reported as 56. Recently, the conventional Giemsa-stained karyotypes of H. nebulosus and H. dunni were described by Mores- calchi [7] and Morescalchi er al. [8], though the shape of small-sized chromosomes of their karyotypes was still not clear. The conventional Giemsa-stained karyotypes of H. nebulosus from the Chugoku and Kinki districts were also de- scribed by Ikebe and Kohno [9], and those from Accepted March 31, 1987 Received January 20, 1987 ' This paper corresponds to “Cytogenetic Studies of Hynobiidae (Urodela). VII”. other sites in the Chugoku district by Seto et al. [10]. However, the banded karyotype has been reported only in H. nebulosus from Nagasaki-shi by Kuro-o et al. using R-banding method [11]. In the present paper, the karyotypes of H. nebulosus and H. dunni from two localities were analyzed by the conventional Giemsa-staining and C-banding methods using embryos. Intraspecific chromosome variations were detected in both species. MATERIALS AND METHODS Materials used in this study were late tail-bud stage embryos. The specimens of H. nebulosus were collected in Nagasaki-shi (Nagasaki Prefec- ture in the Kyushu district) and Minabe-cho (Wakayama Pref. in the Kinki). Those of H. dunni were collected in Takamori-cho (Kuma- moto Pref. in the Kyushu) and Oita-shi (Oita Pref. in the Kyushu). Detailed data from these two species are summarized in Table 1. The methods of chromosome preparation have been described previously [9,12], and the C- banding was accomplished by the BSG method BBIE The chromosomes were designated following the nomenclature for centromeric position on 622 C. IKEBE, K. AOKI AND S. KOHNO TABLE 1. Sites and years of collection of Hynobius nebulosus and Hynobius dunni, and numbers of egg-capsules, metaphases and embryos analyzed No. of metaphases / No. of embryos : Collection No. of S 5 a SHE Ge Venu eee capsules Giemsa C-banding Total Nagasaki-shi H. nebulosus 89. 83, 84 4 19/9 43/16 62 / 23 ae 2 2417 BT 61/10 : Takamori-cho H. dunni 80, °82 5) 16/9 — 16/9 eee 1 34/9 20/4 54/9 chromosomes by Levan et al. [14]. RESULTS Two conventional Giemsa-stained karyotypes of H. nebulosus from Nagasaki-shi and Minabe- cho are shown in Figures 1 and 3, respectively. A C-banded karyotype of H. nebulosus from Naga- saki-shi is shown in Figure 2. All embryos ana- lyzed from two localities had 56 chromosomes as a diploid set, in agreement with previous data [4, 6-10]. Some structural chromosome variations were detected in the karyotypes from the two localities. The morphology of chromosome 10 from Naga- saki-shi was subtelocentric, while that from Minabe-cho was acrocentric, in agreement with previous data of H. nebulosus [7,9, 10]. Second- ary constrictions were observed in the long arm of chromosome | and the short arm of chromosome 8 in specimens from Nagasaki-shi. In chromo- some pairs 1 and 8, all three possible combina- tions, namely homomorphic pair with the secon- dary constriction, homomorphic pair without the secondary constriction and heteromorphic pair with and without the secondary constriction, were observed. The secondary constriction in the long arm of chromosome 5 was observed in specimens from Minabe-cho. We observed the chromosome pair 5 only in two combinations, namely heter- omorphic pair with and without the secondary constriction and homomorphic pair without the secondary constriction. Intraspecific variations in chromosomes 5, 8, 10 and 13 of H. nebulosus are shown in Figure 4. Chromosome 5 from Minabe-cho specimens and chromosome 8 from Nagasaki-shi specimens had a C-positive band corresponding to the region of the secondary constriction. In the two chromo- somes without secondary constrictions, no C- positive band was observed in that region. We could not ascertain whether or not the secondary constriction in the long arm of chromosome 1 from Nagasaki-shi specimens corresponded to the C-positive band. As to chromosome 10, the specimen from Nagasaki-shi showed C-positive bands in the short arm and in the centromere region, and those from Minabe-cho showed C- positive bands in the centromere region and in the proximal region of the long arm. The lengths of the C-positive bands of the specimens from these two localities were almost the same. Chromo- some 13 from Minabe-cho specimens had an especially dark C-positive band near the terminal end of the long arm. This band was not detected by the conventional Giemsa-staining method. We observed all three possible combinations in chromosome pair 13 by C-banding analysis, namely homomorphic pair with this band, homo- morphic pair without the band and heteromorphic pair with and without the band. A conventional Giemsa-stained karyotype and a C-banded karyotype of H.dunni from Oita-shi are shown in Figures 5 and 6. All embryos analyzed from the two localities had a diploid set of 56 chromosomes, which corre- sponded to previous data [5-8]. Six of 9 embryos from Takamori-cho and 4 of 9 embryos from Oita-shi had the same karyotype shown in Figure 5. The remaining 3 embryos from Takamori-cho C-banded Karyotypes of Two Species of Hynobius 623 \Fs , / ! \ Wt iia at i} an kt R% AA Oe MK we eK hs la 14— 19 AA QA af @0n RR mR we An er 1 —__———— 20 — 28 ———_————_- Fic. 1. A conventional Giemsa stained karyotype of Hynobius nebulosus from Naga- saki-shi. 7 4 6 l tf gh i) ay ee ee me WW al 2s 14 Daal 4? A %3 < * « a 22 € # e A 72) Jaf) 23) 743 2 Fic. 2. A C-banded karyotype of Hynobius nebulosus from Nagasaki-shi. 249°; SAO} S6 WI 00 ag Mi re GHRAR 8H wu xx Bs 14g AO @@ an an pa an va An th 3 eee NS —E_ SSS Fic. 3. A conventional Giemsa-stained karyotype of Hynobius nebulosus from Mina- be-cho. and 5 embryos from Oita-shi had a sub- somes (10-13), and one acrocentric small-sized metacentric marker chromosome which was chromosome was absent. We consider the slightly smaller than the midium-sized chromo- karyotype which has no marker chromosome as 624 C. IKEBE, K. AOKI AND S. KoHNO Fic. 4. Chromosomes 5, 8, 10 and 13 of Hynobius nebulosus from Nagasaki-shi (N) and Minabe-cho (M). The left hand chromosomes from specimens in each locality were stained by the conventional Giemsa-staining method and the right hand chromosomes by C-banding method. Arrowheads indicate the secondary constrictions and the variant bands. typical of H. dunni. C-banding analyses of 4 embryos from Oita-shi revealed that this marker chromosome originated in chromosome 20. As shown in Figure 7, one of chromosomes 20 is absent and the long arm of the marker chromo- some had the same C-banding pattern as chromo- some 20. The centromere region of the marker chromosome stained darker than that of chromo- some 20. Comparing the karyotype of H. dunni with that of H. nebulosus, we detected some interspecific variations. The secondary constriction was not detected in H. dunni. The small-sized chromo- somes (14-28) consisted of 5meta- or submeta- centric and 10 acrocentric pairs in H. dunni, while there were 6meta- or submeta-centric and 9 acrocentric pairs in H. nebulosus. Chromosome 10 of H. dunni was subtelocentric as was that of H. nebulosus from Nagasaki-shi. C-banding patterns of all the 17 identified chromosome pairs (chromosomes 1-13 and 4 pairs of small-sized chromosomes) of H. dunni were similar to those of H. nebulosus, except for the variant bands on chromosomes 5, 8 and 13 of H. nebulosus. DISCUSSION This study indicates that intraspecific chromo- some variations exist in both H. nebulosus and H. dunni. These variations are related to the C- positive region. The difference in chromosome 10 of H. nebulosus between Nagasaki-shi specimen and Minabe-cho specimen revealed to be pericentric inversion of the C-positive segment. The variant bands on chromosomes 5, 8 and 13 of H. nebulosus were C-positive. The variant bands on chromosomes 5 and 8 corresponded to the secondary constrictions; however, that on chromosome 13 did not correspond to the second- ary constitution and stained especially intensely by C-banding method. Thus, the variant band on chromosome 13 may be different from those on chromosomes 5 and 8 at the molecular level. The short arm of the marker chromosome of H. dunni stained as deeply as the C-positive region of the long arm of chromosome 20. The short arm of the marker chromosome might have arisen by tandem duplication of the C-positive region in chromosome 20. It suggests that the C-positive region contains constitutive heterochromatin and scarcely in- cludes structural genes. As all embryos used in this study developed normally to the late tail-bud stage as judged by their external morphology, C-positive variant bands of these two species apparently did not affect their development. By comparing the karyotypes of H. nebulosus in the present study with those of H. nebulosus previously published [9, 10], a difference in the constitution of the small-sized chromosomes (14- 28) was detected. We observed 6meta- or sub- meta-centric and 9 acrocentric pairs in the present study, as opposed to 7meta- or submeta-centric and 8 acrocentric pairs [9] and 5meta- or sub- meta-centric and 10 acrocentric pairs [10] in previous studies. In H. nebulosus from Nara-shi (Nara Pref. in the Kinki), Izumo-shi (Shimane Pref. in the Chugoku) and Sakaide-shi (Kagawa Pref. in the Shikoku), we observed the same constitution of the small-sized chromosomes as that in H.nebulosus from Nagasaki-shi and Minabe-cho in the present study (Ikebe ef al., unpublished data). Therefore, the typical con- C-banded Karyotypes of Two Species of Hynobius 625 \) Lied Davse A\ O08 GE OR WS ka we ne on ae I 2 3 UU peep ml tha 14—18 aa Arne RR AK AA aa ee a ee ees 19 — 28 A ) Fic. 5. A conventional Giemsa-stained karyotype of Hynobius dunni from Oita-shi. “ Meh a) 7 8 g AA ad BA as ae oe eT 10 11 12 13 14 15—18 > m x = mf em. Mm -s hi -- ae me 6 19 20 21 N= 7H3 Fic. 6. A C-banded karyotype of Hynobius dunni from Oita-shi. & * “ ae 54 8% we aw 10 15—18 =e 22 Ae mm be ©? 19 7h) ] Fic. 7. A partial C-banded karyotype of Hynobius dunni from Oita-shi. An arrow- head indicates the marker chromosome. stitution of the small-sized chromosomes in H. nebulosus may be 6meta- or submeta-centric and 9 acrocentric pairs. The C-banding patterns of chromosomes 1-13 (except for the variant bands) of H. nebulosus and H. dunni were compared with the previously published C-banding data of H. tokyoensis from Yokose (Saitama Pref.), Yokosuka (Kanagawa Pref.) and Chosei (Chiba Pref.) [15]. H. tokyoen- sis has sometimes been regarded as subspecies of H. nebulosus {2, 3]. While the C-banding patterns of H. nebulosus and H. dunni were similar, those of H. tokyoensis differed from the patterns in these two species in chromosomes 2 and 10. The differences in chromosomes 2 and 10 between H. nebulosus and H. tokyoensis were already de- tected by R-banding analysis [11]. On the basis of the karyological studies, the phylogenetic rela- 626 C. IkEBE, K. AOKI AND S. KoHNO tionships between H. nebulosus and H. dunni appear to be closer than those between H. nebulosus and H. tokyoensis. ACKNOWLEDGMENTS We are grateful to the following people for their help in collecting specimens: Mr. M. Itoh, Mr. M. Miyamo- to, Mr. K. Numata and Mr. S. Tamai. This study was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (No. 58540484). REFERENCES 1 Sato, I. (1943) A Monograph on the Japanese Urodeles, Nippon Shuppan-sha, Osaka, pp. 41-84. (In Japanese) 2 Nakamura, K. and Ueno,S.-I. (1963) Japanese Reptiles and Amphibians in Colour, Hoikusha, Osaka, pp. 6-8. (In Japanese) 3. Thorn, R. (1968) Les Salamandres d’Europe, d "Asie et d ’Afrique du Nord, Paul Lechevalier, Paris, pp. 43-49, 58-59. (In French) 4 Makino, S. (1934) The chromosomes of Hynobius leechii and H. nebulosus. Trans. Sapporo Nat. Hist. Soc., 13: 351-354. 5 Makino, S. (1935) The chromosomes of Hynobius dunni and H. kimurae. Jpn. J. Genet., 10: 243-244. (In Japanese, with English Résumé) 6 Sato, I. (1936) On the chromosomes in some hynobiid salamanders from southern Japan. J. Sci. Hiroshima Univ., Ser. B, Div. 1, 4: 143-154. 7 Morescalchi, A. (1973) Cytotaxonomy of the Amphibia. In “Cytotaxonomy and Vertebrate Evolution”. Ed. by A. B. Chiarelli and E. Capanna, 11 12 13 14 15 Academic Press, London & New York, pp. 240- 244. Morescalchi, A., Odierna, G. and Olmo, E. (1979) Karyology of the primitive salamanders, family Hynobiidae. Experientia, 35: 1434-1435. Ikebe, C. and Kohno, S. (1979) Cytogenetic studies of Hynobiidae (Urodela). I. Karyotypes of Hyno- bius nebulosus nebulosus (Schlegel) and Hynobius nebulosus tokyoensis Tago. Proc. Japan Acad., 55B: 436-440. Seto, T., Utsunomiya, Y. and Utsunomiya, T. (1983) Karyotypes of two representative species of hynobiid salamanders, Hynobius nebulosus (Schlegel) and Hynobius naevius (Schlegel). Proc. Japan Acad., 59B: 231-235. Kuro-o, M., Ikebe, C. and Kohno, S. (1987) Cytogenetic studies of Hynobiidae (Urodela). VI. R-banding patterns in five pond-type Hynobius from Korea and Japan. Cytogenet. Cell Genet., 44: 69- x Kohno, S., Kuro-o, M., Ikebe, C., Katakura, R., Izumisawa, Y., Yamamoto, T., Lee,H.Y. and Yang, S. Y. (1987) Banding karyotype of Korean salamander: Hynobius leechii Boulenger. Zool. Sci., 4: 81-86. Sumner, A. T. (1972) A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res., 75: 304-306. Levan, A., Fredga, K. and Sandberg, A. A. (1964) Nomenclature for centromeric position on chromo- somes. Hereditas, 52: 201-220. Kohno, S., Ohhashi,T. and Ikebe,C. (1983) Cytogenetic studies of Hynobiidae (Urodela). II. Banding karyotype of Hynobius tokyoensis Tago. Proc. Japan Acad. 59B: 271-275. ZOOLOGICAL SCIENCE 4: 627-634 (1987) © 1987 Zoological Society of Japan The Distribution of A and B Blood Group Antigens in Tissues of the Frog, Rana catesbeiana MASAYOSHI KarIno and Ikuo ISHIYAMA Department of Forensic Medicine, Faculty of Medicine, Tokyo University, Hongo, Tokyo 113, Japan ABSTRACT—The distribution of blood group antigens A and B in tissues of the frog, Rana catesbeiana, was demonstrated by light and electron microscopic immunohistochemistry. Antigen B was localized on blood cells and vascular endothelium throughout the body. This distribution pattern is similar to that of human ABO blood group substances. This was not, however, the case for A antigen, which was specifically localized in the pancreas and adult epidermis. These facts clearly indicate that the expression of antigen A is not the allelomorph to that of antigen B in the bullfrog. In the pancreas, antigen A was confined to the excretory acinar cells. plasmalemma showed the reaction. INTRODUCTION Glycoproteins and glycolipids are generally present in animal cell membranes, and they extrude carbohydrate chains with a specific sequ- ence on the cell surface [1]. They have been suggested to be closely related to differentiation phenomena because some differentiated cells or tissues can be distinguished based on differential lectin bindings [2]. In addition, marked changes in carbohydrates are known to occur in malignant cells [1]. It has also been reported that the alteration of the cell surface carbohydrates seems to be responsible for changes in cell adhesion properties and cellular interactions. The antigen on the red cell surface is usually referred to as a blood group when it is possessed by only some individuals of a species. The most popular examples are antigens of the ABO system in man. The biological significance of the blood group antigens is not clearly understood, but it is well known that they are composed of glycopro- teins or glycolipids [1]. Although the substances of the human ABO blood group system are found most commonly on erythrocytes, they are also distributed in some other kinds of cells and tissues Accepted February 27, 1987 Received October 31, 1986 Their secretory granules and including the endothelium, mucous epithelium and mucus secretions [3-10]. Furthermore, the antigens of the ABO system are not confined to human bodies, but are widely distributed in other animals [11,12] and even in some kinds of bacteria [13, 14]. Yada and Yamazawa [15] and Yada er al. [16] reported the presence of the antigen B on blood cells and in many other tissues of the frog, Rana catesbeiana, by the absorption and mixed agglu- tination tests. Antigen A was also found to occur in trace amounts in the cloaca and epidermis of the adult bullfrog. These findings suggest the possibility that A and B antigens are simul- taneously present in different cells or tissues in the same body. This is not the case in human bodies because, phenotypes A and B are in the relation of allelomorphs in man and the A and/or B antigens are normally expressed in the same site in the body. Although the histological localization of the blood group antigens in human and rodent tissues has been described by light and electron microscopic immunohistochemistry [3, 5-10, 12, 17], no such study has been made with regard to frog tissue. In the present study, we examined the immunohistochemical demonstration of A and B antigens in bullfrog tissues to investigate the above-mentioned possibility. 628 M. KaAIHO AND I. ISHIYAMA MATERIALS AND METHODS An anuran amphibian bullfrog, Rana cates- beiana, was used in this study. Adult male and female frogs and tadpoles (stages 15-23) of this species were obtained in fields near Tokyo. They were pithed and dissected to expose the heart. For conventional light microscopy and immuno- histochemistry, the animals were perfused from the heart with a frog Ringer’s solution and then with 10% neutralized formalin. The excised organs and tissues were further fixed by immer- sion in fresh fixative and embedded in paraffin. The A and B antigens were immunohistochemi- cally detected in 4m sections according to the method of Nakane and Pierce [18] with some modificatins. Deparaffinized sections were im- mersed in 0.3% H,O, in methanol and then treated with normal serum (goat or rabbit, diluted 1:10 with phosphate buffered saline (PBS; pH 7.4). Thereafter, they were incubated with anti-A antiserum (rabbit, Takeda) or anti-B antiserum (goat, Takeda), and further reacted with perox- idase-labeled antirabbit IgG (goat, Dako) or anti- goat IgG (rabbit, Dako). Primary antisera and peroxidase-labeled second antibody were diluted 1:5 and 1:25, respectively, with PBS. After the diaminobenzidine (DAB) reaction, some sections were counterstained with hematoxylin. For elec- tron microscopic immunocytochemistry, tissues were fixed by perfusion with a fixative containing 2% paraformaldehyde and 1% glutaraldehyde in 0.1M cacodylate buffer (pH7.4) and further immersed in fresh fixative for an hour at 4°C. Subsequently, they were treated with 0.1M NH,Cl in PBS [19] for 30min and then 15 um thick sections were cut with a Microslicer DTK- 1000 (Dosaka EM). The sliced sections of the pancreas were treated with bovine serum albumin (BSA; Sigma) solution (1% in PBS). Thereafter, they were incubated in anti-A antiserum (1:5) for an hour at 20°C and then in protein A-colloidal gold (15nm) complex solution (1:20 with PBS containing 1% BSA; Janssen Pharmaceuticals) for 20 min. The section of the liver, on the other hand, were treated with normal rabbit serum (1:10) and then in peroxidase labeled antigoat IgG solution (1:20) for 20min. After rinse, peroxidase was histochemically detected by the DAB reaction. Following thorough washing in PBS, both sections were postfixed in 1% OsO, and embedded in an Epon mixture. Thereafter, specimens were processed and observed according to the routine method [20]. In control experiments, anti-A or anti B anti- sera, which had been adsorbed with human erythrocytes of the respective blood groups, were used in the primary incubation in both light and electron microscopic immunohistochemistry. RESULTS Based on the previous data [15, 16] indicating that B antigen is distributed widely in the body of the bullfrog, we first surveyed the distribution of the antigen in the liver, pancreas, kidney, spleen, gallbladder, testis, stomach, heart and skin from adult frogs and tadpoles light microscopically. Consequently, we found that the B antigen is distributed on blood cells and vascular endothelial cells throughout the body (Fig. 1a). Not only the erythrocytes, but all free cells in the blood vessels were positive. In addition, macrophages in the spleen and in intestine of metamorphosing tad- poles (Stages 20-23) and the sperm in adult testes expressed the antigenicity. Light microscopically, only the outer surface of the blood cells appeared to be stained. But in the case of macrophages and endothelial cells, their cell bodies seemed to be stained (Fig. 1a). Kupffer’s cells in the liver Fic. 1. Light micrographs showing the distribution of antigen B in an adult frog liver. Sections were immunohistochemically treated with anti-B antiserum (1a) or with control antiserum (1b) and counterstained with hematoxylin. 1,200. In Fig. 1a, the DAB reaction product is localized on the blood cell surface (arrowhead) and sinusoidal endothelium. Kupffer’s cells are indicated by arrows. Fic. 2. Electron microscopic demonstration of antigen B in an adult frog liver. The luminal surface (double arrows) of the endothelial cell (E) is more densely stained than the other side (arrow) facing Disse’s space. The hepatocyte (H) is negative. Antibodies or reagents were thought to have surely reached the surface of the hepatocytes, because continuity between the external part and Disses’s spaces is certain at the fractured site (arrowhead). 14,000. Blood Group Antigens in Frog Tissues 629 contained dense materials regarded as the secon- dary lysosomes in their cytoplasm (Fig. 1b), but the brown color of the peroxidase reaction could be distinguished from the material, and it was decided that these cells were positive for the B antigen. The liver parenchymal cells lie almost in contact with the positive sinusoidal endothelial cells. Therefore, it was difficult to decide at the 630 M. KAIHO AND I. ISHIYAMA 3b), respectively. The section in Fig. 3b was counterstained with hematoxylin. Tadpole liver (L) and pancreas (P) are contained in the section, but positive reaction is observed only on the pancreas (Fig. 3a). Dark dots as seen on the liver portion (arrowheads) are secondary lysozomes in Kupffer’s cells. 120. Fic. Fic. 4. A part of a tadpole pancreas. The antigen A was immunohistochemically detected. The section was counterstained with hematoxylin. Most regions except for the islet of Langerhans (IL) and connective tissue area are positively stained. 300. 5. Distribution of antigen A in an adult frog pancreas in larger magnification (1,200). The reaction is confined to exocrine acinar cells. The surface and secretory granules of the acinar cells are stained. The centroacinar cells (arrows) are not stained. light microscopic level whether the parenchymal cell surface facing the Disse’s space were positive or not. To clarify this problem, we attempted to demonstrate B antigen in the liver electron mi- croscopically. As a result, we confirmed that the hepatocytes were negative (Fig. 2). In addition, the endothelial cell surface facing the Disse’s space was more weakly stained than the luminal Blood Group Antigens in Frog Tissues 631 Fic. 6. Electron micrograph to show the localization of antigen A in the adult frog pancreas. Colloidal goid particles (arrowheads) are distributed on the surface of exocrine acinar cells (A), but not on the endocrine cell (E). 17,000. Fic. 7. The basal (upper right) and apical (lower left) parts of acinar cells and two duct cells (D) are observed. Colloidal gold particles (arrowheads) are localized on the apical and basolateral surface of acinar cells and on a secretory granule (SG), which are supposed to have been fractured during the slicing procedure of the material. Duct cells are not labeled. 21,000. 632 M. KAIHO AND I. ISHITYAMA surface. In our method, glutaraldehyde was employed in the fixation procedure, and it was impossible to demonstrate the intracellular loca- tion of antigens, possibly because reagents could not penetrate the well fixed plasmalemma. The same organs or tissues as listed above were also examined for A antigen, and we recognized the positive reaction only in the pancreas (Fig. 3a) and epidermis in adult frogs. In tadpoles also, the pancreas expressed the antigenicity, but the epidermis was negative. (The detailed results concerning the epidermis will be presented in a separate paper.) In the pancreatic tissues, many kind of cells, such as endocrine cells, exocrine acinar cells, centroacinar cells, duct cells, connective tissue cells etc., are present. Among them, only the exocrine acinar cells were stained, the other cells being negative (Figs. 3-5). The surface of the acinar cells and secretory granules contained in their cytoplasm were ascertained to be positive. We further investigated the localization of A antigen in pancreatic acinar cells electron micro- scopically. Interestingly, all surface areas involv- ing both the apical and basolateral sides were positive (Figs.6 and 7). The presence of the antigen in the secretory granules was confirmed by the labeling of fractured ones (Fig. 7). The limited distribution of the antigen in the excretory acinar cells was also confirmed. All the above- presented results concerning the localization of A and B antigens were proved specific by the control experiments, which were performed using antisera absorbed with A or B group blood cells. DISCUSSION In the present study, we demonstrated the distribution of blood group A and B antigens in frog tissues by the light and electron microscopic immunohistochemistry. An antigenicity is gener- ally thought to be carried by the antigen determi- nant part of a molecule. Therefore, it should be emphasized that what we detected were the A and B antigens, and not the blood group sub- stances themselves. According to our results, B antigen was localized on the blood cells and endothelium of blood vessels throughout the body. This pattern of distribution is similar to that of human blood group substances in the human body [4-7, 9, 10]. Based on this distribu- tion similarity between the two species, antigen B in the bullfrog is thought to play still not fully clarified roles comparable to human blood group substances. So far as we have observed (more than 10 animals were tested), all the tadpole and adult bullfrog individuals had only B antigen on their erythrocytes, in contrast to the fact that A and/or B, or no antigen(s) are expressed on human erythrocytes according to the corresponding blood groups of A, B, AB or O. We have no data at present as for the presence of their own blood group(s) in the frogs. The results concerning the distribution of A antigen were of much interest. It was confined to the pancreas and adult epidermis. The existence of blood group antigens in the pancreas or epidermis has previously been described by some workers in man and rodents [3, 12,21]. In the case of the bullfrog, only the exocrine acinar cells were stained with anti-A antiserum in the pan- creas, and no other components were positive. Antigen B was also present in the pancreas, but it was localized on the endothelial cells as men- tioned above and not on the acinar cells. From these observations, it is clear that A and B antigens are expressed in different sites in the bullfrog body. This fact indicates that A and B antigens in this animal cannot be regarded as allelomorphs to each other. This means that the expression of the A antigen in the frog is possibly a phenomenon with no phylogenetical relation to human blood group substance. In man, A and B substances are known to be synthesized from a common precursor (H substance). Therefore, it seems important to make clear whether the two antigens in the frog are synthesized in a similar way. We are now proceeding our investigation along this line. The pancreas contains many kinds of cells. The excretory portion is composed of three different types of cells—acinar, centroacinar and duct cells. Among them, only the acinar cells expres- sed the A antigen, although the three cell types appear to arise from a _ morphologically Blood Group Antigens in Frog Tissues 633 homogeneous cell population in the developing pancreatic rudiment [22]. We further examined the expression of A antigens in pancreatic orga- nogenesis during the early (external gill stage) development of the bullfrog larva and confirmed its simultaneous appearance with the formation of the secretory granules in the acinar cells (unpub- lished observations). From these findings, the specific expression of the A antigen in the acinar cells appears to be closely related to their dif- ferentiation. Similar results indicating that spe- cific saccharides are expressed in the exocrine acinar cells have been presented in the rodent pancreas with the use of lectins [23]. According to the present results, the antigen A-positive sites in acinar cells were the plas- malemma and secretory granules. The plas- malemma of epithelial cells is thought to be divided by tight junctions into two spatially and functionally separate regions comprised of the apical and basolateral zones. Exocytosis in acinar cells normally occurs solely in the former [24]. It was, therefore, surprising that the apical- and basolateral-plasmalemmal zones both reacted with the anti-A antibody. Maylie-Pfenninger and Jamieson [25] reported similar findings in dispersed rodent pancreatic acinar cells. According to them, lectins labeled both the apical and basolateral zones uniformly. In their experiments, however, artifactitious effects of cell dissociation conditions in the plane of the membrane were considered [26]. In our study, we used intact tissue. Thus, our data indicate that some kind of cell surface saccharides (A antigen determinants) are uniformly distrib- uted on the surface of acinar cells even in the intact pancreas. It is also suggested, at the same time, that the A antigen probably does not participate in the secretion phenomena (exocyto- sis) of these cells. From the observation that the central region of the secretory granule was labeled, secretory mate- rials are thought to have a moiety with the A antigenicity. However, we cannot decide whether the limiting membranes of the secretory granules are positive or not, but the possibility cannot be ruled out that the A antigen is synthesized on the cisternal surface of the intracellular membrane system and conveyed to the apical surface via the process of exocytosis of secretory granules [27, 28]. The antigens A are, therefore, considered to be carried by at least two different kinds of mole- cules, a membrane-bound type and a nonbound type, that are contained in the secretory granules. Why these different molecules with identical anti- genicity are coexpressed in the acinar cells is not yet known. With regard to their chemical nature, both of them are thought to be glycoproteins, because glycolipids appear to be extracted by the dehydration process with ethanol [25], and excre- tory substances in the pancreas are glycoproteins in nature. However, additional studies using proteolytic enzymes such as papain are required to confirm this point conclusively. Carbohydrate moieties of blood group sub- stances are known to be composed of branched sequences of saccharides and are synthesized stepwise by several glycosyltransferases [29]. An antibody specific to a blood group substance appears to recognize such a sequence. Thus, antibodies are regarded to have higher specifici- ties than lectins, which recognize only one or two terminal saccharides. In practice, we have experi- enced that a lectin of Helix promatia which recognizes the terminal saccharide (GalNAc) of A substance staines wider regions than the anti-A antiserum. To our knowledge, not very many studies concerning the cell surface molecules using anti- bodies have been performed [3, 12]. We recom- mend their use, in addition to lectins, for the detection of cell surface saccharides in the field of cell and developmental biology. REFERENCES 1 MHakomori, S. (1981) Glycosphingolipids in cellular interaction, differentiation and oncogenesis. Ann. Rev. Biochem., 50: 733-764. 2 Watanabe, M., Muramatsu, T., Shirane, H. and Ugai, K. (1981) . Discrete distribution of binding sites for Dolichos biflorus agglutinin (DBA) and for peanut agglutinin (PNA) in mouse organ tissues. J. Histochem. Cytochem., 29: 779-790. 3 Dabelsteen, E., Vedtofte, P., Hakomori, S. and Young, W. W. (1982) Carbohydrate chains specific for blood group antigens in differentiation of human 10 11 1D 13 14 US 16 7) 18 634 oral epithelium. J. Invest. Dermatol., 79: 3-7. Davison, I. and Stejskal, R. (1972) Tissue antigens A, B and H in health and disease. Haematologia, 6: 177-184. Glynn, L.E. and Holborow, E.J. (1959) Distribu- tion of blood group substances in human tissues. Br. Med. Bull., 15: 150-153. Holborow, E.J., Brown, P.C., Glynn. L.E., Hawes, M.D., Gresham, G. A., O’Breien, T. F. and Cooms, R.R. A. (1960) The distribution of the blood group A antigen in human tissues. Br. J. Exp. Pathol., 41: 430-437. Szulman, A.E. (1960) The histological distribution of blood group substances A and B in man. J. Exp. Med., 111: 785-800. Szulman, A.E. (1962) The histological distribution of the blood group substances in man as disclosed by immunofluorescence. II. The H antigen and its relation to A and B antigens. J. Exp. Med., 115: 977-996. Szulman, A. E. (1965) The ABH antigens in human tissues and secretions during embryonal develop- ment. J. Histochem. Cytochem., 13: 752-754. Szulman, A. E. (1971) The histological distribution of the blood group substances in man as disclosed by immunofluorescence. Human Pathol., 2: 575-585. Joysey, V.C. (1959) The relation between animal and human blood groups. Br. Med. Bull., 15: 158- 164. Reibel, J., Dabelsteen, E., Hakomori, S., Young, W. W. and Mackenzie, I. C. (1984) The distribution of blood group antigens in rodent epithelia. Cell Tissue Res., 237: 111-116. Iseki, S. (1952) Blood group substances in bacteria. Gumma J. Med. Sci., 1: 7. Springer, G. F., Williamson, P. and Brandes, W. C. (1961) Blood group activity of gram-negative bac- teria. J. Exp. Med., 113: 1077-1093. Yada, S. and Yamazawa, K. (1962) Distribution of the ABO blood group antigens in various tissues of Rana catesbeiana. Jpn. J. Legal Med., 16: 62-64. Yada, S., Yamazawa, K. and Mori, S. (1962) The ABO blood group antigens in various tissue cells of Rana catesbeiana shown by mixed agglutination. Jpn. J. Legal Med., 16: 233-237. Hinglais, N., Bretton, R., Rouchon, M., Oriol, R. and Bariety, J. (1981) Ultrastructural localization of blood group A antigen in normal human kidneys. J. Ultrastruct. Res., 74: 34-45. Nakane, P.K. and Pierce, B. (1967) Enzyme- labeled antibodies for the light and electron micro- scopic localization of tissue antigens. J. Cell. Biol., 19 20 21 Mp 23 24 25 26 Zi) 28 29 M. KaIHo AND I. ISHIYAMA 33: 307-318. Roth, J. (1983) The colloidal gold marker system for light and electron microscopic cytochemistry. In “Techniques in Immunocytochemistry, Vol. 2”. Ed. by G. R. Bullock and P. Petrusz, Academic Press, London, pp. 217-284. Kaiho, M. Nakamura, T. and Kumegawa, M. (1975) Morphological studies on the synthesis of secretory granules in convoluted tubules of mouse subman- dibular gland. Anat. Rec., 183: 405-420. Ito. N., Nishi, K., Nakajima, M., Matsuda, Y., Ishi- tani, A., Mizumoto, J. and Hirota, T. (1986) Loca- lization of blood group antigens in human pancreas with lectin-horseradish peroxidase conjugates. Acta Histochem. Cytochem., 19: 205-218. Pictet, R. and Rutter, W. (1972) Development of the embryonic endocrine pancreas. In “Handbook of Physiology, Sect. 7. The Endocrine Pancreas, Vol. 1”. Ed. by D. F. Steiner and N. Freinkel, Am. Physiol. Soc., Washington, D. C., pp. 25-66. Maylie-Pfenninger, M. F., Doyle, C. M. and Jamieson, J.D. (1977) Differential lectin receptor appearance during the development of the pancreas. J. Cell Biol., 75: 67a. Jamieson, J.D. and Palade, G.E. (1977) Produc- tion of secretory proteins in animal cells. In “Inter- national Cell Biology”. Ed. by B. R. Brinkley and K.R. Porter, The Rockefeller University Press, New York, pp. 308-317. Maylie-Pfenninger,M.F. and Jamieson, J.D. (1979) Distribution of cell surface saccharides of pancreatic cells. II Lectin-labeling patterns on ma- ture guinea pig and rat pancreatic cells. J. Cell Biol., 80: 77-95. Pisam, M. and Ripoche, P. (1976) Redistribution of surface macromolecules in dissociated epithelial cells. J. Cell Biol., 71: 907-920. Leblond, C. P. and Bennett. G. (1977) Role of the Golgi apparatus in terminal glycosylation. In “Inter- national Cell Biology”. Ed. by B. R. Brinkley and K.R. Porter, The Rockefeller University Press, New York, pp. 326-336. Pestalozzi,D.M., Hess,M. and Berger, E.G. (1982) Immunohistochemical evidence for cell sur- face and Golgi localization of galactosyltransferase in human stomach, jejunum, liver and pancreas. J. Histochem. Cytochem., 30: 1146-1152. Watkins, W. M. (1977) The glycosyltransferase pro- ducts of the A, B, H and Le genes and their relationship to the structure of the blood group antigens. 5th Int. Convoc. Immunol., Buffalo, N. Y., pp. 134-142. ZOOLOGICAL SCIENCE 4: 635-639 (1987) Further Observations on Division Pattern of Binucleate Fish Embryonic Cells SETSURO MIZUKAMI Depatment of Biological Science, Yamanashi Gakuin University, Sakaori 2-4-5, Kofu 400, Japan ABSTRACT — In order to study changes in physiological and cytological properties of embryonic cells during embryogenesis of the killifish, Oryzias latipes, blastomeres were dissociated from morula and blastula stage embryos in Yamamoto’s saline solution. During the following division of the isolated cells, about 30% of them failed in cytokinesis, resulted in formation of a cell with two individual nuclei or binucleate cell. When further divisions of the binucleate cells were observed, a binucleate cell divided into either four daughter cells with one nucleus each (Type A), three daughter cells, two of them with a nucleus each and a cell with two individual nuclei (Type B), two daughter cells with two individual nuclei each (Type C), or gave rise to a cell with four individual nuclei (Type D). Relationship of the frequency of occurrence of each of these four types with the developmental stages © 1987 Zoological Society of Japan at which the cells were dissociated was examined. decreased with the progress of developmental time. The frequency of Type-A division pattern Instead, the frequency of Type D increased during the period from the late morula to the late blastula stage. INTRODUCTION In a series of experiments [1-8], I have been studying formation of binucleate cells in dissoci- ated fish embryonic cells, nuclear fusion in the binucleate cells, and further divisions of the binucleate cells. _Blastomeres are easily dissoci- ated in Yamamoto’s saline solution from morula and blastula stage embryos of the teleost, Oryzias latipes [4]. When isolated embryonic cells are brought into physical contact each other within about 30sec after dissociation, cell fusion could be induced in more than half of the paired cells [4, 6], resulting in formation of a cell with two nuclei or binucleate cell. Binucleate cell forma- tion is also induced by abortive mitosis, in which normal karyokinesis is followed by incomplete cytokinesis [1, 2,8]. When I observed further behavior of two nuclei during interphase of the binucleate cells, I discovered a frequent fusion of the two individual nuclei in the cell [2,3]. In addition, it was noticed that there are four types Accepted February 16, 1987 Received December 19, 1986 of division pattern in binulceate cells [5] (cf. Fig. 1). In this study, I examined whether the frequency of occurrence of four types of division pattern is correlated with developmental stages at which the cells are dissociated. The result clearly showed an existence of a certain correlation between them. MATERIALS AND METHODS Developing eggs of the orange-red variety of the medaka, Oryzias latipes, were used in this study. Handling of eggs, dechorionation and dissociation of blastomeres were carried out as described previously [4]. After dechorionation, whole blastoderms of the morulae and blastulae were mechanically dissociated into their consti- tuent cells with sharp watchmaker’s forceps. The medium used was Yamamoto’s saline solution [9]. (0.75% NaCl, 0.02% KCl, 0.02% CaCl, 0.002% NaHCO;3, pH7.4). Fish blastoderm at these stages consists of cells of the enveloping layer, deep cells and cells of the periblast [10]. Only deep cells were used in this study. The whole procedure of cell dissociation, followed by 636 S. MIZUKAMI observation of the formation of binucleate cells and further divisions of the induced binucleate cells was performed in a deep depression glass slide with the aid of an inverted microscope, without exchanging the medium, at room temper- ature (23-28°C). Although in the saline solution dissociated cells showed surface adhesivity to the glass substrate or to each other, the adhesivity of cells was not so great that cells could not be dissociated or handled. Skillful handling allowed nearly complete cell dissociation without injuring cells. If the isolated cells were brought into physical contact each other within 30 sec after dissociation, cell fusion could be easily induced (3, 4, 6], re- sulted in formation of binucleate cells. On the other hand, binucleate cells were also formed by abortive cytokinesis [1, 2,8]. In this study, divi- sion pattern of binucleate cells formed by abortive mitosis was exclusively followed. RESULTS Blastomeres were dissociated from morulae and blastulae during interphase of divisions (Fig. 1a). Several minutes after dissociation, division of the isolated cells began. The nuclear membrane became invisible (Fig. 1b), followed by reappear- ance of two nuclei (Fig. lc). Usually, the karyo- kinesis is followed by normal cytokinesis. Howev- er, in nearly 30% of the cells, the cleavage furrow progressed to some extent but did not completely divided the cell (Fig. 1c). Instead, the furrow was retracted, resulting in formation of a binucleate cell or a cell with two separate nuclei in the protoplasm (Fig. 1d). Sometimes, pseudopodia appeared on the surface of dividing cells and they played important roles in the process of binucle- ate cell formation [e.g., 2]. When the cleavage furrow advanced to about a half of the cell diameter, hyaline pseudopodia were produced on the surface. The pseudopodia tended to rotate around the cell circumference. During the “circus movement” of pseudopodia, the dividing cell, passing through various shapes, transformed into a binucleate cell. During the following inter- phase, two individual nuclei frequently closed each other (Fig.le), and sometimes they oo) ele d e f Fic. 1. fish-embryonic cell (a—d), and four types of division pattern of the binucleate cells (e-}). Schematic representation of formation of a binucleate cell by abortive mitosis from a single dissociated In Type A (g), a binucleate cell divides into four daughter cells with one nucleus each. In Type B (h), a binucleate cell divides into three daughter cells, two with one nucleus each and one with two nuclei because of an additional abortive mitosis. In Type C (i), a binucleate cell divides into two daughter cells, each with two nuclei. In Type D (j), a binucleate cell fails in cytokinesis again, but four nuclei appear in the same protoplasm. Binucleate Fish Embryonic Cells 637 coalesced together [3]. About 15min after formation of binucleate cells, the nuclear membrane disappeared again, starting the next cycle of karyokinesis (Fig. 1f). About several minutes thereafter four individual nuclei became visible. However, the extent of cytokinesis varied, producing four types of daugh- ter cells. As schematically shown in Figure 1, in the first case, a binucleate cell divided at a time into four daughter cells with one nucleus each (I designated this pattern of division as Type A; Fig. 1g, Fig. 2A). In the second case, a binucleate cell divided into three daughter cells, two of which had one nucleus each and the other had two Fic. 2. Four types of daughter cells formed by divisions of binucleate cells. (A) Type A; four daughter cells with individual nuclei (Type B; Fig. 1h, Fig. 2B). In the third case, a binucleate cell divided into two daughter cells with two individual nuclei each (Type C; Fig. li, Fig. 2C). In the fourth case, a binucleate cell failed in cytokinesis again, result- ing in production of a cell with four separate nuclei in the protoplasm (Type D; Fig. 1j, Fig. 2D). In order to examine whether the frequency of occurrence of each of the four division patterns mentioned above is correlated with the develop- mental stages at which the cells are originated, blastomeres were dissociated from late morulae, early blastulae and late blastulae. At each de- pcan: eon eee a one nucleus each. (B) Type B; three daughter cells, two with one nucleus each and one with two nuclei. (C) Type C; two daughter cells with two nuclei each. Four nuclei are not in an identical focus. (D) Type D; a formed cell with four nuclei. Scale bar, 504m. n, nucleus. 638 S. MIZUKAMI TABLE 1. Frequency of occurrence of four types of division pattern in binucleate cells originated from three different stages of development Types of division pattern No. of cells Stages . examined A B C D Late morula pil 89 (40.2%) 20 ( 9.0%) 67 (30.3%) 45 (20.3%) Early blastula 233 39 (16.7%) 27 (11.6%) 92 (39.5%) I(G2.'%) Late blastula 236 25 (10.5%) 22 ( 9.3%) 71 (30.0%) 118 (50.0%) eco eee eco eco eco ec5o ooo Ye) eo0e @ ee eco eee eee eee eo0e eco eo0e ece eco eco ece eee ecco ooo eco eee eco ABC OD Late Morula Early Blastula Late Blastula Fic. 3. Frequencies of occurrence of the four types of division pattern of binucleate cells originated from the late morulae, early blastulae and late blastulae, respectively. The ordinate indicates percentages of occurrence of four types of division pattern, while the abscissa shows the three developmental stages at which cells are dissociated. The frequency of Type-A division pattern decreases with the progress of developmental time. Instead, the frequency of Type-D division pattern increases from the late morula stage to the late blastula stage. Frequencies of Type-B and Type-C division patterns are rather constant during the period. velopmental stage, more than 200 binucleate cells were examined. The results were summarized in Table 1 and Figure 3. Nearly 40% of binucleate cells formed by abortive mitosis of dissociated cells which origi- nated from the late morulae, showed Type-A division pattern. About 9% and 30% of them showed Type B and Type C pattern, respectively. Type-D division pattern was observed in about 20% of them. In binucleate cells derived from the early blas- tulae, the frequency of occurrence of Type A decreased; only 16.7% of them showed this pat- tern. Instead, the number of Type D pattern increased; nearly 32% of them showed this pat- tern. This inclination continued as development proceeded. In binucleate cells originated from late blastulae, about half of them showed Type-D division pattern, whereas only 10% of them showed Type A pattern (Table 1, Fig. 3). The frequencies of Type B and Type C were rather constant during the period examined. Type-B division pattern occurred in nearly 10% of the cells, irrespective of the developmental stages. Nearly 30% of them showed Type C pattern, although the frequency increased to a- Binucleate Fish Embryonic Cells bout 39% at the early blastula stage (Table 1, Fig. 3). DISCUSSION As clearly shown in this study, the frequency of occurrence of Type A division pattern decreased with the developmental time. Instead, that of Type D increased as development proceeded. This may be related to development of motile activities of embryonic cells. Kageyama [11] reported that the surface of blastomeres of meda- ka embryos begins to undulate gently at the early morula stage and that blebs or pseudopodia appear at the early blastula stage. He also reported that at the mid-blastula stage blebs or pseudopodia are found in most of the blastomeres [11]. These surface activities of the cells are thought to be involved in morphogenetic move- ments of fish embryos [10,11]. As described previously [2], pseudopodia are sometimes associ- ated with abortive mitosis, resulting in formation of binucleate cells. That is, an increase of the cell-surface activities might bring about the in- crease of frequency of Type-D division pattern of the binucleate cells, where abortive mitosis took place again. In other words, the increase of Type D pattern may be reflected by changes during embryogenesis of physiological properties of blastomeres, although the detail mechanism is a subject of further investigation. Polyploidy cells induced by artificial fusion with HVJ usually retained the polyploidy at least during the following several divisions [12]. As discussed in a previous paper [5], in the case of fused nuclei in binucleate fish embryonic cells, the cells recovered the diploidy from the polyploidy- like condition during the following division. This may be one of features of embryonic cells. ACKNOWLEDGMENTS I thank Dr. Noriyuki Satoh of Kyoto University for discussion and help in preparing the manuscript. 10 i 12 639 REFERENCES Mizukami, S. (1971) On the formation of cells with double nucleus in isolated embryonic cells of Ory- zias latipes (Japanese Medaka). Zool. Mag., 80: 132-136. Mizukami, S. (1976) Binucleate formation in iso- lated embryonic cells in the teleost, Oryzias latipes. Annot. Zool. Japon., 49: 120-130. Mizukami, S. (1978) Fusion of dissociated embryon- ic cells in the teleost, Oryzias latipes. III. Nuclear fusion during interphase in the fused cells. Cell Struct. Funct., 3: 275-277. Mizukami, S. (1979) Fusion of dissociated embryon- ic cells in the teleost, Oryzias latipes. II. On factors affecting the induction of spontaneuos fusion. Zool. Mag., 88: 17-23. Mizukami, S. (1981) Fusion of dissociated embryon- ic cells in the teleost, Oryzias latipes. V. Divisions of fused nuclei. Zool. Mag., 90: 251-253. Mizukami, S. and Satoh, N. (1977) Fusion of dis- sociated fish embryonic cells. J. Embryol. Exp. Morphol., 40: 265-270. Mizukami, S. and Satoh, N. (1979) Fusion of dis- sociated embryonic cells in the teleost, Oryzias latipes. IV. Changes in cell surface morphology related to this fusion: A scanning electron micro- scope study. Cell Struct. Funct., 4: 45-49. Shirakami, K. and Mizukami,S. (1965) Cyto- embryological studies of amphibians. V. On the origin, fate, and biological significance of the “dou- blenucleus”. Mem. Fac. Lib. Arts & Ed., Yama- nashi Univ., 16: 133-138. Yamamoto, T. (1975) “Medaka (Killifish)”, Biology and Strains. Keigaku Publ. Co., Tokyo. Trinkaus, J. P. (1963) The cellular basis of Fundulus epiboly. Adhesivity of blastula and gastrula cells in culture. Dev. Biol., 7: 513-532. Kageyama, T. (1977) Motility and locomotion of embryonic cells of the medaka, Oryzias latipes, during early development. Dev. Growth Differ., 19: 103-110. Ringertz, N.R. and Savage, R.E. (1976) Cell Hybrids. Academic Press, New York. ZOOLOGICAL SCIENCE 4: 641-648 (1987) © 1987 Zoological Society of Japan Speract Binds Exclusively to Sperm Tails and Causes an Electrophoretic Mobility Shift in a Major Sperm Tail Protein of Sea Urchins Norio SUZUKI, MASANORI KuURITA, KEN-ICHI YOSHINO and MASAAKI YAMAGUCHI Noto Marine Laboratory, Kanazawa University, Ogi, Uchiura, Ishikawa 927-05, Japan ABSTRACT— Intact spermatozoa, sperm heads and sperm tails of the sea urchin Hemicentrotus pulcherrimus were incubated with various concentrations of synthetic speract. The results of bioassay of the residual respiration stimulation activity in the supernatant obtained from each incubation mixture showed that more than 99% of speract in the original solution bound to sperm tails as well as intact spermatozoa, but little to sperm heads. Speract caused a mobility change of a major high-molecular weight sperm tail protein in H. pulcherrimus or Anthocidaris crassispina on SDS- polyacrylamide gels. INTRODUCTION It is known that sea urchin egg jelly contains small peptides which affect sperm respiration and motility. These include speract and its derivatives and resact. Speract (Gly-Phe-Asp-Leu-Asn-Gly- Gly-Gly-Val-Gly) is a peptide isolated from the egg jelly of MHemicentrotus pulcherrimus and Strongylocentrotus purpuratus [1-4]. The egg jelly of the sea urchins, H. pulcherrimus, Anthoci- daris crassispina and Lytechinus pictus, contains several speract derivatives ([Thr°]-speract, [Ser>]- speract, [Thr°?, Gln!°]-speract and Des-Gly'- [Thr°, Gln’°]-speract) [1,5, 6]. Speract and its derivatives not only stimulate sperm respiration and motility but also cause a transient increase in sperm cGMP concentrations [2,6]. A peptide named resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys- Val-Gly-Gly-Gly-Arg-Leu-NH;) has been purified from the egg jelly of the sea urchin Arbacia punctulata [7,8]. Resact has recently been shown to be a potent chemoattractant for A. punctulata spermatozoa [9]. Like speract, resact increases the cGMP levels, respiration, and motil- Accepted February 19, 1987 Received January 7, 1987 ity of A. punctulata spermatozoa [7,8]. Both resact and speract are species specific, although the physiological events measured appear to be the same. We have shown that exposing A. punctulata spermatozoa to resact results in a change in the electrophoretic mobility (from 160,000 to 150,000) of a major sperm membrane protein [8]. This protein has been purified to homogeneity and identified as guanylate cyclase [9-14]. Sea urchin spermatozoa contain extremely high guanylate cyclase activity, and this enzyme is localized in the plasma membrane of the flagellum [15, 16]. Guanylate cyclase in the flagellum mem- brane has been reported to exist as a phosphory- lated glycoprotein [12-14]. At fertilization, the enzyme looses 15 phosphate groups per molecule. The loss of phosphate groups is postulated to cause a shift in the mobility of guanylate cyclase on SDS-polyacrylamide gels from 160,000 daltons to 150,000 daltons [13]. In this paper we report that speract binds exclusively to sperm tails and it induces a mobility change of high molecular weight protein in sperm tails. 642 N. Suzuki, M. Kurita et al. MATERIALS AND METHODS Experimental animals and chemicals Sea urchins, H. pulcherrimus and A. crassispina, were collected at Japan Sea Coast (Tsukumo Bay) near Noto Marine Laboratory. Artificial sea water (ASW) containing 454mM NaCl, 9.7mM KCl, 24.9mM MgCh, 9.6mM CaCl), 27.1mM MgSO,, 4.4mM NaHCO; and 10mM_ tris (hydroxymethyl) aminoethane (Tris) or N-(2- acetamido)-2-aminoethane sulfonic acid (ACES) was prepared in the laboratory. Sodium dodecyl sulfate (SDS) (95% pure material), Tris, ACES and, molecular weight standards for gel elec- trophoresis (MW-SDS-200kit) were purchased from Sigma Chemical Co. Speract was custom synthesized for us by Peptide Institute, Inc., Osaka. Gamete collection and determination of respiration rates Spermatozoa obtaind by intracoelomic injection of 0.5M KCl were collected “dry” at room temperature and stored on ice at approximately 630 mg (wet weight)/ml until use. Respiration rates were determined at 20°C using a Yanagimoto PO-100A oxygen consump- tion recorder in 3.0ml capacity chamber fitted with a Clark type electrode at 20°C. Dry sperm (18.9mg wet weight/30l) was added to 2.96 ml of ASW (pH6.8). After recording stable basal respiration rates for several minutes, samples (10u1) were added. The new respiration rates were determined over the next 3min. When respiratory stimulation activity of speract solution incubated with intact spermatozoa, sperm heads or sperm tails was determined, 2.97 ml of super- natant obtained by centrifugation of the incuba- tion mixture was added to the chamber and then dry sperm (18.9mg wet weight/30u1) was added. The initial rate of sperm respiration was calcu- lated from the tangent to the slope recorded after the onset of experiment. Respiration rates were determined by assuming the solubility of pure O, at 1 atm to be 234 nmoles/ml of ASW at 20°C. Preparation of sperm tails and sperm heads Spermatozoa (usually 10-15ml of dry sperm) were diluted with 250 vol of ice cold ASW and centrifuged at 1,000xg for 5min at 4C. The supernatant was then centrifuged at 5,000 xg for 30 min at 4°C. The sperm pellet was suspended in 20 vol of ice cold ASW and homogenized in ice bath with a 50 ml Dounce homogenizer fitted with a teflon pestle [17, 18]. About 15-20 strokes were enough to detach the tails from the heads as monitored by phase-contrast microscopy. The homogenate was centrifuged at 1,000g for 30 min at 4°C to pellet the sperm heads. The sperm head fraction was suspended in 100-150 ml of ice cold ASW and centrifuged at 2,000 xg for 30 min at 4°C. The resultant pellet (sperm heads) was used for experiments. The supernatant containing tails was centrifuged at 6,000g for 30min at 4°C, and the resultant pellet was suspended in 100-150 ml of ice cold ASW. The suspension was centrifuged at 2,000 xg for 30min at 4°C and the supernatant was centrifuged at 6,000xg for 30 min at 4°C. The resultant pellet (sperm tails) was used for experiments. Phase-contrast microscopy was used routinely to monitor the extent of contamination by sperm heads in the tail fraction. As shown in Figure 1, contamination by intact spermatozoa in the head and tail fractions and sperm heads in the tail fraction was little. Other methods To prepare samples for electrophoresis, sper- matozoa (18.9mg wet weight) were incubated with or without various concentrations of speract in normal ASW. At 1min, the reaction was stopped by the addition of trichloroacetic acid (final concentration of 10%) and the samples were centrifuged at 10,000g for 30min at 4°C. The resultant pellet was resuspended in ice cold 90% (v/v) acetone and then centrifuged. The pellet was resuspended in ice cold acetone. After centrifugation, the acetone layer was removed and the pellet was lyophilized. Then, 10% SDS (200-300) was added to the residue, and the mixture was placed in a boiling water bath for 5min and vortexed vigorously. This procedure was repeated until the solution became clear. For Effect of Speract on a Sperm Protein 643 electrophoresis, 10 ug of sperm protein was added per lane. Electrophoresis on a 5.5% polyacryla- mide slab gel was performed in the presence of SDS according to Laemmli [19]. The gel was subsequently stained by the method of Morrissey [20]. The molecular weight of proteins was calculated from the relative mobility of co- electrophoresised standards (myosin, rabbit mus- cle, Mw 205,000; (-galactosidase, E. coli, Mw 116,000; phosphorylase b, rabbit muscle, Mw 97,400; albumin, bovine plasma, Mw 66,000 and albumin, egg, Mw 45,000). Protein was deter- mined by the method of Lowry et al. according to Schacterle and Pollack’s modification [21, 22]. RESULTS Respiratory stimulation activity of speract solution incubated with or without intact spermatozoa, sperm heads or sperm tails As shown in Figure 2 (control), speract half- maximally stimulated H. pulcherrimus spermato- zoan respiration at 0.2 nM. This is comparable to reported values [2, 4]. Spermatozoa (18.9 mg wet weight) were incubated with various concentra- tions of speract in 3ml of ASW (pH 6.8) for 3 min ’ \ ~‘ fs , Pa é . J 7 / \e : } ’ a . 2 ee = . 3 4 > *. i “ Pee = ne : J INTACT Pic. "1: pulcherrimus. Bars indicate 100nm. HEAD at 20°C and then the mixtures were centrifuged at 10,000 x g for 30min at 0°C to remove spermato- zoa. The resultant supernatant was assayed for the respiration stimulation activity. The super- natant obtained from incubation of spermatozoa with lower than 3.7210~°M of speract showed the decreased activity for respiration stimulation compared with the corresponding control speract solutions (Fig. 2, intact spermatozoa). Intact speract was recovered from the supernatant. When the resultant sperm pellet was extracted by 70% ethanol, no speract fragments were found in the extracts. Results obtained by the same experiments using sperm heads or sperm tails are shown in Figure 2 (sperm heads and sperm tails). In these experiments, the added amount of sperm heads or sperm tails was equivalent to that of intact spermatozoa based on the amount of pro- tein. A sperm head contained 83% protein of intact spermatozoon. When sperm heads were used for incubation with various concentrations of speract, the resultant supernatant showed almost the same respiration stimulation activity as that of the corresponding control solution while the su- pernatant obtaind from the incubation mixtures of sperm tails exhibited similar stimulation activity to that with intact spermatozoa. These results = TAIL Phase-contrast microscopic photographs of intact spermatozoa, isolated sperm heads and tails of H. 644 N. Suzuki, M. Kurita et al. Fic. 2. Respiration stimulating activity of various con- centrations of speract before or after incubation with H. pulcherrimus spermatozoa, sperm heads or Stimulation of Respiration / sperm tails. {_]——., supernatant from incuba- SPERM tion mixture without intact spermatozoa; TAILS | , Supernatant from incubation mixture INTACT with sperm tails; ——-@——,, supernatant from in- SPERMATOZOA cubation mixture with sperm heads; ——&—, B supernatant from incubation mixture with intact spermatozoa. 13) Qe Om 9 Oe a) a On ai —Logio(Concentration) (M) Fic. 3. The effect of various concentrations of speract on the mobility of a high-molecular weight protein of H. pulcherrimus spermatozoa. 1, 1.12<1071°M; 2, 1.1210~°M; 3, 1.12<10~°M; 4, 1.12x10~7M; 5, 1.12 10~°M; 6, 1.1210->M; 7, none. strongly suggest that speract binds exclusively to treated with or without speract sperm tails but not or less to sperm heads. P When H. pulcherrimus spermatozoa _ were SDS-polyacrylamide gel electrophoresis of sea treated with synthetic speract higher than 1.12 urchin spermatozoa, sperm heads and sperm tails 10~8M in normal ASW, one new major protein Fic. Effect of Speract on a Sperm Protein 645 4. The effect of speract on the mobility of a high-molecular weight protein of H. pulcherrimus spermatozoa, sperm heads or sperm tails. 1, intact spermatozoa without speract; 2, intact spermatozoa with speract (1.12 x 10~°M); 3, sperm tails with speract (1.1210~'*M); 4, sperm tails with speract (1.12x10~°M); 5, sperm tails with speract (1.12x10~°M); 6, sperm tails without speract; 7, sperm heads with speract (1.12 10~°M); 8, sperm heads with speract (1.12 10~°M); 9, sperm heads without speract; 10, intact spermato- zoa without speract. 136 K “3 iv 131K Fic. 5. The effect of various concentrations of speract on the mobility of a high- molecular weight protein of A. crassispina spermatozoa. The concentrations of speract used in each lane were as follows: 1, none; 2, 3.74x10—°M; 3, 3.74x 10~°M; 4, 3.74x10~7M; 5, 3.7410 °M. 646 N. Suzuki, M. Kurita et al. appeared at an apparent molecular weight of 128,000 which was not found in spermatozoa treated without speract (Fig. 3). Intact spermato- zoa without speract had a protein molecular weight of which was 131,000. Treatment of spermatozoa lower than 1.1210~°M of speract resulted in no change in the mobility of any sperm proteins on an SDS-polyacrylamide gel. The 128,000 dalton protein was found as a major protein in sperm tails isolated in normal ASW, while it was faintly detected in sperm heads (Fig. 4). The 131,000 dalton protein was not detected in sperm tails or sperm heads. However, when sperm tails were isolated in acidified ASW (pH 5.6), the 131,000 dalton protein was detected in sperm tails as a major protein and the 128,000 dalton form was not found (not shown). As shown in Figure 5, in A. crassispina sperma- tozoa treated with speract, a new protein whose mass is 131,000 daltons was observed on an SDS-polyacrylamide gel. This protein was not found in spermatozoa without speract, which had a protein with the molecular weight of 136,000. The conversion of the 136,000-molecular weight form to 131,000 dalton form in A. crassispina spermatozoa occurred completely with 3.74x 10~°M of speract. The 131,000 dalton protein was exclusively found in isolated sperm tails (not shown). DISCUSSION It has been reported that radiolabeled speract binds specifically and in a saturable manner to S. purpuratus spermatozoa [23]. Although we did not use labeled peptide, our results presented here also showed that speract binds to H. pulcher- rimus sperm cells (Fig. 2, intact spermatozoa). In the experiments, we biologically determined the amounts of unbound speract. When a fixed amount (18.9 mg wet weight) of spermatozoa was incubated with speract lower than 3.72 X10~°? M, the residual respiration stimulation activity of speract in the supernatant of the incubation mixture was about one-hundredth of the corre- sponding control speract solution. This suggests that more than 99% speract in the original solution was bound to intact spermatozoa. The spermatozoa did not seem to digest speract since no speract fragments were found and intact sper- act was obtained from the supernatant by HPLC after 70% ethanol extraction of the samples. The same results were obtained from experiments with sperm tails (Fig. 2, sperm tails), while experi- ments with sperm heads showed that almost all speract molecules remained in the supernatant, suggesting that speract incubated with the sperm heads did not bind to the heads. These results indicate that speract binds exclusively to sperm tails but not or little to sperm heads. Although the procedure we used here is not accurate enough for determination of binding numbers of speract to sperm cells, it seems to have some advantages compared with the method using labeled speract. It is more simple and economical and it does not need to use tyrosine-substituted speract or Bolton-Hunter adduct of speract which might be troublesome for the bindings because of a large molecule at the N-terminal part of speract. As shown in Figures 3, 4 and 5, speract induced an electrophoretic mobility change (from 131,000 to 128,000 in H. pulcherrimus and from 136,000 to 131,000 in A. crassispina) in a major sperm protein. This results is essentially the same as that obtained from L. pictus spermatozoa treated with speract derivatives [6]. When sperm tails were isolated from H. pulcherrimus spermatozoa in normal ASW (pH 8.0), there was no detectable protein band on a SDS-polyacrylamide gel at an apparent molecular weight of 131,000. The sperm tails possessed the 128,000 dalton protein, which was found in intact spermatozoa treated with speract. It is known that H* concentration in sperm cells is higher than that in normal ASW [24]. Speract has been shown to induce Na‘t/Ht exchange across the plasma membrane of sea urchin spermatozoa [25]. It is possible that Ht concentrations in sperm tails would be changed by exposing the cut end of the tail to lower Ht concentrations in ASW. In this connection, it should be stressed that when sperm tails were isolated in acidified ASW (pH5.6), the 131,000 dalton protein was detected as a major protein in H. pulcherrimus sperm tails. These suggest that the mobility shift observed in a major sperm protein may be triggered by alkalinization in Effect of Speract on a Sperm Protein sperm cells induced by speract. Ward and Vaquier have reported that the shift in electrophoretic mobility and concomitant loss of phosphate can be induced in vitro by calf intestinal alkaline phosphatase [10]. The in- creased mobility is thought to result from in- creased binding of SDS. Our results show that speract induces the increased mobility of a high molecular weight protein and the protein localizes exclusively in sperm tails (Fig. 4). It is known that a specific protein phosphatase involves in dephosphorylation of many proteins [28]. Al- though it is not proved yet, we presume that the electrophoretic mobility shift of a sperm tail protein from 131,000 daltons to 128,000 daltons is a result of dephosphorylation of the protein and a specific protein phosphatase involves in the pro- cess. It has been reported that resact, another sperm activating peptide purified from the egg jelly of the sea urchin, A. punctulata, causes a mobility shift in a major sperm membrane protein which was identified as guanylate cyclase [8, 11, 12]. The enzyme has been reported to be localized in sperm flagellar membrane [15,16]. Guanylate cyclase in A. punctulata has been suggested to be a receptor for resact [26]. The protein observed here may represent guanylate cyclase, although this is not proven. From the experiments on S. purpuratus spermatozoa incubated with radiolabeled speract analogue and a chemical crosslinking reagent, Dangott and Garbers have suggested that a speract receptor is a glycoprotein whose molecular mass is 77,000 daltons as esti- mated by SDS-polyacrylamide gel electrophoresis [27]. This suggests that receptors for sperm activating peptides are different from one species to another or the molecular weight of guanylate cyclase is different in different species. To solve this we are now purifying several proteins from sperm flagellar membrane. ACKNOWLEDGMENTS We wish to thank Mr. T. Shinya and Mr. M. Matada for collecting the sea urchins. This work was supported by research grants to N.S. from the Ministry of Education, Science and Culture of Japan (No. 59540471 and No. 60480022). 10 1 12 13 647 REFERENCES Suzuki, N., Nomura, K., Ohtake, H. and Isaka, S. (1981) Purification and the primary structure of sperm activating peptides from the jelly coat of the sea urchin eggs. Biochem. Biophys. Res. Commun.., 99: 1238-1244. Garbers, D. L., Watkins, H. D., Hansbrough, J. R., Smith, A. and Misono, K.S. (1982) The amino acid sequence and chemical synthesis of speract and of speract analogues. J. Biol. Chem., 257: 2734-2737. Suzuki, N., Hoshi, M., Nomura, K. and Isaka, S. (1982) Respiratory stimulation of sea urchin sper- matozoa by egg extracts, egg jelly extracts and egg jelly peptides from various species of sea urchins: taxonomical significance. Comp. Biochem. Physiol., 72A: 489-495. Suzuki, N., Ohizumi, Y., Yasumasu, I. and Isaka, S. (1984) Respiration of sea urchin spermatozoa in the presence of a synthetic jelly coat peptide and ionophores. Dev. Growth Differ., 26: 17-24. Nomura, K., Suzuki, N., Ohtake, H. and Isaka, S. (1983) Structure and action of sperm activating peptides from the egg jelly of a sea urchin, Anthoci- daris crassispina. Biochem. Biophys. Res. Com- mun., 117: 147-153. Shimomura, H., Suzuki,N. and Garbers, D.L. (1986) Derivatives of speract are associated with the eggs of Lytechinus pictus sea urchins. Peptide, 7: 491-495. Suzuki, N. and Garbers, D. L. (1984) Stimulation of sperm respiration rates by speract and resact at alkaline pH. Biol. Reprod., 30: 1167-1174. Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C.S., Ward, G.E., Bentley, J. K. and Garbers, D.L. (1984) A peptide associated with eggs causes a mobility shift in a major plasma membrane protein of spermatozoa. J. Biol. Chem., 259: 14874-14879. Ward. G.E., Brokaw, C.J., Garbers, D. L. and Va- quier, V.D. (1985) Chemotaxis of Arbacia punc- tulata spermatozoa to resact, a peptide from the egg jelly layer. J. Cell Biol., 101: 2324-2329. Ward, G.E. and Vaquier, V.D. (1983) Dephos- phorylation of a major sperm membrane protein is induced by egg jelly during sea urchin fertilization. Proc. Natl. Acad. Sci., USA, 80: 5578-5582. Ward, G.E. Garbers,D.L. and Vaquier, V.D. (1985) Effects of extracellular egg factors on sperm guanylate cyclase. Science, 227: 768-770. Ward, G.E., Moy, G.W. and Vaquier, V. D. (1986) Phosphorylation of membrane-bound guany- late cyclase of sea urchin spermatozoa. J. Cell Biol., 103: 95-101. Vaquier, V. D. and Moy, G. W. (1986) Stoichiome- try of phosphate loss from sea urchin sperm guany- 14 ils) 16 17 18 19 20 21 648 late cyclase during fertilization. Biochem. Biophys. Res. Commun., 137: 1148-1152. Radany, E.W., Gerzer, R. and Garbers, D.L. (1983) Purification and characterization of particu- late guanylate cyclase from sea urchin spermatozoa. J. Biol. Chem., 258: 8346-8351. Gray, J. P. and Drummond, G.I. (1976) Guanylate cyclase of sea urchin sperm: subcellular localization. Arch. Biochem. Biophys., 172: 31-38. Sano, M. (1976) Subcellular localizations of guany- late cyclase and 3:5-cyclic nucleotide phosphodies- terase in sea urchin sperm. Biochim. Biophys. Acta, 428 : 525-531. Lee, H.C. (1985) The voltage-sensitive Nat/H* exchange in sea urchin spermatozoa flagellar mem- brane vesicles studied with an entrapped pH probe. J. Biol. Chem., 260: 10794-10799. Tombes, R.M. and Shapiro, B. M. (1985) Metabo- lite channeling: A phosphocreatine shuttle to medi- ate high energy phosphate transport between sperm mitochondrion and tail. Cell, 41: 325-334. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacte- riophage T4. Nature, 257: 680-685. Morrissey, J. H. (1981) Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem., 117: 307-310. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and 22 23 24 25 26 27 28 N. Suzuk1, M. Kurita et al. Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Schacterle, G.R. and Pollack, R.L. (1973) A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal. Biochem., 51: 654-655. Smith, A. and Garbers, D. L. (1983) The binding of an I'*°-speract analogue to spermatozoa. In “Biochemistry of Metabolic Processes”. Ed. by D. L. F. Lennon, F. W. Stratman and R. N. Zalten, Elsevier Biomedical, New York, pp. 15-18. Christen, R., Schackmann, R.W. and Shapiro, B.M. (1982) Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Strongylocentrotus purpuratus. J. Biol. Chem., 257: 14881-14890. Hansbrough, J.R. and Garbers, D.L. (1981) Sodium-dependent activation of sea urchin sperma- tozoa by speract and monensin. J. Biol. Chem., 256: 2235-2241. Shimomura, H., Dangott, L.G. and Garbers, D. L. (1986) Covalent coupling of a resact analogue to guanylate cyclase. J. Biol. Chem., (in press). Dangott, L. J. and Garbers, D. L. (1984) Identifica- tion and characterization of the receptor for speract. J. Biol. Chem., 259: 13712-13716. Ingebritsen, T.S. and Cohen, P. (1983) Protein phosphatases: Properties and role in cellular regula- tion. Science, 221: 331-338. ZOOLOGICAL SCIENCE 4: 649-656 (1987) © 1987 Zoological Society of Japan Purification and Structure of Mosact and Its Derivatives from the Egg Jelly of the Sea Urchin Clypeaster japonicus Norio SUZUKI, MASANORI KuRITA, KEN-ICHI YOSHINO, Hiroko Kaysura!, Konyt Nomura? and MASAAKI YAMAGUCHI Noto Marine Laboratory, Kanazawa University, Ogi, Uchiura, Ishikawa 927-05, ‘National Institute for Basic Biology, Okazaki 444, and *Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173, Japan ABSTRACT—Peptides (mosact and its derivatives) associated with the eggs of the sea urchin, Clypeaster japonicus, which stimulate C. japonicus sperm respiration rate but neither Anthocidaris crassispina nor Glyptocidaris crenularis sperm respiration rate, were purified and their amino acid sequences were determined. The sequences of mosact, Des-Gln®, Asn’-[His°]-mosact and Des-Gin°, Asn’-[Phe®]-mosact were found to be Asp-Ser-Asp-Ser-Ala-Gln-Asn-Leu-Ile-Gly, Asp-Ser-Asp-Ser- Ala-His-Leu-Ile-Gly and Asp-Ser-Asp-Ser-Ala-Phe-Leu-Ile-Gly, respectively. Mosact and its deriva- tives half-maximally stimulate C. japonicus sperm respiration rate at 0.5nM at slightly acidic pH values (6.6-6.8) and cause a shift in the apparent molecular weight (126,000-123,000) of a sperm plasma membrane protein at extracellular pH value of 8.0. Sodium ions were essential for the formation of 123,000 dalton protein. INTRODUCTION The sea urchin egg jelly is composed mainly of polysaccharide-protein complex which is separated into a sialoglycoprotein and a fucose polysac- charide [1-3]. The latter is considered to induce the sperm acrosome reaction [3]. In addition to these major macromolecular components, the egg jelly contains several oligopeptides so-called sperm activating peptides [4-11]. These include speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and its derivatives ({Thr°]-speract: Gly-Phe-Asp- Leu-Thr-Gly-Gly-Gly-Val-Gly; [Ser?]-speract: Gly-Phe-Asp-Leu-Ser-Gly-Gly-Gly-Val-Gly; Des- Gly!-[Thr°, Gin'®]-speract: Phe-Asp-Leu-Thr-Gly- Gly-Gly-Val-Gln; [Thr°, Gln'°]-speract: Gly-Phe- Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln) and _ resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly- Gly-Arg-Leu-NH 2). Speract stimulates sperm res- Accepted February 19, 1987 Received January 7, 1987 piration with concomitant Na*/H* exchange across the sperm plasma membrane and increases the concentrations of cAMP and cGMP in sperm cells [12, 13]. Resact which is specific for Arbacia punctulata spermatozoa has been reported to cause a shift in the apparent molecular weight of guanylate cyclase in the sperm membrane of A. punctulata with dephosphorylation of the enzyme [10, 13-17]. Recently resact was identified to act as a potent chemoattractant for A. punctulata spermatozoa [18]. We have reported previously that the egg jelly of the sea urchin C. japonicus contains a respiration stimulating peptide(s) specific for C. japonicus spermatozoa [19]. The peptide named mosact causes a mobility change (from 126,000 daltons to 123,000 daltons) of the sperm protein of C. japonicus. Here we purify and present the amino acid sequence of the peptide and its derivatives. 650 N. Suzuk1, M. Kurita et al. MATERIALS AND METHODS Experimental animals and chemicals Sea urchins, Clypeaster japonicus, Hemicentro- tus pulcherrimus and Anthocidaris crassispina, were collected at Japan Sea Coast (Tsukumo Bay) near Noto Marine Laboratory. The sea urchin G. crenularis was collected at the Aomori Bay near the Asamushi Marine Biological Station. Artificial sea water (ASW) containing 454mM NaCl, 9.7 mM KCl, 24.9mM MgChL, 9.6mM CaCl, 27.1 mM MgSQ,, 4.4mM NaHCO; and 10 mM tris (hydroxymethyl) aminoethane (Tris) or 10mM N-(2-acetamido)-2-aminoethane sulfonic acid (ACES) was prepared in the laboratory. Na‘ -free ASW had the same composition except that choline chloride was substituted for NaCl and NaHCO; was for KHCO3. Sodium dodecyl sulfate (SDS) (95% grade material), Tris, ACES and molecular weight standards for gel electrophoresis (MW-SDS-200 kit) were obtained from Sigma Chemical Co. Acetonitrile (ACN) of liquid chro- matography grade and trifluoroacetic acid (TFA) were from Wako Pure Chemical Co. Speract was custom synthesized for us by Peptide Institute, Inc., Osaka. [Thr°]-speract and [Ser>]-speract were purified from the egg jelly of H. pulcherrimus and A. crassispina, respectively. Synthetic resact and synthetic Des-Gln®, Asn’-[Asn’, His°]-mosact (Asn-Ser-Asp-Ser-Ala-His-Leu-Ile-Gly) were gen- erous gifts from Dr. H. Shimomura and Dr. D. L. Garbers, respectively. Other reagents were of analytical grade. Gamete collection and determination of respiration rates Spermatozoa or eggs were obtained by intra- coelomic injection of 0.5M KCl. Eggs were colleted in sea water and then treated with acidified sea water (pH 5.0) to remove the jelly coat rapidly. Spermatozoa were collected “dry” at room temperature and stored on ice at approx- imately 330 mg (wet weight)/ml until use. Respiration rate of sea urchin spermatozoa was determined as described previously [19]. Isolation of sperm heads and tails Spermatozoa (usually 10-15 ml of dry sperm) were diluted with 250 vol of ice cold ASW and centrifuged at 1,000xg for 5min at 4°C. The supernatant was then centrifuged at 5,000 xg for 30 min at 4°C. The sperm pellet was suspended in 20 vol of ice cold ASW and homogenized in an ice bath with a 50 ml-Dounce homogenizer fitted with a teflon pestle [20, 21]. About 15-20 strokes were enough to detach the tails from the heads as monitored by phase-contrast microscopy. The homogenate was centrifuged at 1,000Xg for 30 min at 4°C to pellet the sperm heads. The sperm head fraction was suspended in 100-150 ml of ice cold ASW and centrifuged at 2,000 x g for 30 min at 4°C. The resultant pellet (sperm heads) was used for experiments. The supernatant fraction containing tails was centrifuged at 6,000 x g for 30 min at 4°C, and the pellet was suspended in 100- 150 ml of ice-cold ASW. The suspension was centrifuged at 2,000 xg for 30 min at 4°C and the supernatant was centrifuged at 6,000 x g for 30 min at 4°C. The resultant pellet (sperm tails) was used for experiments. Phase-contrast microscopy was used routinely to monitor the extent of contamina- tion by sperm heads in the tail fraction. In some experiments, Na*-free ASW was used for prepa- ration of sperm heads and tails. Preparation of sperm plasma membrane Dry sperm were washed twice with millipore- filtered sea water (MFSW) by centrifugation at 2,000 x g for 15 min at 4°C. The sperm pellet was suspended in 20 vol of MFSW containing 20 mM Tris-HCl (pH9.0), 2mM __ benzamidine-HCl, 0.01% (w/v) streptomycin sulfate and 0.01% (w/ v) penicillin G [22]. The suspension was incubated for 12 hr at 4°C and then centrifuged at 7,000 xg for 30 min at 4°C. The supernatant was collected and re-centrifuged under the same conditions. The second 7,000 xg supernatant was then centrifuged at 105,000 x g for 60 min at 4°C to pellet the sperm plasma membrane. Purification of mosact and its derivatives from C. japonicus egg jelly The egg jelly solution of C. japonicus was mixed Mosact and Its Derivatives 651 with a 2-fold volume of 95% ethanol and then centrifuged at 10,000xg for 30 min at 4°C. The supernatant fraction was concentrated under re- duced pressure and delipidated by chloroform extration. The sample was then lyophilized. The residue was dissolved in distilled water. Mosact and its derivatives were purified by sequential high pressure liquid chromatographies. High pressure liquid chromatography (HPLC) HPLC was performed using a Shimadzu model 6A chromatography system with octyl columns (Shimpack C-8 Prep, 104m, 20250 mm _ for step-wise elution; Unisil C-8, 5 wm, 4.6250 mm for gradient elution). The column effluent was monitored for absorbance at 225nm _ using a Shimadzu model SPD-6AV spectrophotometer equipped with a variable wavelength detector. In general, separations were carried out using a combination of the following programs: Program I: flow rate was 9.9 ml/min, the column (Shimpack C-8 Prep) was equilibrated with 5% ACN in 0.1% TFA in water and eluted for 15 min, and then eluted for next 15 min with 60% ACN in 0.1% TFA. Program II: flow rate was 1.0 ml/min, the column (Unisil C-8) was equilibrated with 10% ACN in 0.1% TFA and eluted for 10 min, fol- lowed by a linear gradient from 10% ACN to 50% ACN in 0.1% TFA over a 50-min time period. Amino acid and sequence analysis Amino acid analysis was carried out with an automatic amino acid analyzer Hitachi 835-50 after hydrolysis with constant-boiling HCl. Nor- mally, 2-Snmoles of peptide were hydrolyzed. Sequence analysis was performed by automated Edman degradation using a 470A Protein Se- quencer (Applied Biosystems, Inc.) with 100 pmoles of peptide. Other methods Polyacrylamide gel electrophoresis was _per- formed essentially as described by Laemmli [23]. A gel was run for 8 hr at 10 mA/slab gel at room temperature. The gel was subsequently stained by the method of Morrissey [24]. Protein was deter- mind by the method of Lowry et al. according to Schacterle and Pollack’s modification [25, 26]. RESULTS Purification of mosact and its derivatives The egg jelly solution was obtained by the treatment of C. japonicus eggs with acidified (pH 5.0) sea water for 15 min and by centrifugation to remove eggs. Five liters of the egg jelly solution were mixed with 10 liters of 95% ethanol and the suspension was then centrifuged at 10,000 xg for 30 min at 4°C. The resultant supernatant fluid was concentrated with a rotary evaporator at 50°C. The concentrated sample after being centrifuged to remove salt was delipidated by chloroform extraction and then lyophilized. The residue was dissolved in a minimum volume of distilled water and filtered through a millipore-filter (0.45 um). The filtrate was applied onto a Shimpack C-8 Prep column equilibrated with 5% ACN in 0.1% TFA. The column was eluted first with equilibrating solvent for 5 min and then eluted according to Program I. The fractions were assayed for their ability to stimulate sperm respiration rate. The fractions eluted with 60% ACN in 0.1% TFA were found to contain respiration stimulation activity (Fig. 1A). The fractions were pooled and lyophi- lized. The residue was dissolved in 10% ACN in 0.1% TFA and was further purified by repeated HPLC with a Unisil C-8 column using Program II (Fig. 1B-H). Three active materials were obtained. Amino acid composition and sequence The purified peptides were analyzed for amino acid composition. As shown in Table 1, peptide b was consisted of 10 amino acids while peptide a and c contained 9 amino acids, respectively. Peptide b had the same amino acid composition of the peptide named as mosact in our previous report [19]. The compositions of peptide a and c were similar to that of peptide b while peptide a had three residues of aspartic acid and one residue of glutamic acid and peptide c contained one residue of phenylalanine. These peptides were then subjected to sequence analysis. The se- quences of peptide a and c were similar to that of 652 Relative Absorbance (225 nm) 0 20 were injected onto a reverse-phase (Shimpack C-8 Prep, 20250mm) column. elution. Solid bar indicates active fractions; B: Further purification of the active fractions on a reverse-phase (Unisil C-8) column. Program II was used for elution. Fractions indicated by a, b and c were active and used for further purification; C-—H: Re-HPLC of the active fractions (a, b and c). Elution conditions used 40 were the same as Fig. 1B. TABLE 1. N. Suzuki, M. Kurita et al. € D Or 0 Time (min) Fic. 1. High pressure liquid chromatographs of peptides. A: Elution profile of crude extracts. Crude extracts (0) 0), 1720), 9 2.0) obtained from Clypeaster japonicus egg jelly His Total residue Peptide a 2.080 (2) 1.914 (2) (P2151) 1.063 (1) 1.000 (1) 1.115 (1) 0.935 (1) (9) Peptide b 2.604 (3) 778") 0.718 (1) 1.148 (1) 0.987 (1) 1.000 (1) 1.040 (1) (10) 0 10 20 30 OME 7.0) 60) Amino acid compositions and sequences of sperm activating peptides Peptide c 2.054 (2) 1.880 (2) 1.239 (1) 1.020 (1) 1.000 (1) 1.092 (1) 1.128 (1) (9) Program I was used for Peptide a (Des—Gln®, Asn’—[His°]|—mosact) Asp—Ser—-Asp-Ser—Ala—His—Leu-Ile—Gly Peptide b (mosact) Asp-Ser-Asp—Ser—-Ala—Gln—Asn-—Leu-Ile—Gly Peptide c (Des-—Gin®, Asn’—[Phe®]-mosact) Asp—Ser-Asp-Ser-Ala—Phe-—Leu-Ile-—Gly Mosact and Its Derivatives 653 peptide b (mosact), thus being named as mosact derivatives to be Des-Gln°, Asn’-[His°]-mosact and Des-Gln°, Asn’-[Phe®]-mosact, respectively. However, when the sequence of peptide c was determined, cycle 6 resulted in no identifiable amino acid and phenylalanine was not detected at any cycle. Sixth residue of the peptide was assigned to phenylalanine since the amino acid analysis data clearly showed that the peptide contained one phenylalanine residue. To confirm the sequence, the peptide was synthesized by liquid phase methods with phenylalanine as the sixth residue. The synthesized peptide had the following amino acid composition: Asp (1.0), Ser (1.9), Gly (1.0), Ala (1.1), Ile (1.0), Leu (1.1) and Phe (0.9) and the following sequence: Asp-Ser- Asp-Ser-Ala-Phe-Leu-Ile-Gly. The synthesized peptide also stimulated C. japonicus sperm re- spiration rate at the same concentration as the native peptide (Fig. 2) Respiration stimulation Mosact and its derivatives stimulated sperm respiration rates at slightly acidic pH values. The effect was specific for C. japonicus spermatozoa. Stimulation of Respiration (nmoles O./min/mg wet weight spermatozoa) ris Boh EI. 9° 68. FO SD -Log,,(Concentration) (M) Fic.2. The effect of mosact (4), Des-Gln®, Asn’- [Phe®]-mosact (©), Des-Gln®, Asn’-[His*]-mosact (MI). synthetic Des-Gln®, Asn’-[Phe*]-mosact (@) and synthetic Des-Gln®, Asn’-[Asn', His®] (A) on C. japonicus sperm respiration rates. Mosact and its derivatives were half-maximally active at about 0.5nM on the stimulation of C. japonicus sperm respiration rate (Fig. 2). These peptides did not show this effect on A. crassispina, H. pulcherrimus or G. crenularis spermatozoa. Speract and its derivatives such as [Ser°]-speract and [Thr]-speract at concentrations as high as 104M failed to elevate respiration rate of C. Japonicus spermatozoa. SDS-polyacrylamide gel electrophoresis of proteins of intact spermatozoa, sperm heads,sperm tails and sperm plasma membrane Crude mixtures of C. japonicus egg factors caused a mobility shift of a sperm protein on a SDS-polyacrylamide gel [19]. When C. japonicus spermatozoa were incubated in normal ASW (pH 8.0) with various concentrations of mosact for 1 min the high molecular weight of a protein of the spermatozoa shifted from about 126,000 daltons to 123,000 daltons (Fig. 3). Mosact failed to cause any detectable mobility change in the other pro- teins of C. japonicus spermatozoa. About 735 nM mosact were enough to cause the complete mobil- ity change of the protein. Sodium ions are essential for respiration stim- ulation by speract [8, 12] and for that by mosact (not shown). Spermatozoa were washed in Na~- free ASW (pH 8.0) and then incubated in Na‘ - free ASW (pH 8.0) with mosact for 1 min. Mosact failed to cause the mobility change of the 126,000 daltons protein (Fig. 4). The continued incubation of sperm cells for 1 hr showed no mobility change of the protein. Sperm heads (nucleus and mitochondrial mid- piece) were separated from sperm tails and sperm plasma membrane fraction was prepared by the procedure of Podell et al. [23]. These fractions (sperm heads, sperm tails and plasma membrane) were analyzed by SDS-polyacrylamide gel elec- trophoresis after incubation of spermatozoa in normal ASW (pH8.0) with or without mosact (Fig. 5). On a per-mg-of-protein basis, the 123,000 dalton protein derived from the modification of 126,000 dalton protein was found exclusively in the sperm tails and sperm plasma membrane fractions. 654 N. Suzuki, M. Kurita et al. 205K— - ee 6fy 6 126K wow ¥ aed KO Go a a « ~~, ~ g2 Fic. 3. The effect of various concentrations of mosact on the mobility of a high-molecular weight protein of spermatozoa. C. japonicus spermatozoa were incubated at 20°C with various concentrations of mosact in normal ASW (pH 8.0) (lane 2: none; lane 3: 7.35x107-°M; lane 4: 7.35x10~°M; lane 5: 7.35X10~°M). At 1 min of incubation, the reac- tion was stopped by the addition of trichloroacetic acid (final concentration of 10%). Then the sam- ples for SDS-polyacrylamide gel electrophoresis were prepared as described previously [19]. Ap- proximately 10 ug of sperm proteins were added per lane. The lane 1 shows co-electrophoresis of mo- lecular weight standards (myosin, rabbit muscle, MW 205,000; @-galactosidase, E. coli, MW 116,000; phosphorylase b, rabbit muscle, 97,400; albumin, bovine plasma, MW 66,000). DISCUSSION The egg jelly of C. japonicus which belongs to the order Clypeasteroida contains three sperm activating peptides named as mosact and _ its derivatives (Des-Gin®, Asn’-[His°]-mosact and Des-Gin®, Asn’-[Phe®]-mosact). The primary structures of mosact and its derivatives are similar to each other, but completely different from Fic. 4. The effect of mosact on the appearance of 123,000 dalton sperm protein in the presence or absence of Na*. C. japonicus spermatozoa were incubated at 20°C with or without mosact (7.35 Xx 10~°M) in the presence or absence of Nat. At 1 min of incubation the reaction was stopped by the addition of trichloroacetic acid (final concentration of 10%). The sample preparation for SDS- polyacrylamide gel electrophoresis and the elec- trophoresis were the same as described in the legend of Fig.3. Approximately 8g of sperm proteins were added to lane 2 (without mosact in Na*-free ASW) and lane 3 (with mosact in Na*- free ASW). Approximately 10 ug of sperm proteins were added to lane 4 (without mosact in normal ASW) and lane 5 (with mosact in normal ASW). The lane 1 shows co-electrophoresis of molecular weight standards. speract, [Thr°]-speract or [Ser°]-speract which were previously characterized from the sea urchins, H. pulcherrimus [4], Strongylocentrotus purpuratus [5] and A. crassispina [7], respectively. They are also different from resact which was purified from A. punctulata egg jelly [10]. HZ. pulcherrimus, S. purpuratus and A. crassispina be- long to the order Echinoida and spermatozoa from these species are activated by speract, [Thr°]- Mosact and Its Derivatives 655 205K — Fic. 5. SDS-polyacrylamide gel electrophoresis of sperm membrane, sperm tails, sperm heads and intact spermatozoa. Intact spermatozoa were incu- bated at 20°C with or without mosact (7.35x10~° M) in normal ASW for 1min. Samples for elec- trophoresis were prepared as described in the legend of Fig.3. SDS-polyacrylamide gel elec- trophoresis was performed according to Laemmli’s procedure [23]. Approximately 10 yg of proteins was added to each lane (lane 1: molecular weight standards; lane 2: sperm membrane without mosact; lane 3: intact spermatozoa with mosact; lane 4: intact spermatozoa without mosact; lane 5: sperm tails without mosact; lane 6: sperm heads without mosact). speract and [Ser’]-speract, respectively. A. punc- tulata belongs to another order Arbacioida which also includes G. crenularis. Speract and its deriva- tives activate neither A. punctulata spermatozoa nor G. crenularis spermatozoa. Mosact and its derivatives do not stimulate sperm respiration of H. pulcherrimus, A. crassispina and G. crenularis. These support a theory that peptides isolated from the eggs of one taxonomic order may interact with sperm cells of other species within the same order, but not within another order [6]. Mosact not only stimulates spermatozoan res- piration rate but also causes the appearance of a newly-stained protein whose mass is 123,000 dal- tons as estimated by SDS-polyacrylamide gel electrophoresis. In previous studies, a membrane protein identified as guanylate cyclase was shown to change in apparent molecular weight from 160,000 to 150,000 in response to resact in A. punctulata spermatozoa [10, 14, 15]. The protein observed here located dominantly in sperm tails and is found in sperm membrane as a major protein. However, the protein remains to be identified. The formation of the 123,000 dalton protein by mosact requires sodium ions. It may suggest that the protein involves in the stimulation of sperm respiration by mosact, since a sperm activating peptide, speract, requires sodium ions for the respiration stimulation action [8, 12]. But this may be controversial by the fact that the conversion of a 126,000 dalton protein to 123,000 dalton protein requires 50-100 fold concentrations of mosact as compared with the respiration stimulation. Although resact, speract and mosact do not crossreact with receptors of spermatozoa of the opposite species, the physiological events meas- ured appear to be the same [4, 6, 9-11, 19]. This extends a theory that the apparent coevolution of peptide and receptor is due to the necessity of the resultant physiological responses [11]. The egg jelly peptides show many physiological activities such as respiration stimulation, increas- ing CGMP and cAMP concentrations, the conver- sion of a high molecular weight sperm membrane protein to a lower molecular weight one and chemoattractant action. At present time, we do not know which is the key function of the peptides. This remains to be determined. ACKNOWLEDGMENTS The authors wish to thank Dr. D. L. Garbers at Vanderbilt University School of Medicine for prelimi- nary sequence analysis of mosact and its derivatives. We also wish to thank Mr. T. Shinya and M. Matada for collecting and culturing sea urchins. We express our appreciation to the Asamushi Marine Biological Station for kindly supplying the sea urchin Glyptocidaris crenu- laris. This work was supported by research grants to N.S. from the Ministry of Education, Science and Culture of Japan (No. 59540471 and No. 60480022). 10 11 12 656 REFERENCES Isaka, S., Hotta, K. and Kurokawa, M. (1970) Jelly coat substances of sea urchin eggs. I. Sperm isoagglutination and sialopolysaccharide in the jelly. Exp. Cell Res., 59: 37-42. Ishihara, K., Oguri, K. and Taniguchi, H. (1973) Isolation and characterization of fucose sulfate from jelly coat glycoproteins of sea urchin egg. Biochim. Biophys. Acta, 320: 628-634. SeGall, G. K. and Lennarz, W. J. (1979) Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Dev. Biol., 71: 33-48. Suzuki, N., Nomura, K., Ohtake, H. and Isaka, S. (1981) Purification and the primary structure of sperm activating peptides from the jelly coat of the sea urchin eggs. Biochem. Biophys. Res. Commun., 99: 1238-1244. Garbers, D. L., Watkins, H. D., Hansbrough, J. R., Smith, A. and Misono, K. S. (1982) The amino asid sequence and chemical synthesis of speract and of speract analogues. J. Biol. Chem., 257: 2734- ZA Suzuki, N., Hoshi, M., Nomura, K. and Isaka, S. (1982) Respiratory stimulation of sea urchin sper- matozoa by egg extracts, egg jelly extracts and egg jelly peptides from various species of sea urchins: taxonomical significance. Comp. Biochem. Physiol., 72A: 489-495. Nomura, K., Suzuki, N., Ohtake, H. and Isaka, S. (1983) Structure and action of sperm activating peptides from the egg jelly of a sea urchin, Anthoci- daris crassispina. Biochem. Biophys. Res. Com- mun., 117: 147-153. Suzuki, N., Ohizumi, Y., Yasumasu, I. and Isaka, S. (1984) Respiration of sea urchin spermatozoa in the presence of a synthetic jelly coat peptide and ionophores. Dev. Growth Differ., 26: 17-24. Suzuki, N. and Garbers, D. L. (1984) Stimulation of sperm respiration rates by speract and resact at alkaline extracellular pH. Biol. Reprod., 30: 1167- 1174. Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K. and Garbers, D. L. (1984) A peptide associated with eggs causes a mobility shift in a major plasma membrane protein of spermatozoa. J. Biol. Chem., 259: 14874-14879. Shimomura, H., Suzuki, N. and Garbers, D. L. (1986) Derivatives of speract are associated with the eggs of Lytechinus pictus sea urchins. Peptide, 7: 491-495. Hansbrough, J. R. and Garbers, D. L. (1981) Sodium-dependent activation of sea urchin sperma- tozoa by speract and monensin. J. Biol. Chem., 256: ie) 14 MS) 16 7 18 19 20 DA jf) Mp) 24 Up) 26 N. Suzuxkt, M. Kurita et al. 2235-2241. Repaske, D. R. and Garbers, D. L. (1983) A hydrogen ion flux mediates stimulation of respira- tory activity by speract in sea urchin spermatozoa. J. Biol. Chem., 258: 6025-6029. Ward, G. E. and Vacquier, V. D. (1983) Dephos- phorylation of a major sperm membrane protein is induced by egg jelly during sea urchin fertilization. Proc. Natl. Acad. Sci., 80: 5578-5582. Ward, G. E., Garbers, D. L. and Vacquier, V. D. (1985) Effects of extracellular egg factors on sperm guanylate cyclase. Science, 227: 768-770. Ward, G. E., Moy, G. W. and Vacquier, V. D. (1986) Phosphorylation of membrane-bound guan- ylate cyclase of sea urchin spermatozoa. J. Cell Biol., 103: 95-101. Vacquier, V. D. and Moy, G. W. (1986) Stoi- chiometry of phosphate loss from sea urchin sperm guanylate cyclase during fertilization. Biochem. Biophys. Res. Commun, 137: 1148-1152. Ward, G. E., Brokaw, C. J., Garbers, D. L. and Vacquier, V. D. (1985) Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer. J. Cell Biol., 101: 2324-2329. Suzuki, N. and Yamaguchi, M. (1986) Species specific respiratory stimulation of sea urchin (Clypeaster japonicus) spermatozoa by an egg associated factor. Zool. Sci., 3: 801-806. Lee, H. C. (1985) The voltage-sensitive Na*/H* exchange in sea urchin spermatozoa flagellar mem- brane vesicles studied with an entrapped pH probe. J. Biol. Chem., 260: 10794-10799. Tombes, R. M. and Shapiro, B. M. (1985) Meta- bolite channeling: A phosphocreatine shuttle to mediate high energy phosphate transport between sperm mitochondrion and tail. Cell, 41: 325-334. Podell, S. B., Moy, G. W. and Vacquier, V. D. (1984) Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface. Biochim. Biophys. Acta, 778: 25-37. Laemmli, U. K. (1970) Cleavage of structural proteins durinig the assembly of the head of bacter- iophage T4. Nature, 257: 680-685. Morrissey, J. H. (1981) Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem., 117: 307-310. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265- 2S. Schacterle, G. R. and Pollack, R. L. (1973) A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal. Biochem., 51: 654-655. ZOOLOGICAL SCIENCE 4: 657-664 (1987) © 1987 Zoological Society of Japan Germ Cell Kinetics in Gonadal Development in the Toad Bufo japonicus formosus AKIHIKO TANIMURA and HISAAKI IWASAWA Biological Institute, Faculty of Science, Niigata University, Niigata 950-21, Japan ABSTRACT—The sexual difference in the number of germ cells in primordial gonads of Bufo japonicus was investigated. The sexual difference in the number was not clear in larvae of stage 41 when sexual differentiation of the gonad itself was occurring. The difference was first recognized on the 6th day after metamorphosis in the gonads propers, and on the 18th day in Bidder’s organs. The number was significantly larger in females. A rapid increase in the proliferation of secondary gonia was occurring in female gonads on the 6th day. It is conceivable that in toads the cause of the appearance of a sexual difference in the number is due to a remarkable proliferation of secondary gonia in female gonads. INTRODUCTION Hardisty [1] reviewed the sexual difference in the number of germ cells in larval and neonatal periods of vertebrates, and pointed out that the number is larger in males in mammals and birds, and in females in anamniotes. He also suggested that the cytodifferentiation and proliferation of germ cells depend on the cortico-medullary induc- tion in gonadal primordia. However, the cause of the appearance of this sexual difference is not clear yet. Toads, irrespective of sex, have rudimentary ovaries, Bidder’s organs, between the fat body and gonad proper. Therefore, toads are an interesting material for the study of sexual differentiation. We suggested the sexual difference in oogenic activity in Bidder’s organs of young toads [2]. In the present paper, the sexual difference in Bidder’s organ and the difference between the ovary and Bidder’s organ are shown quantitatively, and a dependence of sexual difference in the number of germ cells upon the oogenic activity is elucidated. MATERIALS AND METHODS Egg strings of Bufo japonicus formosus were Accepted April 8, 1987 Received March 9, 1987 collected in a suburb of Niigata City. Larvae and young toads derived from these eggs were reared at 18+1°C. The animals were fed on commercial fish meal during the larval period, and on small insects thereafter. Gonadal regions of these larvae and young toads were fixed in Bouin’s solution at various intervals after hatching (Tables 1 and 2). These were serially sectioned transversely in par- affin into 5mm thicknesses, and stained with Masson trichrome. In order to determine the developmental stages of the larval specimens, Ichikawa and Tahara’s normal table of develop- ment [3] was used. Germ cells in gonads and Bidder’s organs were classified into 5 stages, i.e., primary gonia, sec- ondary gonia, oocytes in leptotene-pachytene, oocytes in diplotene and auxocyte-like cells. The number of germ cells in each stage was counted, and the obtained data were divided by the dia- meter of each stage of germ cells/thickness of sections + 1 for the revise of overlapping. The results obtained were tested statistically for sig- nificance by paired t-test, Duncan’s new multiple range test or Mann-Whitney’s U-test. RESULTS Number of germ cells in gonad proper The sexual differentiation of gonads occurred at 658 A. TANIMURA AND H. Iwasawa TABLE 1. Number of germ cells pericnene Numb er of Number of germ cells stages pave Total Primary gonia Secondary gonia te ee hatching male female male female male female male female indifferent indifferent indifferent indifferent 34 6 6 WS5az 6) Taata8 0 34 8 6 74+ 3 74+ 3 0 38 12 6 188 +29 188 + 29 0 40 18 6 261 + 34 261 +34 0 41 DD 6 540 +56 540 + 56 0 41 26 4 4 458+ 48 713+ 160 458+ 48 7134160 0 0 42 30 > 4 714+ 88 953+ 152 714+ 88 915+140 0 38-23 43 34 3 4 Vyas 2 WAlas WMP ORs 23 CMEC @ 1295 @49 45 (0) 39 4 4 DIG 2US) 287 5Sa= 85a 257 =D 2102459 0 762+ 431 (6) 45 4 Aer 2218724520 S532 352) “1859251092193 356) 1010 3069s amlos (12) 51 4 4 $332 9—E134 11385222111 9332542794 2032-272) 7716 + 1754 (18) 57 4 4 * 20434188 12989+2996 * 20164176 1506+205 26+26 3369+ 695 (24) 63 4 4 * 38034312 21209+2516 * 37944316 19614645 10+10 4973+1044 (30) 69 4 Ze SUITE S97= 16137-21439 Se SOlVE 897, sig sie eo5mmmO 3311+ 678 Sexual difference: * 0.01 »- v\ $ ‘ ef rit, iy A * ¥ 4 nu (eles vy west at 4 ‘ P aaa : tA fs Lys 1c? Se | 1 rm c} 4 ° i 2 et J au I \ ] ra a 7" : 4 , ECE i aes ae Y | v Pp ary wy & = , +f Py fl ‘Ge 4 rey vila he) i , ‘oe | oy F "ha | i i teicd : ’ ; eax Wie overpay ‘ : : 3 Puy ha \ } 7 i ‘ P ‘ iss Psst ‘ ‘ : ‘ Fp , ; 4 by hi : ty ae iP : Ae Le oy Pa) =) id i Fecatiect thy. era ‘4th ei me ipecACNe: of if i vas rates a6 ct i Cha We ‘tin? joinwage he it He RA ah ‘eyavtisntente ai mote) waleepning nao ho ol TURE Soe ary, uni eaopedeit a ped. Pua Bieiths a a Ailuagp mv a ese Aeterna: A. ads Seid eats sonny tac Syl eqn aly Mian ‘soo heii Reh etna "gane ay anions satft ea pba wa tab etoccGtIN at nt sealie: wait ie aula. We, AP Fees, bnarieemngalay 0 ais ae aah hen gdh 3 en As % 1 3 d f 7 Pee ZOOLOGICAL SCIENCE 4: 675-681 (1987) Extrahypothalamic Projection of Immunoreactive Vasotocin Fibers in the Brain of the Toad, Bufo japonicus You Jokura! and AKIHISA URANO? Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Saitama 338, Japan ABSTRACT— Extrahypothalamic projection of vasotocin (AVT) fibers in the brain of the toad (Bufo japonicus) was examined immunohistochemically by the avidin-biotin-peroxidase complex (ABC) method. Immunoreactive AVT perikarya are localized in the nucleus preopticus pars magnocellularis. The AVT neurons send their immunoreactive varicose fibers to many discrete brain regions, such as the limbic cortex, the thalamus, the optic tectum and the lower brain stem, in addition to the neurohypophysis. A dense network of AVT fibers was found in the septal nuclei and the anterior part of the preoptic nucleus. AVT fibers which run postero-dorsad project to the nucleus posterocentralis thalami, the nucleus posterodorsalis tegmenti mesencephali, and the nucleus isthmi. Meanwhile, AVT fibers which run through in the dorsal infundibular region and then the mesencephalic reticular formation are distributed in the medulla oblongata. These findings suggest that AVT acts as a neuromodulator or a local hormone in the toad brain. © 1987 Zoological Society of Japan INTRODUCTION It is well established that, in anuran amphibians, vasotocin (AVT) has both antidiuretic and vaso- pressor effects. In addition, AVT shows pro- nounced effects on reproductive behavior in Rana pipiens [1] and Taricha granulosa [2]. In T. granulosa, AVT may be involved in control of sexual behavior by acting neurons in the central nervous system [3]. We have shown in the toad brain that vasotocin neurons project their varicose axons into the anterior part of the preoptic nucleus (APON) which is considered to be the triggering center for male mate calling behavior in anuran amphibians [4]. These results suggest that AVT neurons transmit APON neurons peptidergic in- formation concerned with initiation of sexual behavior. In mammalian brains, the distributions of various neurohormones including arginine-vasopressin (AVP) are not confined only in the hypothalamo- Accepted March 28, 1987 Received February 25, 1987 1 Present Address: Tochigi First Lab., Kaou Co., Akabane, Ichikai-machi, Haga-gun, Tochigi 321-34, Japan. ? To whom reprints should be requested. neurohypophyseal system. They are widely distri- buted throughout in discrete brain loci [5]. Ultra- structural studies showed that axon endings of immunoreactive (ir) luteinizing hormone-releasing hormone (LHRH) fibers [6] and ir-AVP ones [7] form synapses and synaptoid contacts with other neurons. Further, varicose LHRH fibers form en passant synapses in the preoptic area of the guinea pig [6]. Therefore, it is probable in amphibian brains that, in addition to the APON and the neurohypophysis, AVT neurons send their fibers to many extrahypothalamic regions. In this study, we examined immunohistochemi- cally the extrahypothalamic distribution of AVT fibers in the toad brain to learn whether AVT neurons project their axons to the loci which are related to control of mating behavior. Further, we have tried to elucidate phylogenetically fun- damental distributional pattern of AVT in the vertebrate brain, since the amphibian brain is considered to show fundamental structural or- ganization of the vertebrate brain [8]. MATERIALS AND METHODS Adult toads (Bufo japonicus) of both sexes, body weight ranging from 117 to 303 g, and body 676 Y. JOKURA AND A. URANO length (snout to vent) from 11.9 to 14.1 cm, were used. These animals were either obtained from an animal supplier in late September or were cap- tured at the breeding season. Three animals, which were obtained from an animal supplier in late November, were used for immunohistochem- ical staining of thick frozen sections. Immunohistochemical procedure Distribution of AVT was immunohistochemical- ly localized in paraffin sections and thick frozen sections that were cut either transversally, sagittal- ly or horizontally to determine exact loci where ir-AVT fibers were found. As for immunohis- tochemical staining of vasotocin in the paraffin sections of the toad brain, details of fixation, tissue preparation and immunohistochemical procedure, Pict 40 um. Fic. 2. Ir-vasotocin fibers travelling in the nucleus infundibularis dorsalis. Sagittal frozen section. Scale, 40 um. Fic. 3. Plexus of ir-vasotocin fibers in the region anterior to the males isthmi. Scale, 40 um. Fic. 4. in which the Vectastain ABC kit (Vector) was used, have been described previously [9, 10]. For the staining of thick frozen sections, the brains were fixed by transcardial perfusion with a fixative solution containing 1% glutaraldehyde, 2% paraformaldehyde and 4% sucrose in 0.1M phosphate buffer (pH7.4). The brains were removed, postfixed in the same fixative at 4°C overnight, and were washed in 0.1 M phosphate buffer. Frozen sections were cut at 50 um, and were washed in 0.1M phosphate buffered saline (pH 7.4) at room temperature for 30 min. Then, they were stained immunohistochemically by use of the Vectastain ABC kit. The sections were first incubated with the primary antiserum in a reaction medium at 4°C for 12-16hr. The solution for incubation contained anti-arginine-vasopressin Ir-vasotocin perikarya in the nucleus preopticus pars magnocellularis. Transversal paraffin section. Scale, Arrowheads indicate melanin granules. Thick sagittal frozen section. Ir-vasotocin fibers in the floor of the medulla oblongata. Thick sagittal frozen section. Scale, 40 um. Toad Extrahypothalamic AVT Fibers 677 serum (1: 8000 dilution, Bioproducts), which cross- reacts completely with vasotocin but not with mesotocin (cross reactivity, 0.3%), in 0.1% Triton X-100 dissolved in 0.1M phosphate buffered saline (PBS-T). After the incubation with the primary antiserum, the sections were washed in PBS-T at room temperature for 30 min, incubated with biotinylated anti-rabbit Ig-G for 1hr, and were washed in PBS-T. Afterward, the tissue sections were incubated in the avidin-biotin- peroxidase complex in PBS-T for 30 min. After a few rinses, they were incubated in DAB solution including 0.05% 3,3’-diaminobenzidine (Sigma) and 0.01% hydrogen peroxide for 10 min, washed briefly in phosphate buffer, and were mounted on slide glasses with 40% ethanol containing 0.75% gelatin. They were then dehydrated, and were cover-slipped with Permount (Fisher). The tests for specificity of immunohistochemical staining followed the previous study [4, 9]. Nissl stained tissue sections were refered to for describing precise localization of ir-AVT. Nomenclatorial usage in this paper is basically those in Rana pipiens [11] and Bufo japonicus [9]. Fic. 5. (broken lines) and ir-vasotocin perikarya (filled circles). RESULTS Distribution of ir-AVT perikarya and fibers As was described previously [4, 9], ir-AVT perikarya are localized in the ventral (VMC) and dorsal (DMC) magnocellular parts of the preoptic nucleus (Figs.1 and 5). Beaded or varicose ir-AVT fibers were widely distributed among the discrete extrahypothalamic loci in the limbic sys- tem and the brain stem (Figs. 2-4). They were not found in the dorsal and anteroventral regions of the telencephalon. The extrahypothalamic ir-A VT projections can be classified roughly into three groups according to their destinations (see Fig. 5). Neither notable seasonal variation nor sexual difference was found in the distribution of ir-AVT fibers in this study. Projection to the telencephalon (Figs. 5-8) A part of ir-AVT fibers emanating from the VMC project to the posteromedial region of the telencephalon, principally to the nuclei medialis septi and lateralis septi. Ir-AVT fibers are sent out to these loci mainly through the white matter including the medial forebrain bundle which sur- rounds the neuronal cell mass of the APON. A 10 Diagram of the mid-sagittal plane of the toad brain illustrating the distribution of ir-vasotocin fibers The thick line shows the hypothalamo- neurohypophyseal vasotocinergic tract. Each numbered arrowhead shows the level of drawings (Figs. 6-11) illustrating the distribution of ir-vasotocin fibers and perikarya. Ir-vasotocin fibers are assembled into three projection groups: 1, projection to the telencephalon; 2, to the thalamus and the tectum; and 3, to the brain stem. 678 Y. JOKURA AND A. URANO considerable number of varicose ir-AVT fibers that diverge from the projection to the telencepha- lon innervate into the APON and the amygdala medialis. Scattered ir-AVT fibers were often found in the pallium mediale and the nucleus of the diagonal band of Broca. The ir-AVT fibers found in the telencephalon have a varicose form, however, they rarely show Herring bodies which are frequently observed in the magnocellular preopticoneurohypophyseal neurosecretory system. Projection to the thalamus and the tectum (Figs. 5, 8-9). Fics. 6-11. Diagrammatic camera lucida drawings illustrating the distribution of ir-vasotocin fibers and peri- karya (filled circles). The hatched area in Fig. 8 shows the region in which dense ir-vasotocin fibers were observed. Toad Extrahypothalamic AVT Fibers 679 Many fibers arising from both the VMC and DMC run posterodorsad to the dorsomedial thala- mic region, and are distributed mainly in the nuclei dorsomedialis anterior thalami and posterocen- tralis thalami. A few ir-AVT fibers further pro- ceed posteriad to terminate in the optic tectum. Projection to the brain stem (Fig. 5, 10-11). Ir-AVT fibers which project to the brain stem arise from ir-AVT neurons in the VMC. They initially proceed laterad into the white matter in the preoptic region. Then, they turn their destina- tion caudad to the direction of the lower brain stem with many ir-AVT fibers that project to the neurohypophysis. Thereafter, the fibers projecting to the brain stem diverge from the preoptico- neurohypophyseal tract around the dorsal infun- dibular region. A few beaded ir-AVT fibers are localized in the nucleus infundibularis dorsalis (Fig. 2). Many ir-AVT fibers run down to the mesencephalic tegmentum. They project to the nuclei posterodorsalis tegmenti mesencephali and isthmi, and further to the griseum centrale rhom- bencephali. A considerable number of fine beaded fibers gather together to form a plexus in the region anterior to the nucleus isthmi (Fig. 3). Abbreviations for Figs. 5-11. Al amygdala, pars lateralis Am amygdala, pars medialis APON anterior part of the preoptic nucleus AVA area ventralis anterior thalami AVL area ventrolateralis thalami BO bulbus olfactorius CBL cerebellum GC griseum centrale rhombencephali LFB lateral forebrain bundle ME median eminence MED medulla oblongata NAD nucleus anterodorsalis tegmenti mesencephali NAS nucleus accumbens septi NAV nucleus anteroventralis tegmenti mesencephali NCER nucleus cerebelli NDB nucleus of the diagonal band of Broca NDMA _ nucleus dorsomedialis anterior thalami NEP nucleus entopeduncularis NI nucleus isthmi NID nucleus infundibularis dorsalis NIV nucleus infundibularis ventralis NLS nucleus lateralis septi NMNT nucleus mesencephalicus nervi trigemini NMS nucleus medialis septi Some fibers which emanate from this plexus proceed dorsad to the stratum griseum tecti. Many ir-AVT fibers which run posteriad through the white matter in the floor of the mesencephalon were also found (Fig. 4). These fibers, which diverge from the fiber group destining to the isthmic region at the level caudal to the dorsal infundibular region, reach the rhombencephalic reticular formation, and proceed further toward the spinal cord. DISCUSSION The present study showed that ir-AVT fibers are distributed widely among many extrahypothalamic loci in the toad brain. Such regions are the limbic cortex, the thalamus, the optic tectum, the isthmic region and the lower brain stem. The distributional pattern of extrahypothalamic ir-AVT fibers in the toad brain seems to be homologous to those described in the brains of other vertebrate classes. In the rat and the monkey, vasopressin neurons, the mammalian counterpart of AVT neurons, project their im- munoreactive fibers to the hippocampus, the sep- tum, the amygdala and the preoptic area. They NOA nucleus olfactorius anterior NPC nucleus posterocentralis thalami NRI nucleus reticularis inferior NRIS nucleus reticularis isthmi NRS nucleus reticularis superior OC optic chiasma OL optic lobe ON optic nerve PD pallium dorsale Pdis pars distalis hypophysis PI pars intermedia hypophysis PL pallium laterale PM pallium mediale PN pars nervosa hypophysis PONmg preoptic nucleus, pars magnocellularis PPON posterior part of the preoptic nucleus SGC stratum griseum centrale tecti SGP stratum griseum periventricularis tecti SGS stratum griseum superficiale tecti ST striatum ETE telencephalon TS torus semicircularis IIIv third ventricle Vv motor nucleus of the trigeminal nerve 680 Y. JOKURA AND A. URANO project further to the thalamus, the superior colliculus, and the several pontine and medullary nuclei [12]. A similar immunohistochemical distri- butional pattern of ir-AVT fibers was observed in the brain of the lizard Gekko gecko [13] and the eel Anguilla japonica (Fujiwara et al., unpublished). A radioimmunoassay study of microdissected brain areas of rough-skinned newts also showed a similar distributional pattern of AVT [14]. These results indicate that the patterns of extrahypothal- amic projections of AVT and vasopressin are fundamentally homologous in all vertebrate classes. Immunoelectron microscopic studies demon- strated the presence of synapses containing neurophypophysial hormones in the rat brain [7, 15]. In the previous study, we showed that LHRH and AVT fibers may contact synaptically with APON neurons [4]. It is therefore highly probable that, in the toad brain, the extrahypothalamic AVT fibers form ordinary and/or en passant synapses with neurons in the loci where AVT fibers were localized. As was discussed in our previous paper [4], beaded or varicose ir-AVT fibers traveling through the white matter may form such synapses with dendrites of many central neurons, because in the amphibian brain many neurons located in the medial cell mass develop their dendritic fields in the adjacent white matter [8, 16]. Since neurohypophysial hormones could excite unit-spike activity of neurons in the rat supraoptic and paraventricular nuclei [17, 18], the eel preoptic nucleus [19] and the toad APON (Fujita and Urano, unpublished), vasotocin may facilitate activity of many central neurons as a neuromodulator or a local hormone. The latter possibility is supported by the fact that AVP of 10~° M, comparable to effective doses of AVP for peripheral targets, could excite rat paraventricular neurons [18]. However, physiological roles of vasotocinergic transmission in the anuran brain are not clear at present, although an involution in reproductive behavior has been suggested as is described in the introduction. The present study showed that vasotocin fibers are distributed in the loci concerned with repro- ductive behavior. Such regions are the limbic cortex, the preoptic area, the optic tectum and the central gray. In the limbic cortex, a considerable number of ir-AVT fibers were found in the septal nuclei, and also in the nucleus of the diagonal band of Broca. These regions contain the majority of ir-LHRH neurons in the toad brain [4, 10]. A similar projection of vasopressin fibers was found in the organum vasculosum lamina terminalis in the mammalian brain where many ir-LHRH peri- karya are localized [12]. In the eel, vasotocin fibers were found in the preoptic region (Fujiwara et al., unpublished). Meanwhile, ir-LHRH fibers project to the VMC in the toad brain [20]. These observations suggest that the LHRH-ergic and vasotocinergic neurosecretory systems are mutual- ly connected, and that they interact on each other for controlling sexual behavior. ACKNOWLEDGMENT We thank Dr. T. Ichikawa, Tokyo Metropolitan Insti- tute for Neurosciences, for his kind help and technical advice. A part of this study was supported by research grants from the Ministry of Education, Science and Culture, Japan. REFERENCES 1 Diakow, C. (1978) Hormonal basis for breeding behavior in female frog: vasotocin inhibits the release call of Rana pipiens. Science, 199: 1456-1457. 2 Moore, F.L. and Zoeller, R. T. (1979) Endocrine control of amphibian sexual behavior: Evidence for a neurohormone-androgen interaction. Horm. Be- hav., 13: 207-213. 3 Moore, F.L. and Miller,L.J. (1983) Arginine vasotocin induces sexual behavior of newts by acting on cells in the brain. Peptides, 4: 97-102. 4 Jokura, Y. and Urano, A. (1985) Projections of luteinizing hormone-releasing hormone and vasoto- cin fibers to the anterior part of the preoptic nucleus in the toad, Bufo japonicus. Gen. Comp. Endocri- nol., 60: 390-397. Buijs, R. M. (1985) Extrahypothalamic pathways of a neurosecretory system: their role in neurotrans- mission. In “Neurosecretion and the Biology of Neuropeptides.” Ed. by H. Kobayashi, H. A. Bern and A.Urano, Japan Sci. Soc. Press, Tokyo /Springer-Verlag, Berlin, pp. 279-286. 6 Silverman, A.J. (1983) Luteinizing hormone- releasing hormone (LHRH) synapses in the diago- nal band (DBB) and preoptic area (POA) of the nN 10 11 12 {3 Toad Extrahypothalamic AVT Fibers guinea pig. Soc. Neurosci., 9: 1182 (abstract). Buijs, R. M. and Swaab,D.F. (1979) Immuno- electron microscopical demonstration of vasopressin and oxytocin synapses in the limbic system of the rat. Cell Tissue Res., 204: 355-365. Herrick, C. J. (1948) The Brain of the Tiger Sala- mander, Ambystoma tigrinum. The University of Chicago Press, Chicago. Takami, S., Jokura, Y. and Urano, A. (1984) Sub- nuclear organization of the preoptic nucleus in the toad, Bufo japonicus. Zool. Sci., 1: 759-770. Jokura, Y. and Urano, A. (1985) An immunohis- tochemical study of seasonal changes in luteinizing hormone-releasing hormone and vasotocin in the forebrain and the neurohypophysis of the toad, Bufo japonicus. Gen. Comp. Endocrinol., 59: 238-245. Wada, M., Urano, A. and Gorbman, A. (1980) A stereotaxic atlas for diencephalic nuclei of the frog, Rana pipiens. Arch. Histol. Jpn., 43: 157-173. Zimmerman, E. A., Nilaver,G., Hou-yu, A. and Silverman, A. J. (1984) Vasopressinergic and oxyto- cinergic pathways in the central nervous system. Fed. Proc., 43: 91-96. Stoll, C. J. and Voorn, P. (1985) The distribution of hypothalamic and extrahypothalamic vasotocinergic cells and fibers in the brain of a lizard, Gekko gecko: presence of a sex difference. J. Comp. Neurol., 239: 193-204. 14 15 16 17 18 15 20 681 Zoeller, R. T. and Moore, F. L. (1986) Arginine vasotocin immunoreactivity in hypothalamic and extrahypothalamic areas of an amphibian brain. Neuroendocrinology, 42: 120-123. Theodosis, D. T. (1985) Oxytocin-immunoreactive terminals synapse on oxytocin neurons in the su- praoptic nucleus. Nature, 313: 682-684. Urano, A. (1984) A Golgi-electron microscopic study of anterior preoptic neurons in the bullfrog and the toad. Zool. Sci., 1: 89-99. Baker, J. L. (1977) Physiological roles of peptides in the nervous system. In “Peptides in Neurobiology”. Ed. by H. Gainer, Plenum Press, New York, pp. 295-343. Inenaga, K. and Yamashita, H. (1986) Excitation of neurones in the rat paraventricular nucleus in vitro by vasopressin and oxytocin. J. Physiol., 370: 165-180. Sugita, R. and Urano, A. (1986) Responses of magnocellular neurons in in vitro eel preoptic nucleus (PONmg) to acetylcholine, catecholamines, vasotocin, isotocin, angiotensin, and Na~. Zool. Sci., 3: 1081 (abstract). Jokura, Y. and Urano, A. (1986) Extrahypothala- mic projection of luteinizing hormone-releasing hormone fibers in the brain of the toad, Bufo japonicus. Gen. Comp. Endocrinol., 62: 80-88. ‘BR 7 _ ere sin. on) 3 an Aas ite brn. 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HP fa! ear Heshelinat at oe ae ‘apr a ee ee a oo * re ; a a | GRE ED Naat Aono ies eit haiti hah * 1 : ‘ i ae usa 3 7 rare i TUT Re. ts 6 a it ¥ Shek 1 y Le ‘ ui i] ‘ ) on 4 5 1 ae % £ ‘ a et ; t i : I ' ss i. 7 p y 10> ee iy iy y ; \ ‘f 7 i Ta! Fl ee iC pndek eueed: aaa ) " y ey 2 r a ¥ y i 3Fes 7% Lye : hy i , 1h) wy #4 i BVAGS ; ie i SM iA cvs = if! bic Lea x = Wikia te, Ford a rs iy ' 4 “i } whist ‘he ZOOLOGICAL SCIENCE 4: 683-692 (1987) A Circadian Aspect of the Photoperiodic Time-measurement on the Basis of the Larval-ecdysis Rhythm in the Small Copper Butterfly, Lycaena phlaeas daimio Seitz KATSUHIKO ENDO and MARIKO SHIBATA Environmental Biology Laboratory, Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan ABSTRACT—The present study was aimed at showing how the circadian clock controlling the timing of larval ecdysis is entrained to light/dark (L/D) cycles in the small copper butterfly, Lycaena phlaeas daimio Seitz. In addition, experiments were made to investigate how the circadian clock is related to the time-measurement of photoperiods in this butterfly. Larval ecdysis occurred in a specific zone of L/D cycles. The zone was determined by the secretion of prothoracicotropic hormone (PTTH) preceding the larval ecdysis by 32 hr at 25°C. The preparatory period was shortened to 29 hr at 30°C, but the secretion of PTTH occurred in almost the same zone of L/D cycles at these two different temperatures. Furthermore, when a group of larvae were held in 4L-20D and transferred to continuous darkness (DD), they showed a significant rhythm of larval ecdysis in DD following the transfer. The timing of larval ecdysis-zone was advanced (or delayed), when the night was interrupted by a supplementary light pulse of 0.5-hr (or 1-hr). In 4L-20D, a specific 6-hr zone may exist, in which spring morph development is prevented by a supplementary light pulse of 0.5-hr. The results suggest that time-resetting of the circadian clock controlling the timing of PTTH- secretion may occur at light-on and light-off, respectively. The circadian clock itself or an oscillator entrained by the circadian clock is supposed to play a significant role in the time-measurement of © 1987 Zoological Society of Japan photoperiod in L. phlaeas daimio. INTRODUCTION In many multivoltine insects, diapause and deter- mination of seasonal morphs are governed by photoperiod and temperature existing during cer- tain developmental stages [1, 2]. The physiological system underlying the photoperiodic control in- volves a functional component measuring day- or night-length. However, when each species of insects was subjected to either a skeleton photo- period consisting of two light-pulses or light/dark (L/D) cycles of non-24-hour, they were found to show a wide diversity of the responses to the photoperiods [3, 4]. For explaining the diversities in the responses to the photoperiods, several models have been pro- Accepted February 20, 1987 Received October 9, 1986 posed by previous workers on the basis of circa- dian rhythm study [3-8]. The diversities could be mostly explained on the basis of these models, but there have remained many speculations yet to be studied. Larval ecdysis occurs in a particular zone of L/D cycles in several lepidopteran insects. The timing of larval ecdysis has been shown to be determined by the secretion of the prothoracicotropic hor- mone (PTTH) [9, 10]. That is, a specific zone exists in L/D cycles in which the PTTH-secretion is allowed to occur. The timing of the allowed zone for PTTH-secretion has been speculated to be determined by a physiological (circadian) sys- tem involving circadian oscillator(s) or hourglass (es) [9, 10]. In the small copper butterfly, Lycaena phlaeas daimio Seitz, determination of seasonal morphs — spring, intermediate and summer morphs — has 684 K. ENDO AND M. SHIBATA been shown to be controlled by photoperiod and temperature during the larval and/or early pupal stages [11, 12]. The physiological system under- lying the photoperiodic control involves a neuroendocrine system secreting the summer- morph-producing hormone [13]. The present study was aimed at assessing whether or not the PTTH-secretion preceding the larval ecdysis is gated by a circadian system in L. phlaeas daimio. Then, the investigation was extended to see how the circadian clock controlling the timing of larval ecdysis is entrained to L/D cycles and related to the photoperiodic time- measurement in this butterfly. MATERIALS AND METHODS Animals Eggs and larvae of L. phlaeas daimio were divided into groups (n=200-250 for each group) and were held in either a petridish (49 x2 cm*) or a container of transparent plastic (¢9 x5 cm?). They were placed in an L/D cycle of either a complete or an asymmetrical skeleton photoperiod at 20°C or 25°C. The larvae were fed on leaves of Rumex acetosa which were exchanged daily. The petridishes and the rearing containers were placed in a _ photoperiod-controlled cabinet (62X39 X23 cm? or 6239110 cm*) and were illuminated by a 10-W or 20-W white fluorescent tube which was controlled by a 24-hr time-switch. In the light period, light intensity was provided at about 400 lux to the petridishes or to the rearing containers. Photoperiodic regimens In the complete photo- periods, the light period was changed depending on the groups at 2-hr increments from 2L-22D (alternating 2-hr light and 22-hr dark periods) to 18L-6D. In the asymmetrical skeleton photo- periods, the dark component of 4L-20D was interrupted by a light pulse of 0.5 hr. Then, the time of the light pulse was delayed from one group to the next at a 1-hr interval so that different groups received the light pulse at different times of night. Besides, to know how much the timing of larval ecdysis is changed in response to a single light- pulse, larvae were reared under 4L—20D, exposed to a night-interruption of 1 hr on the first night of the 2nd larval ecdysis and transferred to DD. Timing of larval ecdysis L. phlaeas \arvae stopped feeding at about 20hr before larval ecdysis and their head was bent downwards to form an oval posture (head capsule slippage stage). Larvae of the oval form were selected from a stock culture with a red-light illumination less than 5 lux twice a day and were put in a lat- ticework-plate of wood and paper. They were held in continuous light and photographed at a 0.5-hr interval to see how many larvae underwent larval ecdysis in each 0.5-hr period of the day. In each group, larval ecdysis from the 3rd to the 4th instar occurred for a 3- to 4-day period. The rhythmicity of larval ecdysis was analyzed by the method of least-square cosin fitting (LSCF) [14]. When the larval ecdysis was judged to occur with a rhythm of 24 hr, the acrophase-time of the best fitting cosin was obtained and the time was regarded as the acrophase-time of larval ecdysis. Subsequently, the time at which the larval ecdysis occurred at minimum frequency within the day (the minimum frequency-time) was determined. Then, the num- bers of ecdysed larvae were added from the minimum frequency-time to the next and the times at which the percentage of ecdysed larvae reached 20% and 80% were recorded. The times are described hereafter as T-20% and T-80%. To assess whether the larval ecdysis occurs with a rhythm in continuous darkness (DD) following L/D cycles, various stages of larvae were transfer- red to DD from 4L-20D. The peaks of larval ecdysis were punctuated one after another at the minimum frequency-time, and the gravity-time of each peak of larval ecdysis was obtained together with T-20% and T-80%. Neck-ligature of larvae Larvae were raised from the egg stage under 4L-20D at 25°C or at 30°C. Newly molted 3rd-instar larvae (day 0) were selected from the stock culture and were divided into groups (n=50-70). The neck was ligatured at a 2-hr interval. The surgery began 14 hr (or 6 hr) after dawn on day 0 at 25°C (or at 30°C) and continued for 2 days. Five (or four) days later at 25°C (or 30°C), the neck-ligatured (decapitated) larvae were observed for the production of 4th- instar cuticle. Criteria for the classification of seasonal Photoperiodic Time-measurement 685 morphs Classification of the seasonal morphs was based on the number of red scales in the 4th and 7th red spots distributed along the central line of the dorsal side of anterior wing. The classification was carried out in each butterfly by using a method of confident ellipse [8]. RESULTS The timing of larval ecdysis in the complete photoperiods Firstly, ecdysis from the 3rd to 4th instar was examined in groups of larvae (n=200-—250) to see whether or not larval ecdysis occurs in a specific zone of L/D cycles in the complete photoperiods. The photoperiods examined varied from 2L—22D to 18L-6D with photophases differing by 2 hr (25°C). Larval ecdysis was found to occur in a specific zone of the L/D cycles in most regimens (Fig. 1). The zone covered approximately an 8-hr period beginning at about 2 hr before dawn in 2L-22D. When the light period became longer, the acro- phase-time of larval ecdysis came to on an almost parallel line (0.4-hr delay/2 hr against dawn in Photoperiods LSCF-analysis) with a line connecting the dawn. Furthermore, the other three indices characteriz- ing the larval ecdysis-zone —T-20% (the begin- ning time), T—80% (the end time) and the mini- mum frequency-time—were delayed (or ad- vanced) in a fashion almost parallel to the shift of the acrophase-time. When larvae were subjected to 18L-6D, the larval ecdysis occurred with no significant rhythm (P>0.05). The results indicated that L. phlaeas daimio has a specific zone of 5-8 hr in which they undergo larval ecdysis in photoperiods with a photophase shorter than 16hr. The timing of larval ecdysis may be determined so that the zone comes to on an almost parallel zone with the connecting line of dawn. The timing of the larval ecdysis in the asymmetrical skeleton photoperiods of 4L-20D To investigate how the larval ecdysis-zone changes in response to the night-interruption, groups of larvae were subjected to the asymmettri- cal skeleton photoperiods of 4L-20D at 25°C. The night-interruption (supplementary light pulse) of 0.5-hr was delayed depending on the groups at 1-hr intervals and the timing of larval ecdysis was Hours after dawn Fic. 1. Fluctuating pattern in the number of ecdysed larvae in the complete photoperiods. Each histogram shows the number of ecdysed larvae in each 0.5-hr period of the day. Solid and open bars show dark and light periods, respectively. Double sided arrows indicate the larval ecdysis-zone between T-20% and T-80%. Solid and open triangles show the acrophase-time and the minimum frequency-time of larval ecdysis, respectively. 686 K. ENDO AND M. SHIBATA examined in each group. When the night-interruption fell in an 11-hr zone following dusk, the timing of larval ecdysis was delayed (Fig. 2). The delay occurred in the minimum frequency-time, but it was not clear in the acrophase-time, with 0.4-hr/1-hr light-pulse delay. The maximum delay of 4-hr was recorded in the minimum frequency-time when the night- interruption fell on at 11hr after dusk (the end-point of the first 11-hr zone following dusk). The larval ecdysis-zone was advanced in response to the night-interruption falling on in an 8-hr zone preceding dawn. The maximum advance of 6-hr was recorded by the night-interruption coming to at 14 hr after dusk. Thereafter, the advance of the acrophase-time as well as that of the other indices became gradually smaller (0.7 hr/1 hr in LSCF- analysis). Time of light pulse(nrs after dawn) Hours after dawn Fic. 2. Fluctuating pattern in number of the ecdysed larvae in the asymmetrical skeleton photoperiods of 41.-20D. Each histogram shows the number of ecdysed larvae in each 0.5-hr period of the asym- metrical skeleton photoperiods at 25°C. Double- sided arrows indicate the larval ecdysis-zone between T-20% and T-80%. Solid and open triangles show the acrophase-time and the mini- mum frequency-time, respectively. The results indicate that the timing of larval ecdysis may be changed when the night is inter- rupted by a supplementary light pulse. However, the night of 4L-20D may be divided into two zones depending on the response of the larval ecdysis- zone. The first zone covers an 11-hr period following dusk, in which the larval ecdysis-zone was delayed by night-interruption. But, in the second zone covering an 8-hr period preceding dawn, the larval ecdysis-zone was advanced. The timing of the prothoracicotropic hormone (PTTH)-secretion for larval ecdysis from the 3rd to the 4th instar To know how much time the PTTH-secretion precedes the larval ecdysis in L. phlaeas daimio, the neck of the 3rd instar larvae was ligatured at 25°C and 30°C. The surgery was started on the first day of the 3rd instar (day 0) and made for 2 days at a 2-hr interval. As is summarized in Figure 3, a small proportion (2%) of larvae producing the 4th-instar cuticle appeared when the neck was ligatured at mid-night on day 0 at 25°C. However, neck-ligatured larvae which produced the cuticle of the 4th instar increased when the surgery was made at and later than 18 hr after the dawn on day 0. The percent- age of the cuticle-producing larvae rose con- tinuously until the increment once stopped at 60% at the end of light period on day 1. The percentage began to rise again a few hours after the dusk to reach about 100% at mid-night on day 1 at 25°C. At 30°C, a low percentage of larvae already produced the 4th-instar cuticle when the neck was ligatured 2 hr after dusk on day 0. The proportion of the cuticle-producing larvae began to rise earlier than in those held at 25°C and the percentage reached near 90% at dusk on day 1. However, increment in percentage of the cuticle-producing larvae became almost zero for a few hours follow- ing dusk on day 1 as in those held at 25°C. In intact (control) insects, larval ecdysis began to occur on day 1 and ended on day 3 at 25°C (or on day 2 at 30°C). They formed two or three peaks in larval ecdysis. The peaks of the PTTH-secretion seemed to precede the corresponding peaks of larval ecdysis by about 32 hr at 25°C. The time period may be shortened to 29 hr when the rearing Photoperiodic Time-measurement 687 : Jaa) ius . ! i Dl Va Me Fic. 3. Hours after dawn The response of the 3rd instar larvae to neck-ligature as a function of the time of operation at 25°C and 30°C. Upper panel: Solid and open circles (left) show the cumulative percentage of neck-ligatured larvae producing the cuticle of the Sth instar at 25°C and 30°C, respectively. Solid and open circles drawn on right-side show the cumulative percentage of intact larvae undergoing larval ecdysis at 25°C and 30°C, respectively. Straight lines show dark periods. Lower panel: Open (or solid) histograms show the increments in percentage of neck-ligatured larvae producing the cuticle of 4th instar at 2-hr intervals and those in percentage of intact larvae undergoing larval ecdysis in each 0.5-hr period of the day at 30°C (or 25°C), respectively. temperature rises to 30°C. However, the allowed zone for PTTH-secretion is judged to cover about the same zone of L/D cycles at these two different temperatures. Rhythmicity of larval ecdysis in DD following L/D cycles To clarify whether or not the larval ecdysis occurs with the rhythm in DD following L/D cycles, larvae were raised from the egg stage under 4. -20D and were transferred to DD by extending the scotophase from day 0 of the 2nd to day 3 of the 3rd instar. Larval ecdysis was examined and the time of PTTH-secretion was obtained by subtracting 32 hr from the observed time of ecdysis after DD-transfer. The histograms of Figure 3 represent the time of PTTH-secretion, the data of which were pooled according to the time elapsed after DD-transfer. As is evident from Figure 4, larval ecdysis occurred with rhythms in DD following the trans- fer. Periodical cycles of a little shorter than 24 hr (23.6 hr?) was obtained when the analysis was made on larvae undergoing larval ecdysis on the days following the 2nd day of DD (Fig. 4). The results indicate that a circadian (or hour- glass) clock determining the timing of PTTH- secretion (or larval ecdysis) may exist in L. phlaeas daimio. As a result, larval ecdysis occurs with a rhythm of circadian cycles in DD following L/D cycles. However, the rhythm of larval-ecdysis may be damped in DD as it becomes long. Phase response of the PTTH-secretion zone against one night-interruption To clarify how the timing of the PTTH-secretion zone is changed by one night-interruption (a light pulse), larvae were raised from the egg stage under 688 15 QE OF oo Vv) Cc oO oS (GE Cm Ow 28 uw O 6) 24 48 72 Hours counted from the last dawn (%) 1: Oigaeuect ues day elhe wis tat Meee gs yey pa Sythe ee eS 5 1 T A O 0.1 = oO 0.01 2S UG\S) 24 Trial periods (hr) Fic. 4. Fluctuations in the number of larvae deter- Fic. 5. mined to undergo larval ecdysis (upper panel) and time period of its rhythm in DD (lower panel). Each histogram (upper panel) shows the approxi- mate timing of PTTH-secretion (at which time lar- vae is determined to undergo larval ecdysis). A waved line represents the best fitting cosin of the cycles of 23.5-hr. Open and solid bars on the abscissa indicate light and dark periods, respective- ly. Solid and open circles (lower panel) show the fittness (P) and the relative error to the cosin tried to fit. Time of light pulse(hrs after last dusk) K. ENDO AND M. SHIBATA 4L-20D at 25°C, divided into groups (n=250-300) on the first day of the 3rd instar, transferred to DD from the next scotophase, and exposed to a light pulse (night-interruption) of 1 hr at different times with a 2-hr interval. The time of larval ecdysis was observed and the minimum frequency-time of larval ecdysis was calculated. The timing and the minimum frequency-time for PTTH-secretion were obtained by subtracting 32 hr from these data and shown in Figure 5. The minimum frequency-time of PTTH- secretion did not change by the night-interruption falling on in a 4-hr period following dusk. Howev- er, the minimum frequency-time was advanced when the night was interrupted at and later than 6 hr after dusk. The maximum advance in the minimum frequency-time of PTTH-secretion (12 hr) was recorded, when the night-interruption fell on at 8 hr after dusk. Thereafter, the advance of the minimum frequency-time became smaller almost steadily (0.7 hr/1 hr in LSCF-analysis) as the time of light pulse was delayed. Based on the response of the minimum frequen- cy-time of PTTH-secretion, the night of 4L-20D may be divided into two zones as proved by the experiments of the asymmetrical skeleton photo- periods. But, the first zone covers, at this time, about a 6-hr period, in which the minimum frequency-time was independent of the night- interruption. In the second zone covering the Hours after last dusk The phase response of the minimum frequency-time of PT TH-secretion representing as a function of the time of night-interruption. Each histogram shows the number of larvae which are determined to undergo larval ecdysis in each 1-hr period of the day. Solid triangles show the minimum frequency-time of PTTH-secretion (?). Open and solid bars show the photoperiodic regimens. Photoperiodic Time-measurement following 13-hr period, the minimum frequency- time is advanced by the night-interruption in the same manner as the groups subjected to the asymmetrical skeleton photoperiods. Photoperiodic response curves of seasonal-morph determination in the complete and the asymmetrical skeleton photoperiods To investigate how the incidence of the spring morphs is changed by daylength or by a sup- plementary light pulse of 0.5 hr, groups of larvae Vebecetesdapdytagad —— Vobetoesdabgactotag = 689 (n=200-250) were raised from the egg stage under either a complete or an asymmetrical skeleton photoperiod of 4L-20D at 25°C. In the complete photoperiods, length of the light period was changed depending on the groups at 2-hr incre- ments, whereas, in the asymmetrical skeleton photoperiods, the night-interruption of 0.5-hr was delayed at 1-hr intervals. Then, the incidence of spring-morph adults was examined in each group. When larvae were subjected to photoperiods with a photophase shorter than 11 hr, the majority etd mor ph(*/e) oO w oO oO rq LEO | w toa ib =| ss ov mma SS4 a Pe & sl ° a {°) TIAA Yea (2) N w N a emaIC a Clenei2Umeqem: oO Mb ESO 0 6 12 oO =) re Sing as Cc 4 petegbetbetagnegg \( ES AUOUIE Ty PERNT ; , CU I aS; ‘| pibobagegagadee o - li CUM Daa tentne = a —— -_ GC Vhtbeeatt Hoopanae © an | egeree Uippage tt totes Vv . IF Viteetpertteae = Vibaetaganadaeee eS |— CIE ~ | Pehitanaeongenad hed vit WALEED a Vianberpeetonna PINNGt Ss SS ~ Re ee <& Tinea a TR LY ee a eee — fe) a E i= [LIME] 18 0 Hours after dawn 6 12 Fic. 6. The allowed zone for PTTH-secretion and photoperiodic response curves represented on the complete and the asymmetrical skeleton photoperiods. Left panels: Solid circles with bars show the acrophase-time and the allowed zone for PTTH-secretion preceding those of larval ecdysis by 32hr at 25°C. Solid triangles show the minimum frequency-time of PTTH-secretion. Thin straight lines indicate the dark periods. Solid lines (upper and lower panels) show the photoperiodic response curves of spring-morph panels: Right determination, while a broken line (lower panel) shows the response curve obtained in the corresponding daylengths of the complete photoperiods. 690 K. ENDO AND M. SHIBATA (80-95%) of them developed into spring morphs and the rest (5-20%) developed into intermediate morphs. On the other hand, in regimens with a photophase longer than 14 hr, they developed into summer (75-80%) and intermediate (10-20%) morphs in addition to some spring morphs (2-3%). The critical daylength preventing spring- morph development in 50% insects (the short- to the long-day) was about 12 hr 30 min at 25°C (Fig. 6: upper panel). In the asymmetrical skeleton photoperiods of 4L-20D, spring-morph development was pre- vented by a supplementary light pulse of 0.5-hr, when if fell on in about 6-hr zone beginning at about 9 hr after dawn (light-on of the main light period). The maximum prevention of 80% was recorded when the night-interruption fell on at 12 hr after dawn. Two critical daylength in induction of the spring morphs existed in the asymmetrical skeleton photoperiods of 4L-20D. They were 10 hr (the short- to the long-day) and 14 hr 30 min (vice versa) at 25°C. The critical daylength of 10 hr (the short- to the long-day) was shorter than that of the complete photoperiods (12 hr 30 min) by 2 hr 30 min (Fig. 6: lower panel). The results indicated that L. phlaeas larvae may measure the length of light-(or dark-)period with an error of less than 1 hr. The critical daylength resulting in the 50%-prevention of spring-morph development is at about 12hr 30min in the Yamaguchi strain of L. phlaeas daimio. In the photoperiod of 4L-20D, a specific 6-hr zone beginning at around 9 hr after dawn may exist, in which the incidence of spring morphs is lowered by a supplementary light pulse of 0.5-hr. Further- more, the critical daylength (the short- to the long-day) is shortened to 10 hr in the asymmetrical skeleton photoperiods of 4L-20D. DISCUSSION The timing mechanism of larval ecdysis In L. phlaeas daimio, larval ecdysis occurs in a specific zone of L/D cycles. The timing of larval ecdysis-zone is found to be delayed at 0.4-hr intervals against dawn when photoperiod was changed at 2-hr increments (Fig. 1). The larval ecdysis occurs with rhythms in DD following L/D cycles (Fig. 3). However, the rhythm is dampted gradually in DD as day goes by as has been shown in several other insects [9, 10]. The periodical cycles of the larval ecdysis (or PTTH-secretion) rhythm in DD is judged to be about 24-hr (23.6-hr in LSCF-analysis) in this lycaenid butterfly. This evidence suggests that the timing of larval ecdysis is controlled by a circadian oscillator(s) or hourglass(es). Therefore, larval ecdysis occurs with a rhythm of ca. 24-hr cycles in DD following L/D cycles, but the rhythm is damped in DD as day goes by. The timing of larval ecdysis is supposed to be determined by the timing of the PTTH-secretion in L. phlaeas daimio in the same manner as has been demonstrated in several other lepidopteran insects [OO IST The timing of PTTH-secretion for larval ecdysis Based on the evidence of neck-ligature experi- ments, the peaks of PT'TH-secretion are found to precede the corresponding peaks of larval ecdysis by 32 hr at 25°C (Fig. 3), where the allowed zone for PTTH-secretion may exist in L. phlaeas daimio. The timing of the allowed zone is supposed to be determined by a temperature- compensated circadian system since the PTTH- secretion occurred at 30°C in approximate by the same zone of L/D cycles as in those held at 25°C (Fig. 3). Third instar larvae of L. phlaeas daimio are found to require about the same preparatory period (32 hr) as those of Manduca sexta for larval ecdysis [9]. The time period is shorter than the period demonstrated in Papilio xuthus (34 hr) by 2 hr [13]. The L. phlaeas preparatory period seemed to be shortened to 29 hr at 30°C. How is the circadian clock entrained to L/D cycles in the complete and the asymmetrical skeleton photoperiods in L. phlaeas daimio? An insect is supposed to have plural oscillators or plural hourglasses. They may be entrained by master oscillator and mostly synchronized to the environmental cycles in 24-hr L/D-cycles [9, 10]. The timing of the allowed zone for PTTH- secretion may be determined by a physiological Photoperiodic Time-measurement 691 system involving the oscillator(s) [9, 10]. The same supposition may be applicable for L. phlaeas mechanism determining the timing of PTTH- secretion. According to this supposition, the allowed zone can be regarded as indicating where an approximate phase of the master oscillator comes to on the photoperiodic regimens as has been speculated in several other insects [9, 10]. In L. phlaeas daimio, each index of the allowed zone for PTTH-secretion (T-20%, T-80%, the acrophase-time and the minimum frequency-time) may be located on a different phase of the circadian oscillator as speculated in the case of P. xuthus [13]. Each index of larval ecdysis-zone comes to on parallel lines in the complete photo- periods when light period is changed at 2-hr increments (Fig. 6). However, the parallel lines connecting the same indices have a small angle with the line connecting dawn (delay of index /extension of light period=0.2) (Fig. 1). The angle of the lines is significantly smaller than those of P. xuthus (delay of index/extention of light period=0.5) (P<0.01) [13]. Furthermore, in the asymmetrical skeleton photoperiods, the L. phlaeas indices of larval ecdysis-zone were found to show different be- haviors against night-interruption from those of P. xuthus: each index of P. xuthus shows a response in almost the same manner against either main light period or a supplementary light pulse [13]. In contrast, the indices of L. phlaeas daimio showed the response against the two light pulses (4-hr and 0.5-hr) in the same manner as those held in the complete photoperiods (Fig. 6: upper and lower panels). That is, the light period may be regarded as Starting at light-on of the main light pulse of 4-hr (or a supplementary light pulse of 0.5-hr), whereas it may be regarded as ending at light-off of the supplementary light pulse of 0.5-hr (or the main light period of 4-hr) in L. phlaeas daimio. Here, we could not provide enough evidence to solve the question why the two species of insects showed the different responses to the night- interruption in the asymmetrical skeleton photo- periods. The difference is supposed to be due to the differences of the nature of circadian clocks between these two different species. Furthermore, in L. phlaeas daimio, synchro- nization of the circadian clock to the environment is supposed to be achieved in two steps; the first rough adjustment may be made at dawn by advancing it and the subsequent fine adjustment may be done at dusk by delaying it for several days. The supposition is based on the evidence of phase response experiments. That is, the larval ecdysis-zone was advanced in the same manner as the groups held in the asymmetrical skeleton photoperiods by one night-interruption falling on in a 12-hr period preceding dawn. However, the larval ecdysis-zone did not show any response (phase advance or phase delay) against one night- interruption even when it fell on in a 7-hr period following dusk (Fig. 5), in which the ecdysis-zone was delayed by the night-interruption in the asymmetrical skeleton photoperiods (Fig. 2). How is the circadian clock related to time- measurement of photoperiod in L. phlaeas daimio? The time-measurement system underlying the photoperiodic control of seasonal-morph deter- mination is thought to involve (or to work with the close assistance of) a circadian clock(s) and specu- lated to measure day- or night-length to see whether or not the light period exceeds a critical length [3-8]. The present evidence about the photoperiodic control of seasonal-morph determination— shortening of the critical daylength and existence of the specific 6-hr zone in which the short-day effect is eliminated by a supplemetary light pulse —may be mostly explained on the model of external coincidence [5, 7]. On this model, a photosensitive phase eliminating the short-day effects is speculated to exist at around the end time of the allowed zone for PTTH-secretion. When the light period is extended to the length longer than 12.5 hr, the circadian (photosensitive?) phase comes out into light period. In the asymmetrical skeleton photoperiods, the circadian phase (the end time of the allowed zone for PTTH-secretion) showed a larger delay against dawn (0.4 hr/1 hr) than in the complete photoperiods (0.2 hr/1 hr). As the results, the circadian phase is supposed to come out into main light period of 4 hr when night was interrupted in a 6-hr period beginning at 10 hr 692 after dawn. A gradual shift of seasonal morphs occuring at around critical daylength of the complete photo- periods is supposed to be caused by the nature of circadian clock in L. phlaeas daimio. The supposi- tion is based on an evidence that the same phase of circadian clock comes to on a line having a small angle (0.2 hr/1 hr) with the connecting line of dawn in the complete photoperiods. If the photo- sensitive phase in which the short-day effect is elliminated by an exposure to light is circadian, the phase is supposed to gradually come out from the group to the next into light period in the complete photoperiods (Fig. 6). Here, we could not provide any direct evidence with respect to the photosensitive phase in which spring-morph development is prevented by a exposure to light in L. phlaeas daimio. The circadian phase on which the photosensitive phase is located in L. phlaeas daimio may be identified by further study. ACKNOWLEDGMENT The authors wish to express their sincere gratitude to Professor Y. Chiba and to Professor A. Okajima of Yamaguchi University for advice and valuable sugges- tions during the course of this work. REFERENCES 1 Miller, H.J. (1955) Die Saisonformenentbildung von Araschnia levana, ein photoperiodish gesteuer- ter Diapause-effekt. | Naturwissenschaften, 42: 134-135. 2 Danilevskii, A. S. (1961) Fotoperiodism i sezonne razvitie nasekomykh. Lzd. Leningradskogo Univer- siteta, Leningrad. 3 Saunders, D.S. (1966) Insect Clock, Pergamon Press, Oxford, 297 pp. 4 10 11 12 13 14 15 K. ENDO AND M. SHIBATA Beck, S. D. (1980) Insect Photoperiodism, 2nd ed., Academic Press, New York. Bunning, E. (1960) Circadian rhythms and time measurement in photoperiodism. Cold Spring Harbor Symp. Quant. Biol., 25: 245-256. Pittendrigh, C. S. (1960) Circadian rhythm and the circadian organization of living systems. Cold Spring Harbor Symp. Quant. Biol., 25: 158-184. bee Pittendrigh,C.S. and Minis,D.H. (1964) The entrainment of circadian oscillators by light and their roles as photoperiodic clock. Am. Nat., 98: 261-294. Tyschenko, V. B. (1966) Two-oscillator model of the physiological mechanism of insect photoperiodic reaction. Zhur. obshch. Biol., 27: 209-222. Truman, J. W. (1972) Physiology of insect rhythm. I. Circadian organization of the endocrine events underlying the moulting cycle of larval tobacco hornworms. J. Exp. Biol., 57: 805-820. Fujishita, M. and Ishizaki,H. (1981) Circadian clock and prothoracicotropic hormone secretion in relation to the larval-larval ecdysis rhythm of satur- niid Samia cynthia ricini. J. Insect Physiol., 27: 121-128. Sakai, T. and Masaki, S. (1965) Photoperiod as a factor causing seasonal forms in Lycaena phlaeas daimio Seitz. Konchu, 33: 275-283. Endo, K., Maruyama, Y. and Sasaki, K. (1985) Environmental factors controlling seasonal morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz. J. Insect Physiol., 31: 525-532. Endo, K. and Kamata, Y. (1985) Hormonal control of seasonal-morph determination in small copper butterfly, Lycaena phlaeas daimio Seitz. J. Insect Physiol., 31: 701-706. Halberg, F., Johnson, E. A., Nelson, W., Runge, W. and Southern, R. (1972) Autorhythmometry: procedures for physiological self-measurements and their analysis. Physiol. Teach., 1: 1-11. Nakahama,K. and Endo,K. (1986) Time- measurement system underlying the photoperiodic control of pupal diapause in the swallowtail, Papilio xuthus L.: A trial for the application of the external coincidence model. Zool. Sci., 3: 837-846. ZOOLOGICAL SCIENCE 4: 693-697 (1987) © 1987 Zoological Society of Japan Mating Aggregation in the Japanese Treefrog, Rhacophorus arboreus (Anura: Rhacophoridae): a Test of Cooperation Hypothesis Enti KAsuyA, HIDEKO SHIGEHARA and MArIKo HIROTA Laboratory of Biology, Faculty of Education, Niigata University, 2-8050 Ikarasi, Niigata 950-21, Japan ABSTRACT—The hypothesis that the relationship among males in one female—multiple male aggregations in mating and oviposition of the foam-making rhacophorid frogs is cooperative was tested in the Japanese treefrog Rhacophorus arboreus. Joining males were not observed beating the foam with their hind limbs. The size of foam nest tended to increase with the body size of female, but was not correlated with the number of joining males. The duration of oviposition was correlated with the number of joining males but not with the body size of the paired female and male. These results were inconsistent with the hypothesis that the relationship among males in the one female—multiple male aggregations is cooperative. INTRODUCTION Contrasting with conflicts among males at the scene of mating, cooperation among males during mating has been rarely reported, except cases where the high degree of relatedness among males is expected (e.g. [1]). However, in some foam- nesting rhacophorid treefrogs, the relationships among males in one female—multi male mating aggregations have been described as cooperative (e.g. [2] for Chiromantis rufescens; [3] for Rha- cophorus arboreus). Following Coe [2], Wilson [4] considered a one female—three male aggregation of the African rhacophorid Chiromantis rufescens reported by Coe as a case of “cooperative breed- ing”. In their review on the sperm competition in amphibians, however, Halliday and Verrell [5] pointed out that there is no evidence for Wilson’s interpretation except the observation that all the males in the aggregation moved their hind limbs and contributed to the formation of the foam nest. The behavioral sequences of mating of foam- nesting rhacophorid treefrogs in one female—multi male mating aggregations are: the pair in amplexus and some other males (hereafter referred to Accepted March 3, 1987 Received December 9, 1986 joining males) arrive at the oviposition site (join- ing males sometimes arrive after the female begins oviposition). Only one male is in the amplexus position. The female secretes a fluid from which the foam nest is constructed and oviposits eggs into it. The female (and males in a few reports) beats the secretion and constructs the foam nest. After oviposition, the female and males leave the con- structed foam nest. In the present paper, we test two sub-hypotheses (“a larger nest hypothesis” and “less susceptibility of predation hypothesis”, [2]) derived from the assumption that the male—male relationship in one female—multiple male mating aggregations is coop- erative in the Japanese treefrog R. arboreus. MATERIALS AND METHODS The observations were made from May to July, 1984, at the ‘Hyoutan’ pond (26mxX8m) in Iwamuro, Niigata, Japan (altitude about 200 m). Frogs were captured by hand or with a handy net, and were individually marked with colored waist bands for later identification [6]. We measured the snout-vent length (body size, hereafter) and weight of each frog. Observations at night were made with a 6V battery head lamp which seemed not to disturb the behavior of frogs. We observed 694 E. KasuyA, H. SHIGEHARA AND M. Hirota the behavior of solitary females and pairs in amplexus and that of males approaching the observed females or pairs under observation. The length and width of newly deposited foam nest were measured (the method of [7]) with a ruler. The product of length and width was used for the size measurement of foam nest and called it ‘size’ of foam nest hereafter. RESULTS During foam nest construction, we observed no males of beating the foam with their hind limbs (swimming-like behavior) apart from _ passive movements when the hind limbs of females touched those of males. In all the instances (n=14), beating of the foam with hind limbs was done by females. There is no significant correlation between the size of foam nest and the number of males in a mating aggregation (r=—0.194, 0.8>P>0.5, medal) aged) The size of foam nest is not significantly corre- lated with the body size of the female (r=0.451, 0.050.8, n=9, Fig. 3). There is no correlation between the number of males in a mating aggregation and the body size of the male in amplexus (r= — 0.039, P>0.8, n=10) nor the size of the female (r=0.263, 0.5>P>0.4, 10 @ 3 5 E e @ _ © @ 3 a e & ® @ @ t Oe gre gt 80 100 120 140 size of foam nest (Cm X Cm) Fic. 1. The relationship between the size of foam nest and the number of males in mating aggregation. @ @ 140 fe Oo ® ® x< 5 ad ) 120 e%e @ . @ 2 Ee oO © Ss 5 100 e@ @ @ e (le pe 6.5 7.0 7.5 8.0 8.5 body size of female cm Fic. 2. The relationship between the size of foam nest and the body size (snout-vent length) of the female. 160 ~ @ E 140 oO = oO e w 2 « 120 e@ 60 e 2 (reas (o) Xu ia 100 6 @ @ 5.0 355) 6.0 6.5 body size of male em Fic. 3. The relationship between the size of foam nest and the body size (snout-vent length) of the male in amplexus. msl) Figures 4 and 5 show the relationships between the duration of oviposition (from the beginning of construction of foam nest, i.e. the swimming movement of hind limbs of female, to the leaving of the female) and the number of males in mating Test of Treefrog Male Cooperation 695 250 @ 200 e @ e 6 e@ Ss 150 e a = * > Oo % @ Cc o FS: = > 100 v0 0 5 10 15 No. of males Fic. 4. The relationship between the duration of ovi- position (from the beginning of foam nest construc- tion to the departure of the female) and the num- ber of males. asterisk: the amplexus posture of male (grip of his fore limbs) was incomplete. 250 e 200 ® = @ (= (o) Ss az 2 6 & = iG e qe oO = e@ J o 6 =) v0 100 Lk ee eee 80 100 120 140 size of foam nest (¢mxcm) Fic. 5. The relationship between the duration of ovi- position (from the beginning of foam nest construc- tion to the leaving of the female) and the size of foam nest. aggregation and the size of foam nest, respectively. There is a significantly positive correlation be- tween the duration of oviposition and the number of males (r=0.820, P<0.05, n=11) (Fig. 4). However, the correlation between the duration of oviposition and the size of foam nest is not significant (Fig. 5, r=0.077, P>0.8, n=9). The correlation between the duration of oviposition and the body size of female is also not significant (r=0.134, 0.8>P>0.5, n=10). DISCUSSION First, we test the following two sub-hypotheses derived from the main hypothesis that the male— male relationship in mating aggregations is coop- erative. They are “a larger nest hypothesis” [2]: the advantage of using more than one male is that a larger nest can be constructed (and thus a larger number of offspring can be reared), and “less susceptibility of predation hypothesis” [2]: the advantage of using more than one male is that the assistance by a larger number of males render a female less susceptible to predation because the Oviposition activity leaves the female exhausted. The first sub-hypothesis predicts the positive correlation between the size of foam nest and the number of males. However, this is inconsistent with the result of Figures 1 which shows weakly negative (not significant) correlation between them. The results of Figures 1 and 2 suggest that the factor responsible for the size of foam nest is not the number of males but the body size of female. The second sub-hypothesis is not directly tested because we did not measure the degree of exhaus- tion of females after oviposition. We use the duration of oviposition as a substitute. Further- more, the duration of oviposition seems to be a reasonable index of susceptibility of predation during oviposition. R. arboreus was preyed by predators including snakes during oviposition (e.g. Kinefuchi, personal communication). The results of Figures4 and 5 show that the duration of oviposition is strongly positively correlated with the number of males and is not correlated with the size of foam nest. Therefore, both sub-hypotheses of the coopera- 696 E. KasuyA, H. SHIGEHARA AND M. HIROTA tion hypothesis are not consistent with the results of the present study. The cooperation hypothesis is not consistent with the observation by Saitou and Kumaki (un- published) that the hatchability of eggs in cases of one female—multi male mating aggregations is similar to that in oviposition by a single pair in amplexus (96.4% and 98.6%, respectively). Fur- thermore, Kato [8] reported that all the males in mating aggregation secrete sperm with both direct and microscopic observations. We propose alternative hypothesis, “sperm competition hypothesis”, for explaining the signif- icance of one female—multi male mating aggrega- tion in foam-making rhacophorids based on the results of the present study. This suggests that one female—multi male mating aggregations are formed as a consequence of selfish activities of joining males, i.e. releasing sperm into the foam and fertilizing eggs by his own sperm. Because interruption of the oviposition is costly for both female and male of the pair, and because the male in the amplexus can not use his fore limbs [5], both female and male of the pair can not effectively counteract other males which approach the pair to release sperm into the foam. In this hypothesis, the joining of males is regarded as detrimental for both the male and female in amplexus. For the female, the joining males increase the duration of oviposition and hence increase the susceptibility of predation. For the male, they decrease the number of eggs fertilized by his sperm and increase his susceptibility of predation. The “sperm com- petition” hypothesis is consistent with the results of the present study. Males joining the mating aggregation do not increase the size of the foam nest (Fig. 1). The size of foam nest is not correlated with the body size of male in amplexus nor the number of males (Fig. 3), but there is a trend between the foam nest and the body size of female (Fig. 2). The activities of males are not responsible for the size of foam nest. It suggests that the body size of female determines the size of the foam nest. The joining of males is costly in terms of the duration of oviposition (Figs. 4 and 5). The activities of joining males in mating aggregation are thus considered to be obstructing the constructing behavior of female rather than assisting it. In the present study, this is a natural conclusion because we did not observe the swim- ming-like behavior of hind limbs of males in mating aggregations. This does not mean, howev- er, that the male—male relationship is cooperative in species in which the swimming-like behavior of males is observed (e.g. [2, 7]). In such species, the swimming-like behavior of males can be inter- preted either as cooperative behavior or selfish one to increase the chance of fertilization of eggs by his sperm. In conclusion, the selective significance of one female—multi female mating aggregation in foam- making rhacophorids is better explained by selfish- ness of males than by cooperation of males, as in the context of reproductive behavior of other anurans (e.g. [9]). ACKNOWLEDGMENTS We are indebted to A. Arak for his critical reading of the manuscript. Thanks are also due to K. Kinefuchi, Y. Kishi, K. Maekawa, S.Tsurumaki and M. J. West- Eberhard for useful comments. T. Saitou and T. Kumaki showed us their unpublished data. We are grateful to members of our laboratory for their assistance. REFERENCES 1 Bertram, B. C. R. (1976) Kin selection in lions and in evolution. In “Growing Points in Ethology”. Ed. by P. P. G. Bateson and R. A. Hinde, Cambridge Univ. Press, Cambridge, pp. 281-301. 2 Coe, M. J. (1967) Co-operation of three males in nest construction by Chiromantis rufescens. Nature, 214: 112-113. 3 Matsui, M. (1980) Breeding behavior of some Japanese anurans. Collection and Breeding, 42: 132-135. 4 Wilson, E. O. (1975) Sociobiology. Belknap Press of Harvard Univ. Press, Cambridge, Mass. 5 Halliday,T.R. and Verrell,P. A. (1984) Sperm competition in amphibians. In “Sperm Competition and the Evolution of Animal Mating Systems”. Ed. by R. L. Smith, Academic Press, Orlando, Florida, pp. 487-508. 6 Emlen, S. T. (1968) A technique for marking anuran amphibians for behavioral studies. Herpetologica, 24: 172-173. 7 Coe, M. J. (1974) Observations on the ecology and breeding biology of the genus Chiromantis. J. Zool., Lond., 172: 13-34. Test of Treefrog Male Cooperation 697 choice in anuran amphibians. In “Mate Choice”. Ed. by P. Bateson, Cambridge Univ. Press, Cambridge, pp. 181-210. Kato, K. (1956) Ecological notes on the green frogs during the breeding season. 2. Breeding habit and others. Jpn. J. Ecol., 6: 57-61. Arak, A. (1983) Male-male competition and mate nes aes 4 Ree eek ¥ ae jek iat ‘. 4 Cray i kee OCHED ae 2 eta a iki ne 7 oe; P f .- y Crepe +a ’ re ig i Prt 7 i i | ! 14 bh iy A $4 + Yj i f y EP 4 far 4 use pte F ty hagas * tA be 4 HYD Oe ti” : oe ¥ i ii ’ i Net} i Take t Re he ie . ‘4 i 7 sj A aa ee hw j Me} af &” 9 a Meet te . fia ia ¥ # a) i v f > in ait: reed! id | oer ) . t ti ) CS car t Kunuki fais 9 uereyutli ee ai bl th. ap eh I ieee as ‘ci ial Ad =} ¥ pit LEC) Sees exp ri \ vee pagar NH mk pis ZOOLOGICAL SCIENCE 4: 699-709 (1987) © 1987 Zoological Society of Japan What is Scopura longa Uéno, 1929 (Insecta, Plecoptera)? A Revision of the Genus! SHIGEKAZU UcuipaA* and Hiroki MARUYAMA? Department of Natural History, Faculty of Science, Tokyo Metropolitan University, Setagaya, Tokyo 158, and *Laboratory of Entomology, Tokyo University of Agriculture, Setagaya, Tokyo 156, Japan ABSTRACT— Scopura prolifera Kawai is synonymized with S. longa Uéno. S. longa sensu Kawai, 1974 is redescribed as a new species, S. montana Maruyama. Two new species, S. bihamulata Uchida and S. quattuorhamulata Uchida, are described from Hokkaido. One more new species, S. laminata Uchida, is described from Korean nymphs. These species are distinguished from one another by one or more characters: male and female genitalia, lateral margin of adult nota and epiproct of male nymph. Scopura Uéno, 1929 [1] is the only genus of the family Scopuridae Uéno, 1935 [2], which is endem- ic to Japan and Korea. It comprises peculiar wingless stoneflies with many primitive morpho- logical features [3, 4]. The first species, S. longa Uéno, 1929, on which the present genus was established, was described from nymphs collected by the lake Towada-ko, northern Honshu [1]. Subsequently, the first adult of Scopura was collected in 1930 at Takeshi-t6ge, Nagano-ken, central Honshu, and described as /onga by Uéno in the next year [5]. Then, /Jonga was considered to occur in Honshu, Hokkaido and Korea [6-8]. But longa in this broad sense was not based on the detailed comparative studies of adults from differ- ent localities, which were seldom collected except those from central Honshu. In 1974, Kawai Accepted March 31, 1987 Received December 6, 1986 Parts of this paper were presented at the annual meetings of the Entomological Society of Japan, Kan- to Branch, Tokyo, December 1982, December 1983 and the Entomological Society of Japan, Tsukuba, October 1984. Present address: Limnologische FluBstation des Max- Planck-Instituts fir Limnologie, Postfach 260, D 6407 Schlitz, Germany. Present address: Nihon Tokushu Noyaku Seizo K. K., Osaka Branch Office, Furukawa-Osaka Bldg., 1-29, Dojimahama 2-Chome, Kita-ku, Osaka-shi, Osaka 530, Japan. — N Ww divided /onga into two species by describing the second species, S. prolifera, from Sado Is. on the Sea of Japan [9]. He found clear differences between prolifera and his “longa” in male genitalia which were figured with a key to the species. According to his distribution map, “/onga” occurs in most parts of Honshu but prolifera is restricted to Sado Is. and Ibaraki-ken, Honshu. However, he was unable to find a nymphal character which distinguishes these two species from each other, and therefore left the nymphs from Hokkaido and Korea unidentified, where no adult had been collected. Moreover, it is uncertain if he examined the adult male from the type locality of longa by the lake Towada-ko, because he did not show the locality of examined specimens of his “/onga”. Accordingly, it is problematic that he assigned the adults from most parts of Honshu to longa. We found an important nymphal character of Scopura species in the epiproct of the male nymph, and succeeded to acquire many adults from the type locality of longa by rearing nymphs. They are clearly assigned to longa by a nymphal characteris- tic of their epiprocts which was described and figured in the original description. Comparison of the adults from the type locality with those from Sado Is. made it clear that S. prolifera Kawai is a junior synonym of S. longa Uéno, and that the “longa” of Kawai [9] is an unnamed species. The 700 S. UCHIDA AND H. MARUYAMA distribution of /Jonga is confined to Sado Is. and northern Honshu, whereas the unnamed species occurs only in central Honshu. We also acquired the adults from Hokkaido by rearing. They belong to two new species which are clearly distinguished from each other and from the two species of Honshu by the genitalia and the epiproct of the male nymph. The adult of Korean Scopura has not been collected yet. However, the epiprocts of male nymphs from south Korea indicate that they also represent a new species which is the fifth one of the genus. Specimens used in this study are deposited in the collection of the National Science Museum, Tokyo (NSMT), the Laboratory of Entomology, Tokyo University of Agriculture (TUA), the Limnologi- sche FluBstation des Max-Planck-Instituts fiir Lim- nologie, Schlitz (LFS) and the senior author (SU). Localities where only nymphs were collected are not stated in detail in this paper. They will be published elsewhere with the depository of speci- mens. Genus Scopura Uéno, 1929 Scopura Uéno, 1929 [1], 124. Type species: Scopural longa Uéno, original design. and monotypy. Generic characters of Scopura were described by authors (see [10]) as the characters of S. longa in a broad sense. Kawai [9] distinguished the following characters (1-3) of species from the generic ones, to discriminate prolifera from his 1) projection at the base of male cercus, 2) male epiproct, 3) armature of penis. We accept his distinction of characters and add the following (4-8) to the characters of species: 4) epiproct of male nymph, 5) lateral margins of adult thoracic nota, 6) male abdominal tergum 9, 7) vagina, 8) hairs and sclerites around the opening of vagina. We present here some generic features which have never or insufficiently been described. Male Paraproct weakly sclerotized, simple, covered with fine spinules and curved dorsally with rounded tip. “Internal sac of epiproct” (Fig. 1, Fics. 1-7. Internal sac of epiproct and penis — 1, Sco- pura longa from Wainai, internal sac of epiproct, everted, lateral view; 2-3, S. montana sp. nov. from Mité-san, everted penis, lateral (2) and ven- tral (3) views; 4-7, lateral sclerites on penes of S. montana sp. nov. from Mité-san (4), S. longa from Wainai (5), S. bihamulata sp. nov. (6) and S. quat- tuorhamulata sp. nov. (7). Scales, 0.5mm. =“Epiproctblase” of Zwick [4]) eversible, armed with teeth and spinules. Basal part of the sac thick, with a pair of dorso-lateral rows of strong teeth and with ventral scattered teeth; apical part slender, gradually tapered to tip. The whole sac densely covered with small spinules. The sac has no particulars that could serve species distinction, but exhibits minor geographical variation in the number of ventral teeth. The penis (Figs. 2 and 3) consists of basal everted and apical armed (not everted) parts; the apical part with a pair of lateral sclerites (Figs. 4-7) and with fine spinules on the apical expansion around the gonopore. Apical part of the lateral sclerite with many fine longitu- dinal folds; but basal part without fold, concave and forming a longitudinal trough. Female General color paler than in male. Lateral margin of pronotum (Figs. 14-19) with a Revision of Scopura Stoneflies 701 hump. Vagina (Figs. 57-59, 61) with an irregularly branched accessory gland attached to the anterior part of its dorsal side. Distal end of oviduct irregularly folded, connected to apical end of vagina which bears a small sclerite ventrally; the folded part remains after KOH treatment. Nymph _ The female nymph, from young (ca. 10 mm long) to mature ones, is distinguished from the male one by a central notch on abdominal sternum 8 [11]; the notch moves from the anterior margin of the sternum in young nymphs to its posterior part in mature ones. Epiproct and paraproct each with a thread of tapered gill, but the mature male nymph lacks that of the epiproct. The gills persist in adult females, but are reduced in the male. Scopura montana Maruyama, sp. nov. (Japanese name: Mine-towada-kawagera) (Figs. 2, 4, 8,9, 14, 15, 20, 24, 25, 30, 31, 36, 37, 42, 45, 54, 57, 62, 68 and 69) Scopura longa: Uéno, 1931 [5]: 40, fig. 3. In part (adult). Scopura longa: Kawai, 1967 [7]: 4, 178, figs. 105-108. Scopura longa: Zwick, 1973 [4]: 167, fig. 57. Scopura longa: Kawai, 1974 [9]: 275, fig. 2, D-F. Additional references: Scopura longa, [2, 12, 13, 14, 15, 16]. Material Types: Holotype @ with exuvia (NSMT), Mit6-sawa at 1270m, Mité-san, Hino- hara-mura, Tokyo, nymph collected 1.x emerged 11-12.x.1984, S. Uchida; Paratypes, 897? Gri TWA] 302 LFS; 4042 SU))same locality, 1.x.1984, nymph collected 1.x emerged 6-9.x.1984, 1.xi.1984, S.Uchida. Additional specimens: Ishikawa, Yoshinodani-mura, Chigt, nymph collected 6.x emerged 12.xi-11.x11.1982, T. Ito and S. Uchida, 1832 (SU); Shizuoka, Hon- kawane-ch6, Tekari-dake at 2380 m, 14.x.1980, S. Uchida and N. Morihiro, 51 (Specimens re- ported by [14]: 2, LFS; 3$12, SU); Shizuoka- shi, Tekari-dake at ca. 2400m, 13.x.1980, N. Morihiro, 1 (Specimen reported by [14]; LFS) Nagano, Tobira-toge, 11.x.1982, M. Hasegawa, 12 (TUA); liyama, 23.x.1960, collector?, 2912 (LFS); Otari-mura, Tsugaike, 10.x.1982, M. Hase- gawa, 12 (TUA); Omachi-shi, Nakatsuna at 950 m, nymph collected 9.viii emerged 6-18.x.1982, S. Uchida, 12922 (6%, LFS; 6922, SU); Niigata, Itoigawa-shi, Renge-onsen, nymph _ collected 12.viil emerged 6-16.x.1982, S. Uchida, 1 (SU); Kanagawa, Tsukui-machi, Kasagi-zawa at 1290 m, 4.x1.1980, S. Uchida, 17839 (Specimens re- ported sbyi 15)" (or Pessina? OAKS); Tokyo, Hinohara-mura, Mit6-san, nymph col- lected v, ix emerged 28.ix—8.x.1981, H. Maruyama and A. Mase, nymph collected 2.v emerged ix—x. 1982, H. Maruyama, A. Mase and T. Okayama, Das BCIWAN) 25¢X519850 HiSaito 1 219(SU); Okutama-machi, Kawanori-yama, 30.x.1983, K. Takahashi, 1@ (TUA); Yamanashi, Enzan-shi, Ichinose, 1.x.1984, nymph collected 1.x emerged 1-5.x.1984, 8.x.1984, nymph collected 8.x emerged) =) S-ilo:xal984— aS: Uchida, 1348) 9 (3832, LFS; 10$5 2, SU); Tateno-sawa at 1700 m near Sydgen-toge, 18.x1.1985, S. Uchida, 1$22 (SU); Daibosatsu-tége, nymph collected 23.v, 29.vi emerged ix.1983, A.Mase, H. Maruyama, R. Terakoshi, C Tobiyama and M. Furukawa, 12892 (TUA); Tabayama-mura, Hiryt-san, Waru-dani at 1750 m, 22.xi.1984, S. Uchida, 1 (SU); Gumma, Minakami-machi, Doai, 27.x.1982, T. Torii, 12 (SU); Katashina- mura, Tokura, nymph collected 10.ix emerged ix-x.1982, H. Maruyama, 18 (TUA); Tochigi, Nikk6-shi, Hantsuki-yama, nymph collected 12.vi emerged 14—20.1x.1982, A. Mase, H. Maruyama, M. Watanabe and R. Terakoshi, 3$12 (TUA); 968 nymphs (138 nymphs, TUA; 97 nymphs, LFS; 733 nymphs, SU) from Ishikawa, Shizuoka, Naga- no, Niigata, Tokyo, Yamanashi, Gumma, Tochigi and Fukushima. The name l/onga is presumed to have been applied to this species by authors [3, 17, 28, etc.] who treated the Scopura from central Honshu (see longa). But their material was not accessible for re-examination and most of these authors did not describe the characteristics of this species. The male /onga of Uéno [5] is assigned to this species, because he described long posterior pro- jections on the lateral margins of meso- and metanota. Kawai [7, 9] and Zwick [4] also show clearly the characteristics of this species in their descriptions and figures of male genitalia. Male Body 13-25 mm long. Anterior projec- tion on lateral margin of mesonotum (thick arrows in Figs. 8 and 9) slightly shorter than posterior one (fine arrows); anterior one of metanotum (thick 702 S. UCHIDA AND H. MARUYAMA arrows) nearly as long as posterior one (fine arrows). Abdominal tergum 9 (Fig. 20) without spinule patch; postero-mesal part somewhat mem- branous. Epiproct (Figs. 42 and 45) with a pair of hooks, which project dorsally and curve anteriorly to terminate in pointed tips; the hooks are cylin- drical and bear spinules on the dorsal side near the tips. Internal sac of epiproct opens dorsally between hooks; anterior side of its opening sclero- tized, with spinules. Projection at the base of cercus (Figs. 24, 25, 30, 31, 36 and 37) directed rk 14 Fics. 8-19. Lateral margins of thoracic nota: top, pro- nota; middle, mesonota; below, metanota: 8-13, male; 14-19, female — 8-9, 14-15, Scopura monta- na sp. nov. from Tekari-dake (8, 14) and Mit6-san (9, 15); 10-11, 16-17, S. longa from Yamizo-san (10, 16) and Wainai (11, 17); 12, 18, S. bihamulata sp. nov.; 13, 19, S. quattuorhamulata sp. nov. Scale, 1 mm. dorso-mesally with a rounded tip which bears many spinules. Lateral sclerite on penis (Fig. 4) strongly sclerotized, somewhat broad and weakly folded; fine folds on its apical part directed basally to ventral side. Apical expansion of penis (Figs. 2 and 3) with spinules on dorsal and lateral side, but not on ventral side. Female Body 25-35 mm long. Lateral margin of pronotum (Figs. 14 and 15) with a hump on anterior part; the hump is elongate and extends longitudinally. Projections on lateral margin of meso- and metanota (Figs. 14 and 15) similar to those in male in relative length of the anterior (thick arrows) and posterior (fine arrows) projec- tions, but all of them are longer than those of male. Hind margin of abdominal sternum 8 (Fig. 54) shallowly concave. The membrane between vaginal opening and sternum 9 without or at most with a few hairs. Vagina (Fig. 57) large, 1.4-1.6 mm wide at its opening. Male nymph _ Epiproct (Fig. 62) with a pair of sclerotized knobs dorsally; mesal part between the knobs flat and sclerotized. Scopura longa Uéno, 1929 (Japanese name: Towada-kawagera) (Figs: 1, 5,10, 11, 16.175 21526, 27) 32633Nasneo: 43, 44, 46-48, 51, 55, 58, 63 and 69) Scopura longa Uéno, 1929 [1]: 125. Holotype: a nymph, Wainai by the lake Towada-ko, Akita-ken, northern Honshu, deposited in the collection of Dr. T. Kawai (Nara). Scopura prolifera Kawai, 1974 [9]: 275. Holotype @, Myoken-zan, Sado Is. on the Sea of Japan, de- posited at the National Science Museum, Tokyo. Syn. nov. Additional references: Scopura longa, [5], details of type locality; S. prolifera, [16]. Material Niigata, Sado Is., Kanai-machi, My6ken-zan, nymph collected 10—11.ix emerged 24-30.1x.1983, H.Maruyama and A. Mase, 3862 (TUA); Ibaraki, Kita-ibaraki-shi, Hana- zono-san, nymph collected 6.1x emerged 4-11.x. 1982, H. Maruyama and A. Mase, 1932 (TUA); Daigo-machi, Yamizo-san, nymph collected 7-8.vi emerged 1.x—5.x1.1981, N. Gokan, T. Nagashima and H. Maruyama, nymph collected 6.ix emerged 13-27.ix.1982, H.Maruyama and A. Mase, 3492 (TUA); Akita, Kosaka-machi, Wainai by Revision of Scopura Stoneflies 703 Fics. 20-41. Male abdominal tergum 9 (20-23), dorsal views with lateral views, and the bases of cerci (24-41), dorsal views (24-29), posterior views (30-35) and inside views (36-41) — 20, 24-25, 30-31, 36-37, Scopura montana sp. nov. from Nakatsuna (24, 30, 36) and Mit6é-san (20, 25, 31, 37); 21, 26-27, 32-33, 38-39, S. longa from Sado Is. (26, 32, 38) and Wainai (21, 27, 33, 39); 22, 28, 34, 40, S. bihamulata sp. nov.; 23, 29, 35, 41, S. quattuorhamulata sp. nov. Scales, 1mm. the lake Towada-ko, nymph collected 11.ix emerged 14.x.1982, H.Maruyama, 1¢42 (TUA); 21.ix.1984, nymph collected 21.ix emerged 21.ix—2.x.1984, S.Uchida, 449332 (S872, LFS; 399262, SU); 166 nymphs (63 nymphs, TUA; 32 nymphs, LFS; 71 nymphs, SU) from Niigata, Ibaraki, Fukushima, Yamagata, Iwate, Akita and Aomori. This name was previously used for all species of the genus from Honshu, Hokkaido and Korea [8, 10]. Kawai [9] erroneously used this name for the population of central Honshu. According to our study, /onga occurs only in Sado Is. and northern Honshu (Fig. 69). Therefore, most workers of Scopura have presumably applied the name longa to other species, mostly to montana from central Honshu where the genus was intensively studied. Uéno [1] described a mesal knob on the dorsal side of the epiproct of the male nymph in detail. It is clear enough to assign this species to longa. Kawai [9] also showed clearly some of the charac- teristics of this species in his description of prolifera. Male Body 18-25 mm long. Anterior projec- tion on lateral margin of mesonotum (thick arrows in Figs. 10 and 11) longer than posterior one (fine arrows); anterior one of metanotum (thick arrows) far longer than posterior one (fine arrows). Ab- dominal tergum 9 (Fig. 21) with a postero-mesal patch of long spinules, but those in the central light part are short. Epiproct (Figs. 43, 44, 46-48 and 51) with a mesal sclerotized hook at dorsal tip, which curves anteriorly and forms a blunt tip; spinules scattered on dorsal side before the hook. Internal sac of epiproct opens postero-dorsally behind the hook; lateral side of its opening with a pair of sclerotized lobes, which bear spinules postero-dorsally. The lobes of the material from the type locality (Figs. 48 and 51) are less pro- duced than those from Sado Is. (Figs. 43 and 46) and Yamizo-san (Figs. 44 and 47). Projection at the base of cercus (Figs. 26, 27, 32, 33, 38 and 39) 704 S. UcHIDA AND H. MARUYAMA \ Shs =a\ WU *) LU YT! sell | rn Fics. 42-47. Male epiprocts, dorso-lateral (42-44) and posterior (45-47) views — 42, 45, Scopura montana sp. nov. from Mité-san; 43-44, 46-47, S. longa from Sado Is. (43, 46) and Yamizo-san (44, 47). Scale, 0.5mm. directed posteriorly, with a pointed (specimens from Sado Is., Figs. 26, 32 and 38) or rounded (those from the type locality, Figs. 27, 33 and 39) tip which bears some spinules; dorso-mesal side of the base of the projection with hairs. Lateral sclerite on penis (Fig.5) strongly sclerotized, constricted in median part and strongly folded; fine folds on its apical part directed basally to dorsal side. Apical expansion of penis with spinules on all sides around the gonopore. Female Body 23-40 mm long. Lateral margin of pronotum (Figs. 16 and 17) with a hump on median part, which is positioned transversely and is produced laterally. Projections on lateral mar- gins of meso- and metanota (Figs. 16 and 17) similar to male in relative length of anterior (thick arrows) and posterior (fine arrows) projections, but all of them longer than those of male. Hind margin of abdominal sternum 8 (Fig. 55) deeply concave. The membrane between vaginal opening and sternum 9 with many hairs, which point anteriorly. Vagina (Fig.58) smaller than in montana, 1.2-1.3 mm wide at its opening. Male nymph _ Epiproct (Fig. 63) with a mesal sclerotized knob dorsally; both sides of the knob also sclerotized, rounded, but never produced to knobs nor swellings as in montana, bihamulata and laminata. Scopura bihamulata Uchida, sp. nov. (Japanese name: Futakagi-towada-kawagera) (Figs. 6, 12, 18, 22, 28, 34, 40, 49, 52, 56, 59, 64 and 69) Material Types: Holotype ¢ with exuvia (NSMT), Asari-t6ge, 600m, Otaru-shi, Hok- kaido, nymph collected 18.vii emerged 17-23.1x. 1984, S. Uchida; Paratype, $, same locality, nymph collected 18.vii.1984 emerged 2-8.x.1985, S. Uchida (SU). Additional specimens: 45 nymphs (32 nymphs, LFS; 13 nymphs, SU) from Sapporo- shi and Otaru-shi, Hokkaido. Male Body 22mm long. Lateral projections Revision of Scopura Stoneflies 705 on thoracic nota (Fig. 12) poorly developed; ante- rior ones of meso- and metanota (thick arrows) longer than those of montana and quattuorhamula- ta but shorter than those of /onga, in relation to the posterior ones (fine arrows). Abdominal tergum 9 (Fig. 22) with a pair of spinule patches posteriorly; the posterior spinules shorter than the anterior ones. Epiproct (Figs. 49 and 52) with a pair of strong hooks, which are spread like fans. Dorsal margin of the hook forming an elongate disk with spinules; the disk extends anteriorly to a pointed tip. Internal sac of epiproct opens dorsally be- tween the hooks; anterior side of its opening sclerotized without spinules. Projection at the base of cercus (Figs. 28, 34 and 40) directed dorso-mesally, with a rounded tip which bears dense spinules. Lateral sclerite on penis (Fig. 6) weakly sclerotized, slender and weakly folded; fine folds on its apical part directed basally to dorsal side. Apical expansion of penis with spinules on all Fics. 48-53. sides around the gonopore. Female Body 22mm long. Lateral margin of pronotum (Fig. 18) with a hump on posterior part, which is longitudinally elongate. Projections on lateral margins of meso- and metanota (Fig. 18) similar to those of longa, but less pointed. Hind margin of abdominal sternum 8 (Fig. 56) not concave, with a pair of sclerotized humps. The membrane between vaginal opening and sternum 9 with two lip-like folds; the anterior one without hairs, but the posterior one with many hairs; the mesal hairs shorter than the lateral ones. Vagina (Fig. 59) short but as wide as in Jonga, 1.2 mm wide at its opening, with a dorsal sclerite which tapers anteriorly. Male nymph _ Epiproct (Fig. 64) with a pair of dorso-lateral sclerotized swellings which are pro- duced dorsally; the swellings far larger than the knobs of montana. ray 7 Male epiprocts, dorso-lateral (48-50) and posterior (51-53) views — 48, 51, Scopura longa from Wainai; 49, 52, S. bihamulata sp. nov.; 50, 53, S. quattuorhamulata sp. nov. Scale, 0.5mm. 706 S. UcCHIDA AND H. MARUYAMA Gnd Tea 7 VNAN 5 . ees 0 Gulia “4 77,71 N.S NDS er aero eo M aot Bly 2 UY NaN aN 55 Oh roa age al MRR SH otis SALI Sahin a 6° Po follots Maqeeod ck aye ) 7S saaaaiinsaaEEEEEEEEEEEEEmemmememmmemmmnmmemema eee! Fics. 54-59. Female abdominal sterna 8 and 9 (54-56) and vagina (57-59) — 54, 57, Scopura montana sp. nov. from Mit6-san; 55, 58, S. longa from Wainai; 56, 59, Scopura bihamulata sp. nov. Scale, 1mm. Scopura quattuorhamulata Uchida, sp. nov. (Japanese name: Yotsukagi-towada-kawagera) (Figsiy/5 Br Oe 2329 35)04 1 ou 53, 60, 61, 65 and 69) Material Types: Holotype ¢ with exuvia (NSMT), small stream into Niikappu-ko, 450 m, 1.5km WNW of Niikappu-dam, Niikappu-cho, Hokkaido, nymph collected 19.ix emerged 19-20. 1x.1984, S. Uchida and R. Kuranishi; Paratypes, OB MOS Cele, US Zee. ess JeSS, SU), same data. Additional specimens: 48 nymphs (21 nymphs, LFS; 27 nymphs, SU) from Otoinep- pu-mura and Niikappu-cho, Hokkaido. Male Body 18-25 mm long. Anterior projec- tions on lateral margins of meso- and metanota (thick arrows in Fig. 13) longer than those of montana but shorter than those of longa and bihamulata, in relation to the posterior ones (fine arrows). Abdominal tergum 9 (Fig. 23) with a pair of spinule patches posteriorly; the spinules uni- formly short excepting some long ones at the anterior end of the patch. Epiproct (Figs. 50 and 53) with four small hooks; a mesal pair before the opening of the internal sac, another lateral pair on its lateral sides. The mesal hooks curve anteriorly whereas the lateral ones curve antero-laterally; all tips of the hooks pointed. Projection at the base of cercus (Figs. 29, 35 and 41) directed dorsally, mesally and posteriorly, with a rounded tip which bears dense spinules. Lateral sclerite on penis (Fig. 7) strongly sclerotized, slender but broad in apical part, moderately folded; fine folds on its apical part directed basally to dorsal side. Apical expansion of penis with spinules on all sides around the gonopore. Female Body 22-30 mm long. Lateral margin of pronotum (Fig. 19) with a hump on posterior Revision of Scopura Stoneflies 707 sie ye : é . RY erste | Neue WN Qeeess ee Mle? Opel sae on Ch Sa ody fi) [ea Fe aR me d 3 Nc eee Fics. 60-61. Female abdominal sterna 8 and 9 (60) and vagina (61) of S. quattuorhamulata sp. nov. Scale, 1mm. end, which is oval with the longitudinal axis. Projections on lateral margins of meso- and metanota (Fig. 19) similar to those of montana, but anterior projections (thick arrows) slightly longer in relation to posterior ones (fine arrows). Hind margin of abdominal sternum 8 (Fig. 60) more deeply concave than in Jonga. The mem- brane between vaginal opening and sternum 9 with many hairs; the mesal hairs shorter than the lateral ones. Vagina (Fing. 61) nearly as large as in /onga, 1.2 mm wide at its opening, with a dorsal sclerite which tapers anteriorly. Male nymph Epiproct (Fig. 65) without knob nor swelling, smoothly rounded and sclerotized dorso-laterally. Scopura laminata Uchida, sp. nov. (Figs. 66, 67 and 69) Material Types: Holotype, a male nymph (NSMT), small stream beside the temple Pukdae- sa, ca. 1200m, Odae-san, Kangwon-do, Korea, 10.vi.1983, S. Uchida; Paratypes, 9 male nymphs (1 nymph, TUA; 4 nymphs, LFS; 4 nymphs, SU), same data. Additional specimens: 74 nymphs (14 nymphs, LFS; 60 nymphs, SU) from Chiri-san, Kyongsangnam-do and the type locality, Korea. Adult Unknown. Male nymph Epiproct (Figs. 66 and 67) with a pair of lateral membranous swellings which are produced posteriorly and with a dorso-mesal scle- rite which is produced dorsally with a transversely elongated small concavity at its center. The swellings of the specimens from Chiri-san (Fig. 66) are smaller than those from the type locality (Fig. 67) KEYS TO THE SPECIES OF SCOPURA The adult of /Jaminata is not known. Male 1. Epiproct with a mesal hook (Figs. 43, 44 MIT AAS) aay tecise cierto tape ec longa — Epiproct with one or two pairs of hooks (Bigs: 74) (49 xandh50)is..10n Mone. Menenawee y) 2. Epiproct with two pairs of hooks (Fig. 50) Baar ae is me cactcehae mara ete s quattuorhamulata — Epiproct with one pair of hooks (Figs. 42 ra Meech) eso omen SED ee te ee ee ae 3 3. Hooks of epiproct cylindrical (Fig. 42) SER UR ee Urs ae ma emule AV Ato a montana — Hooks of epiproct fan-shaped (Fig. 49) Bed le lal ene ae Nao a bihamulata Female 1. Lateral margin of pronotum with a hump Fics. 62-67. Epiprocts of male nymphs, posterior views — 62, Scopura montana sp. nov.; 63, S. longa; 64, S. bihamulata sp. nov.; 65, S. quattuorhamulata sp. nov.; 66-67, S. laminata sp. nov. from Chiri-san (66) and Odae-san (67). Scale, 0.5mm. 708 S. UCHIDA AND H. MARUYAMA Fic. 68. Scopura montana Maruyama, sp. nov., fe- male, habitus. Scale, 5mm. on anterior or median part (Figs. 14-17) ....2 Lateral margin of pronotum with a hump on posterior part (Figs. 18 and 19)............ 3 Lateral margin of pronotum with a longi- tudinally elongate hump on anterior part (Figs. 14:andila)" 3 3. eee montana Lateral margin of pronotum with a trans- versely elongate hump on median part (Figs: 16 and: 17))....2e:.ce 1.69 longa Lateral margin of pronotum with a longi- tudinally elongate hump on posterior part (Figs 18) es ce ee ee bihamulata Lateral margin of pronotum with an oval hump on posterior end (Fig. 19) CEE Sete Aces Metter: Mt raanele3 quattuorhamulata Male nymph Epiproct without knob nor swelling (Fig. OD) hkies Laat a ee eee quattuorhamulata Epiproct with knob(s) or swellings (Figs. 62-64, 66 and 67)).2..c95et-r oe eee 2 Epiproct with a mesal knob (Fig. 63)... longa Epiproct with a pair of knobs or swellings (Figs: 62, 64,/66) and'67)): 2-5. nee eee 3 Epiproct with a pair of small knobs dorsally; (Fie. 62) en sceeee eee montana Epiproct with a pair of large swellings dorso-laterally or laterally (Figs. 64, 66 wguattuorhamulata Bbihamulata Alonga @montana @laminata Fic. 69. Distribution of Scopura species. Solid lines represent the approximate distribution of the genus (from [8] and our material). Revision of Scopura Stoneflies 4. Swellings of epiproct sclerotized, pro- duced dorsally, without produced sclerite between the swellings (Fig. 64).... bihamulata — Swellings of epiproct membranous, pro- duced posteriorly, with a mesal sclerite which is produced dorsally between the swellings (Figs. 66 and 67)............. laminata ACKNOWLEDGMENT We are very grateful to Professors R. Ishikawa, T. Yamasaki (Tokyo Metropolitan University), H. Sawada and Y. Watanabe (Tokyo University of Agriculture) and Dr. S. Asahina (Tokyo) who gave us helpful advice and encouragement throughout this study. Professor T. Kawai (Nara Women’s University) kindly provided valuable literature and helpful information on this study. We are also grateful to Priv.-Doz. Dr. P. Zwick (Limnol- ogische FluBstation des Max-Planck-Instituts fur Limnol- ogie) for his critical reading of the manuscript and to many people given in the text for their gift of specimens used in this study. REFERENCES 1 Uéno, M. (1929) Studies on the stoneflies of Japan. Mem. Coll. Sci. Kyoto Imp. Univ., Ser. B, 4: 97-155, pl. 24. 2 Uéno, M. (1935) Plecoptera. In, “Aquatic Animals of the Azusa-gawa River System and Kamikéchi”. Iwanami, Tokyo, pp. 30-51. (In Japanese.) 3 Illlies, J. (1962) Das abdominale Zentralnervensys- tem der Insekten und seine Bedeutung fur Phy- logenie und Systematik der Plecopteren. Ber. 9. Wandervers. Dtsch. Entomol., Berlin, 45: 139-152. 4 Zwick, P. (1973) Plecoptera, phylogenetisches Sys- tem und Katalog. Das Tierreich, 94: xxxii+ 465 pp. 5 Uéno,M. (1931) Two apteral stoneflies, Capnia nivalis and Scopura longa. Kontyti, Tokyo, 5: 10 11 12 13 14 iS 16 My] 18 709 38-46, pl. 2. (In Japanese.) Uéno, M. (1938) Recorded localities of Scopura longa Uéno (Plecoptera, Scopuridae). Mushi, Fukuoka, 11: 201-203. (In Japanese.) Kawai, T. (1967) Plecoptera. Fauna Japonica. Biogeographical Society of Japan, Tokyo, 211 pp. Komatsu, T. (1970) Localities of Scopura longa Uéno (Plecoptera) in Japan and Korea. New Insect, 14: 14-28. (In Japanese.) Kawai, T. (1974) The second species of the genus Scopura (Plecoptera, Scopuridae). Bull. Natl. Sci. Mus., Tokyo, 17: 275-281. Komatsu, T. (1970) Bibliography of Scopura longa Uéno (Plecoptera). New Insect, 14: 29-36. (In Japanese. ) Kohno, M. (1951) Notes on Scopura longa Uéno of Aizu, Fukushima-pref. (1), (2). Collect. & Breed., Tokyo, 13: 49-54, 128-130. (In Japanese.) Uéno, M. (1938) Scopuridae, and aberrant family of the order Plecoptera. Ins. Matsum., Sapporo, 12: 154-159. Kobayashi, M. (1981) New locality of Scopura longa Uéno. Nat. Hist. Rept. Kanagawa, Yokohama, 2: 45-46. (In Japanese.) Uchida, S. (1981) Benthic animals of mountain streams in the Oi-gawa Genryubu wilderness area. In “Conservation Reports of the Oi-gawa Genryubu Wilderness Area in the Southern Japanese Alps, Central Japan”. Nature Conservation Society of Japan, Tokyo, pp. 295-319. (In Japanese.) Uchida, S. (1984) Distribution of the large stoneflies in Tanzawa mountains. Nat. Hist. Rept. Kanagawa, Yokohama, 5: 17-25. (In Japanese.) Zwick, P. (1980) Plecoptera (Steinfliegen). Hand- buch der Zoologie, Berlin, 4 (2) 2/7: 115 pp. Kohno, M. (1937) Notes on Perlodes yarizawana Uéno and Scopura longa Uéno. Mushi, Fukuoka, 9: 116-119. Komatsu, T. (1956) On the imago, egg and first instar of Scopura longa Uéno. New Entomol., Ueda, 5: 13-21, 1 pl. (In Japanese.) (aconnqel ab: r a aha ( : Livre ie waved hak ersiqosalt jo zseliege wh nk) Be i ho om RISTO SMS 5 en ign ie Pe, ile nuqul to Walope tnaigrngoayoits RPA +. al ry sar (08 bs ie Y .peleertiA ek MeO “hoo Hg Loe ta thoreast') dat 7 mee: BP Refhonsquh Gi) ACBL pl vO. SH IS ecto eral) agar A VVet) YT isweot ox, Ri eet ece gas fT) | Std, ! outer ery! i Peas? ! tied t't (eissaqanelt) onal } fC ayonnget OND yd Rar win re {128 0) Jando OTe oe ck mash torigierniatecnier i ati ero | al mul NE! 48% MBSE PES EN OO) iC) rl (773 sp thaws whe eee ive THEY ti LPaavi sonal Le oe ty rte usin anh RISIQO" ats Pb) sit’ ee in a a rt satay “210 wiledieieeh U8et) M jideayaded TIMOR vf wrt iio edt 184 coal a ( saonedal, al) at ed phone 1 slung Meee, (BCL). @ .ebiddst ww econ ehlive woiy sti? awag-iO SHt fir KR ATIS ibapiaaty: ey yj) west bey ee nays ‘WOME TRIG” te Qik cehiagay nyse ant im aa pantie | mer HOLeTIOTG. sheidavdd ‘ rigenk Thiel ie iin b.oantanidy eee alts ERE ape OME T eMeRnet By sf) bet FBG Tek oir heniin fiche, hae Ee) ot bitigls rE | i eee yt t4 wht vi HiT BVPRA EL ee i, Sean aay Fe iy ec { » nh ets rd ee ar aualnrsiel ¢ ity is send 1h tw tty ee F CEE RRA rm Pe Kime eae, eat: mi agen h (VERT A i pre ® xa), TAA OS 2- ee IRQS sf; eee ead RAL Tivo Y ‘g Ready? Sst) che Rare] ) ie diane a bovovec ahead © spam ‘ erate Le aig oo ANA we | Sngient AL) flak IRmEY 18 db ~ f By 4 Me 4 %. a ahi . pon LET aa: ome: arene : 2 siitannl. babe ay (BERT), Moasu a a f. er e a Fh! nd dei os a. ED es iets DH Luteral G nate (f ek 7 6 oa Gee rail efi barging ial tina wid Maina ts tan Vth : brs asa bier fire nae bbe St i iisanatth Divs’ cit \' pea seis; stad @ . a ne Ot. bi uh oe desir: ib } ‘sy ‘oF bid, He re. ar: NN ie oh + pl iis ay ; ha se eur Biteve {OVI | n ‘Seon t _ dari Ww ciepay Y enn ieee i ait TH staat “9 eB anki ZOOLOGICAL SCIENCE 4: 711-720 (1987) © 1987 Zoological Society of Japan Taxonomic Notes on Coenomyiidae (Insecta: Diptera)’ AKIRA NAGATOMI Entomological Laboratory, Faculty of Agriculture, Kagoshima University, Kagoshima 890, Japan ABSTRACT—A list of Coenomyiidae of the world is prepared and some notes are given to certain species. A brief review is presented to the recent literature on taxonomy, mouthparts, genitalia, biology, immature stages, and distribution in N. America. RECENT LITERATURE ON TAXONOMY Nagatomi and Saigusa [1] revised the Coenomyi- idae of Japan. Later, Nagatomi [2-4] defined the extent and limit of Coenomyiidae and in these papers, the genera Glutops and Pseudoerinna (=Bequaertomyia) are excluded and the genus Dialysis is newly combined with the Coenomyi- idae. James (in McAlpine et al. [5]) put the genera Arthropeas, Coenomyia, and Dialysis in the Coenomyiinae of the Xylophagidae. Webb [6-8] revised the Nearctic Dialysis, Coenomyia, and Arthropeas, respectively. Webb and Lisowski [9] found the larva and pupa of Dialysis fasciventris from N. America, and rein- forced the assignment of Dialysis into the Coenomyiidae. Webb [10] erected a new genus Napemyia from N. America, a close relative of Dialysis. Nagatomi [11] revised the genus Odontosabula, adding two new species from Japan, and Nagatomi and Naga- tomi [12] revised the genus Arthropeas. Oldroyd [13] revised the genus Coenomyia and recognized three component species, that is, basa- lis (=comans) (Japan), bituberculata (Himalayas), and ferruginea (Europe and N. America). Wein- berg and Bachli [14] doubted the specific validity of Coenomyia basalis and C. bituberculata and considered that “this genus includes a single species”. However, basalis and bituberculata seem to be valid species as shown in this paper. Accepted March 25, 1987 Received January 21, 1987 " Fifth supplement to Nagatomi [17] on Male Genitalia of the Lower Brachycera (Diptera). RELATED FAMILIES AND THEIR AGES The Coenomyiidae belong to the Xylophagidae s. lat. which include the Rachiceridae, Xylophagi- dae, Exeretoneuridae, Heterostomidae, and pos- sibly Pantophthalmidae. The Xylophagidae s. lat. are comprised in the Stratiomyioidea, together with the Stratiomyidae and Xylomyidae (=Solvidae). The sister group of the Stra- tiomyioidea is the Tabanoidea consisting of the Pelecorhynchidae, Rhagionidae, Athericidae, and Tabanidae. The scheme above is chiefly derived from Hennig [15], Nagatomi [16], and McAlpine et al. [5]. Within the Xylophagidae s. lat., the Coenomyi- idae are most closely related to the Heterostomi- dae and Exeretoneuridae, each of which include only one genus. Heterostomus was removed from the Rhagionidae (Nagatomi [16-18]), and Exere- toneura from the Nemestrinidae (Nagatomi [16, 17}). Mackerras [19] wrote, “the distribution of the genus (=Exeretoneura) in Tasmania and on the mountains of Eastern Australia suggests that its nearest relatives will probably be found in South America”. The nearest relative in question will be Heterostomus known from Chile. For geographi- cal distribution of the Xylophagidae s. lat., see Nagatomi [20]. Mackerras [21] discussed the composition and distribution of the fauna of Australia and put Exeretoneura into the archaic element, which is “a small element, consisting of primitive animals that have evidently survived with little change since Palaeozoic or early Mesozoic times”. 712 A. NAGATOMI If Mackerras’ assumption is correct as to the age of Exeretoneura, almost the same will apply to Heterostomus. The genera of Coenomyiidae are obviously more advanced than Exeretoneura and Heterosto- mus, but their origins seem to be old too, judging from morphological evidence, although Dialysis is apparently younger than other genera of Coenomyiidae. FAMILY CHARACTERISTICS The family characteristics of Coenomyiidae were given by Nagatomi [2], to which some notes are added. Palpus is 1 or 2 segmented in Arthropeas species according to individual. It is always 1-segmented in other genera. Midventral part of fused basistyles (=area corresponding to sternum 9 or hypandrium) has a large desclerotized or membranous area in Dialysis iwatai (after Nagatomi [17]) and 8 Nearctic species of Dialysis (after Webb [6]), as in other genera of Coenomyiidae. However, the midventral mem- branous area (=“ventral opening”) is absent in Dialysis kesseli and Napemyia illinoensis (after Webb [6, 10]). MOUTHPARTS AND GENITALIA Nagatomi and Soroida [22] discussed the struc- ture of the mouthparts of the orthorrhaphous Brachycera and described and illustrated those of five genera (and 5 species) of Coenomyiidae. They [22] wrote, “the mouthparts of Therevidae (based on 4 genera and 4 species) are similar to those of Coenomyiidae and may be difficult to distinguish from the latter, although the systematic positions of these two families are evidently very distant”. After the Therevidae, the mouthparts of Coenomyiidae may be similar to those of Rha- gionidae (some genera), Vermileonidae (Ver- mileo), Nemestrinidae (Hirmoneura), and Hilari- morphidae (Hilarimorpha), of which the last two (or three) families belong to the Asilomorpha. It may be said that the structure of the mouth- parts in the families or genera above are general- ized or primitive among the orthorrhaphous Brachycera, apart from the absence of mandibles. The described and illustrated genitalia of the genera of Coenomyiidae are seen in the literature given below. Male genitalia: Anacanthaspis, Nagatomi and Saigusa [1]; Arthropeas, Webb [8], and Nagatomi and Nagatomi [12]; Coenomyia, Nagatomi and Saigusa [1], Webb [7], Weinberg and Bachli [14], and Nagatomi [17]; Dialysis, Hardy [23], Webb [6], and Nagatomi [17]; Napemyia, Webb [10]; Odontosabula, Nagatomi and Saigusa [1], and Nagatomi [17]. Some errors must be pointed out to Nagatomi [17] as follows. P. 114, line 9 under Genus Dialysis, read “mid-anterior”, for “mid-posterior.” P. 115, line 10 (in Odontosabula gloriosa), read “dorso-posterior” for “dorso-anterior.” In Figure 26 by Nagatomi [17] as to Coenomyia basalis, there is “a large mid-anterior membranous or desclerotized part which is not shorter than posterior sclerotized part.” This part was inter- preted as dorsal plate (=dp), but seems to be aedeagal dorso-anterior plate (=adp). The male genitalia of Anacanthaspis bifasciata Japonica are described and illustrated in the forthcoming chapter. Female genitalia: Arthropeas, Webb [8]; Coenomyia, Nagatomi and Iwata [24], and Webb [7]; Dialysis, Nagatomi and Iwata [24], and Webb [6]; Odontosabula, Nagatomi and Iwata [24]. In the genitalia of Coenomyiidae, no definite distinguishing characters are found among genera in both sexes. The male genitalia in each family of the Xylophagidae s. lat. are very distinctive, but are similar between Coenomyiidae and Rhagionidae (which does not belong to the Xylophagidae s. lat.). However, Nagatomi [17] wrote, “the genera of Coenomyiidae, which are almost identical with one another in male genitalia, may easily be separated from each genus of Rhagionidae, which may be characteristic in the details of male genitalia.” On the other hand, the female genitalia are similar among the Xylophagidae s. lat. (except the Pantophthalmidae), although those of each family may be separated (see the keys in Nagatomi and Taxonomic Notes on Coenomyiidae W13 Iwata [24, 25]). Webb [6] shows in Dialysis that the endophallus (=anterior bar of aedeagus) and “endophallic hilt” (=correctly endophallic supporting sclerite) (=aedeagal dorso-anterior sclerite) vary in shape with species. BIOLOGY AND IMMATURE STAGES Very little is known of the biology of Coenomyi- idae. It is introduced below. Arthropeas sibiricum: “The larvae develop in the soil of spruce-fir forests.” (after Krivosheina [26]); Coenomyia fer- ruginea: “Adults feed on fluid matter or the nectar of flowers (Malloch, 1917). ... Larvae have been collected in a field some distance from timber or in decaying wood (Malloch, 1917) and from silty clay loam (pH 4.6) in Connecticut in a red oak and sugar maple forest (C. T. Maier, personal com- munication). The larvae are predaceous and feed upon white grubs and other insect larvae (Malloch, 1917).” (after Webb [7]); Dialysis fasciventris: “The larvae were collected from the top 5 cm of soft organic soil in a beech and sugar maple forest in Vermilion County in eastern Illinois. Six larvae were collected from 42 soil samples (each sample 890 sq. cm), giving an average abundance of 1.57 larvae per square meter.” (after Webb and Lisows- ki [9]). I observed a swarm of Dialysis iwatai, flying low in the air, of which some male individuals were captured by net. It was early in the morning (something past seven), on July 29, 1977 at Ichinose, Mt. Haku, Ishikawa Prefecture, Honshu. The literature on the larval and pupal stages of Coenomyiidae is as follows: Arthropeas sibiricum (larva): Krivosheina [26, 27]; Coenomyia ferru- ginea: Beling (1880) (larva and pupa), Brauer (1883) (larva), Hart (1898) (pupa), Malloch (1917) (larva and pupa), Greene (1926) (pupa), Peterson (1951) (larva), James (in McAlpine et al., 1981) (larva), and Webb (1983a) (larva and pupa) (after Hennig [28], and Webb [7]); Dialysis fasciventris (larva and pupa): Webb and Lisowski [9]. A comparison of the morphological characters of the pupal stages of Coenomyia and Dialysis and that of the larval stages of the three genera above are summarized in Tables1-—2 by Webb and Lisowski [9], who thus reinforced the grouping of these three genera into the family Coenomyiidae. Judging from Table 2 by Webb and Lisowski, it is almost certain that Arthropeas occupies an in- termediate position between Coenomyia and Di- alysis and it is not deniable that Arthropeas is more similar phylogenetically to Coenomyia than to Dialysis. The biology and immature stages of Heterosto- mus and Exeretoneura, close relatives of Coenomyiidae, are not known. DISTRIBUTION IN NORTH AMERICA _ The North American Coenomyiidae may be broadly divided in geographical distribution into eastern and western species, which are shown below, according to Webb [6-8, 10] who prepared a very useful distributional map in each species. Eastern species: Arthropeas americanum; Coenomyia ferrugines; Dialysis species such as elongata, fasciventris and rufithorax; Napemyia illinoensis. Western species: Arthropeas magnum, Dialysis species such as aldrichi, dispar, kesseli, lauta, mentata and reparta. Coenomyia ferruginea, an eastern species, is also distributed in Europe. Arthropeas americanum, an eastern species, is closely related to sibiricum (which is distributed in Siberia, N. China, N. Korea, and Tibet) and sachalinense (Sakhalin). These three species are common in several struc- tural characters and different from magnum, a western species. PHYLOGENETIC RELATIONSHIPS AMONG GENERA The matter titled above must be analized, but there are few definite plesiomorphic or apomor- phic characters which are determined as such in the genera of Coenomyiidae. A number of combinations of the relationships are able to imagine, but only one thing is right and it is difficult to determine. However, one possibility is presented in Figure 1 and the key to genera by Nagatomi [2] is here modified according to more 714 A. NAGATOMI 4 (apomorphic)................. Anacanthaspis - Scutellum trapezoidal or semicircular and with a pair of spine-like processes (which become small and inconspic- uous in C. bituberculata Enderlein) (plesiomorphic); vein Mg, arising from Coenomyia Anacanthaspis Arthropeas Odontosabula discal Cel much beyond m-cu crossvein and distance between bases of M, and Dialysis M; roughly as long as that between bases of M3 and M, (apomorphic); eye distinctly pilose (apomorphic); abdo- men widest at segment 2 or 3 (ple- STOMONPHIC) x5 Bae eee Coenomyia Antennal segment 3 annulated, por- rect, and subulate (as in Anacanthas- pis, Arthropeas, and Coenomyia) (ple- siomorphic); 4th posterior cell widely open and anal cell narrowly open or closed at wing margin (as in Anacan- thaspis, Arthropeas, and Coenomyia) (plesiomorphic); scutellum with a pair of spine-like processes (which are longer than in Coenomyia); female front broader toward antenna (as Fic. 1. Possible phylogenetic relationships among the genera of Coenomyiidae. possible synapomorphic characters. 4(1). KEY TO GENERA OF COENOMYIIDAE (modified from Nagatomi [2]) 1. Hind femur not longer than mesono- tum and scutellum combined (ple- siomorphic); body somewhat slender (in Arthropeas americanum) to robust ....2 ~ Hind femur distinctly longer than mes- onotum and scutellum combined (apo- morphic); Dodyaslendeten-- +a. es-aeteeeeeRee 4 2(1). Antennal segment | distinctly longer in Anacanthaspis, Arthropeas, and than wide and segments 1+2 over 1/2 Coenomyia) (plesiomorphic) ass long, as flagellum. a(apomorphic)sige > \Gikemmseseeee eae eeee teen eee Odontosabula head distinctly narrower than thorax (apomonphic) aes ss... eee Ae eee Antennal segment 1 about as long as wide and segment | +2 less than 1/2 as long as flagellum (as in Dialysis and Odontosabula) (plesiomorphic); head nearly as wide as thorax (as in Dialysis Antennal segment 3 conical and with a long arista (apomorphic); 4th posterior cell (when M; is present) and anal cell closed before wing margin (sometimes the latter closed at wing margin) (apo- morphic); scutellum unarmed (as in Arthropeas); female front nearly par- and Odontosabula) (plesiomorphic) allel-sided (apomorphic) ............. Dialysis Buster iterah eee aoe ae eee Arthropeas 3(2). Scutellum unarmed, much wider than long, and its hind margin distinctly LST OF CEO Oa concave at middle and consisting of two, more or less rounded portions (apomorphic); vein My, arising from discal cell at or near m-cu crossvein (as in Arthropeas, Dialysis, and Odontosa- bula) or sometimes arising from m-cu crossvein (plesiomorphic); eye bare or practically so (as in Arthropeas, Dialy- sis, and Odontosabula) (plesiomor- phic); abdomen widest at segment 3 or A list of Coenomyiidae is presented below and some notes are given to certain species. The synonyms given by Kertész [29] are omitted. Genus Anacanthaspis R6der Anacanthaspis Réder, 1889, Wien Ent. Ztg., 8: 8. Type species: Anacanthaspis bifasciata Roder, 1889 from Siberia (Amur). For diagnosis of Anacanthaspis, see Nagatomi and Saigusa [1] and Nagatomi [3]. Taxonomic Notes on Coenomyiidae 715 Anacanthaspis contains only one species, con- sisting of A. bifasciata bifasciata RO6der, 1889 from E. Siberia and Manchuria and A. bifasciata japoni- ca Shiraki, 1932 from Japan (Honshu). Shiraki [30] distinguished bifasciata japonica from bifasciata bifasciata “by the wing-patterns and by the proportionally long second joint of the antennae.” Anacanthaspis bifasciata RO6der Anacanthaspis bifasciata Roder, 1889, Wien Ent. Ztg., 8: 8. Type locality: Siberia (Amur). There are 1f, 12 from Manchuria (=NE. China) (=bifasciata bifasciata) which differ from the description of bifasciata japonica (based on TL &, 1% from Honshu (Yamanashi and Nagano Pref.)) by Nagatomi and Saigusa [1]. Between the continental and the Japanese forms, the difference in wing pattern may be of subspecific importance. It remains undetermined whether or not the differences in head structure and the relative lengths of leg segments are significant, simply because the material examined is small. Description of bifasciata bifasciata, based on 1%, 12 from Manchuria is given below. Male. Head: pile on cheek partly black; relative lengths of antennal segments 1, 2, and segment 3+ flagellum 100: 86: 329 (measured along mid- inner surface) and segment 1, 1.1 times as long as wide (in bifasciata japonica 1.3-1.5 times). Thorax: pile on mesonotum and scutellum partly black. Legs: hairs on coxa and femur chiefly black; relative lengths of segments (excluding coxa and trochanter) of fore leg 223 : 261 : 100: 39:29:23: 48, of mid leg 229:245 :81:32:26:23:48, of hind leg 335 : 332: 103:39:29:23:45 and in hind leg, rela- tive thickness of femur, tibia, and tarsal segments f3e 2 oo 23: 19: 16: Wing: ist and 2nd basal cells without any darkened basal spot, and anal cell and axillary not darkened. Abdomen: pile in middle of terga 2-3 may be black. Genitalia: not examined. Length: body 12.3mm; wing 10.7 mm; fore basitarsus 1.2 mm. Female. Head: hairs on head wholly black; in 1 specimen measured, width of one eye on a mid line from a direct frontal view equals distance from antenna to median ocellus (in bifasciata japonica 1.2 times), and 0.8 times width of face at lowest portion from a direct frontal view (in bifasciata Japonica 1.1 times); width of front just above antenna 1.6 times that at median ocellus (in bifasciata japonica 1.8 times); space between antennae 0.7 times width of ocellar triangle (in bifasciata japonica 0.5 times); antenna 1.4 times distance from antenna to median ocellus (in bifasciata japonica 1.7 times); relative lengths of antennal segments 1, 2, and segment 3+ flagellum (measured along midinner surface) 100:100:325 (in bifasciata japonica 100:71:247); antennal segment 1, 1.1 times as long as wide (in bifasciata Japonica 1.4 times). Thorax: hairs on mesonotum and scutellum chiefly black; pile on lower parts of metapleura black (as well as that on propleura). Legs: relative lengths of segments of fore leg 247 :274:100:32:29:24:47, of mid leg 262:265: 82 :29:29:21:44, of hind leg 371: 362: 115 :38:32: ?:? and in hind leg, relative thickness of femur, tibia, and tarsal segments 1-3, 56:35:26:21:21. Wing: Ist and 2nd basal cells without any darkened basal part, and anal cell and axillary not darkened. Length: body (with ovipositor) 15.2 mm; wing 12.2 mm; fore basitarsus 1.3 mm. Distribution. E. Siberia and Manchuria. Specimens examined: 1, 12, Ersentientze, Manchuria, 15. vi. 1941, V. N. Alin. Anacanthaspis bifasciata japonica Shiraki (Fig. 2) Anacanthaspis bifasciata var. japonica Shiraki, 1932, Trans. Nat. Hist. Soc. Formosa, 22: 489. Type locality: Honshu (Kamikochi or Ibuki). This subspecies was redescribed and illustrated by Nagatomi and Saigusa [1]. Some notes are given to the male genitalia, of which dorsal aspect is here illustrated (Fig. 2). Their ventral aspect, and the dorsal aspect of tergum 9, cerci, and sternum 10 were figured by Nagatomi and Saigusa [1]. Basistyle biuntly attenuate distally; dististyle (from dorsal or ventral 716 A. NAGATOMI AR Fic. 2. Anacanthaspis bifasciata japonica, g. Genitalia (excluding tergum 9, cerci, and sternum 10), dorsal view. AA, anterior bar of aedeagus; ADP, aedeagal dorsoante- rior plate; ADS, aedeagal dorsoanterior sclerite; B, basistyle; BDP, basistylar dorsoinner anterior process; D, dististyle; DP, dorsal plate. view) widest before apex; anterior bar of aedeagus almost parallel-sided; anterior margin of dorsal plate deeply concave horizontally and its midante- rior part transparent; dististyle with dorsal haris which are absent on inner apical part and outer basal part, and with ventral hairs which are absent on midinner part except inner margin; basistyle haired, except dorsoinner basal part and area corresponding to sternum 9; tergum 9 at posterior part, cerci, and sternum 10 at ventrtal surface with hairs. Distribution. Japan (Honshu). Genus Arthropeas Loew Arthropeas Loew, 1850, Stettin. Entomol. Zeitg., 9: 304. Type species: Arthropeas sibirica Loew. By monotypy. For diagnosis of Arthropeas, see Nagatomi [3] and Webb [8]. Leonard [31] and Webb [8] revised the North American species and Nagatomi and Nagatomi [12] reviewed the species from Far East and N. America. americanum Loew, 1861, Berl. Entomol. Zeits., 5, p. 316 — Northern Wisconsin; “Virginia to Maine and west to Minnesota” (after Webb [8]). magnum Johnson, 1913, Can. Ent., 15: 11 — Manitoba; Manitoba to Oregon (after Webb [8]). sachalinense Matsumura, 1916, Thous. Ins. Jpn., Addit., 2: 361 — Sakhalin. sibiricum Loew, 1850, Stettin. Entomol. Zeitg., 9: 305 — Siberia; Siberia, China, and Korea. var. fenestrale Malloch, 1932, Stylops, 1: 119 — Tibet (Tatsienlu, 8,900 feet). var. semifuscum Malloch, 1932, Stylops, 1: 119 — Tibet (Ja-Ze Pass, 16,000-17,150 feet). Genus Coenomyia Latreille Coenomyia Latreille, 1796, Precis d. caract. génér. d. Ins. p. 159. Type species: Musea ferruginea Scopoli (subsequent monotypy by Latreille, 1802). For diagnosis of Coenomyia, see Oldroyd [13] and Nagatomi and Saigusa [1]. Coenomyia basalis Matsumura Coenomyia_ basalis Matsumura, 1915, Kontyu- bunruigaku, Part 2, p. 47. Type locality: Japan. Coenomyia_ apicalis Matsumura, 1915, Kontyu- bunruigaku, Part 2, p. 47. Type locality: Japan. Coenomyia comans Enderlein, 1927, SitzBer. Ges. naturf. Fr. 1927, p. 47. Type locality: Japan. Coenomyia japonica Séguy, 1955, Boll. Lab. Ent. agr. Portici, 14: 290. Type locality: Japan. Matsumura [32, 33] described this species twice as “Coenomyia basalis sp. n”. In 1915 he [32] gave the name basalis for ? and apicalis for § and in 1916 he [33] used the name basalis in the text and grandis by mistake in the explanations of appended figures. Taxonomic Notes on Coenomyiidae ply) This species was redescribed by Nagatomi and Saigusa [1]. Shiraki [30] synonymized basalis with ferru- ginea. Weinberg and Bachli [14] noticed consider- able colour variations of legs and abdomen within ferruginea and found no difference in the male genitalia between ferruginea and basalis. Howev- er, it seems that basalis differs specifically from ferruginea in the following respects: antenna and palpus in both sexes distinctly longer than in ferruginea; hairs on eye and occiput near cerebrale in both sexes and those on male mesonotum much longer than in ferruginea; eyes of male not contiguous but separated; pair of knobs at midpos- terior margin of mesonotum in both sexes some- what larger than in ferruginea. In basalis antenna (1) 1.2-1.4 times in # and (2) 1.8-1.9 times in ?., and palpus (3) 0.9-1.2 times in § and (4) 1.4-1.7 times in § distance from antenna to median ocellus respectively; width of $ front just above antenna (5) 0.6-0.7 times and that at median ocellus (6) 0.3-0.4 times half width of head from a direct frontal view respectively. Whereas in ferruginea, (1) 0.9 times, (2) 0.9 times, (3) 0.6 times, (4) 0.8 times, (5) 0.7 times, and (6) 0.4 times respectively. The width of $ front is not different significant- ly between basalis and ferruginea. We have seen 9S S, 6? $ of basalis from Japan and1 7, 1 of ferruginea from N. America. Distribution. Japan (Hokkaido, Honshu, Shiko- ku, and Kyushu). Coenomyia bituberculata Enderlein Coenomyia bituberculata Enderlein, 1921, Mitt. Zool. Mus. Berlin, 10: 213. Type locality: Sikkim. This species was redescribed in detail by Old- royd [13]. Pair of spine-like processes on scutellum become small, inconspicuous tubercles in bituberculata. “Wings (fig.5) show a distinct difference in shape from other two species” (after Oldroyd [13])”. Distribution. Sikkim, Tibet, and Nepal. Coenomyia ferruginea Scopoli Musca ferruginea Scopoli, 1763, Entomol. Carniol., p. 340. Type locality: Europe. For synonyms, see Leonard [31] and Oldroyd [13]. This species was redescribed in detail by Leonard [31], Oldroyd [13], and Webb [7]. Distribution. Europe and North America. Genus Dialysis Walker Dialysis Walker, 1850, Ins. Saund., Dipt. 1, p. 4. Type species: Dialysis dissimilis Walker, 1850 (from N. America). By monotypy. Triptotricha Loew, 1872, Berl. Ent. Zeits., 14: 59. Type species: Triptotricha lauta Loew, 1872 (from Califor- nia). By designation of James, 1965. For diagnosis of Dialysis, see Leonard [31], Nagatomi [3], and Webb [6]. The North American species were revised by Leonard [31] and Webb [6]. A revision of the Formosan and Japanese species is necessary. The list of the Nearctic species given below is quoted from James [34] and Webb [6]. A total of 12 known species are distributed as follows: N. America (9 species), Japan (2), and Formosa and S. China (1). aldrichi Williston, 1895, Kans. Univ. Quart., 3: 265 — Idaho; California to British Columbia east to Idaho. arakawae Matsumura, 1916, Thous. Ins. Jpn. Addit., 2: 351 — Japan (Honshu); Japan (Honshu and Shi- koku). cispacifica Bezzi, 1912, Ann. Mus. Nat. Hung., 10: 444 — Formosa; Formosa and S. China. dispar Bigot, 1879, Ann. Soc. Ent. France, ser. 5, 9: 197 — California; California to British Columbia. disparilis Bergroth, 1879, Wien. Ent. Zeitg., 8: 296 — British Columbia. elongata (Say), 1823, J. Acad. Nat. Sci. Phila., 3: 41 (Stygia) — Pennsylvania; Georgia to Quebec. dissimilis Walker, 1850, Insecta Saundersiana Dipt. 1, p. 4— North America. fasciventris (Loew), 1874, Berl. Ent. Zeits., 18: 380 (Triptotricha) — Pennsylvania; “North Carolina and Tennessee to New York west to eastern Kansas.” iwatai Nagatomi, 1953, Mushi, 25: 13— Japan (Honshu). kesseli Hardy, 1948, Wasmann Coll., 7: 129 — Cali- fornia. lauta (Loew), 1872, Berl. Ent. Zeits., 16: 59 (Tripto- tricha) — California. discolor (Loew), 1874, Berl. Ent. Zeits., 18: 379 (Triptotricha) — San Francisco. mentata Webb, 1978, J. Kansas Ent. Soc., 51: 424 — California; California to Washington. reparta Webb, 1978, J. Kansas Ent. Soc., 51: 428 — California. rufithorax (Say), 1823, J. Acad. Nat. Sci. Phila., 3: 36 (Leptis) — Pennsylvania; “northern Florida to 718 A. NAGATOMI Pennsylvania and New Jersey, west to southern Illinois.” Genus Napemyia Webb Napemyia Webb, 1983, Proc. Entomol. Soc. Wash., 85: 822. Type species: Napemyia illinoensis Webb, 1983 (from Illinois), by original designation. This genus contains only one species. Judging from the original description and illustrations, Napemyia is very similar to Dialysis but differs from the latter by having the side of face haired, posterior margin of sternum 10 (=“ventral plate of proctiger”) narrowly concave at middle, area corresponding to sternum 9 (=hypandrium) with- out a desclerotized area (=“ventral opening”), and wing without thyridium. Webb [6] wrote as to Dialysis kesseli, “basistyle (Fig. 4) without ventral opening”, which is present in other 8 Nearctic species of Dialysis. It is suspected whether or not the distinguishing characters above are of generic value. However, Napemyia is retained as a distinct genus at present. Napemyia illinoensis Webb Napemyia illinoensis Webb, 1983, Proc. Entomol. Soc. Wash., 85: 823. Type locality: Illinois (Vermilion County). The original description of this species was based on a single male specimen. The female is unknown. Webb [10] wrote, “I have made several trips each year for the last six years and have not collected additional specimens, nor have I found additional specimens in 67 museum, university, and private collections that I have examined”. Distribution. N. America (Illinois). Genus Odontosabula Matsumura Odontosabula Matsumura, 1905, Thous. Ins. Jpn., 2: 78. Type species: Odontosabula gloriosa Matsumura, 1905 from Japan (Kyushu). By monotypy. Stratioleptis Pleske, 1925, Encyc. ent. B. I. Dipt., 2: 182. Type species: Stratioleptis czerskii Pleske, 1925 from East Siberia. By monotypy. For diagnosis of Odontosabula, see Nagatomi and Saigusa [1] and Nagatomi [3]. Nagatomi [11] revised this genus and wrote, “this genus is distributed in East Siberia, Manchur- ia, Korea, and Japan (Honshu and Kyushu) and contains 5 species, that is, czerskii, decora sp. n., gloriosa (=pleskei), fulvipilosa sp. n. and licenti. But licenti is possibly a synonym of czerskii. It is not necessarily certain whether the distinction is specific or subspecific among them”. Odontosabula czerskii (Pleske) Stratioleptis czerskii Pleske, 1925, Bull. Soc. ent. France, 1925: 166 or Encyc. ent B. II. Dipt., 2: 182. Type locality: East Siberia (“Province Littorale”). It is necessary to examine the type specimen or the specimens from the type locality for exact identification of czerskii. Nagatomi [11] described and illustrated 1~, 4° $ from Korea (Quelpart I.) as czerskii. Some additional notes are given below, based on 4 female specimens whose collect- ing data were given in Nagatomi [11]. Width of ocellar triangle 0.7—1.0 times as wide as long and that of front almost fits the description of decora mentioned in this paper. Distribution. East Siberia, (=Manchuria), and Korea. NE. China Odontosabula decora Nagatomi Odontosabula decora Nagatomi, 1985, Kontyu, 53: 220. Type locality: Japan (Fukui Pref., Honshu). Some additional notes are given below, based on 8 female specimens whose collecting data were given in Nagatomi [11]. Width of one eye on a mid line from a direct frontal view 0.9-1.1 times width of front just abvove antenna; width of ocellar triangle 1.0—1.2 times its length; width of front at median ocellus 1.8-2.0 times width of ocellar triangle, 0.6-0.7 times width of front just above antenna, and 0.6-0.8 times distance from antenna to median ocellus. There is additional 1 ¢ , Norikura Kégen (1,800 m), Nagano Pref., 25. vii. 1979, K. Ohara. Distribution. Japan: Honshu (Fukui, Ishikawa, and Nagano Prefectures). Odontosabula fulvipilosa Nagatomi Odontosabula fulvipilosa Nagatomi, 1985, Kontyu, 53: 223. Type locality: Japan (Yamagata Pref., Honshu). The female structural characters of decora given in this paper almost apply to fulvipilosa. In 4? ? measured, width of one eye on a mid line from a Taxonomic Notes on Coenomyiidae 719 direct frontal view 1.1—1.3 times width of front just above antenna; width of ocellar triangle 0.9-1.1 times its length; width of front at median ocellus 1.8-2.1 times width of ocellar triangle. There is additional 1 2 (Yumoto, Nikko, 27. vii. 1984, T. Kumazawa) which is newly recorded from Tochigi Prefecture. In this specimen, anterior black border of tergum 2 is wider than its posterior yellowish brown border (as in decora) and sternum 3, except posterior narrow border, dark brown to black (as in decora). Distribution. Japan: Honshu (Yamagata, Tochigi, Nagano, and Gifu Prefectures). Odontosabula gloriosa Matsumura Odontosabula gloriosa Matsumura, 1905, Thous. Ins. Jpn., 2: 78. Type locality: Japan(Kyushu). Stratioleptis pleskei Séguy, 1926, Encyc. ent. B. II. Dipt., 3: 11. Type locality: Japan (Kyushu). The female structural characters of decora given in this paper almost apply to gloriosa (N=10). There is 1 2 (6. v. 1966, K. Kusigemati) which is newly recorded from Amami Oshima. Distribution. Japan: Kyushu (Fukuoka, Oita, Kumamoto, Miyazaki, and Kagoshima Prefec- tures) and Amami Oshima. Odontosabula licenti (Séguy) Stratioleptis licenti Séguy, 1952, Rev. fr. Ent., 19: 243. Type locality: Manchuria (“Kirin”). It is possible that licenti is a synonym of czerskii. Distribution. NE. China (=Manchuria). CONCLUDING REMARKS 1. A brief review is given to the recent literature on taxonomy, mouthparts, genitalia, biology, immature stages, and distribution in N. America. 2. Phylogenetic relationships among _ the genera are discussed, and one possibility is shown in Figure 1. 3. Anacanthaspis bifasciata Anacanthaspis bifasciata japonica is perhaps valid as subspecies on the basis of the wing marking. 4. The validity of the 2 Coenomyia species, i.e. basalis and bituberculata is not deniable at present. 5. The genus Napemyia may be a synonym of is redescribed. Dialysis, because the distinguishing characters are slight. However, it is hasty to treat so now. 6. It was observed that a swarm of Dialysis iwatai (g\ 3g) flew low in the air early in the morning. 7. A list of Coenomyiidae is prepared. ACKNOWLEDGMENTS I express my deepest respect for the late Dr. Mortimer D. Leonard and Dr. D. Elmo Hardy, whose works have influenced me very much. My sincere thanks are offered to Dr. PaulH. Arnaud, Jr. (California Academy of Sciences, San Francisco), Dr. D. W. Webb (Illinois Natural History Survery, Champaign), Mr. T. Kumaza- wa, and Mr. K. Ohara (Tokushima Prefectural Office) for their generous gift or loan of material. Professor T. Saigusa (College of General Education, Kyushu Uni- versity, Fukuoka) has helped me in many ways. His help is greatly appreciated. REFERENCES 1 Nagatomi, A. and Saigusa, T. (1970) The Coenomyiidae of Japan (Diptera). Mem. Fac. Agric. Kagoshima Univ., 7: 257-292. 2 Nagatomi, A. (1975) Definition of Coenomyiidae (Diptera). I. Diagnoses of the family. Proc. Japan Academy, 51: 452-456. 3. Nagatomi, A. (1975) Definition of Coenomyiidae (Diptera). II. Genera of the family. Proc. Japan Academy, 51: 457-461. 4 Nagatomi, A. (1975) Definition of Coenomyiidae (Diptera). III. Genera excluded from the family. Proc. Japan Academy, 51: 462-466. 5 McAlpine, J.F., Peterson, B. V., Shewell, G. E., Teskey, H. J., Vockeroth, J. R. and Wood, D. M. (1981) Manual of Nearctic Diptera. Res. Br., Agric. Can. Monogr., 27(1), 674 pp. 6 Webb, D. W. (1978) A revision of the Nearctic genus Dialysis (Diptera: Rhagionidae). J. Kans. Entomol. Soc., 51: 405-431. 7 Webb, D. W. (1983) The genus Coenomyia (Dip- tera: Coenomyiidae) in the Nearctic region and notes on generic placement. Proc. Entomol. Soc. Wash., 85: 653-664. i 8 Webb, D. W. (1983) A revision of the Nearctic species of Arthropeas (Diptera: Coenomyiidae). Proc. Entomol. Soc. Wash., 85: 737-747. 9 Webb, D. W. and Lisowski, E. A. (1983) The im- mature stages of Dialysis fasciventris (Loew) (Diptera: Coenomyiidae). Proc. Entomol. Soc. Wash., 85: 691-697. 10 Webb, D. W. (1983) A new genus and species of 11 12 13 14 1S) 16 7 18 19 20 Za 22 23 720 Nearctic Coenomyiid (Diptera: Coenomyiidae). Proc. Entomol. Soc. Wash., 85: 822-825. Nagatomi, A. (1985) The genus Odontosabula (Dip- tera, Coenomyiidae). Kontyt, Tokyo, 53: 216-228. Nagatomi, A. and Nagatomi, H. (1987) A revision of Arthropeas (Diptera, Coenomyiidae). Kontyd, Tokyo. (in press) Oldroyd, H. (1966) Notes on Coenomyia Latreille (Diptera: Coenomyiidae). Beitr. Ent., 16: 953-963. Weinberg, M. and Bachli, G. (1984) The review of some families of the infraorder Asilimorpha (Dip- tera) from the collections of the museum of Ziirich University. Travaux du Muséum d’Histoire naturelle Grigore Antipa, 25: 191-201. Hennig, W. (1973) Ordung Diptera (Zweifligler). Handb. Zool., 4(2), 2/31 (Lfg. 20), pp. 1-337. Berlin. Nagatomi, A. (1977) Classification of lower Brachycera (Diptera). J. nat. Hist., 11: 321-335. Nagatomi, A. (1984) Male genitalia of the lower Brachycera (Diptera). Beitr. Ent., 34: 99-157. Nagatomi, A. (1985) Redescription of Heterostomus curvipalpis (Diptera, Heterostomidae) and some notes on my paper of the male genitalia of the lower Brachycera (Diptera). Kontyi, Tokyo, 53: 699-710. Mackerras, I. M. (1925) The Nemestrinidae (Dip- tera) of the Australasian region. Proc. Linn. Soc. N. S. W., 50: 489-561. Nagatomi, A. (1982) Geographical distribution of the lower Brachycera (Diptera). Pac. Insects, 24: 139-150. Mackerras, I. M. (1970) Composition and distribu- tion of the fauna. In “The Insects of Australia”. Ed. by Division of Entomology, CSIRO, Melbourne Univ. Press, pp. 187-203. Nagatomi, A. and Soroida, K. (1985) The structure of the mouthparts of the orthorrhaphous Brachycera (Diptera) with special reference to blood sucking. Beitr. Ent., 35: 263-368. Hardy, D. E. (1948) New and little known Diptera 24 JUS) 26 27 28 78) 30 31 32 3) 34 A. NAGATOMI from the California Academy of Sciences Collection (Rhagionidae and Dorilaidae). Wasmann Coll., 7: 129-137. Nagatomi, A. and Iwata, K. (1976) Female termina- lia of lower Brachycera (Diptera). I. Beitr. Ent., 26: 5-47. Nagatomi, A. and Iwata, K. (1978) Female termina- lia of lower Brachycera (Diptera). II. Beitr. Ent., 28: 263-293. Krivosheina, N. P. (1967) Comparative characteris- tics of the larva of Arthropeas sibilica Loew (Dip- tera, Xylophagidae). Zool. Zhurnal, 46: 954-956. (in Russian) Krivosheina, N. P. (1971) The family Glutopidae, Fam. n. and its position in the system of Diptera Brachycera Orthorrhapha. Ent. Obozr., 50: 681-694. (in Russian) Hennig, W. (1952) Die Larvenformen der Dipteren, Vol. 3. 628 pp. Akademie-Verlag, Berlin. Kertész, K. (1908) Catalogus dipterorum hucusque descriptorum. Vol. 3, 367 pp. Hungarian National Museum, Leipzig, Budapest. Shiraki, T. (1932) Some Diptera in the Japanese Empire, with descriptions of new species GC): Coenomyiidae. Trans. Nat. Hist. Soc. Formosa, 22: 487-492. Leonard, M. D. (1930) A revision of the Dipterous family Rhagionidae (Leptidae) in the United States and Canada. Mem. Am. Ent. Soc., 7: 1-181. Matsumura, S. (1915) (=Taxonomic Entomology). Tokyo. Matsumura, S. (1916) Thousand Insects of Japan, Additamenta 2. Keiseisha, Tokyo. James, M. T. (1965) Family Rhagionidae. In “A Catalog of the Diptera of America North of Mex- ico”. Ed. by A. Stone, C. W. Sabrosky, W. W. Wirth, R. H. Foote, and J. R. Coulson, U. S. Dept. of Agriculture, Washington, D. C. pp. 342-348. Kontyu-bunruigaku Part 2. Keiseisha, ZOOLOGICAL SCIENCE 4: 721-729 (1987) Further Studies on Cestodes of Japanese Bats, with Descriptions of Three New Species of the Genus Vampirolepis (Cestoda: Hymenolepididae)! ISAMU SAWADA Biological Laboratory, Nara Sangyo University, Sango, Nara 636, Japan ABSTRACT —Three new and three known species of hymenolepidid cestodes are recorded from bats collected at various places in Japan in 1986. Vampirolepis rikuchuensis sp. n. from Myotis hosonoi from Ichinohe-ché, Iwate Prefecture, closely resembles V. ogaensis Sawada, 1974, but differs from it in smaller rostellar hooks (0.018 vs. 0.021 mm), thicker outermost chorion (tough vs. thin), surface structures of eggs (smooth vs. rough) and position of genital pores (located a little anterior to middle vs. middle). V. kaguyae sp. n. from M. frater kaguyae from the same locality, closely resembles V. yoshiyukiae Sawada, 1980, but differs from it in larger suckers, larger rostellum, larger rostellar sac, and longer rostellar hooks (0.032 vs. 0.0245mm). V. yakushimaensis sp. n. from Murina aurata ussuriensis from Yakushima, Kagoshima Prefecture closely resembles V. iriomotensis Sawada, 1983, but differs from it in shape of ovary (pentalobate or hexalobate vs. trilobate), morphological feature of egg inner membrane (provided with polar filaments vs. no polar filaments) and longer embryonic © 1987 Zoological Society of Japan hooks (0.018 vs. 0.014mm). INTRODUCTION As a continuation of my serial studies on the cestode parasites of the Japanese bats, the present paper reports three new and three known hyme- nolepidid cestodes from bats collected at various localities, except Hokkaido and Shikoku, in Japan in 1986. MATERIALS AND METHODS The bats were captured alive and autopsied immediately at the collecting sites (Fig. 1). Their alimentary canals were cut open to extract en- doparasites as soon as possible and fixed in Carnoy’s fluid and bought back to my laboratory. After being soaked in 45% acetic acid for about 1 hr for expanding, they were stored in 70% alcohol. Cestodes obtained from these alcohol-preserved guts were stained with alcohol-hydrochloride- carmine, dehydrated in alcohol, cleared in xylene, and mounted in Canada balsam. Measurements Accepted March 26, 1987 Received February 23, 1987 ' This paper corresponds to “Helminth Fauna of Bats in Japan XXXVII”. are given in millimeters. RESULTS The localities and dates of bats examined and cestodes obtained are shown in Table 1. Vampirolepis Spassky, 1954 Vampirolepis rikuchuensis sp. n. (Figs. 2-5) From June 17 to July 7, 1986, five specimens of Hosono’s whistered bat, Myotis hosonoi, were captured by Mukooyama around street lamps at Ichinohe-ch6, Iwate Prefecture. On dissection, one of them was found infected with two mature and one juvenile specimens of this cestode. Description: Medium-sized hymenolepidid; ma- ture worms 24—43 in length and 0.8—1.1 maximum width. Metamerism distinct, margins serrate. Proglottids wider than long. Scolex 0.245—-0.280 long and 0.273-0.287 wide, not sharply demar- cated from strobila. Rostellum pyriform, 0.126 long by 0.070-0.091 wide, armed with a crown of 29-30 spanner-shaped hooks measuring 0.018 long. 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(7) Myotis hosonoi 40 V. rikutsuensis sp. n. 5) June 17-July 1 Ichinohe-cho, Iwate Pref. 1) Street lamps 3) Forest July 30 Ooyama-cho, Toyama Pref. (8) Myotis frater kaguyae V. kaguyae sp. n. 20 100 June 17-20 July 30 1) Street lamps 3) Forest (9) Murina aurata ussuriensis V. sp. (juvenile, unidentified) V. yakushimaensis sp. n. 100 Dec. 26 Kamiyaku-ch6, Yakushima, Kagoshima Pref. (10) Murina leucogaster hilgendorfi 13) Abandoned gold mine Three New Species of Bat Cestodes 723 Nov.9 mine 7) Abandoned Nose-cho, Osaka Pref. ' Locality numbers correspond to those in Fig. 1. prominent, round at its end, slightly shorter than blade; blade sharp at its end. Rostellar sac slightly elongate, 0.189 long by 0.133-0.161 wide, extend- ing posterior to suckers. Suckers discoid, 0.091- 0.105 in diameter. Genital pores unilateral, located a little anterior to the middle of proglottid margin. Testes three in number, ovoid, 0.098-0.112 long by 0.056-0.070 wide, arranged in a transverse row, one poral and two aporal. Cirrus sac club-shaped, attaining a size of 0.154 long by 0.042 wide, filled with internal seminal vesicle measuring 0.070-0.077 long by 0.035 wide. External seminal vesicle 0.112 long by 0.065 wide. Ovary transversely elongate, 0.308- 0.315 wide, located anterior half of proglottid. Vagina opening in genital atrium, extending medially, then enlarging and forming seminal receptacle. Seminal receptacle 0.070-0.077 long by 0.049-0.056 wide. Vitelline gland compactly lobate, 0.140 by 0.056, located posterior to ovary. Uterus arising directly from ovarian lobe as a lobed sac, gradually enlarging, filling all available space in senile proglottids. Eggs spherical or ellipsoidal, 0.049-0.053 by 0.042-0.046, sur- rounded by four envelopes; outermost chorion thick and with smooth surface. Onchospheres spherical 0.032-0.035 in diameter; embryonic hooks 0.014 long. Type host: Myotis hosonoi Imaizumi, 1954. Site of infection: Small intestine. Type locality and date: Ichinohe-ché, Iwate Prefecture; June 17, 1986. Type specimen: Holotype: NSU Lab. Coll. No. 8701. Paratypes: 8702. Remarks: The present new species, V. riku- chuensis closely resembles V. ogaensis Sawada, 1974 [1] from Rhinolophus ferrumequinum nippon in the shapes of scolex, rostellum, rostellar sac and rostellar hooks. However, it differs from V. ogaensis in smaller rostellar hooks (0.018 vs. 0.021), thicker outermost chorion of eggs (tough vs. thin), the surface structure of eggs (smooth vs. rough) and the position of genital pores (located a little anterior to the middle vs. the middle). 724 I. SAWADA ete, oo PL ee TOOHOKU : bee a 2 te eee” . . . a = = = a Ge ee ow ee oe ee ee em a aw ok a = TANEGA SHIMA\ ®%11 YAKU SHIMA 12 Zeococeao Seen eee ea one eee ae ane 7-2 BS OOOO OES oes w 4 i OKINOERABU ue SHIMA OKINAWA SHIMA Fic. 1. Map showing the collection sites of bats. For the locality number, see Table 1. to 20, 1986, one was found infected with one mature specimen of this cestode. Description: | Medium-sized hymenolepidid; Of five long-leged whistered bat, Myotis frater strobila length 45; maximum width 1.6. Metamer- kaguyae, captured by Mukooyama around street ism distinct, craspedote, margins serrate. Proglot- lamps at Ichinohe-ch6, Iwate Prefecture, June 17 __ tids wider than long. Scolex 0.280 long by 0.135 Vampirolepis kaguyae sp. n. (Figs. 6-9) Three New Species of Bat Cestodes 725 0.012 Fics. 2-5. Vampirolepis rikuchuensis sp. n. 2: Scolex. 3: Rostellar hooks. 4: Mature proglottid, dorsal view. 5: Ripe egg. Scales in mm. wide, slightly set off from neck. Rostellum pyriform, 0.140 long and 0.112 wide, armed with a single row of 35 spanner-shaped hooks measuring 0.032 long. Hook handle long, attenuate; guard prominent, round at its end, slightly shorter than blade; blade sharp at its end. Rostellar sac 0.217 long by 0.182 wide, when the rostellum is invagin- ated. Suckers round, 0.105 in diameter. Genital pores unilateral, located a little poste- rior to the middle of proglottid margins. Testes 726 I. SAWADA a 0.03 Fics. 6-9. Vampirolepis kaguyae sp. n. 6: Scolex. 7: Rostellar hook. 8: Mature proglottid, dorsal view. 9: Ripe egg. Scale in mm. three in number, spherical, 0.077-0.091 long by 0.063—-0.077 wide, arranged in a transverse row, one poral and two aporal. Cirrus sac slightly elongate, 0.154-0.175 long and 0.049-0.056 wide, extending to longitudinal excretory canals. Inter- nal seminal vesicle 0.112-0.119 long by 0.049- 0.056 wide, enlarging to fill proximal portion of cirrus sac. External seminal vesicle slightly elon- gate, 0.104-0.175 long by 0.070-0.77 wide, ex- tending to poral testis and dorsal to seminal receptacle. Vagina opening in genital atrium, extending to median field, then enlarging to Three New Species of Bat Cestodes 727 forming voluminous seminal receptacle measuring 0.112-0.133 long by 0.070-0.084 wide. Ovary transversely elongate, 0.280-0.287 wide. Vitelline gland compact, directly posterior to ovary, 0.077- 0.091 long by 0.035-0.049 wide. Eggs spherical, 0.042 in diameter, surrounded by four envelopes; outermost chorion thick and with smooth surface. Onchospheres spherical, 0.028 in diameter; em- bryonic hooks 0.011 long. Type host: Myotis frater kaguyae Imaizumi, 1956. Site of infection: Small intestine. Type locality and date: Ichinohe-cho, Iwate Prefecture: June 17, 1986. Type specimen: Holotype NSU Lab. Coll. No. 8703. Remarks: The present new form, V. kaguyae, closely resembles V. yoshiyukiae Sawada, 1980 [2] from the same species bat collected at Oze, Gunma Prefecture, in the shape and number of rostellar hooks. However, it differs from V. yoshiyukiae in larger suckers (0.105 in diameter vs. 0.056-0.070), larger rostellum (0.140 by 0.112 vs. 0.070 by 0.056), larger rostellar sac (0.217 by 0.180 vs. 0.046 by 0.054), and longer rostellar hooks (0.032 vs. 0.0245). Vampirolepis yakushimaensis sp. n. (Figs. 10-14) On December 26, 1986, one lesser tube-nosed bat, Murina aurata ussuriensis, was collected in an abandoned gold mine at Kamiyaku-ché6, Yaku- shima, Kagoshima Prefecture. The bat was found infected with 13 mature specimens of this cestode. The cestode is the first to be reported from this species bat from Japan. Description: Small-sized hymenolepidid; ma- ture worms 25-30 long and 1.0-1.2 maximum wide. Metamerism distinct, craspedote, margins serrate. Scolex clavate, 0.105-0.154 long and 0.259-0.315 wide, not sharply demarcated from strobila. Rostellum 0.049-0.077 long and 0.063- 0.084 wide, armed with a single circle of 28-35 spanner-shaped hooks measuring 0.021 long. Hook handle slender; guard prominent, round at its end, slightly shorter than blade; blade remark- ably sharp at its end. Rostellum retractable into rostellar sac measuring 0.119-0.140 long and 0.119-0.126 wide. Unarmed suckers round, 0.070-0.084 in diameter. Neck region behind scolex 0.22—0.28 long and 0.28 wide. Mature and gravid proglottids much broader than long. Genital pores unilateral and located a little anterior to middle. Testes three in number, subspherical, 0.070-0.084 long by 0.084—-0.105 wide, arranged in a form of triangle, one poral and two aporal, not in contact with longitudinal osmoregulatory canals laterally. Cirrus sac cylin- drical, 0.154-0.270 long and 0.032-0.056 wide, positioned anteromedial from genital atrium, ex- tending beyond osmoregulatory canals. Internal seminal vesicle 0.124—-0.138 long by 0.056 wide, occupying almost whole of cirrus sac. External seminal vesicle 0.111-0.126 long by 0.042-0.055 wide. Ovary pentalobate or hexalobate, 0.182- 0.189 wide. Vitelline gland lying posterior to ovary, irregularly lobate, 0.028-0.035 long by 0.070-0.077 wide. Seminal receptacle saccate, 0.126-0.140 long by 0.070-0.084 wide, overlap- ping poral testis. Uterus arising directly from ovarian lobes as a lobe sac, gradually enlarging, filling whole gravid proglottid. Ripe eggs elliptical; outermost chorion thin, 0.056—0.060 in major axis and 0.042-0.046 in minor axis; inner membrane 0.032—0.035 by 0.028-0.032, with at each pole a round projection provided with polar filaments. Onchospheres_ subspherical, 0.025-0.028 by 0.028—0.032; embryonic hooks 0.018 long. Type host: Murina aurata ussuriensis Ognev, 191s Site of infection: Small intestine. Type locality and date: Kamiyaku-ché, Yakushi- _ma, Kagoshima Prefecture; December 26, 1986. Type specimen: Holotype: NSU Lab. Coll. No. 8704. Paratypes: No. 8705. Remarks: The present new species, V. yakushi- maensis closely resembles V. iriomotensis Sawada, 1983 [3] from Rhinolophus imaizumii in the number and length of rostellar hooks. However, it differs from V. iriomotensis in the shape of ovary (pentalobate or hexalobate vs. transversely elon- gated and trilobate), the morphological feature of egg inner membrane (provided with polar fila- ments vs. no polar filaments) and in longer embryonic hooks (0.018 vs. 0.014). 728 I. SAWADA 0.01 aS 0.03 Fics. 10-14. Vampirolepis yakusimaensis sp. n. 10: Scolex. 11: Rostellar hook. 12: Mature proglottid, ventral view. 13: Ripe egg, showing a projection (arrow) of the inner membrane provided with polar filaments. 14: Ripe egg, showing polar filaments (arrow). Scale in mm. Three New Species of Bat Cestodes 729 Vampirolepis isensis Sawada, 1966 [4] Host: Rhinolophus cornutus cornutus and R. c. orii. For localities, see Table 1 and Figure 1. Vampirolepis hidaensis Sawada, 1976 [5] Host: Miniopterus schreibersii fliginosus and M. s. blepotis. For localities, see Table 1 and Figure 1. Hymenolepis Weinland, 1858 Hymenolepis rashomonensis Sawada, 1972 [6] Host: R. ferrumequinum nippon. For localities, see Table 1 and Figure 1. ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and a grant from the Nissan Science foundation. I am indebted to Mr. Mitsuru Mukooyama (Sannohe High School) for supplying the alimentary canals of bats; and to Mr. Masashi Harada (Laboratory of Experimental Animals, Osaka City Uni- versity Medical School), Dr. Kimitaka Funakoshi (De- partment of Anatomy, Faculty of Medicine, Kyushu University), Mr. Teruo Irie (Kumamoto Education Center) and Mr. Masahide Nakaji (Gyokusen-d6, Oki- nawa) for their generous cooperation. REFERENCES 1 Sawada, I. (1974) Helminth fauna of bats in Japan XV. Annot. Zool. Japon., 47: 103-106. 2 Sawada, I. (1980) Helminth fauna of bats in Japan XXII. Annot. Zool. Japon., 53: 194-201. 3 Sawada, I. (1983) Helminth fauna of bats in Japan XXIX. Annot. Zool. Japon., 56: 209-220. 4 Sawada,I. (1966) On a new tapeworm, Vampiro- lepis isensis found in bats with the table of the morphological features of tapeworms in Vampiro- lepis. Jpn. J. Med. Sci. Biol., 19: 51-57. 5 Sawada, I. (1976) Helminth fauna of bats in Japan II. Jpn. J. Parasitol., 16: 103-106. 6 Sawada, I. (1976) Helminth fauna of bats in Japan XI. Bull. Nara Univ. Educ., 21: 27-30. above ifesietd. aM oi bow fed io itroles cima 4q0} yi waiseQ tomas letpmiegen to. Golgodit “Aly ideosamt nisin el ,(ootze mie A samba je 4 = rye BORDA Ores omit 3 ie . HO Abc ais sox +) ieade yt wa} tae PEM E DEE RT reat ai add Wo ane Atmmioht (bVer) 7 atewee { ' aOT-207 NB noast loa jonuk VR oy nine of ated to sack gininieH (0801) A abawee 2 lane 5 (OS£07 £2! noget’ tooS toma OX etal ni ued ky eeust Hats unisht {28Cly) goawed foo. i amet! a (SLOWS 182 L aogat looS Gonnk KURK Joel we: ¥ Aa a rosin wom umowsest wea se WO. (caRit Jbyabiwat k= om teen roe afi to aide? of} ine eied as bawol aha pics 3 is OUGINN ml anradwenl ho edhe. drobeeleate erate ' Ne~{t 41 lol ioe be Ace all foqet at ad Ye sins! Balls (Overy A abewad 4? bin. fil biline | a O-EOT Ot, hate? TV agh etinouha ts euleiin aasid eos ol eter To wai ral Gaitioh (OFC) LV ahiewse 8 asani anit mint ite ey bee G£-SE 32 ;.awbd vinky aie tet 04 wingilé AM GY herdebal” rw 4 7 b \ , : reel "hae, caging dl ae AERRIEPR ls SDN yal i a ite? iy } S ie 9 ,} ‘7 _ ii - ; é i “ é = i ¥ re ote ee : r Jiegp lenny ended 24 ateya ype S . ZOOLOGICAL SCIENCE 4: 731-734 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Origin of Binucleate Cells in the Neural Gland of the Ascidian Halocynthia roretzi (Drasche) MizuHo Ocawa, KiyosHi TERAKADO! and JIN OKADA Department of Biology, Faculty of Liberal Arts and Science, and ‘Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Saitama 338, Japan ABSTRACT—The origin of binucleate cells in the neural gland of the simple Ascidian, Halocynthia roretzi (Drasche) was examined by light- and electron- microscopy. The central area of dorsal wall of the gland is a simple columnar epithelium composed of rather compact mono-nucleate cells. Peripheral to the center of the dorsal wall, however, the epithelium expands and becomes stratified containing binucleate cells. The binucleate cells seem to proliferate in this limited area of the dorsal epithelium and slough off into the lumen of the gland to form a loose parenchymatous tissue. INTRODUCTION Many investigators have examined possible en- docrine functions of the neural complex, reasoning that there is a definite structural similarity between the vertebrate hypothalamus-pituitary-Rathke’s pouch pattern and the tunicate cerebral ganglion- neural gland-neural gland duct pattern [1]. Re- cently, cells containing immunoreactive substances have been demonstrated after incubation with antisera raised against some mammalian pituitary and gastrointestinal hormones [2-5]. However, the true function or functions of the neural gland remain uncertain. In an earlier paper we reported that most of the cells of the neural gland in Halocynthia are binucleate [6]. However, the origin of these binucleate cells remained uncertain as does their significance in the neural gland and indeed the function of the neural gland itself. Accepted April 22, 1987 Received March 26, 1987 The aim of the present work is to describe the origin of binucleate cells in the neural gland of the ascidian Halocynthia roretzi. MATERIALS AND METHODS Specimens of the ascidian Halocynthia roretzi were purchased from the market in February and every other month for a year. They were kept in aquaria for at least 2days before use. After dissection, the neural glands were fixed in Bouin’s or Zenker’s solution, then dehydrated, embedded in paraffin and sectioned at 6 wm using routine procedures. For electron microscopy, small pieces of the neural gland were excised and immediately fixed with 2% OsQ, in 0.15 M collidine buffer (pH 7.3) at 4°C for 1 hr. They were dehydrated in graded concentrations of ethanol and embedded in epoxy resin. Thin sections were cut with glass knives, doubly stained with aqueous uranyl acetate and lead citrate, and examined with a Hitachi H 700H electron microscope operated at 100 KV. RESULT The neural gland of Halocynthia roretzi appears to be composed of acinous lobules. The dorsal wall of the neural gland is composed of a simple columnar epithelium. The same epithelium not only extends dorso-posteriorly as a dorsal cord, but also connects anteriorly with a neural gland duct opening into the pharyngeal cavity via a M. OGawa, K. TERAKADO AND J. OKADA 732 Origin of Binucleate Cells of Neural Gland 733 ciliated funnel. The opposite, ventral wall of the gland, is surrounded by a thin cell layer and a connective tissue. The central area of the dorsal epithelium is composed of simple columnar, rather compact and mono-nucleate cells (Fig.1). These mono- nucleate epithelial cells are relatively high co- lumnar (about 10 um high) with an oval nucleus; they are held together laterally. No mitotic figures were observed in this area. By _ electron- microscopy, these cells show a relatively small Golgi body, some small mitochondria and vesicles, and a poorly developed terminal bar; they lack microvilli (Fig.3). Some cells contained two nucleoli located on the opposite sides of the nucleus. In contrast to the free surface, the surface of basal membrane was covered with a diffuse coating. Peripheral to the center of dorsal wall, the epithelium expands and becomes irregularly strat- ified. In this region binucleate cells are observed (Fig. 2). By electron-microscopy, the simple co- lumnar epithelium is seen to be gradually trans- formed into a pseudostratified form in the areas around the center of the dorsal wall; the trans- formed cells lack uniformity and binucleate cells are encountered (Fig. 4). Some of the cells lose their connection with the free surface and become round in shape. These rounded cells are also binucleated with some mitochondria and vesicles. No seasonal change occurs in these epithelial features so far examined. DISCUSSION It has been reported that the epithelium of the neural gland undergoes cyclical changes of activity. In the neural gland of Ciona intestinalis, according to Georges [7-9], there are two different morpho- logical phases, showing 24 hours rhythmical secre- tory cycle: a loose reticulated form (epithelial structure) and an expanded compact form (mesen- chymal structure). Whether or not the neural gland of Halocynthia shows such a circadian rhythm is not known at present. However, in the adults examined in this study, the neural gland was filled with binucleate cells appearing like parenchymatous tissue with a compact form at every season so far examined. The central region of the dorsal epithelium is composed of simple columnar, mono-nucleate cells. However, peripheral to the center of the dorsal epithelium, binucleate cells are observed. How the binucleate cells arise in the limited area of the dorsal epithelium remains uncertain. No mitotic figures were observed in the present study. Although the lumen of the gland was filled with binucleate cells, no cell division was observed. At present it is believed that the binucleate cells originate in a limited, peripheral area of the dorsal epithelium and slough off into the lumen of the gland to form loose parenchymatous tissue. In some sense, the neural gland seems to be classified into a holocrine gland. The available data still do not show whether the neural gland is homologous with the pituitary of vertebrate. The possible role of the neural gland, therefore, requires further study. ACNOWLEDGMENTS We wish to express our gratitude to Prof. W. S. Hoar, University of British Columbia, for his encouragement and critical reading of the manuscript. Fic. 1. Cross section of the neural gland. Arrow shows the center of dorsal epithelium from which the dorsal cord branches. Dense, tubular structures seen on the upper side of the figure are cut-planes of the dorsal cord. Scale, 50 «am. Fic. 2. High magnification of the area surrounding the central region of dorsal epithelium. Binucleate cells occur here. Scale, 20 um. Fic. 3. Electron-micrograph of the central region of dorsal epithelium, being a typical simple columnar epithelium. Arrow shows the basement membrane. Scale, 2 um. Fic. 4. Electron-micrograph of the area peripheral to the central region of dorsal epithelium, expanding and turning into a stratified epithelium. There is a binucleate cell (under right hand corner) just sloughing from the epithelium, but a poor terminal bar is still existent (arrowhead). Arrow shows the basement membrane. Scale, 2 um. 734 M. Ocawa, K. TERAKADO AND J. OKADA REFERENCES Goodbody, I. (1974) Adv. Mar. Biol., 12: 1-149. Pestarino, M. (1984) Gen. Comp. Endocrinol., 54: 444-449, Pestarino, M. (1984) Acta Zool. (Stockh.), 65: 113- 118. Pestarino, M. (1985) Gen. Comp. Endocrinol., 60: 293-297. Pestarino, M. (1985) Cell Tissue Res., 24: 497-500. 6 Ogawa, M., Orikasa,C. and Terakado, K. (1985) ZS SO, 28 ASAT, Georges, D. (1970) C.R. Acad. Sci. Paris, 270: 3137-3140. Georges, D. (1971) Acta Zool. (Stockh.), 52: 257- DAs Georges, D. (1978) In “Comparative Endocrinolgy”. Ed. by P. J. Gaillard and H. H. Boer, Elsevier/North Holland Biomedical Press, Amsterdam, pp. 161-164. ZOOLOGICAL SCIENCE 4: 735-737 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Asynchronous Expression of Alleles at the Alcohol Dehydrogenase Locus during Oryzias Hybrid Development JACK S. FRANKEL Department of Zoology, Howard University, Washington, D.C. 20059, U.S.A. ABSTRACT—The expression of alleles encoding the enzyme alcohol dehydrogenase (ADH; EC 1.1.1.1) was investigated in Oryzias melastigma, O. javanicus and in O. melastigma 2 X O. javanicus JS hybrids by acryla- mide gel electrophoresis. It was not possible to investi- gate the expression of ADH in reciprocal hybrids as these fry failed to develop past the stage of embryonic body formation. The delay in appearance of isozymes of paternal ADH subunit composition is suggestive of an incompatibility between maternal cytoplasmic regulatory factors and the paternal regulative element. INTRODUCTION The expression of gene loci during development is both the result of, and impetus for, cellular differentiation and specialization. The appearance of specific enzymes during embryogenesis reflects metabolic and, thus, biochemical changes associ- ated with increasing developmental complexity. Furthermore, since different enzymes have distinct physiological roles, their expression during de- velopment may indicate times of unique metabolic requirements in certain cells, tissues or organs. While many enzymes have general “housekeep- ing” functions, serving basic metabolic needs in most cells and, therefore, usually found through- out embryogenesis, others are restricted spatially and/or temporally; appearing in specifiic tissues at specific times during development. Enzymes ex- hibiting such tissue and/or temporal specific pat- terns have been the focal point of numerous ontogenetic studies [1-19]. Isozymes have proven to be ideally suited to Accepted April 10, 1987 Received March 6, 1987 studies designed to analyze patterns of allelic expression during interspecific hybrid develop- ment [6-8, 12-14, 20-22]. Genetic variation in parental alleles, reflected in isozymes with differ- ent electrophoretic mobilities, permits the deter- mination of the temporal expression of maternal and paternal contributions toward development. In instances where a delay or total repression of paternal isozyme expression occurs, the asynchro- nous activation of alleles is attributed to incom- patibilities of the maternal species effector mole- cules with the paternal gene; thereby reducing the efficiency of allelic recognition and delaying activa- tion [16]. The present communication reports on the activation and expression of the gene locus encoding alcohol dehydrogenase (ADH; EC 1.1.1.1) in Oryzias javanicus, O. melastigma and their hybrid. MATERIALS AND METHODS Healthy adults of O. javanicus and O. melastig- ma were obtained from Mid-Atlantic Distributors, Inc., Springfield, VA, USA. Intra- and inter- specific embryos were obtained by artificial ferti- lizations under conditions and procedures de- scribed by Iwamatsu et al. [23], except that fishes were incubated in fingerbowls measuring 105 mm in diameter and 45 mm in depth. At selected stages/times during development, pre- and posthatching, intra- and interspecific fishes were collected and screened electrophoreti- cally for the presence of ADH activity. The ADH patterns of the hybrids were compared with those of intraspecific fry at corresponding times during 736 J. S. FRANKEL development (posthatching). In determining the temporal spacing of ADH gene expression, fry were sampled at 2-hr intervals. The time indicated for the appearance of a given ADH isozyme is an average of four separate observations. Individual observations varied no more than + or — 2 hr from their mean. For each assay, individual fry were randomly chosen from a time-monitored fingerbowl and homogenized at 4°C in a 0.01M K,PO,/0.3 mM EDTA solution adjusted to pH 7.0 with 0.01 M KH,PO,. Fifty percent homogenates, by volume, were prepared for all samples. Homogenates were centrifuged for 10 min at 4°C in a Beckman 152 Microfuge. Twenty-yl aliquots of the clear supernatant were electrophoresed. Liver tissue was excised from adult fishes decapi- tated after immobilization in ice water. Tissue samples were prepared for electrophoresis as described for fry samples. Electrophoresis was conducted employing an acrylamide gel vertical slab system (E. C. Appa- ratus Co.), maintained at 4°C by a recirculating water bath (Lauda K-—2/R, Brinkman _Instru- ments). Six percent acrylamide gels were run at 300 volts for 5 hr using the 0.155 M tris-citrate (pH 7.0) buffer system of Shaw and Prasad [24]. Gels were stained with an incubation medium from Shaw and Koen [25], and fixed in 7% acetic acid. RESULTS AND DISCUSSION The ADH phenotypes of liver tissue for O. melastigma, O. javanicus and the O. melastigma ? X O. javanicus § hybrid are shown (Fig. 1). As has been observed in a previous investigation on the interspecific hybridization of O. javanicus and O. melastigma [23], O. javanicus eggs insemi- nated with O. melastigma sperm failed to develop past embryonic body formation and, as a result, it was not possible to determine the timing of ADH expression in these hybrids. ADH is dimeric in nature, being encoded in the majority of verte- brates at a single gene locus [3, 8-11, 26, 27]. The enzyme occurs at its highest concentrations in liver, but has also been reported to be present to a lesser extent in stomach and kidney [3, 26]. Zymograms of ADH of O. javanicus and O. melastigma exhibit single, cathodal zones of en- AB C De ce F Fic. 1. Zymogram of Oryzias ADH isozymes of liver tissue and fry samples. Channel A—O. javanicus liver. Channel B—O. melastigma liver. Channel C—O. melastigma ? XO. javanicus & hybrid liver. Channel D—hybrid fry at 104hr posthatching (p. h.). Channel E—hybrid fry at 122 hr p.h. Channel F—hybrid fry at 134hr p.h. zymatic activity, restricted in expression to liver (Fig. 1, channels A and B, respectively). The zymogram of the hybrid displays both O. melastig- ma and O. javanicus homodimers, along with a heterodimer of intermediate electrophoretic mobility (Fig. 1, channel C). Evidence of Adh locus expression in intraspecific O. javanicus and O. melastigma fry was observed at 100 and 104 hr posthatching (p.h.), respectively, by the initial appearance of ADH activity on the gel. By 128 hr p.h., ADH activity of intraspecific fry appeared to reach a maximum level. However, zymograms of hybrid fry at 104hr p.h. exhibited a zone of maternally derived ADH along with an extremely faint zone of activity corresponding to the hybrid allodimer (Fig.1, channel D). The paternal homodimer was not detected at this time. Hybrid zymograms at 122hr p.h. showed two distinct zones of ADH activity corresponding to the maternally derived homodimer and the hybrid allodimer, along with a faint zone of activity corresponding to the paternal homodimer (Fig. 1, channel E). Zymograms of hybrid fry at 134 hr p.h. (Fig. 1, channel F) revealed an ADH pattern indicative of a 1:2: 1 ratio in the concentrations of the three isozymes; suggestive of full expression of Adh alleles of maternal and paternal origin. It would seem that at the time when the Adh locus is expressed in intraspecific Oryzias fry (104 hr p.h.), preferential expression of the melastigma allele Allelic Asynchrony in Oryzias occurs in the O. melastigma ° X O. javanicus § hybrid. The marked temporal shift in the expression of the paternal Adh allele in the Oryzias hybrid is consistent with the observed effects of gene regula- tory divergence among species [17]. As has been noted in other studies [21, 22], both developmental abnormalities at the morphological level and aber- rant expression of allelic isozymes in differentiated tissues increase concomitantly with the greater evolutionary distance of the parental species used in forming a given hybrid. Indeed, the asymmet- rical developmental response of reciprocal Oryzias hybrids is suggestive of a “significant” degree of gene divergence between O. melastigma and O. Javanicus. In conclusion, the observations from the present study are in keeping with reports on ADH expression in other hybrids [8, 11, 26, 27] and support postulated mechanisms of allelic expres- sion [16, 18] where alleles of maternal origin effectively interact with maternally derived effec- tor molecules, whereas alleles of paternal origin may interact less effectively, if at all. Indeed, allelic asynchrony during Oryzias hybrid develop- ment may be a consequence of varying degrees of affinity between maternal regulatory molecules and maternal and paternal regulative elements controlling the expression of structural gene loci. ACKNOWLEDGMENT The author thanks Joan Ann Frankel for her help with the photography. REFERENCES 1 Holmes, R.S. and Whitt, G.S. (1972) Biochem. Genet., 4: 471-480. 2 Lindahl,R. and Mayeda, K. Biochem. Physiol., 45B: 265-273. 3 Shaklee,J.B., Champion,M. and Whitt, G. S. (1974) Dev. Biol., 38: 356-382. 4 Champion, M. J. and Whitt, G.S. (1976) J. Exp. (1973) Comp. 17 18 19 20 74 22 748) 24 25 26 27 TM Zool., 196: 263-282. Champion, M. J. and Whitt, G. S. (1976) Biochem. Genet., 14: 723-737. Hart, N. H. and Cook, M. (1976) Comp. Biochem. Physiol., 54B: 357-364. Frankel, J. S. and Hart, N. H. (1977) J. Hered., 68: 81-86. Frankel, J. S. (1978) J. Hered., 69: 57-58. Frankel, J. S. (1980) J. Hered., 71: 430-431. Frankel, J.S. (1981) Comp. Biochem. Physiol., 70B: 643-644. Frankel) Ji.s. (1983) J. Hered:, 74: 31/1/=3127 Frankel, J.S. and Wilson, R. V. (1985) Comp. Biochem. Physiol., 80B: 463-466. Frankel, J.S. (1985) Comp. Biochem. Physiol., 81B: 635-639. Frankel, J.S. (1985) Comp. Biochem. Physiol., 82B: 413-417. Philipp, D. P., Childers, W.F. and Whitt, G. S. (1979) J. Exp. Zool., 210: 473-488. Philipp, D.P., Parker,H.R. and Whitt,G.S. (1983) In “Isozymes: Current Topics in Biological and Medical Research”. Ed. by M. C. Rattazzi, J. C. Scandalios and G. S. Whitt, Alan R. Liss, New York, Vol. X, pp. 193-237. Whitt, G. S. (1981) Am. Zool., 21: 549-572. Whitt, G. S. (1983) In “Isozymes: Current Topics in Biological and Medical Research”. Ed. by M. C. Rattazzi, J. C. Scandalios and G. S. Whitt, Alan R. Liss, New York, Vol. X, pp. 1-40. Frankel, J.S. and Wilson, R. V. (1984) Biochem. Physiol., 78B: 179-182. Whitt, G. S., Cho, P. and Childers, W. F. (1972) J. Exp. Zool., 179: 271-282. Whitt, G. S., Childers, W. F. and Cho, P. (1973) J. Hered., 64: 55-61. Whitt, G.S., Philipp,D.P. and Childers, W. F. (1977) Differentiation, 9: 97-109. Iwamatsu, T., Watanabe, T., Hori, R., Lam, T. J. and Saxena, O. P. (1986) Zool. Sci., 3: 287-293. Shaw, C.R. and Prasad,R. (1970) Biochem. Genet., 4:297-320. Shaw, C. R. and Koen, A. L. (1968) In “Chroma- tographic and Electrophoretic Techniques”. Ed. by I. Smith, John Wiley, New York, Vol II, p. 347. Hitzeroth,H., Klose,J., Ohno, S. and Wolf, U. (1968) Biochem. Genet., 1: 287-300. LeVine, J.P. and Haley, L. E. (1975) Biochem. Genet., 13: 435-446. Comp. ¥ 3 1s sie a ii F ‘Giveewsded! Pookaielige Meseeaeanniton he = canataetene SS {QR nga evens fe Mi vpememedh: fey tate’ Ah Dae ligne ys ESE hte teaiitaiod: : keaton. Brae (age. fe EMD Dist as ow, ees oa - cute i Otte SRE SARE. bibs +2a.. baats : (et ie. bay €, Samant f, ate seaithad donset ttReOT oe sy oc ‘ Abe GPT i THBP : i Rey” ee PES eit i) as pian PERSE) th Ce Pride’ hi i SY T Ay WR eS y Lc Ae eS eV Gercki My (Rinne Edednatutike CUTIE ity heme f bieg aM ae Tikad chee bre oe, onary Gl a Qn ETM Be, mi bales ord pumatocith ! { Wedd matio9 if, 3m nes (CHEE A tote Bai ee “ eae Bisy ek 8, womyaa™ Nerd eto mine Pw (tet; 2 ' ray Ci Se SSG: WS Ie OE. OB Lit die ic US ANGE TRO TUE ep re ota ee Bary te ER ME LO LR DEORE bs Ce eA GE os CRG Lhhyadvel> 9 Lagi ol ik BOG Tl i aeatsecy I aT Ad A ATMS et HH, LEBYr ) “7 f iseatie ht any, yO .Oc} . apne a id nity ah iar pd a Se ae HH or bike 2Odsbunee tue Oe CD eae eee +e IOC er a SEMOS ETAL CLC Why AME OO eA Pate aig | nivaepaT ine reeniinslt lobar hc 2 Orin Ot ae, me: ehnehs.) avigars Si tesihe art is inrigo loth; i BALA. fit it td DOG AED 9% bed | uaa ” 2 aD bg Alo ot veal eid mock TEOUEY PE how bas 2 leunerd ROOU_ SRP LOPE HRY OY add Banshee b (ote) Ay evel bas Tod 2 0 ew - OS Dine i SBR ETS any HORS! Red | | (eves) Pon ahe AW Heese ei ts : 4 ec if Some Ai yubaee H vali?’ }. tren 40 on ih ld of ae pee ves VOi~TE -@ notletas iit (vee ly i Hd. ede Tee cag vil, ES SORT HR OR ee aa eeassar Dak” 2 eh WO Saeed ORL wane ms Ch aitestiena (RRO. does seca: gesnrastt oe ake Leas ant Yer ROPE EES 3, cee Wie Ves. Sipe wad ol adgats alg Lote MEADS, Gg alio¥ .drat wo, KOE gio} ftdinrie, y ve UP ow bce 2 oni Ser. cd thier “Shit Os er: | Wen Nee ai ‘Shhacy st wAG 8 et} - Wreidhngh: feet ey, pis tg Sy AR et wey ot vat ‘ve 7. ‘ a So pKa? BURSEERL-EES gonzo ) ie” sig ag ’ ry? bee pi Cds. Je " 5 of aval wok Ss, wie wearin Sy : A Tere 2 ae ee ini oe pie ll ae 90K pdr 1onRtG aff ps wala sedp Sno ere aie Ande tayen ii aOnyy he Ap eee , igtple Sips aise teil arghsd: bared dine tee Heat hae ies WP ahe? ethane Geni ss bby hit" deine ey to aately Heebind, Whe 1 WD ulewk webby itty rou gore id erwin? Topps eoaretadn yn islinpes serena strong wvitebant casing ak Hos Sahai whincapedse fiw ns Mot teh ibaa 810 ay od ae a peng’ n BF he: “ae Rea ry lata ZOOLOGICAL SCIENCE 4: 739-742 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Cloning of Stage Specific Gene Sequences from a cDNA Library Representing Poly(A)" RNA of Sea Urchin Prism-Embryos Kon AKASAKA, MASANOoRI TarRA!, TsuUGIO SHIROYA and HIRAKU SHIMADA Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, and ‘Department of Biochemistry, Chiba University School of Medicine, Inohana, Chiba 280, Japan ABSTRACT—A cDNA library was constructed using poly(A)* RNA from sea urchin embryos (H. pulcherri- mus) at the prism stage. Four cDNA clones correspond- ing to the RNA population abundant at the prism stage but rare at the morula stage were selected by differential hybridization technique. The relative abundance of individual RNAs were measured by Northern hybridiza- tion analysis. The size of the RNA population repre- sented by these cloned cDNAs was small in the unfertil- ized eggs, decreased further until blastula stage, and thereafter, increased sharply. However, the different RNA species started to increase in their amount at different developmental stages. Moreover, the amount of each of these RNA species fluctuated in a different manner. INTRODUCTION It has been reported that the RNA synthesis is not necessarily needed for the development of sea urchin embryos up to the blastula stage [1], and that most of the genes become active at the gastrula stage [2]. The mechanisms of the onset of gene activation during early development have not been clarified as yet. To study the molecular mechanism of the regulation of gene expression during blastula-gastrula transition of the sea urchin development, we constructed a cDNA library and selected the genes which mRNAs begin to accumu- late at the stage of blastula-gastrula transition. Accepted April 18, 1987 Received January 26, 1987 MATERIALS AND METHODS Embryo culture Eggs and sperm of Hemicentrotus pulcherrimus were collected by artificial spawning induced with a 0.5M KCI solution. Eggs were fertilized and developed in filtered sea water at 15°C. Preparation of RNA Total cellular RNA was isolated from embryos by the guanidinium/hot phenol method [3]. Polyadenylated RNAs were isolated by oligo(dT)- cellulose chromatography [4]. Preparation of a library of cloned cDNAs from H. pulcherrimus prism embryos (\HpPR library) DNA complementary to the polyadenylated RNA of the prism embryo was prepared by the use of reverse transcriptase [5]. Double-stranded cDNA was formed in a DNA synthesizing system containing E.coli ribonuclease H and DNA polymerase I [6]. Construction of a cDNA library in Agt10 was carried out according to the methods developed by Huynh et al. (7). E. coli C600hfi* was used as a host. Preparation of cDNA probe Polyadenylated RNA from morula-stage or prism-stage embryos was used for the synthesis of cDNA probes. The primary single stranded cDNA copies were synthesized using reverse transcriptase 740 K. AKASAKA, M. Tarra et al. in the presence of a random primer [8] and [a—*’P] dCTP. cDNA probes were obtained after the hydrolytic removal of RNA from the RNA-DNA hybrids by incubating with 0.6 N NaOH at 37°C for 1 hr. Isolation of phage recombinants containing stage- specific gene and preparation of the cDNA from the clones The library was screened by the method of plaque hybridization using morula-cDNA probes and prism-cDNA probes. Each of the stage- specific clones was amplified and isolated from the culture medium [9]. The cloned DNAs were extracted with phenol/chloroform, digested with EcoRI and then electrophoresed on a low melting- point-temperature agarose. The cDNAs were extracted from the gel slices with hot phenol and precipitated with ethanol. RNA determination To eliminate the color development by uronic acid and sialic acid, polysaccharides were removed from the samples by ethanol precipitation after hydrolysis with 0.3 N KOH. A 10% trichloroacetic acid-insoluble fraction of 5 x 10* eggs or embryos was defatted with ethanol and ether, and then hydrolized with 0.3 N KOH at 37°C for 12 hr to liberate ribonucleotides. After neutralizing the pH of the hydrolysate with 0.3 N HC1O,, polysacchar- ides were precipitated with two volumes of etha- nol. The amount of RNA in the supernatant was determined by the orcinol-FeCl; method [10]. Northern blot hybridizations Ten microgram each of total cellular RNA prepared from the embryos at various stages using the guanidinium/hot phenol method [3] was elec- trophoresed on formaldehyde-agarose gels and transferred to nitrocellulose filters [11]. Then RNA was immobilized on nitrocellulose filters and hybridized with nick-translated cDNA clones. RESULT AND DISCUSSION Preparation of acDNaA library from prism embryos To construct a library of cDNA clones which were expected to contain the RNA species that accumulated during development, the total cellu- lar RNA was prepared from prism embryos. About 1.5 mg of RNA was obtained from 1 ml of prism embryos. The amount of poly(A)*RNA obtained after oligo(dT)cellulose column (1 ml) chromatography was 30 ug, which corresponds to 2% of the total cellular RNA. The cDNA library was constructed from 40 ng of the double-stranded cDNA as described in Materials and Methods, yielding 6 X 10° plaques. Screening of stage specific genes About 2 X 10° clones were plated out on E. coli C600hfl*. The screening was carried out by differential plaque hybridization using the morula- cDNA and the prism-cDNA as probes. Among the prism-stage specific clones, four clones (AP1, AP2, AP3 and AP16) were selected and purified for further analysis. AP16 contained only one EcoRI fragment of cDNA (0.6 kb), while two size classes of the EcoRI fragment were found in the cDNA inserts of AP1, AP2 and AP3. The predicted sizes of the fragment by gel mobilities were 1.1 kb and 0.53 kb in AP1, 1.3 kb and 0.48 kb in AP2, and 0.80 kb and 0.36 kb in AP3. It is not clear at present whether the presence of two fragments in one cDNA clone reflects the multiple EcoRI sequences in a stretch of mRNA or different fragments recombined incidentally during the ligation process. The EcoRI fragments were tentatively designated here as AP1-1.1, AP1—-0.53, AP2-1.3, }P2-0.48, AP3- 0.80, AP3-0.36 and AP16-0.60. Northern analysis Prior Northern analyisis, the RNA content of sea urchin embryos during development was deter- mined. The amount of RNA was about 0.1 ng per embryo at all the stages examined in this experi- ment. It seems that the fraction of newly synthe- sized RNA was negligibly small in comparison with that of stored RNAs. As any significant changes in the RNA content of the embryos were not observed during development, a definite amount of RNA (10 yg) was loaded on each slots. Total RNA was extracted from the embryos at nine different developmental stages, and subjected Stage Specific Gene of Sea Urchin 741 3 S) e --— Blastula Gastrula Prism Pluteus RNA ( yig/10%embryos ) 0 10 20 30 407) 50a 00 Development (hr ) Fic. 1. Ontogenic change in RNA content of embryos during early development of H. pulcherrimus. The amount of RNA was assayed as described in the Materials and Methods. to Northern analysis as described in Materials and Methods. Since the AP3-0.36 probe scarcely hybridized with RNA from the embryos at any stages examined, it was difficult to determine the relative content of this RNA species, and the further experiments were carried out using the other clones. There was a single size class of mRNA corre- sponding to each clone with molecular lengths predicted by gel mobilities of 1.7 kb (AP1-1.1), 1.7 kb (AP1-0.53), 2.0kb (AP2-1.3), 2.0kb (AP2- 0.48), 1.8 kb (AP3-0.80) and 0.9 kb (AP16-0.60). The relative intensity of the bands in Northern blots was quantified by microdensitometric scan- ning of the autoradiograms (Table 1). Unfertilized eggs contained the RNA which hybridized with all of the clones used as probes, though the content of these mRNA species per egg was small. The intensity of hybridization decreased until the embryos reached the 32-cell or unhatched-blastula stage. RNAs corresponding to AP1—0.53, AP2-1.3, AP2-0.48 and AP16—0.60 began to increase at the unhatched-blastula stage while RNAs correspond- ing to AP1—-1.1 and AP3-0.38 started to increase later at the hatched-blastula stage. The concentra- tion of RNAs corresponding to AP1-0.53, AP2-1.3 and AP2-0.48 RNA decreased once at the mesen- chyme-blastula stage and then increased again. It seems that the RNAs detected in the early stages of development may be derived from the maternal stored mRNAs and those found in the later stages correspond to newly synthesized RNAs. General- ly, the level of RNAs represented by the present clones decreased by the blastula stage and thereaf- ter increased sharply. However, the onset of the increase in the individual RNA species occurred at differnt developmental stages, and the amount of each RNA species fluctuated in a different man- ner. The reproducibility of the data shown in Table 1 was confirmed by two to three separate experiments. It seems that the accumulation of the different RNA species is under a different pattern of control. TABLE 1. Abundance of individual mRNA species during development Clone AP1-1.1 AP1-0.53 AP2-1.3 AP2-0.48 AP3-0.80 AP16-0.60 Stage Percentage of specific RNA content in prism stage Oocyte 168) 0.60 1.0 1.8 i5) 13 2-cell 0.57 0.28 0.72 0.73 12 Ded) 32-cell 0.56 0.15 0.56 0.61 6.0 1:3 Unhatched bestia 0.51 12 4.0 3.0 0.47 Dal Hatched Blistala 9.8 ip) i ai} 10 2 Mesenchyme Binetala 17 15 7) 18 27 43 Gastrula 45 32 45 56 37) 96 Prism 100 100 100 100 100 100 Pluteus 95 69 86 is 80 94 The intensities of the bands in Northern blots were quantified by densitometry of the auto- radiograms. 742 The stage-specific difference in the complexity of the mRNA population during sea urchin de- velopment has been clearly shown by Galau et al. [12], suggesting that the expression of many kinds of genes is _ switched on-and-off during embryogenesis. Though the sea urchin genes for histone [13], actin [14] and collagen [15] have been cloned, the mechanism underlying the stage spe- cific expression of these genes during development has not been elucidated. In the present study, we have constructed a cDNA library from the sea urchin embryos at prism stage, and cloned some of the genes which mRNAs show a stage-specific mode of accumulation. Shepherd et al. have previously selected the stage-specifically expressed genes from a cDNA library of sea urchin embryos by colony differential hybridization using in vivo [**P]labeled RNA [16]. This method, however, seems inefficient for the screening of these genes. Here we have employed the plaque differential hybridization technique using in vitro [?*P]labeled cDNAs for screening of the genes more effective- ly. The cDNA clones obtained in the present experiment would be helpful for the molecular study of the regulation of gene expression during blastula-gastrula transition. ACKNOWLEDGMENTS We would like to thank Professor Masamiti Tatibana of the Chiba University for his encouragement. The present study was supported by Grant-in-Aid 61304009 for Scientific Research from the Japan Ministry of Education, Science and Culturte. REFERENCES 1 Giudice, G., Mutolo, V. and Donatuti, G. (1968) 16 K. AKASAKA, M. Taira et al. Wilhelm Roux’ Arch. Entwicklungsmech. Org., 161: 118-128. Giudice, G. (1986) The Sea Urchin Embryo. Sprin- ger-Verlag, Berlin, Heidelberg, New York, Tokyo. Feramisco, J. R., Helfman, D.M., Smart, J. E., Burridge, K. and Thomas, G. P. (1982) J. Biol. Chem., 257: 11024-11031. Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. USA, 69: 1408-1412, Verma, I.M., Temple, G.F., Fan, H. and Balti- more, D. (1972) Nature (New Biol.), 235: 163-167. Gubler, U. and Hoffman, B. J. (1983) Gene, 25: 263-269. Huynh, T.V., Young,R.A. and Davis, R. W. (1984) In “DNA Cloning”. Ed. by D. M. Glover, IRL Press, Oxford, Washington DC, Vol. 1, pp. 49- 78. Taylor, J. M., Illmensee, R. and Summers, J. (1976) Biochim. Biophys. Acta, 442: 324-330. Yamamoto, K.R., Alberts, B. M., Benzinger, R., Lawhorne, L. and Treiber, G. (1970) Virology, 40: 734-744. Mejbaum, W. (1936) Z. Physiol. Chem., 258: 117- 120. Lehrach,H., Diamond,D. Wozney,J.M. and Boedtker, H. (1977) Biochemistry, 16: 4743-4751. Galau, G. A., Klein, W. H., Davis, M. M., Wold, B.J., Britten, R.J. and Davidson, E.H., (1976) Cell, 7: 487-505. Mauron, A., Kedes, L., Hough-Evans, B., R. and Davidson, E. H. (1982) Dev. Biol., 94: 425-434. Merlino, G. T., Water, R. D., Moore, G. P. and Kleinsmith, L. J. (1981) Dev. Biol., 85: 505-508. Venkatesan, M., Pablo, F., Vogeli,G. and Simp- son, R. T. (1986) Proc. Natl. Acad. Sci. USA, 83: 6351=9355) Shepherd, G. W., Rondinelli, E. (1983) Dev. Biol., 96: 520-528. and Nemer, M. ZOOLOGICAL SCIENCE 4: 743-746 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan Met-Enkephalin-like Immunoreactivity in the Nervous System of Helix aspersa TRINIDAD OrTIZ, JOAQUIN PINERO and RAFAEL COVENAS Departamento de Biologia Celular de la Facultad de Biologia, Universidad de Sevilla, Avd. Reina Mercedes s/n, 41012-Sevilla, Spain ABSTRACT—By means of indirect immunocytochemi- cal method, met-enkephalin-like immunoreactivity was demonstrated in the cerebral and visceral ganglia of the snail, Helix aspersa. Immunoreactive neurons and nerve fibers of the neuropile were located in the ganglia. INTRODUCTION Enkephalin and (-endorphin were demon- strated in the neurons of the earthworm by Alumet et al. [1]. Rémy and Dubois [2] also reported that f8-endorphin immunoreactive cell bodies exist in the subpharyngeal ganglion of the earthworm and since then there have been investigations that have established the existence of immunocytological detection of methionine-enkephalin-like neuro- peptides in invertebrates [1]. In molluscs, by immunocytochemistry, met- enkephalin has been localized in the cerebral and pedal ganglia of Lymnea stagnalis, in the pedal ganglia of Mytilus edulis and Octopus vulgaris nerves [3, 4]. On the other hand, enkephalin-like immunoreactivity in a pulmonate gastropod, Achatina fulica, has been demonstrated [5]. In Helix aspersa, Williamson and Emson [6], in extracts of the nervous tissue, have detected met-enkephalin by radioimmunoassay. In the present study, we have localized met-enkephalin- like material by indirect immunocytochemical method in the perikarya and fibers of the snail Helix aspersa. Accepted March 16, 1987 Received December 27, 1986 MATERIALS AND METHODS The snails, Helix aspersa, were collected from Aljarafe, south Spain region, in October. Cere- bral and visceral ganglia were removed and fixed for 2-4hr in paraformaldehyde in 0.15M phos- phate buffer at pH 7.4 containing 0.342% L-lysine and 0.055% sodium periodate, embebbed in paraf- fin and cut serially at 7 4m. All the sections were treated for 10 min with 10% H>,O, in methanol, to inactivate endogenous peroxidase activity before incubation with immunohistochemical reagents. Several sections were pretreated for 20 min at 37°C in a solution of pepsin (4mg/ml), in 0.01N HCl, to eliminate background reaction. The sections were stained according to the immunoperoxidase indirect method. Met- enkephalin antibodies were prepared as previously described by Arluison eft al. [7]. Briefly, the peptide was coupled to haemocyanin with glutaral- dehyde and injected repeatedly every month into rabbits. Antibodies were purified from collected sera by affinity chromatography onto a met- enkephalin—CH-sepharose 4B column. With the radioimmunoassay conditions of Cesselin et al. [8], we found that the met-enkephalin antibodies presently used exhibited only very limited cross- reactivity with other peptides: 0.4% with leu- enkephalin, 1.7% with $-endorphin, less than 0.01% with substance P, cholecystokinin- octapeptide, vasoactive intestinal peptide, dynor- phin and FMRF-amide. The sections were incubated for 20hr in a solution containing met-enkephalin-antiserum (1:10°), Triton X-100 (0.3%) and normal goat 744 T. Ortiz, J. PINERO AND F. CovENAS serum (1%) in 0.15M phosphate buffer at pH 7.4. The site of antigen-antibody reaction was re- vealed by goat anti-rabbit IgG, labelled with peroxidase, at a concentration of 1: 250. The specificity of the reactions was verified by a) omitting the first antiserum in the incubation medium and b) by using a previously absorbed antiserum with excess of antigen (250ug of met- enkephalin in 1 ml of antiserum diluted to 1: 10°) for 24 hr at 4°C. RESULTS Met-enkephalin-like immunoreactivity (ELI) was found in the nervous system of the adult Helix aspersa. Many neuronal cell bodies of different ae ere, ~ oe = : ‘as f Cree Vo * i * a < ; e . if ce # th, ? +7, Ry v4 + 4 : ‘oO Fic. 1. noreactive cell bodies. 200. * ~ 4» € oe y— Fic. 3. Cerebral ganglion. Met-enkephalin-like immu- Fic. 2. Cerebral ganglion. sizes were seen in the cerebral ganglia. Here, the perikarya showed intense enkephalin im- munoreactivity and several axonic eminences were also immunostained (Figs. 1 and 2). In the visceral ganglion several cell bodies showed enkephalin-like immunoreactivity as well as the fibers in the neuropile (Figs. 3 and 4). The majority of ELI cell bodies presented a narrow peripheral band of immunoreactivity (Fig. 5), but also, some neurons showed immunoreac- tivity in whole cytoplasm (Fig. 4). The enkephalin immunoreactive material appears in granular form. Both nuclei and fibers did not show significant immunoreactivity in control sections (Figs. 6 and Dye Higher reveals darkly immunoreactive perikarya. magnification x 500. , Visceral ganglion. Small (~@ ) and medium (*) size cell bodies showing immunoreactivity. 100. Met-Enkephalin in Helix aspersa 745 «a th. : par: tet tile . .< * - a - * é. De eine ue : > —— oS ~ ts = : 4 : ey 7‘ se Fic. 4. Visceral ganglion. Enkephalin-like immunore- activity in whole cytoplasm and fibers in the neuro- pile. 500. Fic. 6. Visceral ganglion. sorbed antiserum showing no Xx 100. DISCUSSION In the last few years, at various times, the presence of neuronal peptides in molluscs has been demonstrated [3, 4, 9]. However, by im- munocytochemical methods, the enkephalin was only found in the pedal ganglia of Mytilus edulis [4] and in the nerves of Octopus vulgaris [10]. In this paper, we describe the population of peptidergic neurons containing met-enkephalin-like substance in the cerebral and visceral ganglia of Helix aspersa. Many recent studies have demonstrated the functional role that the met-enkephalin plays in vertebrates, indicating that it can be related to Fic. 5. Section treated with preab- immunoreactivity. Visceral ganglion. Higher magnification of narrow peripheral band of immunoreactivity. x 500. > 2 a: <2 ae 2s } se aX : Soe hei ee ; 255 7 a RS : : ae pe hs Fic. 7. Visceral ganglion. Magnification from the neuropile demonstrating no ELI fibers. x 500. stress-analgesia, inhibiting nociceptive neurons lo- cated in the dorsal horn of the spinal cord [7, 11]; however, up to now, the function of this in molluscs has been a subject of controversy. In Helix pomatia it has been demonstrated that an increase of met-enkephalin increases the level of dopamine and so also increases the dopaminergic nervous transmission [12]. Gromov et al. [13] have demonstrated that enkephalin altered the effect of acetylcholine on postsynaptic acetylcholine recep- tors in the neurons of Helix pomatia. This suggests that opioid peptides take part in the regulation of synaptic transmission of some biogenic amines. Also, a naloxone-reversible action of met- enkephalin on the activity of single identified 746 neurons in Helix pomatia has been reported [14]. The study of Salanki et al. [9] strongly suggests that substance P has its own receptors in Helix pomatia, and also suggests that substance P and met- enkephalin antagonizes each other activity by separate mechanisms. This suggests that some of the activities of both peptides, substance P and met-enkephalin, occur in invertebrates in the same way as in mammals. However, the analysis of nervous systems like that of the snail, to gain more precise understanding of concepts such as neuro- transmission and neuromodulation, may prove to be a good means for providing the answer to many questions, such as how different neuropeptides act. REFERENCES 1 Alumets,J., Hakanson,R., Sundler,F. and Thorell, J. (1979) Nature, 279: 805-806. 2 Rémy, Ch. and Dubois, M. P. (1981) Cell Tissue Res., 218: 271-278. 3) Schot, 7k G. Boer, Hv He Swaab).Dak> and Van Noorden, S. (1981) Cell Tissue Res., 216: 373- T. Ortiz, J. PINERO AND F. CovENAS 378. Stefano, G. B. and Martin, R. (1983) Cell Tissue Res., 230: 147-153. Van Noorden, S. (1980) Gen. Comp. Endocrinol., 40: 375-379. Williamson, M.E. and Emson, P. C. Biochem. Soc. Trans., 10: 384-385. Arluison, M., Conrath-Verrier, M., Tauc, M., Mail- ly, P., De la Manche, I. S., Cesselin, S., Bourgoin, S. and Hamon, H. (1983) Brain Res. Bull., 11: 555- Swi. Cesselin, F., Hamon, M., Bourgoin, S., Buisson, N. and Devitry, F. (1982) Neuropeptides, 2: 351-369. Salanky,J., Vehouszky, A. and Stefano, G. B. (1983) Comp. Biochem. Physiol., 75C: 387-390. Martin, R., Frosch, D., Kiehling, C. and Voigt, K. H. (1981) Neuropeptides, 2: 141-150. Basbaum, A. I. and Fields, H. L. (1984) Ann. Rev. Neurosci., 7: 309-380. Juel, C. (1982) Neuropharmacology, 21: 1301- 1303; Gromov, L. A., Krivorotov, S. V. and Skryma, R. N. (1983) Neuroscience, 8(4): 855-860. Stefano,G.B., Vadasz,I. and Hiripi, L. (1980) Experientia, 36: 666-667. (1982) ZOOLOGICAL SCIENCE 4: 747-749 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan A Method of Measuring the Volume of Soft Tissue E11 YOSHIOKA Seto Marine Biological Laboratory, Kyoto University, Shirahama, Wakayama 649-22, Japan ABSTRACT—A convenient method for measuring the volume of soft tissue is reported. The volume can be calculated from the area x the thickness if it is flattened evenly. This was tried successfully for the gonad of chiton Acanthopleura japonica using a metal frame of 1 mm thickness. On the example of the chiton’s gonads, the calculated volume as well as the wet weight was shown to be correlated to the dry weight and the volume can be taken as a quantity of the tissue. When dealing with the majority of small inverte- brates, it is not an easy task to measure the weight of the organ, and a most precise balance and a laboratory free of vibrations are often necessary. Nevertheless, the wet weight is not so reliable because the extra water on the organ cannot be removed uniformly and with certainty. Desicca- tion of the material for taking the dry weight naturally makes it unsuited as an_ histological sample. In these respects, the volume is thought to be a good indicator of the size of the tissue. The rough volume can be grasped visually, but to measure it quantitatively some device must be introduced. In this paper, a method for measuring the volume of tissue, for example, as the numer- ator quantity of the gonad index is reported. The gonad of the chiton, Acanthopleura japonica was used for the experiment, which was easily sepa- rated from other tissues and so was appropriate for this method. A gonad placed on a glass slide was soaked in sea water, the isotonic medium to the body fluid of the marine invertebrate, to avoid problems which may result from osmotic pressure and drying. A Accepted March 16, 1987 Received February 17, 1987 metal frame of 1 mm thickness was secured encir- cling it and a cross-ruled glass slide for plankton counting was pressed over it so as to make the gonad evenly 1 mm thick (Fig. 1). The area the gonad covered was measured against the 1mm lattice. When the gonad is large enough, the number of the lattice points it covers may be counted, which mathematically appropriates the number of squares divided into the area of the gonad (for example, 22 points in the case shown in Fig. 1). The volume of the gonad was calculated simply by multiplying the area (in mm’) by the thickness (1mm). The wet weight of the gonad was measured after excess water was removed with filter paper, and its dry weight was measured after desiccation (90°C, 6 hr) to 10 mg. Figure 2 showed the relationships of the dry weight of the gonad to its volume and wet weight. The dry weight is generally admitted to be the most meaningful value among the three as an expression of the substantial size of the gonad. According to the values of correlation coefficient (r), the volume was shown to be correlated to the dry weight as well as the wet weight was shown. The balance used in this study (Parkin Elmer; Autobalance AD-2) is very expensive and re- quires careful handling. However, the apparatus here described is proof against rough handling in the field, and the cross-ruled glass slide is easily obtained from oceanographic warehouses or planktological laboratories. Moreover, the proce- dure of measuring the volume does not damage the organ for histological preparation. The thickness of the inserted frame and the square size of cross-rule can be modified to measure various organs of different sizes. 748 E. YOSHIOKA Imm Fic. 1. The apparatus and procedure for measuring the volume of the gonad. = (es) dry weight (mg) (Sa) 10 20 30 volume of gonad (mm?) (a) dry weight (mg) 20 40 wet weight of gonad (mg) (b) Fic. 2. The relationships of the dry weight to the volume (a), and to the wet weight (b) of the gonad of the chiton Acanthopleura japonica. (a) r=0.970; (b) r=0.963. As Grant and Tyler [1] pointed out, studies of invertebrate reproduction are expensive in both time and money. Many researchers of invertebrate reproduction use the gonad index, or the ratio of wet or dry weight of gonad to the total animal weight, as the indicator of reproductive activity [2]. The method in this study, as expected, allows studies in reproductive biology to be effectively and economically carried out. REFERENCES 1 Grant, A. and Tyler, P. A. (1983) Int. J. Invertebr. Reprod., 6: 259-269. Measuring Volume of Tissue 749 2 Giese, A. C. and Pearse, J. S. (1979) In “Reproduc- Giese and J. S. Pearse, Academic Press, New York, tion of Marine Invertebrates, Vol. 1”. Ed. by A.C. pp. 1-49. m quem ; ee An Invitation to Join the Zoological Society of Japan The Zoological Society of Japan was founded in 1878 for the purpose of discussing and exchanging information pertaining to the biology of animals. 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(Photo: by courtesy of Olympus Optical CO., LTD) | SOME FEATURES of MO-102 and MO-103: | * The manipulator head is so small that it can be mounted directly on the | microscope stage. There is no need for a bulky stand. | * Hydraulic remote control ensures totally vibration-free operation. * 3-D movements achieved with a single joystick. Micromanipulators Microelectrode pullers Stereotaxic instruments NARISHIGE SCIENTIFIC INSTRUMENT \ eas / LABORATORY CO.,LTD. 4-9-28, Kasuya, Setagaya-ku, Tokyo 157 JAPAN Telephone: 03-308-8233 Telex: NARISHG J27781 (Contents continued from back cover) Morphology Yoshioka, E.: A method of measuring the volume of soft tissue (COMMUNICATION) 747 Environmental Biology Endo, K. and M. Shibata: A circadian aspect of the photoperidoic time-measurement on the basis of the larval-ecdysis rhythm in the small copper butterfly, Lycaena phlaeas daimio Seitz Ecology Kasuya, E., H.Shigehara and M. Hirota: Mating aggregation in the Japanese treefrog, Rhacophorus arboreus (Anura: Rhacophor- idae): a test of cooperation hypothesis ...693 Taxonomy Uchida S. and H. Maruyama: What is Sco- pura longa Uéno, 1929 (Insecta, Pleco- ptera)? A revison of the penus ......... 699 Nagatomi, A.: Taxonomic notes on Coenomyiidae (Insecta: Diptera) ........ Tila Sawada, I.: Further studies on cestodes of Japanese bats, with descriptions of three new species of the genus Vampirolepis (Cestoda: Hymenolepididae) ZOOLOGICAL SCIENCE VOLUME 4 NUMBER 4 AUGUST 1987 CONTENTS REVIEWS Grunz, H.: The importance of inducing fac- tors for determination, differentiation and pattern formation in early amphibian de- velopment; | ie. 2a) carey er ener SI Uéno, S.-I.: The derivation of terrestrial cave SMiMAlS: dad Ae Ao 593 ORIGINAL PAPERS Physiology Higuchi, T., H. Nakamura, K. Sawauchi and H. Okumura: Frequency block in the giant axons of a sabellid worm, Pseudopotamilla occelata Cell Biology Gomi, T., A. Kimura, H. Tsuchiya, T. Hashi- moto, K. Higashi and S. Sasa; Electron microscopic observations of the alveolar brush cell of the bullfrog Ogawa, M., K. Terakado and J. Okada: Ori- gin of binucleate cells in the neural gland of the ascidian Halocynthia roretzi (Drasche) (COMMUNICATION) Genetics Ikebe, C., K. Aoki and S. Kohno: analysis of two Japanese salamanders, Hyno- bius nebulosus (Schlegel) and Hynobius dun- ni Tago, by means of C-banding Frankel, J.S.: Asynchronous expression of alleles at the alcohol dehydrogenase locus during Oryzias hybrid development (COM- MUNICATION) Karyotype Immunology Kaiho, M. and I. Ishiyama: The distribution of A and B blood group antigens in tissues of the frog, Rana catesbeiana INDEXED IN: Current Contents/LS and AB & ES, Science Citation Index, ISI Online Database, CABS Database Developmental Biology Mizukami, S.: Further observations on divi- sion pattern of binucleate fish embryonic cells Suzuki, N., M. Kurita, K. Yoshino and M. Yamaguchi: Speract binds exclusively to. sperm tails and causes an electrophoretic mobility shift in a major sperm tail protein of sea urchins Suzuki, N., M. Kurita, K. Yoshino, H. Ka- jiura, K.Nomura and M. Yamaguchi: Purification and structure of mosact and its derivatives from the egg jelly of the sea urchin Clypeaster japonicus Tanimura, A. and H.Iwasawa: Germ cell kinetics in gonadal development in the toad Bufo japonicus formosus Hori, R., V. P. E. Phang and T. J. Lam: Pre- liminary study on the pattern of gonadal development of the sea urchin, Diadema setosum, off the coast of Singapore Akasaka, K., M. Taira, T. Shiroya and H. Shi- mada: Cloning of stage specific gene sequ- ences from a cDNA library representing poly(A)*RNA of sea urchin prism embyros (COMMUNICATION) Endocrinology Jokura, Y. and A. Urano: Extrahypothala- mic projection of immunoreactive vasotocin fibers in the brain of the toad, Bufo japonicus Ortiz, T., J. Pinero and R. Covenas: Metenkephalin-like immunoreactivity in the nervous system of Helix aspersa (COM- MUNICATION) (Contents continue on inside back cover) Issued on August 15 Printed by Daigaku Printing Co., Ltd., Hiroshima, Japan ZOOLOGICAL SCIENCE The official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Hideshi Kobayashi (Tokyo) Toshin-building, Hongo 2-27-2, Bunkyo-ku Managing Editor: — ae Tokyo 113, Japan. Tel. (03) 814-5675 Seiichiro Kawashima (Hiroshima) Officers: Assistant Bditors: cen President: Nobuo Egami (Tsukuba) Takeo Machida ; (Hiroshima) Secretary: Yasuto Tonegawa (Urawa) Sumio Takahashi (Hiroshima) Treasurer: Tadakazu Ohoka (Tokyo) Librarian: Shun-Ichi Uéno (Tokyo) 5) Editorial Board: Howard A. Bern (Berkeley) Walter Bock (New York) Aubrey Gorbman (Seattle) Horst Grunz (Essen) Robert B. Hill (Kingston) Yukio Hiramoto (Tokyo) Susumu Ishii (Tokyo) Yukiaki Kuroda (Mishima) Koscak Maruyama (Chiba) Roger Milkman (Iowa) Hiromichi Morita (Fukuoka) Kazuo Moriwaki (Mishima) Tokindo S. Okada (Okazaki) | Andreas Oksche (Giessen) Hidemi Sato (Nagoya) Hiroshi Watanabe (Shimoda) | Mayumi Yamada (Sapporo) Ryuzo Yanagimachi (Honolulu) ZOOLOGICAL SCIENCE is devoted to publication of original articles, reviews and communications in the broad field of Zoology. The journal was founded in 1984 as a result of unification of Zoological Magazine (1888-1983) and Annotationes Zoologicae Japonenses (1897-1983), the former official journals of the Zoological Society of Japan. ZOOLOGICAL SCIENCE appears bimonthly. An annual volume consists of six numbers of more than 1000 pages including an issue containing abstracts of papers presented at the annual meeting of the Zoological Society of Japan. 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New subscriptions and renewals begin with the first issue of the current volume. All rights reserved. No part of this publication may be reproduced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1987, The Zoological Society of Japan Publication of Zoological Science has been supported in part by a Grant-in-Aid for Scientific Publication from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 4: 751-761 (1987) REVIEW Fine Structure of Ascidian Smooth Muscle KIyvosHI TERAKADO Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 338, Japan I. INTRODUCTION The body-wall muscle of the ascidian Halocyn- thia roretzi is a vertebrate-type (paramyosin-free type) smooth muscle [1], possessing an actin- linked regulatory system of contraction, which is the case with that of vertebrate striated muscle. In this review, observations on the structural or- ganization of the ascidian smooth muscle, with special reference to contractile structures, will be discussed, and comparisons are made between the organization of vertebrate smooth and striated muscles (for vertebrate smooth muscle, see ref. [2-5]). II. GENERAL ARCHITECTURE The body-wall muscle consists of two layers, an inner longitudinal layer and an outer circular one [1]. Both layers of muscle consist of numerous bands of muscle separated from each other by a thick layer of connective tissue filaments. Each band is about 50-150 ~m in width and contains about 10-100 bundles of muscle fibers. Each bundle is further composed of several muscle cells arranged longitudinally and is completely covered by a thin layer of connective tissue filaments and a basement membrane (Fig. 1) [6]. The cells within each bundle are arranged side by side in very close proximity, in a type of simple apposition and behave like a single cell at contraction. This grouping of cells into bundles in the ascidian smooth muscle contrasts with the situation in most vertebrate smooth muscles which consist of indi- Received May 25, 1987 © 1987 Zoological Society of Japan Cc Cc = NO co ‘Oo co co vidual cells separated by collagen fibrils and the basal iamina. These recent findings indicate that the bundles of cells, rather than the individual cells, are the structural and functional units in the muscle tissues. il. CELLULAR FEATURES 1. Multiple Nuclei The cells in the body-wall muscle are very long, cylindrical structures, tapering at each end. Most individual, isolated cells are about 1.5-2 mm in length and fixed cells are about 1.3-1.7 mm in length. Each cell contains about 20-40 nuclei, the number being proportional to the length of the cell [6]. Multinucleate smooth muscle cells, which are very rare in vertebrates and invertebrates, were found first in the ascidian smooth muscle [7], and their presence was confirmed in the same material by Shinohara and Konishi [8] by a three- dimensional reconstruction method. Aside from the ascidian body-wall muscle, the giant smooth muscle cells of ctenophores are the only known examples of such multinucleate smooth muscle cells [9, 10]. The presence of multiple nuclei is apparently related to elongation of the cells, as in the case of vertebrate skeletal muscles, but the mechanism for the generation of multiple nuclei remains to be clarified. 2. Myofilaments Recent ultrastructural studies of smooth muscle revealed that both actin and myosin are persistent- ly present in filamentous form. Thick (myosin) and thin (actin) filaments are about 14-16 nm and 52 K. TERAKADO Fic. 1. muscle (double fixation). Cross-section of a small bundle of muscle fibers from the outer layer of the ascidian body-wall Glycogen particles have been lost from the cytoplasmic matrix. Basement membrane (arrow) and connective tissue filaments fully cover the bundles of fibers, but not individual cells. Contractile elements tend to lie toward the outer surface of the bundle. Bar, 1 wm. (From Terakado and Obinata [6]). 6-7nm in diameter, respectively, which are dimensions very similar to those of vertebrate smooth and striated muscles. In transverse sec- tions of relaxed muscle cells, the population densities of thick and thin filaments are about 220-330 and 1300-2300 filaments per pm7, respec- tively (Fig. 2) [6]. The former value is much higher than in vertebrate smooth muscles, but is lower than in striated muscles (Table 1). The latter value is nearly identical to that in vertebrate smooth muscles. The ratio of thick to thin filaments is about 1:6, which is intermediate between the ratios of those filaments in vertebrate smooth and striated muscles. The thick filaments are often arranged in a quasi-rectangular or quasi-hexagonal lattice; each thick filament is always surrounded by a single row of 5-9 thin filaments, to produce a rosette in transverse section (Fig. 2, inset) [6]. The distances between the thick filaments arranged in the lattice are about 30-50 nm, distances more similar to those in striated muscles than those in vertebrate smooth muscles. Cross-bridges, with a repeat period of 14.5 nm or 29nm, between thick and thin filaments, can also be identified. In smooth muscles, it was noted sometime ago that the identification of cross- bridges in sections is generally difficult, presum- ably because of the less regular alignment and greater spacing between adjacent thick filaments, in addition to other reasons [11]. The ease with which cross-bridges can be observed in the present material probably reflects the population density of thick filaments which is much higher than in vertebrate smooth muscles. The periodicity of about 14.5 nm is identical with the results from X-ray diffraction [12, 13], optical diffraction [14], and electron-microscopic observations of isolated native and synthetic myosin filaments [13]. The exact length of the thick filaments has not been determined in smooth muscles. In the ascidian smooth muscle, the length appears to be about 2-2.4 um, based on the length of A-zone (see below) and on measurements of some long filaments in longitudinal sections (Fig. 3) [6]. Structure of Ascidian Smooth Muscle 753 ERRORS i ERAGE NG Orit toa J a a by hE. seni gitan en ye 2 Ay ee 2 ¢ st os] y) komt ns ede ARN” “yae.k Sex a CA a: ny > ty vy * * a Ke. AES iat ba fi Pie Webby Sah: au gcc a *. * igs shy xe 2, Ri y 2s Oa Zon A See Hg: % 3 Fes CBX Ne n ys + 4 Pea rea ERE ALY at 2 << . * RA. SO ey ee ue . ~ * ee. ve eal y AIP WER Ket TAN Sas kaha Ae BVA Rap./ } y Bar a hay + ghey Semel Ke Wait bes * ee os Ws vo a it By 3 iy frees K j hes rae pe ‘ p hd aes a¥ : Sk eee ia cs ie hy SE aac. ue be ia ‘* AD ‘i - Aid ‘ ne 3 Wes M are it 3 uke a a, z i rem, gk mi Fic. 2. Cross-section of a cell from the outer layer of the ascidian body-wall muscle in a relaxed state. Dense bodies (DB) are oval structures in which numerous thin filaments are evident. Groups of thin filaments (circled), having diameters similar to those of the dense bodies, are also found. Intermediate filaments (arrowheads) either lie between the groups of thick and thin filaments or are associated with the dense bodies. Note the abundance of thick filaments. Bar, 0.2 ~m. Inset: Each thick filament is surrounded by a single row of several thin filaments. Cross-bridges are seen between them. Bar, 20nm. (From Terakado and Obinata [6]). x a5 ue 4s Si eee 5B tte gd Ne op MT 754 K. TERAKADO This value is similar to that estimated from intermediate high voltage electron microscopy [15]. However, isolated thick filaments from guinea pig taenia coli are of variable length, but most are less than 3 um in length [14]. However, thick filaments of smooth muscles are evidently longer than those of striated muscles. The exact length of thin filaments has also been unsettled. The isolated, native, thin filaments of the ascidian smooth muscle are about 1-2 um long as measured by electron microscopy, and 1.4—2.2 um in solu- tion, as estimated by a flow birefringence method [1]. In some vertebrate smooth muscles, isolated thin filaments have been found to be at least 1.2 ym [16] and as much as 4 wm [17] long. It has not been ascertained whether the variability in length of isolated filaments is a result of fragmentation during isolation procedures, or whether it reflects Fic. 3. on both sides of an I-zone (I). true variability among filaments. Other possible reasons for the variability in length may be related to differences in tissues and/or species, and cannot yet be eliminated. 3. Contractile Apparatus Presence of myofibrils in smooth muscle cells was suggested by earlier observations under the light microscope (see [4]). However, such fibril- lar structures have not commonly been noted by electron microscopy, in spite of the great number of investigations; these structures are only found in trypsinized cells or in trypsinized and detergent- treated cells (see [4]). It is probable that the proteolytic and/or detergent treatments enhance the fibrillar structures by extraction of some filaments. In the ascidian smooth muscle, distinct fibrillar structures (myofibrils) with a diameter of Longitudinal-section of part of a group of myofibrils from a cell of the outer layer, showing formation of an A-I zone. Thin filaments which emerge from dense bodies are inserted into small groups of thick filaments (A) Intermediate filaments run through spaces in the continuous network of myofibrils and run across myofibrils along I-zones exclusively (arrowheads). Bracket indicates width of a single myofibril. Bar, 0.2 um. Structure of Ascidian Smooth Muscle pays) about 0.2—0.6 «am are observed without such treat- ments, and are seen more easily in contracted cells. The myofibrils are repeatedly branched and fused, and run along almost the total length of the cell. They are separated from each other by interfibrillar spaces and a network of intermediate filaments which run through the spaces (Fig. 3) [6]. Dense bodies, with a diameter of about 0.06-—0.1 yam, are evenly scattered throughout the myofi- brils, but very often the dense bodies are located near the lateral surface of the myofibrils. From these structural features, it is apparent that the myofibrils exist as distinct structural units of the contractile elements in the ascidian smooth muscle cells, and presumably such is also the case in vertebrate smooth muscles. Within the myofibrils, sub-groups of more reg- ularly aligned, parallel, thick and thin filaments can be identified (Fig.3) [6]. In longitudinal sections, it can be seen that the thin filaments are inserted into both sides of the dense bodies, and that the thin filaments which emerge from the dense bodies enter into adjacent groups of thick filaments on both sides, to form a sarcomere-like structure (Fig. 3) [6]. In a given sarcomere-like structure, the group of thick filaments (with the inserted thin filaments) is termed the A-zone, and the group which contains only thin filaments is termed the I-zone, by analogy with the A- and I-bands of myofibrils of striated muscle, even though they are not arranged exactly in parallel. A dense body lies in the center of each I-zone. However, areas containing thick filaments exclu- sively (H-zone) are not found. The A-zone is about 2.2—2.5 ~m in length and 0.1-0.2 um in width in the relaxed state. The length of the I-zone is about 1-2 ~m. In transverse sections, many groups of about 25-50 thin filaments, which are apparently cut-planes of the I-zones, can be identified (Fig. 2) [6]. In vertebrate smooth muscles, the association between dense bodies and thin filaments, which Suggests that the cytoplasmic dense bodies are functionally equivalent to the Z-bands of striated muscle, was proposed sometime ago but has only recently been unequivocally demonstrated [18, 19]. The finding of an A-I band pattern [6, 18], together with the association of thin filaments with dense bodies, indicates that the minimum func- tional units of the contractile apparatus in smooth muscle are the sarcomere-like structures, which consist of interdigitating thick and thin filaments with attached dense bodies. Dense patches (surface dense bodies) on the cell surface, where the thin and intermediate filaments are attached, vary greatly in shape and in the extent of differentiation, from area to area and from cell to cell. In general, in cells of the outer circular muscle, the dense patches are only thin, narrow layer of cell surface with indistinct borders, and, therefore, it seems that the thin filaments are attached to a lesser extent to the lateral surface of the middle part of these cells. However, in cells of the inner longitudinal muscle, the inner surface of the cell membrane is richly covered with dense fibrous material, which increases in abundance toward the tapering ends of the cells, and to which numerous thin filaments are attached. With respect to establishment of the association of thin filaments with dense bodies, an important question arises concerning the presence or absence of “free” thin filaments which do not associate with cytoplasmic dense bodies or surface dense patches. From the ratio of the cut-plane area of a sarco- mere-like structure to that of a myofibril, calcu- lated from the widths of myofibrils (about 0.2-0.6 yan) and sarcomere-like structures (about 0.1-0.2 yan), it seems that there may be about 2-16 sarcomere-like structures in the cut-plane of a single myofibril. This value is very similar to the number of groups of 25-50 thin filaments (about 3-13) in a cut-plane of a single myofibril, calcu- lated from the ratio of number of thin filaments in a group of thin filaments (25-50) to the number of thin filaments in a myofibril (72-650, estimated from population density, 2300/m7, in areas ex- cluding interfibrillar spaces). This agreement means that the number of thin filaments in a single I-zone corresponds to that in a single sarcomere- like structure, and that practically all the thin filaments are attached to dense bodies. From this, it is further suggested that the thin filaments emerged from opposing dense bodies are not much overlapped (or separated) in the central region of the sarcomere-like structure in relaxed state, if 756 K. TERAKADO they have a definite length. This is compatible, in a large measure, with the lengths of isolated, native, thin filaments [1]. In spite of the fact that practically all thin filaments are presumed to be attached to the dense bodies, many “excess” thin filaments, which do not participate in the formation of rosettes, occur between the rosettes and around the lattices of thick filaments in transverse sections (Fig. 2). This result may be explained as follows. Since most thick filaments do not lie in isolation, but are predominantly arranged in quasi-rectangular [11] or quasi-hexagonal lattices, in which each thick filament is surrounded by a single row of about 7 (mean value) thin filaments which form a rosette in transverse section (Fig. 2, inset) [6], some “excess” thin filaments, of which the number would depend on the size of the thick filament lattice, should be present. However, such a clear separation of thin filaments into rosette-forming and “excess” fila- ments is unlikely in such a system. The observed “excess” of thin filaments may actually be due, ina large measure, to the partial association of each thin filaments with a thick filament(s), which may occur to a variable extent (Fig. 4) [6]. From these data, a tentative model of the sarcomere-like structure in the smooth muscle cell has been proposed [20]. In this model, each thin filament is partially associated with two or more thick filaments, via cross-bridges. At contrac- tion, the sarcomere-like structure shortens from about 3.5-4.5 ~m to about 2.5 um, as a result of the shortening of the I-zone. This reduction in length indicates the presence of a sliding filament mechanism as a fundamental aspect of smooth muscle contraction. 4. Intermediate Filaments It is well known that intermediate filaments in smooth muscles occur in significant quantity and associate with dense bodies [6, 14, 15, 18, 19, 21-26]. Recent investigations [6, 15, 18, 19] have provided further evidence that the dense bodies Fic. 4. Longitudinal-section of part of myofibrils from a cell of the outer layer, showing the presence of a long thick filament (arrows indicate both ends). Cross-bridges occur along the entire length of the thick filament. By contrast, each thin filament associates only partially with a thick filament(s). Arrowheads indicate thin filaments partially associ- ated with two thick filaments in different regions. Bar, 0.1 um. (From Terakado and Obinata [6]). Structure of Ascidian Smooth Muscle 757 are not only the sites of lateral attachment for intermediate filaments, but are also the sites of attachment of thin actin filaments, through which the contractile apparatus is mechanically inte- grated within the smooth muscle cells. Neverthe- less, the relationship between the localization or passage of intermediate filaments and the actual organization of the contractile apparatus was not well understood, mainly because of the paucity of information about the structure of the contractile apparatus of smooth muscle cells. In the ascidian smooth muscle, an extensive network of in- termediate filaments, which interconnect the cyto- plasmic dense bodies and connect the dense bodies to the cell surface, is easily visualized in double- fixed, tannic acid-stained preparations. The intermediate filaments are approximately 9-10 nm in diameter and usually occur in groups in interfibrillar spaces (Fig. 2) [6]. A small number of intermediate filaments is also present in the intrafibrillar spaces. In transverse sections, there are about 35-65 (mean value 50) intermediate filaments per «am of the area occupied by filaments (about 2.5% of all filamentous components). The number of intermediate filaments attached to the dense bodies varies greatly from 3 to 20 (mean value 9). In general, large numbers of intermedi- ate filaments are attached to the dense bodies which are located on the surface of myofibrils, but fewer filaments are attached to the dense bodies in the interior of the myofibrils. The patterns of interconnection between the dense bodies also vary, apparently because of the irregular arrange- ment of dense bodies in the myofibrils. The network of filaments runs through spaces in the network of myofibrils, connecting them longitudi- nally, obliquely and transversely in the vicinity of the dense bodies, to form an intimately associated, dual network. In their transverse passage, the intermediate filaments run across myofibrils, along the I-zones exclusively, interconnecting successive dense bodies (Fig. 3). The attachment of intermediate filaments to dense bodies does not neccessarily occur along the long axis from one end to the opposite end, but is frequently partial and oblique, and occasionally transverse. Furthermore, in most cases, the fila- ments associate with only one side of the lateral surface of dense bodies. The predominantly one- sided association of intermediate filaments with cytoplasmic dense bodies is different from that in vertebrate smooth muscles, where intermediate filaments often form a circle around the dense body in transverse sections [15, 18, 25]. The one-sided association seen in the ascidian smooth muscle seems to be primarily related to the organization of distinct myofibrillar structures and the predominant interfibrillar passage of in- termediate filaments, which makes possible the lateral, one-sided association of the filaments with dense bodies. Such a mode of association may be effective for the establishment of an efficient contractile apparatus, since the non-contractile, intermediate filaments are clearly separated from the contractile apparatus, although the filaments are associated laterally in the regions of the dense bodies. Thus, the intermediate filaments should disturb only minimally the cycles of contraction and relaxation and remain folded and unfolded in interfibrillar spaces [6]. The transverse and oblique interconnection of neighboring myofibrils or sarcomere-like struc- tures in the regions of the dense bodies, and the longitudinal interconnection of adjacent dense bodies by the network of intermediate filaments, reveal close similarities in structure and possibly in function to striated muscles (see [27]). The most probable explanation for such a continuous net- work of intermediate filaments in the ascidian smooth muscle cells may be that it is involved in the mechanical maintenance of the ordered arrangement of the contractile apparatus, and that it acts to facilitate the restoration of the original arrangement after contraction, via the intimate association of the network with the organized contractile apparatus and the cell surface. 5. Sarcoplasmic Reticulum Sarcoplasmic reticulum is poorly differentiated in the cells of ascidian smooth muscle and is almost absent in the filamentous area. However, many small tubules and oval vesicles are characteristical- ly found just beneath the cell membrane. The tubular elements of the sarcoplasmic reticulum are agranular, with filamentous material in the lumen, and are always coupled to the cell membrane. gjqeis AJOAe]OI [61] ‘[gt] (sonbeyd ssuap) 0RjINs [[90 puke soIpog ssueq 21qeI1S aoejins [Jao pue spueq-7 wu ge == [oe] 0€-Sz:T Gal [Se=CG Se Bl ell SI —OF ap U7 /00TT—-006 Unt /00bZ-O00T wT [LT] wr7p 0) dn ‘[gq] ur/ ZT] jsvaq ye wu /-9 wu /-9 91qe1s oTIqe| WU ¢"pT wu p'p1-P1 zur7 /OSb-OSE [te] -ur//ost-o8 ‘[IT] -4//09T oo irl ar deuce acd ioe WU 9[-ZI wu 8[—-CI K. TERAKADO (y)8us] ut u/ ¢-7) [st] (yy3ua] ut w/ g-¢—¢°Z) dJIDWOOIe DINJONIAJS JYlT]-sIOWIOIIC S [st] 4H S (peyuuopt jou Ayje1ouo3) [6z ‘pt] (ue/s-0-€'0) Tqyokyy [129 & Jo IajUaD (wr Z—¢°0) THQyokW ][99 eB Jo IawUudD = ‘Aaydited Jaq (w/ 001 /1-S'0) ‘wr NOT /OI-€ (we 001 /1-Z'0) I ‘spuesnoy} 0} spolpuny iI un (7-S ‘u/ OOT-OT ww (07-7 ww [°0-S0'0 “WU (S-T WU ¢‘0-T'0 S][29 JENPIAIpUI Jo s}sIsuOd «5][29 [ENPIAIPUI Jo S}sIsuOd AT]SOU ‘WI OBIpIe| “WI [2}9[94S QPSNUI poles 91e1GQ9119A spPsNnu YJOOWS dJ¥IGIIIA 758 gqeis ATOAeIOI SO[DISOA pue oorjins [[99 ‘soIpog osueq [9¢] wu g¢ 9:1 zw /00E€T-ODET [1] w/77-p] 10 we 7-] [g] wug ‘wu/-9 aTIqey WU Cp] zur /(QLZ URDU) YEE-O7ZZ uw 7-7 [g] wugz-gt ‘wu 9-41 (yIpim ul u/7'—-T'0 pue ‘ysuo] ul ur/¢-p—-C"¢) dINJONIYS dy¥I]-I1IOWIOIIVS (opm ww 99-70) TqQyosW Azaydited a9 ww NOT /Z Or-02 [g] wr O¢-O1 [g] wuz ‘wu 7-T S][99 JO so[pung jo S}SISUOD “uy 07-S¢ QJSNUI YJOOUIS UeIPIOSy SOPSNU Pojelljs PUB YJOOWS 9}eIQILIOA JO 9SOY} YIM J[ISNUI YIOOUIS ULIPIOSe 9} JO SONSIa}JOeIeYS [eINJONIyS 9WOS JO uostieduroo V Ayyiqeis SOPS JUSUTYILNV uluodol} Jo UONezIeI0] JO [eAIO}U] SJUOWIe[Y uly 0} YoIy Jo oneY Ayisuop uoneindog yysuaT Io}oUIeIG sjuswely (ude) UI], AyyIqeis uondofoid je1rajey Jo AyoIposg Ayisuop uonejndog yysuaT] I9}oWIeIG sjusweyy (UIsOAW) YoIUL, snjeredde g]t0e1]UOD Jo JUN WINUTTUTYY snjyeredde anoe1U0D Tgjonu JO UOTeZI[eIOT lajonu jo Ayisuap uonejndog ]Jo0 Jad tojonu jo Joquinyy YIPIM [19D ysus] [12D 91N}99}TYoIe ONSSLT, ‘T a1av 759 Structure of Ascidian Smooth Muscle ‘So[DSNUI Pd}eII]S PUL YJOOWS 93¥.1Q9}I9A IOj BIep [TV (wia3shs_ ulsoAwiodo.-utu0dod}) poyuly-unse ysty (popunolins) spueg-Z wooelpe joouU0DI93UT Ayquenb ut poyuwiy sjliqyoAul 0} Ajasoyo poyoene ‘payenuorayyip ATysIY BUIWIZJOOIVS 9y} YeIUNqG pue s[liqyoAul Us9MjOq JURpUNGe ‘[8z] s]]Je9 Jo sajpunq Jo jsIisuod pue uoNdasxo ue ole SofOSNU JOYyeoJ URIAV , "SOJOIIL MOIIADI PUL SYOOQ}X9} WOIJ Udye} o1e ‘pdyeoIpul ssoyuN (uleyo 1YsI] UIsOAU ® Jo Wajshs uoe|AIoydsoydap -uolje[Aroydsoyd) poyurly-ulsoAw Jo (wi9}sKs ulsoAwodo1}-uoWsopyed IO -UIU0}019]) payuT]-uyoe porea [Sz ‘81 ‘ST] popunosins SdIpOg asUap jO9UU0II9}UI juepunqe Jood Ajje1oue3 Axoydised |ja0 pue UOIda1 IBdTONULIOd UI pa}zed0] ‘AUeUT [st] suo] Ww ¢ [-¢°0 pue opIM wT" [g¢] wu QZ-0T [ge] wu cpr "WI OvIpPIeD “WI [219]94S QJISNUI Poj}eII}S 9}e1Q9}I9A, [11] wu6 SFL sz [L¢] wu 0g x09 QJOSNUI YJOOUIS 9}e1G9}19A (penunuoD) [{] (uroqsAs ulsoAuiodo4}-utu0do1}) poyury-uoe snowoud pepis-ouo ApUeUTWOpoid SoIpoq ssusp jD9UU0D19}UT juepunqge ueIquIOUI [[29 943 03 paydnos Apsour ‘100d Azaydiiod |[99 Ul pojes0] Joquinu Ul Moj opiM wi7/T"9-90'0 “Teao wu 9¢-81 wu (S-O¢ gOsnuUl YJOOWS uUeIPlsy ‘[ a1av yb ‘[9] eyeUIQO pue opeyeiay, WoOIJ UdyZe} O1e ‘pojedIpUT Sso[UN ‘o]DSNUI YJOOUIS ULIprOse ay} jnoge eIeVq (wi9}sAs A10}e[NSdy) saponied uasod/]{s Jo }U9}U0D, SOIPO dSUSpP YIM UOT}LINOSSe JO SPO|| sJUSWe]Y 9}eIpsw3}uy WIN]NdIVa1 stuIse[dooses BLIPUOYION IY SoIpog ssusq s}usWIe]Y UI) pue Yyory) UdIMJ9q DOULISIG s}USWIeTY Yor) U99Mj9q JoURISIG 760 K. TERAKADO The coupled elements of the sarcoplasmic reticu- lum are sometimes continuous with uncoupled elements which are often granular. Vesicles that open to the cell surface, which are common in cells of vertebrate smooth muscle, are rarely found. Oval vesicles are also found in groups in the perinuclear region. Information on the structure of ascidian smooth, vertebrate smooth and vertebrate striated muscles is summarized in Table 1. IV. SUMMARY 1) The cells of the ascidian smooth muscle are multinuclear. Each cell contains 20-40 nuclei, the number of nuclei being proportional to the length of the cell. 2) The population density of thick filaments and the ratio of thick to thin filaments in the ascidian smooth muscle are intermediate between those in vertebrate smooth and striated muscles. 3) The contractile apparatus consists of numer- ous myofibrils which are separated from each other by a network of intermediate filaments. 4) The myofibrils further consist of many irregu- larly arranged, sarcomere-like structures. 5) Almost all the thin filaments are attached to the dense bodies, and the thin filaments which emerge from dense bodies associate partially with thick filaments via cross-bridges. 6) Most or all of the dense bodies are intercon- nected by intermediate filaments in networks, through which the myofibrils are folded in each cell, in a manner that permits the cycles of contractions and relaxations to be minimally dis- turbed. 7) The agranular, tubular elements of sarcoplas- mic reticulum are situated just beneath the cell surface coupled to the cell membrane. REFERENCES 1 Toyota, N., Obinata, T. and Terakado, K. (1979) Comp. Biochem. Physiol., 62B: 433-441. 2 Shoenberg, C. F. and Needham, D. M. (1976) Biol. Rev., 51: 53-104. 3 Somlyo, A. P. and Somlyo, A. V. (1977) In “Ex- citation-Contraction Coupling in Smooth Muscle”. Ed. by R. Casteels, T. Godfraind and J. C. Reugg, 12 13 14 15 16 17 18 19 20 21 22 23 24 M5) 26 2) 28 29 Elsevier/North Holland, New York, pp. 317-322. Small, J. V. and Sobieszek, A. (1980) Int. Rev. Cytol., 64: 241-306. Fay, F.S., Rees, D. D and Warshaw, D. M. (1981) In “Molecular Structure and Function”, Vol. 4. Ed. by E. E. Bitter, John Wiley and Sons, New York, pp. 79-130. Terakado, K. and Obinata, T. (1987) Cell Tissue Res., 247: 85-94. Terakado, K. and Obinata, T. (1977) Zool. Mag., 86: 491. (Abstract, In Japanese) Shinohara, Y. and Konishi, K. (1982) J. Exp. Zool., 221: 137-142. Hernandez-Nicaise, M.-L., Mackie,G.O. and Meech, R. W. (1980) J. Gen. Physiol., 75: 79-105. Hernandez-Nicaise, M.-L., Nicaise,G. and Malav- al, L. (1984) Biol. Bull., 167: 210-228. Somlyo, A. P., Devine, C. E., Somlyo, A. V. and Rice, R. V. (1973) Phil. Trans. R. Soc. Lond., B265: 223-229. Lowy, J., Vibert, P.J., Haselgrove,J.C. and Poulsen, F. R. (1973) Proc. R. Soc. Lond., B265: 191-196. Shoenberg, C. F. and Haselgrove, J. C. (1974) Na- ture, 249:152-154. Small, J. V. (1977) J. Cell Sci., 24: 327-349. Ashton, F. T., Somlyo, A. V. and Somlyo, A. P. (1975) J. Mol. Biol., 98: 17-29. Panner, B. J. and Honig, C. R. (1967) J. Cell Biol., 35: 303-321. Marston, S. B. and Smith, C. W. J. (1984) J. Musc. Res. Cell Motility, 5: 559-575. Bond, M. and Somlyo, A. V. (1982) J. Cell Biol., 95: 403-413. Tsukita, S., Tsukita,S. and Ishikawa, H. (1983) Cell Tissue Res., 229: 233-242. Terakado, K. (1986) J. Electron Microsc., 35, Suppl. 4: 3117-3118. Campbell, G. R., Uehara, Y., Mark, G. and Burn- stock, G. (1971) J. Cell Biol., 49: 21-34. Cooke, P. H. and Chase, R. H. (1971) Exp. Cell Res., 66: 417-425. Uehara, Y., Campbell,G.R. and Burnstock, G. (1971) J. Cell Biol., 50: 484-497. Cooke, P. H. and Fay, F. S. (1972) J. Cell Biol., 52: 105-116. Cooke, P. (1976) J. Cell Biol., 68: 539-556. Small, J. V. and Sobieszek, A. (1977) J. Cell Sci., 23: 243-268. Price, M. G. and Sanger, J. W. (1983) In “Cell and Muscle Motility”, Vol. 3. Ed. by R. M. Dowben and J. M. Shay, Plenum Press, New York, pp. 1-40. Drenckhahn, D. and Jeikowski, H. (1978) Cell Tissue Res., 194: 151-162. Small, J. V. (1974) Nature, 249: 324-327. 30 a1 32 33 34 Structure of Ascidian Smooth Muscle 761 Tsukita, S., Tsukita, S., Usukura, J. and Ishikawa, H. (1982) Eur. J. Cell Biol., 28: 195-201. Heumann, H.-G. (1973) Phil. Trans. R. Soc. Lond., B265: 213-217. Devine, C.E. and Somlyo, A. P. (1971) J. Cell Biol., 49: 636-649. Heumann, H.-G. (1971) Cytobiologie, 3: 259-281. Bois, R. M. (1973) Anat. Rec., 177: 61-78. 35 36 37 38 Somlyo, A. V., Butler, T. M., Bond, M. and Som- lyo, A. P. (1981) Nature, 294: 567-569. Endo, T. and Obinata, T. (1981) J. Biochem., 89: 1599-1608. Rice, R. V., McManus, G. M., Devine, C. E. and Somlyo, A. P. (1971) Nature, 231: 242-243. Huxley, H. E. (1957) J. Biophys. Biochem. Cytol., 3: 631-647. Fat ahs ta i oe, on uit el ita iol LtRee yb rea. bow { i ih \ fa ‘ i Tea) 4 a 4 Me yp we Wy ny } a S oi i i iy | } “ i ay } ¢ —* Ten oe it ej ‘ne — aa eax A Spates ViM i idl tet 2 POO SOR Pate eee ee Ree ceinsbed chen: ney’ aed ou i ¥ 1 ‘ u } DAS CRIA et ad 44h GAA ul 24 , ’ *, iy of bine i iad iy " , v na ae i i! a i H ey ya? } . 1 se ‘>? ie 4t a) : yy Ma by ‘ tA.) ty chi a yay bt f a ee iby ee be v ) Ri The tA ie * De ee - Aras ; q q ‘ re , a , ¥ ‘ 5 CN hh He eka eee AL ae a i , i oa At : ha} a4 4 4 @ ‘ y ‘ ’. J y i ‘ ie i r Se a ik rélrinehowoplaaninamibienee ve ORR RAR emia, (kL): Snduupted aA ay pee wich are coma ae phages Moh an ne et rv) a lol | it ct oeiveeth OWN aith eae reels ta BS ee r t aisciatt ait a ett Hy wi pe Tale. Shah Te i* a cipean 4 ¢ t us : hk r i iy } vi Big ’ UP Le : | a t ; wu Kune ea ee i man j , oe (° , ’ Mes ‘ ZOOLOGICAL SCIENCE 4: 763-779 (1987) © 1987 Zoological Society of Japan REVIEW Cell Rearrangement in Morphogenesis Ray KELLER Department of Zoology, University of California at Berkeley, Berkeley, CA 94720, U.S.A. INTRODUCTION There is strong evidence that tissue cell rear- rangement, the local repositioning of cells with respect to one another, plays a major role in metazoan morphogenesis. Cell rearrangement is defined here, after Fristrom’s definition [1], as the local repositioning of cells with respect to one another, such that the overall shape of the tissue array is changed (Fig. 1). This rearrangement may be passive, accomodating the changes in embryonic shape generated by other mechanisms, or active, resulting in new arrays and driving the overall change in shape of the cell population. Rearrangement is likely to be a major morphoge- netic process in those situations where cell popula- tions undergo dramatic changes in shape which are not reflected in the shapes of individual cells, and cannot be attributed to oriented cell divisions or cell death (see p. 109 of [2]; [3]). Cell rearrangement is a common phenomenon. It has been shown by indirect morphometric data to occur during Drosophila imaginal disc evagina- tion [1], during neurulation in amphibians [4], during epiboly in amphibians [5, 6], and during segment shortening in insect metamorphosis [7, 8}. It also occurs during pronephric duct elongation [9], during teleost epiboly [10], during Hydra regeneration [11, 12], during secondary elongation of the sea urchin archenteron [13, 14], and during scale primordia alignment in the moth wing [15]. It has been analyzed directly with time-lapse mi- crography of cell behavior during amphibian gas- trulation and neurulation [16-18], during Received June 20, 1987 notochord formation in ascidians [19] and amphib- ians [17, 18], and during teleost epiboly [20]. In some of these systems, cell rearrangement appears to be a passive accomodation to external forces, wheras in others it appears to be an active, force-generating morphogenetic process. Cell rearrangement is a powerful and efficient mechanism of generating changes in tissue form. A short movement on the part of individual cells, between adjacent cells, exercised over the whole population, can produce dramatic effects. For example, if the cells in a 2X5 array actively intercalate themselves between their neighbors to form a 1X10 array, no cell actively moves more than a cell diameter but some in the population Fic. 1. A schematic diagram shows how local cell rear- rangement can produce large changes in shape of the cell population. From Keller [21] with permis- sion. 764 R. KELLER may be displaced great distances with respect to one another by virtue of their membership in the tissue array (Fig. 1). Although these endogenous- ly driven transformations in tissue shape have prominent roles in embryogenesis and have fascin- ated generations of embryologists, their mechan- ism has remained obscure [21]. In this paper, I will review the major features of what is known about the occurrence, the function, and the mechanism of cell rearrangement in several systems. REARRANGEMENT WITHIN THE PLANE OF AN EPITHELIAL SHEET In this type of rearrangement, epithelial cells connected to one another by a circumapical junctional complex, exchange neighbors within the plane of the epithelium during a tissue deforma- cy & © = ©) ra a ‘ O o O N (e) et) 2) 5 Sah SNe hours/ stage tion in which the epithelial sheet takes part. These situations appear to be of two types: those in which the epithelial cells generate the force for tissue deformation and those in which the epithelial sheet passively deforms. Enveloping Layer Cells Rearrange to Accomodate Changes in Shape of the Embryo during Teleost Fish Epiboly The following is based on the work of Kageyama [10] on Oryzias latipes and Keller and Trinkaus [20, 22] on Fundulus heteroclitus. In the develop- ment of these teleost fish, cleavage is incomplete, resulting in a cap of cells at one end and uncleaved yolk at the other. The cap is composed of a surface layer of epithelial cells, called the enveloping layer, and a deep mass of non-epithelial cells. At its margin, the enveloping layer is connected to the unsegmented portion of the egg, the yolk syncytial 46 40 Fic. 2. The number of marginal cells of the enveloping layer (EVL) of Fundulus is plotted against hours and developmental stage. The arrows show movement of the margin of the EVL. The embryonic shield (ES) is darkly shaded. Modified from Keller and Trinkaus [20] with permission. Cell Rearrangement in Morphogenesis 765 layer. As gastrulation occurs, the enveloping layer undergoes epiboly and spreads vegetally over the yolk while the deep cells collect on the dorsal side and make an embryonic shield from which the embryo will be derived (Fig. 2). This epiboly occurs by spreading and thinning of the original population of cells since few, if any, cells are added by cell division [23]. The marginal and submargin- al regions of the enveloping layer must increase their circumference as they pass from near the animal pole toward the equator and decrease their circumference as they pass beyond the equator and approach the vegetal pole. Cell counts in silver-stained blastoderms [10, 22] show decreased numbers of marginal cells during epiboly (Fig. 2), and direct time-lapse cinemi- crography shows that marginal cells decrease their boundary with the yolk syncytial layer, lose con- tact with it, and then recede from the margin [20] (Fig. 3). The adjacent cells on either side to come into contact immediately, and thus the continuity of the epithelial sheet is never broken. Submargin- al cells likewise rearrange by separating from one another along the animal-vegetal axis and allowing cells on either side come into contact simul- taneously [20] (Fig. 3). As the connection between two submarginal cells or a marginal cell and the yolk syncytial layer narrows, the apices of the cells become rounded, phase-dense, and convex in profile. When separation occurs, no apical protru- sive activity is seen in the case of marginal separations, but “flowers” of apical microvilli appear, immediately adjacent to the sites of submarginal separation. The cells of the enveloping layer are attached to one another and to the yolk syncytial layer by a circumapical junctional complex consisting of con- tinuous tight junctions, desmosomes, and gap junctions [23, 24]. Thus this epithelial sheet forms a high resistance physiological barrier [25], one that is even nearly impermeable to water [26]. Nevertheless, these epithelial cells can and do exchange neighbors in the course of epiboly. Thus the junctional complex has the seemingly imcom- patible properties of allowing relative movement of cells and maintaining a physiological barrier. But such incongrous properties might be expected of embryonic epithelial sheets, which at once must Fic. 3. Progress of the rearrangement of marginal and submarginal cells is shown in line tracings of time- lapse recordings of the EVL at 0, 33, 65, and 95 minutes. The margin of the EVL is below in all figures. Note the narrowing of the boundary of cells a, b and d with the yolk syncytial layer (point- ers +1, +2, and #4). Note also that cell b narrows its common boundary with the cell above it and eventually separates from it (pointer #3). From Keller and Trinkaus [20] with permission. protect the milieu interieur and at least permit, if not actually generate the dramatic changes in shape of morphogenesis. This is particularly true of Fundulus, an esturine fish, which must develop in a variety of salinities. The enveloping layer is under meridional and circumferential tension, and thus its component cells might respond to these tensions by moving past one another. Alternatively, the cells may actively participate in bringing about their own rearrangement with appropriate motile activity at their basal surfaces. They do bear underlapping protrusions at their basal ends [23, 24]; whether or not these protrusions function in rearrangement is not known. 766 R. KELLER Passive Epithelial Cell Rearrangement Accomo- dates Convergence and Extension of the Dorsal Marginal Zone during Xenopus Gastrulation The dorsal marginal zone of the amphibian embryo undergoes extreme narrowing (conver- gence) and elongation (extension) in the course of gastrulation and neurulation. Time-lapse cinemi- crography of this region of the gastrula and neurula shows that the superficial epithelial cells rearrange to form a longer, narrower array [16] (Fig. 4). As the marginal zone elongates, the Fr269:075hr 500pm7cell 530 pm/cell Fr 527. 1.46hr Pl=1.39 Fic. 4. Tracings of cell apices in the dorsal marginal zone of a Xenopus gastrula, as they appear in time-lapse cinemicrography at 0.75 hours and 1.46 hours, shows the rearrangement of these epithelial cells (shaded cells) as the region undergoes con- vergence (narrowing) and extension (lengthening). The axis of convergence is horizontal; the axis of extension is vertical. From Keller [16] with permis- sion. apices of the individual cells also elongate and then return to their original isodiametric shape as they rearrange. Such behavior suggests a passive rear- rangement in response to stretching by external forces. In fact, the underlying deep cell population produces the force for convergence and extension [27, 28]. However, the junctions in the marginal zone epithelium may be specialized to allow cell rearrangement. Epithelial cells from the animal cap, a region which normally does not undergo convergence and extension, will show convergence and extension when grafted to the dorsal marginal zone, but in contrast to the native epithelium of this region, the grafted cells elongate greatly and tear apart from one another in an apparent reluctance to rearrange [27]. Research should be done to determine if the junctional complex is uniform throughout the gastrula or whether it is specialized in some fashion in those regions des- tined to undergo rearrangement. Active Epithelial Cell Rearrangement May Func- tion in Tissue Elongation during Newt Neurulation During neurulation in the urodele amphibian, the neural anlage undergoes an elongation that is important in transforming its originally circular outline into a keyhole shape [4] and may contrib- ute to neural tube closure as well [29]. The neural anlage consists of two regions, the notoplate, which lies immediately superficial to the notochord, and the neural plate, which lies lateral to the notoplate and superficial to the somitic mesoderm. The boundary of the notoplate and neural plate seems to play a special organizing role in producing the elongation of the neural anlage. This boundary elongates during neurulation, prob- ably by a mechanism in which cells in the interior of the notoplate move outward to the notoplate- neural plate boundary and thus elongate the boundary [30]. Jacobson [29, 30] favors the notion that this movement is driven by adhesive rela- tionships along the boundary; his idea is that the notoplate-neural plate interface is a region of maximum adhesion and the notoplate cells move to attach themselves to this favorable adhesive boundary. Jacobson et al. [31] proposed that this movement is driven by a “cortical tractor” mechanism in which a fountainoid flow of cell Cell Rearrangement in Morphogenesis 767 cortex and adhesions to other cells would function to move cells between one another. In the case of the notoplate cells, their lateral surfaces would be the only ones free to engage in cortical tractoring, because of motility-inhibiting contacts at their other surfaces, and thus they would tend to intercalate in the plane of the epithelium. Lamelli- podia, sometimes spanning 3 or 4 cell diameters, have been seen among these cells [31]. It is worth noting that isolated amphibian cells are notorious for showing amoeboid locomotion of one type or another, during which they may exert traction on the substratum by movement of the cortex [32, 33]. The cortical tractor is in many ways an elaboration of this well-travelled observation. Active Cell Rearrangement Brings about Evagina- tion of the Drosophila Imaginal Disc Although cell rearrangement was suggested on indirect evidence in early work (see p. 109 of [2]) and implied in the results of others [34, 35], its modern definition and the revival of serious interest in it as a morphogenetic mechanism is largely due to the pioneering work of Fristrom and her colleagues [1, 3, 36, 37] on the role of cell rearrangement in evagination of the Drosophila imaginal disc. The leg imaginal discs of Drosophila consist of a monolayered epithelium forming a circular “disc” of concentric folds. After exposure to B-ecdysone in vitro, the discs evaginate to form a leg structure resembling, in general form, a cylindrical tube. The geometry of such a trans- formation involves decreasing the circumference and increasing the length of the disc such that it forms a cylinder, with more peripheral parts of the disc (prospective proximal parts of the leg) show- ing greater change in shape. The cells maintain specialized junctional contacts throughout the process [3]. Cell division is not necessary for the process of evagination since it occurs in the absence of DNA synthesis [38] and in the presence of colchicine [3]. Likewise cell death does not account for the change in shape [3]. Theoretically, a fixed number of cells could either change their shape or their arrangement to produce such a shape change of the cell population. Fristrom [1] found that the latter was the case. In the basitarsal region, which is the easiest to analyze, the number of cells comprising the circumference decreases and the number of cells comprising the length increases, with little or no change in cell shape. In the tarsus the number of circumferential cells is reduced to three fifths the original (Fig. 5) and in the tibia to less than half the original number. Moreover, the number of cells does not change significantly. Analysis of cell contacts shows that unlike adult epidermal cells, the cells of the disc show departures from strict hexagonal packing during rearrangement, with more cells having 5 and 7 neighbors instead of 6. Little is known of the actual cell motility involved in rearrangement of these epithelial cells other than the fact that in time-lapse videomi- crography they show more pulsatile activity throughout the period of rearrangement than earlier (Dr. D. Fristrom, personal communica- tion). It seems likely that this activity may have some function in rearrangement but there is no direct evidence that this is the case or how it works. Trypsin (0.1%) will accelerate the rate of evagination in vitro but the mechanism of action is not known [39]; the chracteristic septate junctions are found in trypsin-accelerated discs, so it does not appear to be a simple loosening of junctions. Ultrastructural work by Fristrom [37] has shown how the individual cells maintain a junctional seal at their apical ends while exchanging neighbors. Each cell is viewed as having a lateral surface, or contact domain, with each adjacent cell (Fig. 6). Fic. 5. Schematic diagrams show the transition from partly evaginated to fully evaginated leg imaginal disc (1a) and the corresponding change in the arrangement of the cells in the first tarsal segment (b). From Fristrom [37] with permission. 768 R. KELLER Fic. 6. Fristrom’s scheme for redistribution of septa during cell rearrangement is shown diagramatically. Cell B is intercalating between cells A and C. The contact domains are shaded and the septate junc- tions (s) and tricellular plug (tp) are indicated. Note that the septa adjust to the decreasing width of the contact domain between cells A and C by folding and to the increasing width of the contact domain between cells B and C by unfolding. The tricellular plug forms a seal between the extensions of the septate down the margins of the contact domains where three cells meet. From Fristrom [37] with permission. As expected, each contact domain has a septate junction forming a band immediately beneath the apex of the cell, but contrary to expectation, these junctions turn basally and run along the lateral edge of the contact domain (Fig. 6). The space where three cells join is occupied by a tricellular plug, a structure consisting of electron-dense ladder-like arrays of lenticular bars (Fig. 6). According to Fristrom’s model, neighbor exchange occurs by adjacent cells decreasing their common contact domain; as they do so, the septate junc- tions fold. As the contact domain nears zero, and exchange of vertices involving the tricellular plugs would have to occur, and it is not yet clear how this might occur. Active Cell Rearrangement Functions in Secondary Invagination of the Sea Urchin Archenteron Sea urchin embryos show two phases of archen- teron formation. Primary invagination involves the bending the vegetal plate to form a short stubby archenteron, spanning one third to one half the diameter of the blastocoel. During secondary invagination, the archenteron narrows and extends across the blastocoel to the animal region, where it attaches to the ectoderm on the ventral side of the embryo and later forms the mouth. In this second phase of invagination, secondary mesenchyme cells send out long filipodia, which contact the underside of the ectoderm. These were thought to develop tension and stretch the archenteron into its elongate shape (see [40] for a review). The evidence that archenteron elongation in- volves active cell rearrangement is based on several facts. Ettensohn [13] showed that archen- teron elongation is accompanied by a decreased number of cells in any transverse cross-section, a fact that must be attributed to a cell rearrange- ment. Moreover, the junctional complex linking these cells at their apices remained intact during the cell rearrangement. Mechanical simulations of pulling by filopodia on the roof of the blastocoel showed that if this were the sole mechanism of archenteron elongation, the blastocoel roof should deform and the archenteron should acquire a shape not seen in embryos [14]. Moreover, the archenteron shows elongation to nearly normal lengths in lithium chloride-induced exogastrulae, in which the archenteron is directed outward into the medium where secondary mesenchyme cells are not in position to exert traction on the archenteron [14]. Recently, Hardin has killed the secondary mesenchyme cells with a laser mi- crobeam at the end of primary invagination and found that the archenteron nevertheless elongates (Jeff Hardin, personal communication). Thus it seems that a major part of the force producing elongation is generated within the archenteron, and that the process underlying elongation may involve active cell rearrangement. Archenteron elongation does not require DNA synthesis or cell division [41]. Given that cell rearrangement functions in archenteron elongation, how do the cells rearrange themselves? Gustafson and Kinnander [42] and Kinnander and Gustafson [43] recorded pulsatile activity at the basal ends of the archenteron cells, but this protrusive activity could not be resolved. There are stage-specific changes in the type of protrusions and cell shapes in the archenteron (Hardin and Benson, personal communication). Early in secondary elongation, the basal ends of the cells bear large lamellipodia oriented toward the archenteron tip and overlapped shingle-fashion (Fig. 7; Hardin and Benson, personal communica- tion; [44, 45]). As elongation procedes, the lamellipodia disappear and the cells become more rounded and are connected by short, basal protru- Cell Rearrangement in Morphogenesis 769 Ay Fic. 7. Early in secondary invagination of the sea urchin gastrula, archenteron cells show elongation parallel to the long axis of the archenteron and have large basal lamellipodia (pointers) overlapping the basal ends of cells nearer the closed end of the archenteron (upper right of photograph). The micrograph was provided by Jeff Hardin. Magnification is 200. sions. Then, toward the end of secondary in- vagination, the cells again elongate slightly. How these changes in protrusive activity and cell shape function in rearrangement, if at all, is not known. Undoubtedly changes in the cytoskeleton or in the adhesions of these cells to one another underlies the observed changes in protrusive activity. What- ever these might be, intact microtubules do not appear to be necessary for archenteron elongation or cell rearrangement [46]. Epithelial Feet May Function in Cell Rearrange- ment during Formation of Scale Spacing Patterns in the Manduca Wing Epithelial cell rearrangement may be mediated by filopodia during the formation of rows of scale primordial cells in the development of the wing of the moth Manduca sexta [15]. The pupal wing of this organism consists of two epithelial monolayers closely applied to one another and initially com- posed of morphologically homogeneous cells. By two and half days after pupation, when the epithelium retracts from the overlying cuticle, two cell types are seen—smaller general epithelial cells of the wing and larger scale primordial cells fated to form scale and socket cells by subsequent divisions. After the primordial scale cells are freed from the cuticle, they begin shifting their positions within the epithelial sheet and transform their initially random distribution into rows parallel to the anterior-posterior axis of the wing (Fig. 8). As they rearrange, the scale primordial cells become polarized, with their anterior and posterior edges being longer than their proximal and distal edges. Moreover, during this period from day 2.5 to 4, these cells, and the general epithelial cells as well, form basal filopodia in all directions and contact cells as far as several cell diameters away (Fig. 8). In the fifth day, the basal filopodia become aligned along the anterior-posterior axis of the wing, and by the end of day 5, the rearrangment is complete and all basal filopodia are retracted. In this case of cell rearrangement, modulation of junctions appears to occur. The epithelial cells lose most of their hemidesmosomal connections with the underlying basal lamina, and the basal 770 R. KELLER Fic. 8. A whole mount of the upper epithelial monolayer of a Manduca wing, stained by the procedure of Locke and Huie [7, 8] shows the primordial scale cells that have begun to align in regularly spaced rows oriented perpendicular to the proximal distal axis of the wing. Anterior is at the top, proximal is to the right. At this stage (day 4) the protrusions of the cell margins extend both between rows and along transverse rows. From Nardi and Magee-Adams [15] with permission. lamina becomes highly convoluted perhaps as a result of pulling filopodia during rearrangement. Later in development the epithelial cells reestab- lish contact with the cuticle and a smooth basal lamina. Interestingly, the generalized epithelial cells also show numerous short filopodia on their apical surfaces during the period of cell rearrange- ment, whereas the scale primordial cells do not. Nardi and Magee-Adams [15] suggest that these basal filopodia function to generate the physical forces that bring about rearrangement and perhaps impose the control necessary to bring cells into such precise alignment. Since the filopodia span from one to several cell diameters, both short and long range interactions are possible by direct contact [15]. These workers favor the notion that cells rearrange within the epithelium until cells of similar or identical adhesive values are maximized to form the most stable arrangement, which in the case of the moth wing is a series of anterior- posterior bands. They point out that this notion is consistent with the evidence that there is a proximal-distal adhesive gradient in the wing [47, 48], with the fact that alignment and homotypic contacts of protrusions occur at the end of rear- rangement, and that the density of primordial scale cells is maximized, relative to earlier states, within a transverse row in the final state. Epidermal Feet in Pupal Segment Morphogenesis Research by Locke and Huie [7, 8] and Locke [49] suggests that specialized protrusions or “epidermal feet” at the basal ends of insect epidermal cells play a role in driving the cell rearrangement that occurs during abdominal seg- ment shortening in Calpodes ethlius (Lepidop- tera). During the last larval (Sth) stage in this insect, the body of the animal shortens to 60 percent of its previous length as a result of the shortening of individual segments. This shortening can not be explained as the elastic recoil of the inflated larval segments as hemolymph pressure is Cell Rearrangement in Morphogenesis 771 reduced and thus appears to require generation of new axial forces. It is accompanied by epidermal cell rearrangement [49]. The epidermal cells show protrusions of their basal margins which undergo a sequence of stage-specific changes in morphology. Initially, the epidermal cells have a typical poly- gonal morphology and bear a few short basal protrusions. Then numerous protrusions form at their basal ends, which extend across adjacent cells to form an interlaced fringe around each cell. Thus several layers of feet form between the basal ends of the cells and the basal lamina beneath the epidermis. However, all the protrusions are connected by hemidesmosomes to the basal lamina and are mechanically united by desmosomes con- necting them to one another and to adjacent cells. These protrusions contain axially oriented micro- tubules and microfilaments. Initially, the epidermal feet are distributed uniformly in all directions but later they become progressively longer and oriented axially. Some cells show polarity as well, with the anteriorly directed feet being more finely divided. As the segment begins to shorten, the feet straighten and appear to lose some of their branching, and the epidermal cells become columnar. Finally, the feet shrink back to the basal perimeter of the cell and form a slightly stellate basal apron. The relative shortening of the segment occurs by cell rearrangement, since all the component cells are isodiametric at the start and at the end of the process. As the cells begin to rearrange, the original junctions seems to be maintained, and the margins of the cells are pulled into extensions to accomo- date the shift in relative positions of the cells. Later, the junctions are broken down and new stable connections made between cells. As the feet shorten the basal lamina forms folds, presumably due to compression. A new basal lamina then appears to form below the stabilized pupal cell arrangement. Locke [49] has some evidence that the initiation, extension, and retraction of the epidermal feet may be controlled by ecdysteroid levels. The epidermal feet are not found in larval moults but only in the moult generating the pupa, during which segment shortening occurs. Thus there appears to be strong circumstantial evidence that segment shortening involves an active rear- rangement of component epidermal cells, prob- ably driven by the protrusion, attachment and retraction of epidermal feet. Changes in Proportions of the Body Column of Hydra Involve Cell Rearrangement The Hydra body column consists of two epithe- lial cell layers, the ectoderm and the endoderm, separated by a non-cellular mesogleal layer. The epithelia are attached to the mesogleal layer by basal muscle processes aligned parallel to the body axis in the ectoderm and perpendicular to it in the endoderm [51]. In regeneration of Hydra from isolated fragments of the body column, or in normal asexual reproduction by budding, changes in body form occur which cannot be attributed to cell division (see [51]), nor to permanent change in cell shape [11, 52]. Bode and Bode [12] have made a detailed study of the reshaping of the body column during regeneration. Pieces of body tissue of various sizes and numbers of cells parallel and perpendicular to the long axis of the body column were excised, allowed to heal into a sphere, and monitored in subsequent stages for changes in axial and circum- ferential cell number, shape of individual cells, and shape of the regenerating body column. Pieces of approximately the same cell number but differing in initial dimensions were compared. These classes of explants were studied: circumferential rectang- les (long dimension along the body circumfer- ence), Squares, and axial rectangles (long dimen- sion along the length of the body column) (Fig. 9). Instead of forming short fat cylinders, intermedi- ate cylinders, and tall narrow cylinders as might be expected from their original shapes, after 6 days these pieces formed cylinders of the same propor- tions with the same cell arrangements (Fig. 9). This was undoubtedly accomplished by cell rear- rangement. Remimiscent of what was seen in Drosophila imaginal disk evagination, the rear- rangement of cells in Hydra regenerates was accompanied by increased numbers of cells having 4, 5 and 7 neighbors rather than the usual 6. The explant affected the proportions of the regener- ated body column, with small pieces being shorter and wider than large pieces. This difference is related to the presence of a head. Those without 772 R. KELLER DAY | DAY 6 Fic.9. These diagrams show cell rearrangements in the ectoderm during regeneration of pieces of Hy- dra of nearly the same size but different shapes. The shapes are: (A) circumferential rectangle, (B) square, and (C) axial rectangle. The cell rearrange- ment is shown schematically with each block repre- senting a 5 by 5 array of cells. From Bode and Bode [12] with permission. heads remain spherical whereas those with heads form cylinders with a constant ratio of head diameter to body diameter for all regenerate sizes. The head consists of a larger part of the total tissue mass in small regenerates. Thus the reproportioning of the regenerating body column of Hydra during regeneration occurs by cell rearrangement, although it is not known how the cells move between one another. A likely possibility is that these cells use their basal muscle processes to bring about these movements [SO, 53], perhaps in a manner similar to the proposed function of the epidermal feet in insects. The fact that the head appears to control the diameter of the body cylinder, a parameter determined by cell arrangement, suggests that the head controls cell rearrangement. Bode and Bode [12] favor the notion that a gradient of adhesive differences can drive conversion of an epithelial sheet into a cylinder [54]. There is, in fact, evidence consistent with the notion of an adhesive gradient in Hydra: cells in the upper part of the body column are small in area and columnar, suggesting strong lateral adhesions, whereas those in the lower part of the body have large areas and are squamous in shape, suggesting weaker lateral adhesions [12]. INTERCALATION OF NONEPITHELIAL CELLS INTO AN EPITHELIAL SHEET Intercalation of Deep Cells into the Superfical Epithelial Layer during Epiboly Hatta [34] noted in histological sections of the lamprey embryo that the animal cap was trans- formed from a double-layered structure to a single-layered one in the course of epiboly. Moreover, this appeared to occur by gradual interdigitation of the tapered, adjacent ends of the two tiers of cells until the deep cells had interca- lated themselves into the outer layer. Such behavior implies the entry or ingression of none- pithelial cells into an epithelial sheet. Holtfreter [32] contended that a similar phe- nomenon occurs during epiboly in the urodele amphibian, based on his observation that the animal cap region is initially dark and later becomes lighter in color, in consequence of the intercalation of deep, non-pigmented cells be- tween the darker epithelial cells to form a single layer. This is in fact the case. Surface cells marked with Bolton-Hunter reagent in the blastula stage are later joined in this layer by unlabelled cells that must have been deep when the surface cells were marked [6]. It is not known how nonepithelial cells manage to insinuate themselves between epithelial cells, despite the fact that the latter are presumably Cell Rearrangement in Morphogenesis 173 connected to one another by circumapical junc- tional complexes. The nonepithelial cells are presumably able to invade an epithelial layer and, after doing so, become true epithelial cells them- selves. These events deserve further study. Intercalation of Inner Cell Mass Cells into the Trophectoderm of Mouse Blastocyst Work by Cruz and Pedersen [55] and Winkel and Pedersen (personal communication) shows that the same phenomenon occurs in the mouse blastocyst. The mouse blastocyst consists of an outer, epithelial monolayer, the trophectoderm, and a nonepithelial region, the inner cell mass. The inner cell mass is located eccentrically, im- mediately beneath the trophectoderm, and con- sists of several layers of cells. Cells of the polar trophectoderm (trophectoderm immediately above the inner cell mass), marked with a cell lineage tracer, were found to be displaced peripherally, at later stages, away from the polar trophectoderm into the mural trophectoderm [55]. These workers proposed that inner cell mass cells were moving outward into the polar trophec- toderm and displacing those cells already there. Winkel and Pedersen (personal communication) produced proof of this; they labelled inner cell mass cells and found that these nonepithelial cells, or their progeny, move outward and intercalate themselves into the epithelial polar trophec- toderm. BOUNDARY SHORTENING METHOD OF ANALYZING BEHAVIOR OF CELLS IN A MONOLAYER Honda and his associates [56-61] have de- veloped the boundary shortening (BS) method to analyze the behavior of cells in monolayers. This method may be useful in analyzing cell rearrange- ment. The BS procedure predicts how much shortening of the boundaries of cells in a mono- layer is possible, and it involves using a computer to perform iterations of a boundary shortening procedure beginning with outline tracings of cell perimeters. In each iteration, the total length of five boundaries, the common boundary between two cells and the two sides adjacent to this boundary on both of its ends, are shortened without changing the areas of the polygons in- volved (see [60]). The procedure is repeated, selecting cell boundaries at random, until no further significant shortening occurs. The amount of shortening, s, is expressed as a fraction of the original boundary length. In analysis of cultured epithelial and nonepithelial cells, the former were found to have small s values and the latter large ones [60]. These workers developed two other useful parameters, a motility area and a motility center. The former is the area swept out by marginal fluctuations of the cell over a period of time as a fraction of total area, and the latter is displacement of the apparent center of gravity of the cell outline over that period of time; the two show high correlation. On the basis of these parameters, these investigators classified the cells into several categories: stable tensile monolayers (small motility and s values); stable non-tensile monolayers (small motility and large s values); and fluctuating monolayers (large motility and s values). These parameters have been used to monitor developmental changes in epithelia. The starfish embryo changes from a non-tensile to a tensile state in the course of formation of the blastula [58], and during wound healing in the corneal endothe- lium the endothelium shows a transient change from tensile to non-tensile state [57]. Honda and his coworkers propose that cell rearrangement occurs by boundary shortening. As healing occurs the cells are deformed; the circumapical microfila- ment system actively contracts, shortening the boundaries and resulting in neighbor changes. Computer simulations, using the boundary shortening procedure and allowing vertex changes, mimic the cell rearrangement seen in the cornea. In an analysis of cell patterns in the bird oviduct, Honda and others [61] were able to conclude that boundary shortening and adhesive differentials between two types of cells are sufficient to produce a stereotyped repacking of cells. The immature oviduct epithelium of the Japanese quail consists of two cell types, large ciliated cells (C cells) and smaller gland cells (G cells) arrayed in a kagome (star) pattern in which the former are surrounded by the latter (Fig. 10). This pattern matures to 774 R. KELLER form a rectangular block pattern resembling a checkerboard but deformed towards a honeycomb pattern (Fig. 10). On theoretical grounds, the authors argue that the modified checkerboard pattern is a result of strong adhesion between unlike cells and boundary contraction. Using computational analysis they were able to estimate the value of the difference of adhesion between like and unlike cells. Using this parameter and the BS procedure, they successfully simulated the generation of the rectangular pattern from the kagome pattern. This work makes specific predic- tions about how relatively simple modulations of adhesion and contraction can maintain or generate complex patterns of cell arrangement in a mono- Fic. 10. Light micrographs show the luminal surface of an immature oviduct epithelium (1A) and a mature oviduct epithelium (B) after staining with silver- nitrate to reveal the boundaries of the cells. Cor- responding line drawings show the large C cells (stippled) surrounded by G cells (unstippled) in the kagome pattern of the immature oviduct (C) and in the rectangular pattern of the mature oviduct (D). The bar is 10um. From Honda et al. [61] with permission. layer. Such an approach should be equally productive when applied to other systems. REARRANGEMENT OF NONEPITHELIAL CELLS DURING MORPHOGENESIS Deep Cells Actively Intercalate To Form a Longer, Narrower Array during Gastrulation and Neurula- tion of the Xenopus Embryo As mentioned above, the deep, nonepithelial cells of the dorsal marginal zone of anuran amphibian Xenopus laevis appear to be the active cell population in producing narrowing (conver- gence) and elongation (extension) of this region of the embryo during gastrulation and neurulation [17, 18, 27, 28]. Both the involuting and noninvo- luting parts of the dorsal marginal zone show convergence and extension. The deep cells of the involuting marginal zone are _ prospective mesodermal cells [62]. Tracing cells with a cell lineage tracer showed that the deep cells rearrange to form a narrower, longer array during conver- gence and extension, and direct time-lapse cine- micrography of explants of the dorsal marginal zone showed that the deep cells actively move between one another to form a longer, narrower array [17, 18]. Such intercalation occurs from the midgastrula stage and continues through neurula- tion. Intercalation involves pulsatile advance and retraction of the deep cell margins, which results in gradual movement of cells between their neigh- bors. During notochord formation in the early neurula stage, the notochord cells extend protru- sions transverse to the long axis of the notochord and gradually intercalate (Fig. 11); ultimately each cell spans nearly the entire diameter of the notochord. Deep cells are connected to one another by numerous filiform and lamelliform protrusions which are undoubtedly involved in the intercalation process. Individudal cells may move rapidly between others by using large lobopodia formed and advanced by the amoeboid-type cyto- plasmic flow described by Holtfreter [33] and others, but this is a rare event [45]. It is not known what agents direct the intercalation to be mediolateral and thus result in a longer, narrower array. The dorsal noninvoluting marginal zone also Cell Rearrangement in Morphogenesis 775 56min Tracings of cells from time-lapse films of the dorsal mesoderm in explants of Xenopus gastrulae, show three rows of notochord cells rearranging to form two rows of cells during the development of the notochord. The long axis of the notochord is vertical. From Keller et al. [17] with permission. Pic. 11. shows convergence and extension, but the under- lying cellular activities may be different from those occurring in the involuting marginal zone. Con- vergence and extension in the noninvoluting and involuting dorsal marginal zones involve different cell morphologies; the rate of extension is greater in the former; the convergence and extension of the former is dependent on basal contact with itself or with dorsal mesoderm (Keller and Danilchik, unpublished results). This latter property makes it impossible to observe cellular behavior directly in the noninvoluting marginal zone, since in order to do so the basal surface must be exposed. Thus we know little about the motility of the cells in this region. It does appear, however, that there are two different mechanisms of convergence and extension in the Xenopus gastrula and neurula. Intercalation of deep cells also occurs during epiboly of the Xenopus gastrula. Several layers of deep cells of the animal cap and noninvoluting marginal zone intercalate between one another to form fewer layers of greater area [5]. Although it is important in generating the increased area during epiboly, nothing is known about the mechanism of this intercalation. Notochord Cells Rearrange during Morphogenesis of the Ascidian The ascidian embryo offers good material for the study of cell rearrangement because the clarity of the embryo allows direct observation of mor- phogenesis. After blastopore closure the notochord rudiment in the ascidian lies above the dorsal lip of the closed blastopore. The notochord narrows and elongates by a process similar to that seen in the amphibian. The component cells elongate transverse to the long axis of the notochord, become wedge-or spindle-shaped and intercalate to form a single row of rectangular cells [19, 63]. The rearrangement of the notochord cells occurs with a pulsatile advance and retraction of the tapered protrusions at the medial borders of these intercalating cells [19]. Here, as in the amphibian, nothing is known about how this protrusive activity is organized, but this organism warrants further study, given the simplicity of its notochord and its favorable optical properties. Pronephric Duct Cells Rearrange during Elonga- tion of the Rudiment The development of the pronephric duct in urodele amphibians has been described in detail by Poole and Steinberg [9]. The pronephric duct rudiment consists of an oblong tissue mass which segregates from the adjacent mesoderm near the ventral boundary of somites 2 through 7 in the early tailbud stage. Thereafter it narrows and elongates posteriorly to reach nearly twice its original length. Vital dye marking experiments show that its elongation is due to reshaping of the component tissue rather than recruitment of addi- tional tissue along its route. Morphometric analy- sis shows that the duct narrows from about 6 to 8 cells in diameter to 2 or 3 cells in diameter in the course of its elongation, without significant change in size or shape of the component cells. The posterior tip of the duct, which leads its migration, has lamellipodia, lobopodia, and filopodia in 776 R. KELLER intimate contact with the mesoderm at the ventral edges of the somites, and results of experiments in which the duct rudiment was grafted to abnormal positions are consistent with the notion that its tip migrates in response to a gradient of adhesion in the lateral mesoderm [64, 65]. Although the migration of the tip makes a major contribution to the guidance of the duct, it is not clear to what extent the component cells in the body of the duct are passively rearranged as a result of tension generated by the duct tip, or whether these cells actively rearrange and contribute to the forces bringing about elongation of the duct. It would seem that the minimum contribution of cell rear- rangement to duct elongation would be to allow elongation in response to the tension generated by the cells at the tip. Interestingly, cell rearrangement may play another role in duct elongation. Gillespie and Armstrong [65] found that the lateral mesoderm is temporarily transformed from two layers of cuboidal cells to one layer of columnar cells as the duct migrates across it. These changes in height and number of cell layers occur as a wavefront over the entire lateral mesoderm. These authors suggest that such changes may be related to the apparent adhesive gradient controlling the direc- tion of migration of the duct tip. MECHANISMS OF CELL REARRANGEMENT There are Probably Several Mechanisms of Cell Rearrangement Although cell rearrangement is not understood in any system, there is enough evidence to suggest that several mechanisms exist. Cell rearrangement in the moth wing and insect segment epidermis differs in detail from that seen in the sea urchin archenteron and the insect imaginal disc, even though the rearrangement occurs in epithelial sheets in all cases. In the first two cases, the long filiform protrusions apparently involved in displac- ing the cells undergo one or at most a few cycles of extension and retraction, whereas in the last two cases there is rapid, pulsatile activity. In the gastrulation of Xenopus there are at least two mechanisms of convergence and extension, which appear to generate active, force-producing cell rearrangement by different rules of cell motility. The intercalation of nonepithelial cells into an epithelial sheet during amphibian and lamprey epiboly and during blastocyst development in mice undoubtedly differs from intercalations involving only epithelial cells or nonepithelial cells. Un- doubtly there are similarities of underlying cellular behavior in these various types of cell rearrange- ment, but at present there is not enough evidence to know what these are, beyond the obvious and trivial ones, or to evaluate their significance. Motile Activity, Orientation, Directionality, and Adhesion These properties of cells are undoubtedly in- volved directly or indirectly in cell rearrangement. Cells undergoing rearrangement are connected by filiform or lamelliform protrusions. In the case of the insect epidermal cells, cell rearrangement seems to involve one cycle of extension, perhaps across many cell diameters, attachment, and re- traction of protrusions. The same may be true in the case of the primordial scale cells. In Xenopus, pulsatile protrusive activity repeated on the order of minutes appears to be involved in cell rear- rangement, and the same is true of notochord cells in the ascidian. It is worth considering the possibility that mechanisms of rearrangement fall into two categories: those which involve one or a few cycles of extension, attachment, and retraction of protrusions over great distances; and those which involve rapidly repeated cycles of short- range protrusive activity. In some cases, such as the insect epidermis, the displacement of cells during rearrangement un- doubtedly involves contraction of protrusions which have become attached to surfaces of other cells some distance away. In other cases, such as the deep cells of Xenopus, advance of the cell by cytoplasmic flow may be involved. On the other hand, Honda and his associates have argued cogently that a general, circumferential contrac- tion of the cell margin can function in cell rearrangement by the boundary shortening mechanism. On the limited evidence available, it seems likely that several kinds of motile activity are likely to be involved in the various types of cell Cell Rearrangement in Morphogenesis WT rearrangement, or in various aspects of a single type of rearrangement, and that the mechanical context in which these activities are exercised may be as important as the character of the activity itself. More data on the motile activity involved in cell rearrangement is badly needed; we have time-lapse records of actual cell rearrangement at reasonably high resolution only in the ascidian notochord, the cornea during wound healing, the fish enveloping layer, and the deep and superficial cells of Xenopus gastrulae. None of these systems have been analyzed in sufficient detail. Cell rearrangement appears to be an oriented process (cell rearrangement along an axis, in either direction) and may be directional (cell rearrange- ment in one direction along an axis) as well. In the imaginal disc and in the sea urchin archenteron the orientation of rearrangement is circumferential. In the Xenopus gastrula rearrangement appears to be directional and generally towards the dorsal side. In the moth wing it is exercised in the proximal- distal axis and thus produces aligned cell in anterior-posterior rows. In the notochord, inter- calation of the central cells is oriented at right angles to the long axis with activity at either end; those notochord cells at the notochord-somitic mesoderm boundary show directed intercalation, with all activity confined to their inner ends. In this case, the notochord boundary may exert a polarizing influence. In most cases, however, it is not known whether oriented intercalation results from a bias in the direction of protrusive activity, or from a bias in the motile effectiveness of certain protrusions over others. If protrusive activity is biased, is this bias an intrinsic property of the cell, perhaps based on its cytoskeletal organization, or is it a bias imposed on the cell by its extracellular environment, such as the extracellular matrix or a diffusible chemotactic molecule? Another possi- bility is differential adhesion within a_heter- ogeneous population of cells. Mittenthal and Mazo [54] have proposed a model for the conver- sion of a disc into a cylinder, based on blocks of cells which have differential adhesion for one another and within which cells are free to rear- range, but between which they cannot. Related ideas involve collection of cells at boundaries of greater adhesiveness and thus enlarging the boundary [12, 15, 30]. The timing of disassembly of old adhesions and the formation of new ones during neighbor exchange is also an important unresolved issue in most systems. It is clear that rearrangement can occur even among epithelial cells joined with sealing tight or septate junctions. In general, there are many ideas about how cell rearrangement works. Some of these ideas, if not correct themselves, may lead to others that are useful. But there is too little direct information about the intercellular adhesions, and the nature, organization, and timing of motile activity during cell rearrangement. Thus there is little evidence on which to decide among current ideas or to suggest new ones. Given the prevalence and importance of cell rearrangement in development, these basic questions of mechanism should be pursued with vigor. ACKNOWLEDGMENTS Many thanks to Paul Wilson and Jeff Hardin for their insightful comments, and to Paul Tibbetts for help in preparing the manuscript. This was prepared under the support of NIH Grant HD18979 to the author. REFERENCES 1 Fristrom, D. (1976) The mechanism of evagination of imaginal discs of Drosophila melanogaster. III. Evidence for cell rearrangement. Dev. Biol., 54: 163-171. 2 Waddington, C. H. (1940) Organizers and Genes. Cambridge University Press, Cambridge. 3 Fristrom, D. and Fristrom, J. (1975) The mecha- nism of evagination of imaginal discs of Drosophila melanogaster. 1. General considerations. Dev. Biol., 43: 1-23. 4 Jacobson, A. and Gordon, R. (1976) Changes in the shape of the developing nervous system analyzed experimentally, mathematically and by computer simulation. J. Exp. Zool., 197: 191-246. 5 Keller, R. E. (1980) The cellular basis of epiboly: A SEM study of deep cell rearrangement during gastrulation in Xenopus laevis. J. Embryol. Exp. Morphol., 60: 201-234. 6 Smith, J. and Malacinski, G. M. (1983) The origin of the mesoderm in an anuran, Xenopus laevis, and a urodele, Ambystoma mexicanum. Dev. Biol., 98: 250-254. 7 Locke, M. and Huie, P. (1981) Epidermal feet in insect morphogenesis. Nature, 293: 733-735. 8 10 11 12 13 14 15 16 17 18 19 20 21 778 Locke, M. and Huie, P. (1981) Epidermal feet in pupal segment morphogenesis. Tissue Cell, 13: 787-803. Poole, T. and Steinberg, M. (1981) Amphibian pronephric duct morphogenesis: segregation, cell rearrangement and directed migration of the Ambystoma duct rudiment. J. Embryol. Exp. Morphol., 63: 1-16. Kageyama, T. (1982) Cellular basis of epiboly of the enveloping layer in the embryo of the Medaka, Oryzias latipes. I1. Evidence for cell rearrangement. J. Exp. Zool., 219: 241-256. Graf, L. and Gierer, A. (1980) Size, shape and orientation of cells in budding hydra and regulation of regeneration in cell aggregates. Wilhelm Roux’s Arch., 188: 141-151. Bode, P. M. and Bode, H. R. (1984) Formation of pattern in regenerating tissue pieces of Hydra attenuata. III. The shaping of the body column. Dev. Biol., 106: 315-325. Ettensohn, C. A. (1985) Gastrulation in the sea urchin embryo is accompanied by the rearrangement of invaginating epithelial cells. Dev. Biol., 112: 383-390. Hardin, J. D. and Cheng, L. Y. (1986) The mechan- isms and mechanics of archenteron elongation dur- ing sea urchin gastrulation. Dev. Biol., 115: 490-501. Nardi, J. and Magee-Adams, S. M. (1986) Forma- tion of scale spacing patterns in a moth wing. I. Epithelial feet may mediate cell rearrangement. Dev. Biol., 116: 278-290. Keller, R. E. (1978) Time-lapse cinemicrographic analysis of superficial cell behavior during and prior to gastrulation in Xenopus laevis. J. Morphol., 157: 223-248. Keller, R. E., Danilchik, M., Gimlich, R. and Shih, J. (1985) Convergent extension by cell intercalation during gastrulation of Xenopus laevis. In “Molecu- lar Determinants of Animal Form. UCLA Sympo- sium on Molecular and Cellular Biology, New Series”, Vol. 31. Ed. by G. M. Edelman, Alan R. Liss, New York, pp. 111-142. Keller, R. E., Danilchik, M., Gimlich, R. and Shih, J. (1985) The function of convergent extension during gastrulation of Xenopus laevis. J. Embryol. Exp. Morphol., 89: Suppl., 89: 185-209. Miyamoto, D. M. and Crowther, R. (1985) Forma- tion of the notochord in living ascidian embryos. J. Embryol. Exp. Morphol., 86: 1-17. Keller, R. E. and Trinkaus, J. P. (1987) Rearrange- ment of enveloping layer cells without disruption of the epithelial permeability barrier as a factor in Fundulus epiboly. Dev. Biol., 120: 12-24. Keller, R. E. (1986) The cellular basis of amphibian gastrulation. In “Developmental Biology: A Com- R. KELLER 22 23 24 25 26 27 28 29 30 Sil 3Z 33 34 35 36 prehensive Synthesis, Vol. 2, The Cellular Basis of Morphogenesis”. Ed. by L. Browder, Plenum Press, New York, pp. 241-327. Keller, R. E. and Trinkaus, J. P. (1982) Cell rear- rangement in a tightly joined epithelial layer during Fundulus epiboly. J. Cell Biol., 95: 325a. Betchaku, T. and Trinkaus, J. P. (1978) Contact relations, surface activity, and cortical microfila- ments of marginal cells of the enveloping layer and of the yolk syncytial and yolk cytoplasmic layers of Fundulus before and during epiboly. J. Exp. Zool., 206: 381-426. Trinkaus, J. P. and Lentz, T. (1967) A fine struc- tural study of cytodifferentiation during cleavage, blastula and gastrula stages of Fundulus heteroclitus. J. Cell Biol., 32: 121-138. Bennett, M. V. L. and Trinkaus, J. P. (1970) Elec- trical coupling of embryonic cells by way of ex- tracellular space and specialized junctions. J. Cell Biol., 44: 592-610. Dunham,P. B., Cass, A., Bennett ,M. V.L. and Trinkaus, J. P. (1970) Water permeability of Fun- dulus eggs. Biol. Bull., 139: 420-421. Keller, R. E. (1981) An experimental analysis of the role of the bottle cells and the deep marginal zone in gastrulation of Xenopus laevis. J. Exp. Zool., 216: 81-101. Keller, R. E. (1984) The cellular basis of gastrula- tion in Xenopus laevis: Active, postinvolution con- vergence and extension by mediolateral interdigita- tion. Am. Zool., 24: 589-603. Jacobson, A. G. (1981) Morphogenesis of the neu- ral plate and tube. In “Morphogenesis and Pattern Formation”. Ed. by T. G. Connelly, L. L. Brinkley and R. M. Carlson, Raven Press, New York, pp. 233-263. Jacobson, A. G. (1985) Adhesion and movement of cells may be coupled to produce neurulation. In “The Cell in Contact: Adhesions and Junctions as Morphogenetic Determinants”. Ed. by G. M. Edel- man and J.-P. Thiery, John Wiley and Sons, New York, pp. 49-65. Jacobson, A., Oster, G., Odell, G. and Cheng. L. (1986) Neurulation and the cortical tractor model for epithelial folding. J. Embryol. Exp. Morphol., 96: 19-49. Holtfreter, J. (1943) A study of the mechanics of gastrulation. Part I. J. Exp. Zool., 94: 261-318. Holtfreter, J. (1947) Structure, motility, and locomotion in isolated embryonic amphibian cells. J. Morphol., 79: 27-62. Hatta, S. (1907) Gastrulation in Petromyzon. J. Coll. Sci. Imp. Univ. Tokyo, 21: 3-44. Holtfreter, J. (1944) A study of the mechanics of gastrulation. Part II. J. Exp. Zool., 95: 171-212. Fristrom, D. and Chihara, C. (1978) The mecha- 37 38 39 40 41 42 43 45 46 47 48 49 50 51 Cell Rearrangement in Morphogenesis nism of evagination of imaginal discs of Drosophila melanogaster. V. Evagination of disc fragments. Dev. Biol., 66: 564-570. Fristrom, D. (1982) Septate junctions in imaginal discs of Drosophila melanogaster: A model for the redistribution of septa during cell rearrangement. J. Cell Biol., 94: 77-87. Fristrom, J. W., Logan,W.R. and Murphy, C. (1973) The synthetic and minimal culture require- ments for evagination of imaginal discs of Drosophi- la melanogaster in vitro. Dev. Biol., 33: 441-456. Fekete, E., Fristom, D., Kiss, I. and Fristrom, J. W. (1975) The mechanism of evagination of imagin- al discs of Drosophila melanogaster. II. Studies on trypsin-accelerated evagination. Wilhelm Roux’ Archiv. Entwicklungsmech. Org., 178: 123-138. Gustafson, T. and Wolpert,L. (1967) Cellular movement and contact in sea urchin morphogenesis. Biol. Rev., 42: 442-489. Stephens, L., Hardin, J., Keller, R. and Wilt, F. (1986) The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo. Dev. Biol., 118: 64-69. Gustafson, T. and Kinnander, H. (1956) Méicro- aquaria for time-lapse cinematographic studies of morphogenesis in swimming larvae and observations on gastrulation. Exp. Cell Res., 11: 36-57. Kinnander, H. and Gustafson, T. (1960) Further studies on the cellular basis of gastrulation in the sea urchin larva. Exp. Cell Res., 19: 276-290. Ettensohn, C. A. (1985) Mechanisms of epithelial invagination. Q. Rev. Biol., 60: 289-307. Keller, R. E. and Hardin, J.D. (1987) Cell be- havior during active cell rearrangement: Evidence and speculations. J. Cell Sci., (in press). Hardin, J. D. (1987) Archenteron elongation in the sea urchin is a microtubule-independent process. Dev. Biol., 121: 253-262. Nardi, J. and Kafatos, F.C. (1976) Polarity and gradients in lepidopteran wing epidermis. I. Changes in graft polarity, form, and cell density accompanying transposition and reorientation. J. Embryol. Exp. Morphol., 36: 469-487. Nardi, J. and Kafatos, F.C. (1976) Polarity and gradients in lepidopteran wing epidermis. II. The differential adhesiveness model: Gradient of a non- diffusible cell surface parameter. J. Embryol. Exp. Morphol., 36: 489-512. Locke, M., (1985) The structure of epidermal feet during their development. Tissue Cell, 17: 901-921. Otto, J. (1977) Orientation and behaivor of epithe- lial cell muscle processes during Hydra budding. J. Exp. Zool., 202: 307-322. Campbell, R. D. (1967) Tissue dynamics of steady- state growth in Hydra littoralis. 1. Patterns of cell 52 58, 54 55) 56 37) 58 ay) 60 61 62 63 64 65 (1) division. Dev. Biol., 15: 487-502. Campbell, R. D. (1968) Cell behavior and mor- phogenesis in hydroids. In Vitro, 3: 22-32. Campbell, R. D. (1980) Role of muscle processes in hydra morphogenesis. In “Developmental and Cellular Biology of Coelenterates”. Ed. by P. Tardent and R. Tardent, Elsevier/North Holland, New York, pp. 421-428. Mittenthal, J. and Mazo, R. (1983) A model for shape generation by strain and cell-cell adhesion in the epithelium of an arthropod leg segment. J. Theor. Biol., 100: 443-483. Cruz, Y. P. and Pedersen, R. A. (1985) Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers. Dev. Biol., 11: 73-83. Honda, H. and Eguchi, G. (1980) How much does the cell boundary contract in a monolayered cell sheet? J. Theor. Biol., 84: 575-588. Honda, H., Ogita, Y., Higuchi,S. and Kani, K. (1982) Cell movements in a living mammalian tissue: long-term observation of individual cells in wounded corneal endothelia of cats. J. Morphol., 174: 23-39. Honda, H., Dan-Sohkawa, M. and Watanabe, K. (1983) Geometrical analysis of cells becoming organized into a tensile sheet, the blastular wall, in the starfish. Differentiation, 25: 16-22. Honda, H. (1983) Geometrical models for cells in tissues. Int. Rev. Cytol., 81: 191-248. Honda, H., Kodama, R., Takeuchi, T., Yamanaka, H., Watanabe, K. and Eguchi,G. (1984) Cell behavior in a polygonal cell sheet. J. Embryol. Exp. Morphol., 83: Suppl., 313-327. Honda, H., Yamanaka, H. and Eguchi, G. (1986) Transformation of a polygonal cellular pattern during sexual maturation of the avian oviduct epithelium: computer simulation. J. Embryol. Exp. Morphol., 98: 1-19. Keller, R. E. (1976) Vital dye mapping of the gastrula and neurula of Xenopus laevis. I1. Prospec- tive areas and morphogenetic movements in the deep region. Dev. Biol., 51: 118-137. Cloney, R.A. (1964) The development of the ascidian notochord. Acta Embryol. Morphol. Exp., 7: 111-130. Poole, T. and Steinberg, M. (1982) Evidence for the guidance of pronephric duct migration by a cra- niocaudally traveling adhesion gradient. Dev. Biol., 92: 144-158. Gillespie, L. L. and Armstrong, J. B. (1986) Mor- phogenetic waves in the development of the lateral mesoderm in the Mexican axolotl (Ambystoma mexicanum) and their relationship to pronephric duct migration. J. Exp. Zool., 237: 327-338. ‘2 48 fide 454 Wah 1 SRR yh NG | a ek Rae he ert bk Enis SHOE: FRR hegyensy! re i Siw Giety. i ee ae ON a RTE: it ebloues: iL Saat ogotg, at abemiedney! y : Lesa Hy giabe neh) ti eb 3 chee brik ncn siti ii wre! : Scatperioningta: anya pe? 4 yin SKE Tear tineteedyy aman tinetalie ihe ou att ise eoeel eng ek A Dae) ae oe , Sy LE RR ie Ey epee at aaa ad ave Daag Api ye ners pone Pe bet Bb cieira web meteeh bee canines Rea aR boil ca Sessa vhs gy ise COG JF Si i el ih Gott Papas) |e PY cee Merde UE, deh et 1 HUONG evOePAG iis TRH TCT ee a may | er Bit id ae TRESS I ees Ba pi fae PC, ee Rao OG ABET ER SOU Wo ; spel sisted : once | Rioldg oh Aiink 1 1 Dre ae he oye TH hddeeudh SS ee ee PR Pls spent wruneses Ay caine a ee ee seo et ea Pam tibet Th Gd Piy Biat) Haha te fhohoeierittyta rae) ¢ Wea PS? 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Syrhe is ah hes : Sa EP enh SS. me how ‘ghd at spedlcanioles ‘Morais ame, a SR oct uriwep rial bai, ‘Vivhaster | ARGH EaLD | Leanna” pee shee ey Hie Ga bee Hie iinet val eed re Seo yo fen home 40 Widibe Dh ayn SS heeonenees Unis, “cena {foot ZOOLOGICAL SCIENCE 4: 781-788 (1987) Geotactic Behavior in Paramecium caudatum I. Geotaxis Assay of Individual Specimen Kost TANEDA Department of Biology, Faculty of Science, Kochi University, Akebonocho 2-5-1, Kochi 780, Japan ABSTRACT—Geotactic behavior of an individual specimen of Paramecium caudatum was examined by analyzing its swimming path in a vertical glass tube. The path was recorded by means of a specially designed apparatus. The specimen swam upward to the top of the tube and stayed there (negative geotaxis), when the glass tube was thicker than 1.8 mm in its inner diameter. In thinner tube the specimen did not show the negative geotaxis, because upward orientation of the specimen was somehow inhibited in the tube. Inhibition of ciliary activity caused a decrease in the upward swimming velocity. This seems to indicate that the upward orientation of the specimen is independent of the ciliary activity. Change in hydrostatic pressure on the specimen did not affect the geotactic behavior. Thus a hydrostatic pressure difference between upper and lower portion of the specimen can not be a cause for the upward orientation of the specimen. Change in the hydrostatic pressure with time was also eliminated from possible causes for the negative geotaxis. The specimen showed negative geotaxis in a heavy water-containing solution, whose specific gravity was larger than that of © 1987 Zoological Society of Japan the specimen. orienting specimen caused its negative geotaxis. INTRODUCTION Many ciliated and flagellated protozoans exhib- ited negative geotaxis. Since specific gravity of the cells is usually higher than their surrounding medium, they exhibit negative geotaxis with ex- penditure of energy. Several hypotheses for the mechanism of the negative geotaxis have been proposed [1-4]. All the hypotheses were based on the analysis of distribution of the cells in gravity field. For better understanding of the mechanism it is important to know locomotor behavior of an individual organism in gravity field. Present paper will deal with precise description of swimming behavior of a single specimen of Paramecium caudatum in a vertical glass tube. The effects of some environmental factors on the behavior will also be presented. Some hypotheses proposed by previous workers will be confuted on the basis of present findings. Accepted June 6, 1987 Received October 18, 1985 In highly viscous solution, frequent occurrence of ciliary reversal in a downward MATERIALS AND METHODS Specimens of Paramecium caudatum (wild type) were cultured in bacterized (Klebsiella aerogenes) dried lettuce infusion at 22-24°C. The specimens were washed with a diluted Herbst’s modified artificial sea water (ASW), which was osmotically equivalent to 2mM NaCl and buffered to pH 7.2-7.4 with NaHCO3. All the chemicals ex- amined were dissolved in the diluted ASW. In some experiments heavy water (D,O; C.E.N. Sacley, France, 99.8%) was substituted for natural water. Specimens obtained from various cultures with different culture age were rinsed with the diluted ASW at least three times. As shown in Figure 1, a single specimen was pipetted into the bottom of a vertical glass tube (T1; 15cm in length) filled with a test solution through a thin glass tube (O; 3 mm in inner diameter, 5 mm in length) branched at the bottom of Tl. The specimen (C) in the tube was observed through a magnifying glass with a horizontal mark line (L). The swimming specimen in the tube was tracked by moving the line along the tube. The movement 782 K. TANEDA 2r r 172 |e 2v | C 5 | c ee Vv oO \\ Ww Oli pas ©) [I (oc) G Fic. 1. ah) Schematic representation of the experimental set-up for recording the swimming behavior of an individual specimen of Paramecium caudatum. 11; a vertical glass tube where a single specimen of Paramecium (C) is introduced through an opening (O). Position of Paramecium in the tube is followed by moving a small magnifying glass with a horizontal mark line (L) through which the specimen is observed. G; a finger-grip for moving the magnifying glass. The movement of the glass is recorded on a paper on a kymograph drum (D) by means of a pen (P). T; a transformer for adjusting rotation rate of the drum (1.5 cm/min). T2; a vertical glass tube connected to T1 with silicone rubber tube (S) for applying extra hydrostatic pressure to the specimen. The tube is kept lay down when the extra hydrostatic pressure is not needed. See the text for details. W; a weight for smooth movement of pulley-systems. of the line was transferred into vertical movement of a recording pen (P) by a pulley-system to record the movement of the specimen on a sheet of section paper lapped around a kymograph drum (D). Running speed of the drum was adjusted to be 1.5 cm/min by a voltage controller (T). Other details of the experimental methods will be de- scribed in Results section. All the experiments were performed at room temperature ranging from 18s Coane: RESULTS Effect of thickness of the glass tube on the swim- ming behavior A series of experiments was carried out to examine influence of bore size of the glass tube on swimming behavior of a specimen in the tube. Some typical results on the specimens obtained from 6-day culture are shown in Figure 2. When . N\\ : 1:8 4-2 E 2s Imin Fic. 2. Influence of bore size of the glass tube on the swimming behavior of P. caudatum in the tube. Each record represents time-change in the position of a single specimen in the tube. The specimens were obtained from 6-day culture. Each number indicates inner diameter of the glass tube in mm. Geotaxis in Paramecium I. 783 inner diameter of the tube was thinner than 1.8 mm, the specimen swam upward and downward alternatively over the full length of the tube. Ina glass tube thicker than 2.4 mm the specimen swam upward to the top of the tube then turned downward, but soon began to swim upward again. Thus they tended to stay near the upper portion of the tube (negative geotaxis). In the following experiments the geotactic behavior was examined in a glass tube of 4.2 mm in inner diameter. Effect of culture age on the swimming behavior Swimming behavior in the vertical tube was examined in several specimens with different culture age. Typical results are shown in Figure 3. The specimens in the logarithmic growth phase (1-3-day culture) exhibited weak negative geo- taxis, while those in the early stationary phase (6—7-day culture) showed strong negative geotaxis. In order to show degree of the negative geotaxis 1min APS 7 Fic. 3. Effect of the culture age on the swimming behavior of P. caudatum in a vertical tube with inner diameter of 4.2mm. Each record represents time-change in the position of a single specimen in the tube. Each number indicates the culture age in day. quantitatively, period of time when the specimen was present in upper 1/5 portion of the tube during 30-min observation was determined and expressed in percentage (Gs). Moreover, the period of time from the moment when a specimen at the top of the tube began to swim downward to the moment when it reoriented upward (Td) was measured. Td corresponds to the tendency of the specimen to swim downward. In Figure 4 mean values of Gs and Td obtained from 5 measurements with 5 different specimens were plotted against culture age together with the growth curve of the speci- mens in the culture. Gs was minimum in 1-2-day culture, increasing gradually and reached its max- imum in 6—7-day culture. Td was inversely related with Gs, e.g., it was maximum in 1-2-day culture, while minimum in 6-7-day culture. (0) 30 20 Td (sec) Population density (cells/ml) CFO wae a er ee Culture age (days) Fic. 4. Relation of the culture age with degree of the negative geotaxis (Gs; solid circles) and with tendency of the specimen of P. caudatum to down- ward swimming (Td; open circles) in the vertical tube. For the definitions of Gs and Td see the text. Cell density in the culture (open triangles) is also presented as a function of the culture age. Effect of starvation on the swimming behavior When the specimens had been kept unfed, the negative geotaxis became unconspicuous. As shown in Figure 5, tendency of the specimen. to stay in the upper portion of the tube decreased with starvation period. Changes in both Gs and Td with starvation period are shown in Figure 6. Td increased slightly by 4-day starvation and greatly after then. Gs decreased to zero in 4-day starva- tion. 784 K. TANEDA | is hal hd ginger pain Imin Fic. 5. Effect of starvation on the swimming behavior in the specimen of P. caudatum obtained from 6- day culture. Each record represents the position of a single specimen in the vertical tube. Each num- ber indicates period of starvation in day. ARO Ooo ae oem Starvation period (days) Fic. 6. Relation of the period of starvation (days) with Gs (solid circles) and Td (open circles) of P. cauda- tum in the vertical glass tube. See the legend of Fig. 4. Effects of hydrostatic pressure and of density of the external solution on the swimming behavior Hydrostatic pressure on the specimen in the tube decreases as the specimen swim upward. In order to examine a possible dependency of the negative geotaxis on the decrease in the hydrosta- tic pressure with time, the experimental apparatus was arranged so that the pressure increased as the specimen swam upward. As shown in Figure 1, the glass tube, T1, was connected to another glass tube, T2. T2 moved upward twice as fast as the swimming speed of the specimen in T1 by means of a pulley-system, so that the hydrostatic pressure on the specimen increased with the specimen swam upward. The increased hydrostatic pressure can be removed suddenly by laying T2 down. Three typical examples of geotactic behavior of the specimens under the influence of increasing the hydrostatic pressure are shown in Figure 7. The behavior was essentially similar to that in normal case, where the pressure decreased as the speci- men swam upward (Figs. 3-6). The specimens stayed at the top of the tube even after removing the increased hydrostatic pressure. aE Imin Fic. 7. Time-change in the position of a single speci- men of P. caudatum in the vertical tube under the influence of increasing the hydrostatic pressure. C indicates the time when the extra hydrostatic pres- sure was removed. In order to examine the resistance hypothesis for the negative geotaxis proposed by Davenport [3], behavior of the specimens in a solution with a density higher than that of Paramecium (d=1.04) was examined. Heavy water (d=1.10) was em- ployed to make the density of the solution higher. Viscosity of the solution was almost the same with that of normal solution. When the specimens were transferred into the solution, their swimming speed decreased gradually with time and finally Geotaxis in Paramecium I. 785 Control ci Imin Fic. 8. Time-change in the position of a single speci- men of P. caudatum in the vertical tube filled with a heavy water-containing solution. Upper record (control); the time-change in the normal water test solution. Middle records; the specimens were in- troduced into the bottom of the tube. Lower records (T); the specimens were introduced into the top of the tube. they stopped the swimming. As shown in Figure 8, some specimens showed clear negative geotaxis. When the specimens were introduced into top of the tube instead of into bottom’, they did not show clear tendency to swim downward (lower records labelled as T in Fig. 8). Effects of NaN3 and other inhibitors of motility The effect of NaN3, a metabolic inhibitor, on the geotaxis of Paramecium was examined. As shown in Figure 9, NaN3 produced little effect on the geotaxis when its concentration was below 5xX10-?M. With increasing NaN; concentration upward swimming velocity of the specimen de- creased gradually. However, tendency of the * Introduction of the specimen inte the top of the tube was performed by turning the glass tube; T1 (see Fig. 1) upside down, so that the opening (O) for introduc- ing the specimen was located at the top of the tube. Control 5x10“M 10°73 M 5x10°M 10°°M Fic. 9. Concentration effect of NaN; on the time- change in the position of a single specimen of P. caudatum in the vertical tube. Control 107’ M 5x10’ M 10°° M rast Gin] Nile ae ee de a 8 5 1min Fic. 10. Concentration effect of NiSO, on the time- change in the position of a single specimen of P. caudatum in the vertical tube. 786 K. TANEDA Fic. 11. tion of a single specimen of P. caudatum in the vertical tube. Control 0. 25°. (500cP) 0-5 %/.(1000 cP) 1-0 %/e( 2000 cP) E -L Imin Concentration (%) effect of methylcellulose on the time-change in the posi- An approximate value of the viscosity of each solution is presented in parenthesis. specimen to go upward remained even in a solution with 5xX10°*M NaN3. The specimen stopped its swimming in a solution with NaN; higher than 10-7 M. It consequently did not show the upward swimming. Effect of an addition of NiSO, or methylcellu- lose into the external solution on the geotaxis was examined and compared with that of NaN3. Ni? ions are well known to inhibit ciliary beating and thereby decrease swimming velocity [5]. Methyl- cellulose decreases swimming velocity of the speci- men due to increased viscosity. Some typical results are shown in Figures 10 (Ni?*) and 11 (methylcellulose). The specimen showed negative geotaxis in the presence of Ni*t or methylcellu- lose. The upward swimming velocity of the specimen decreased with increasing the concentra- tion of Ni*t or methylcellulose. The negative geotaxis in a solution with high concentration of Ni?+ or methylcellulose became unconspicuous due to decreased swimming velocity. The specimens in a highly viscous methylcellu- lose-containing solution (2000 cP) migrated up- ward slowly, while they showed frequent alterna- tion of upward and downward swimming (Fig. 11, the lowest trace). In order to understand the mechanism for the negative geotaxis in the highly viscous medium, frequency of ciliary reversal was determined in a specimen oriented either upward or downward in a methylcellulose solution. A D U , D ME A | Cn Imin Fic. 12. Spontaneous ciliary reversal recorded in three different specimens of P. caudatum in a highly vis- cous solution (8000cp by 4% methylcellulose). Black parts of each column indicate that the speci- men shows ciliary reversal. D; the specimen oriented downward. U; the specimen oriented upward. specimen was introduced into a small hanging drop of 4% methylcellulose solution (8000 cP) and placed on a stage of a horizontal microscope. The specimen was oriented upward or downward by rotating the stage. Three typical results are shown in Figure 12. The downward-oriented specimens showed ciliary reversal more frequently than the upward-oriented specimens. DISCUSSION Bean [6] found that Chlamydomonas did not show negative geotaxis in a thin glass tube, because of restriction of turning of the cell in the tube. Inhibition of the negative geotaxis of Geotaxis in Paramecium I. 787 Paramecium in a thin glass tube seems to be also due to restriction of its turning in the tube (Fig. 2). An increase in degree of the negative geotaxis, Gs, with culture age was correlated with a decrease in Td (Fig. 4). A decrease in Gs by starvation was correlated with an increase in Td (Fig.6). A decrease (increase) in Td corresponds to an increase (decrease) in the probability that the specimen swimming downward turns upward. The present findings, therefore, confirmed the previous idea that the upward orientation of the specimen is an important factor for its negative geotaxis [6]. Four hypotheses have been proposed for the mechanism of the negative geotaxis in protozoans as reviewed by Kuznicki [7]. According to the pressure hypothesis by Jensen [2], a small differ- ence in hydrostatic pressure between the upper and lower portions of the organism causes the negative geotaxis, because ciliary activity is assumed to be higher when the pressure is higher. However, Lyon [8] demonstrated that a change in the hydrostatic pressure as small as the hydrostatic pressure difference between upper and lower portions of Paramecium did not produce change in the ciliary activity. Present finding that the negative geotaxis is not affected by subjection of the specimen to an increasing hydrostatic pressure as it moved upward (Fig. 6) denies a possibility that decrease in the hydrostatic pressure with time causes the negative geotaxis. An abrupt decrease in the hydrostatic pressure did not affect the negative geotaxis. This finding is also unfavorable to the pressure hypothesis. In order to understand the mechanisms by which the negative geotaxis, positive centrotaxis and negative rheotaxis are governed, Davenport [3] proposed the resistance hypothesis. According to his hypothesis, Paramecium orients and moves in a direction so as its energy consumption to be the highest. Since Paramecium is heavier than its surrounding water, upward swimming requires more energy than downward swimming. It was found that the specimens of Paramecium showed clear negative geotaxis in the heavy water solution with a density higher than that of Paramecium (Fig. 8). This finding is unfavorable to the resist- ance hypothesis. Platt [9] and Lyon [5] reported that ciliates such as Paramecium and Spirostomum exhibited negative geotaxis in a gum-arabic con- taining high density medium. This is also unfavor- able to the resistance hypothesis. Kanda [4] and Lyon [5] proposed the statocyst hypothesis for the negative geotaxis of Para- mecium. They assumed that inclusions (granules, crystals or food vacuoles) and nuclei of the cell of Paramecium play a role essentially similar to the statolith in metazoan statocyst. Mechanical stim- ulation of inner surface of the cell membrane by the cell inclusions or nuclei might produce upward orientation of the specimen. In a methylcellulose- containing viscous solution the specimens migrated upward, while they showed frequent alternation of upward and downward swimming (Fig. 11). The specimens oriented downward in the viscous solu- tion showed change in the swimming direction more frequently than those oriented upward (Fig. 12). This apparently causes denser distribution of the specimens in the upper portion of the tube. As shown in Figure 11, swimming velocity was higher in the specimens oriented downward than upward. The viscous resistance experienced by the speci- men with its anterior end is presumably larger when it swims downward, and thereby produces more frequent ciliary reversal due to activation of mechanosensitive Ca channels in the anterior membrane [10]. This mechanism for the negative geotaxis is, however, applicable only to the case in highly viscous solution. Ciliary reversal was not necessary for establishing the negative geotaxis. Specimens of the CNR mutant [11], which is incapable of showing ciliary reversal, also exhib- ited the negative geotaxis as normal specimens did. All the three hypotheses mentioned above involve some stimulus-transduction mechanisms in the cell. They therefore can be classified as physiological hypotheses. According to Bean [6] negative geotaxis in Chlamydomonas was inhibited by NaN3. The inhibition, however, was found to occur before motility of the cell was inhibited. Thus he assumed a gravity-force-sensory mechanism for the upward orientation of the cell which is more sensitive to NaN; than the motile mechanism. However, in Paramecium the negative geotaxis was inhibited in parallel with inhibition of ciliary motile activity by NaN; or by Ni?* ions (Figs. 9 and 10). 788 Present results are not favorable to all the three hypotheses except for the case in a highly viscous medium. Verworn [1], Dembowsky [12, 13] and recently, Fukui and Asai [14] proposed the me- chanical hypothesis. According to them, the posterior end of Paramecium is heavier than the anterior end. Thus a force which orients the specimen upward is produced in a gravity field. Applicability of this hypothesis will be discussed in the succeeding paper. ACKNOWLEDGMENT The author wishes to express his thanks to Professor Y. Naitoh of Tsukuba University for critical reading of the manuscript. REFERENCES 1 Verworn, M. (1889) Psychophysiologische Protis- tenstudien, G. Fischer Verlag, Jena. 2 Jensen, P. (1893) Uber den Geotropismus niederer Organismen. Pfltiger’s Arch. Gesamte Physiol., 53: 428-480. 3. Davenport, Ch. B. (1897) Experimental Morpholo- gy, Macmillan Co., New York. 4 Kanda, S. (1914) On the geotropism of Paramecium and Spirostomum. Biol. Bull., 26: 1-24. K. TANEDA 5 10 12 13 14 Kuznicki, L. (1963) Reversible immobilization of Paramecium caudatum evoked by nickel ions. Acta Protozool., 6: 301-312. Bean, B. (1977) Geotactic behavior of Chlamydo- monas. J. Protozool., 24: 394-401. Kuznicki, L. (1968) Behavior of Paramecium in gravity fields. I. Sinking of immobilized specimens. Acta Protozool., 6: 109-117. Lyon, E. P. (1904) On Jensen’s theory of geotrop- ism in Paramecium. Am. J. Physiol., 13: 15-16. Platt, J. B. (1899) On the specific gravity of Spiro- stomum, Paramecium and the tadpole in relation to the problem of geotaxis. Am. Nat., 33: 31-38. Naitoh, Y. and Eckert, R. (1969) Ionic mechanism controlling behavioral response of Paramecium to mechanical stimulation. Science, 164: 963-965. Takahashi, M. and Naitoh, Y. (1978) Behavioural mutants of Paramecium caudatum with defective membrane electrogenesis. Nature, 271: 656-659. Dembowski, J. (1929) Die Vertikalbewegungen von Paramecium caudatum. 1. Die Lage des Gleich- gewichtscentrumus in K6rper des Infusors. Arch. Protistenkd., 66: 104-132. Dembowski, J. (1929) Die Vertikalbewegungen von Paramecium caudatum. II. Einfluss einiger Aussen- faktoren. Arch. Protistenkd., 68: 215-260. Fukui, K. and Asai, H. (1980) The most probable mechanism of the negative geotaxis of Paramecium caudatum. Proc. Japan Acad., Ser. B., 56: 172-177. ZOOLOGICAL SCIENCE 4: 789-795 (1987) Geotactic Behavior in Paramecium caudatum II. Geotaxis Assay in a Population of the Specimens Kom TANEDA, SEvI Miyata! and AKIKo SHIOTA Department of Biology, Faculty of Science, Kochi University, Akebonocho 2-5-1, Kochi 780, Japan ABSTRACT—The negative geotaxis exhibited by the specimens of Paramecium caudatum in an assay tube was examined. The negative geotaxis was conspicuous in the specimens from 1-2-day culture. Number of the Ni?*t-immobilized specimens which oriented upward in a solution with the specific density identical with that of the specimen during centrifugation was the highest in the specimens showing the highest negative geotaxis. However, no difference in the number of the upward-oriented specimens was found between two groups of the specimens, one from upper half and the other from lower half of the assay tube. This suggests less important role of the gravity-buoyancy torque in the negative geotaxis. Anterior half of the specimens showing negative geotaxis was thinner than posterior half. However, no difference in the cell shape was found between two groups of the specimens, one from upper half and the other from lower half of the assay tube. This indicates that a torque produced by difference in the drag force between the anterior and posterior halves of the specimen is not an essential factor for the negative geotaxis. The propulsion-gravity model is a © 1987 Zoological Society of Japan remaining probable candidate for the mechanism of the negative geotaxis. INTRODUCTION In the preceding paper, we examined swimming path of an individual specimen of Paramecium caudatum in a vertical glass tube to analyze its geotactic behavior. Our results could not be satisfactorily explained on any _ physiological hypotheses, such as the pressure hypothesis [1], the resistance hypothesis [2] and the statocyst hypothesis [3]. We, therefore, examined applica- bility of physiological hypotheses to the geotactic distribution of the specimens of Paramecium in a vertical tube. To examine if the specific density is different between the anterior and the posterior halves of the specimen, orientation of Ni**- immobilized specimens [4] subjected to a centri- fugal force was determined. The difference, if it presents, causes upward orientation of the speci- men in the gravity field. We also determined the cell shape to examine the difference in the drag force between the anterior and the posterior halves Accepted Jnue 6, 1987 Received October 18, 1985 " Present Address: Toyama Toyama 930, Japan. Chemical Industry, of the specimen as a possible cause for the upward orientation of the specimen. We found that neither the gravity-buoyancy model of Verworn [5], which was recently supported by Fukui and Asai [6], nor the drag-gravity model of Roberts [7] was explainable for our results. MATERIALS AND METHODS Specimens of Paramecium caudatum (strain G-3) were cultured in a test tube filled with bacterized (Klebsiella aerogenes) lettuce infusion at 22-24°C. Specimens obtained from culture were washed three times with a standard saline solution [8] containing 1 mM KCI, 1 mM CaCl, and 1 mM Tris-HCl (pH 7.2), and kept equilibrated in the solution for more than 10min prior to experi- mentation. Geotactic behavior of an individual specimen was examined by the method similar to that described in the previous paper [9]. Geotactic distribution of a population of the specimens was quantitatively determined by the following methods. Suspension of the specimens (10 cells /ml) was introduced into a vertical glass tube (3 cm 790 K. TANEDA, S. MIYATA AND A. SHIOTA in inner diameter, 30 cm in length), and kept for a given period of time. The suspension in the tube was divided into two fractions, e.g. upper and lower halves, and the number of the specimens in each fraction was counted. Then the geotaxis index (Gt) was calculated according to Bean [10]. Gt is defined as: Gia Nb—(Nb+Nt)/2 (Nb-+-Nt)/2 where Nt is number of the specimens in the upper fraction, and Nb is that in the lower fraction. All the experiments were done at a constant temperature of 24+1°C. Some details of the experimental procedures will appear in Results section. a b c d e f Fic. 1. RESULTS Geotactic responses in an individual specimen Swimming path of the specimens obtained from different cultures with different culture age (1-5 days) in the vertical tube was recorded. As shown in Figure 1, the specimens from 1- or 2-day culture introduced into the bottom of the vertical tube swam upward to the top of the tube and stayed there (negative geotaxis). Most of the specimens from 3-day culture showed upward and downward swimming alternately over the full length of the glass tube. Some specimens from 4-day culture first showed negative geotaxis then showed down- ward swimming to the bottom of the tube and Ara ia ah fh | [ Swimming path of an individual specimen of Paramecium caudatum in a vertical tube. Five groups of the specimens from 5 different cultures with different culture age (1-5-day; indicated by corresponding numbers at the left of each group of the records) were examined. Each group consists of 6 different specimens (labeled a-f). Geotaxis in Paramecium II. 791 stayed there (positive geotaxis), as shown in Figure 1, 4-f. Some of them showed first positive and then negative geotaxis (Fig. 1, 4-e). Number of specimens to show the positive geotaxis increased in 5-day culture (Fig. 1, 5—d, e, f). Geotactic responses in a population of the speci- mens Time-change in distribution of the specimens in the geotaxis-assay tube was examined. Homo- geneous suspension of the specimens was intro- duced into the tube and kept undisturbed for various period of time (1-20 min). Then, the negative geotaxis index (—Gt) was determined. Time-change in —Gt is shown in Figure 2. —Gt increased quickly and reached a definite saturated level (0.84) in 5 min. Therefore, determination of of; 5 10 15 20 Time in min Fic. 2. Time-change in the negative geotaxis index (—Gt) of a populaiton of the specimens of P. caudatum. Each point is the mean obtained from 5 measurements with the specimens from 2-day cul- ture. Vertical bars; standard deviations of the mean. e o iS rr) ro) Oo 1 Culture age (days) Fic. 3. Change in the negative geotaxis index (—Gt) of a population of P. caudatum as a function of culture age (day). —Gt was carried out after 10-min assay. Thigmo- taxis of the specimen was not observable in the present assay condition. Change in —Gt as a function of culture age is shown in Figure 3. —Gt reached its maximum value (0.87) by the 2nd day of culture, then decreased to zero on the 5th day. The change in —Gt with culture age appears to correspond to the geotactic activity in each individual specimen of the population (see Fig. 1). To find correlation between the individual behavior and populational behavior, geotactic activity of individual specimen (Gs) was calculated from the data presented in Figure 1 according to Taneda [9], and the value was compared with Gt. Gs corresponds to the probability that the specimen stay in upper half of the assay tube. As shown in Figure 3, Gs changed with culture age in parallel with —Gt. Orientation of Ni-immobilized specimens under influence of a centrifugal force In order to examine localized difference in the specific density of the protoplasm in the cell as a possible cause for the upward orientation in the negative geotactic specimens, they were first im- mobilized by Ni** (0.0075% NiSO,, w/v), then introduced into a vessel (40201 mm?*) with a solution whose specific density was adjusted to be the same with that of the specific density was adjusted to be the same with that of the specimen (d=1.042) by adding gum arabic. They were subjected to a centrifugal force of 110 G for 30 sec. Then, the direction in which the anterior end of the specimen pointed was determined by the aid of a horizontal microscope. As shown in the upper left inset of Figure 4, a circle around each speci- men was divided into 9 directional divisions (A to I) with reference to direction of the centrifugal force (an arrow labelled with g). Percent number of the specimens which pointed in each directional division was counted, and shown in Figure 4. As shown in the figures, most of the specimens from 1- or 2-day culture pointed their anterior end up- ward. Number of the upward-oriented specimens decreased with culture age, and the specimens from 4- or 5-day culture oriented more or less at random. If the upward-oriented specimens swim upward 792 K. TANEDA, S. MIYATA AND A. SHIOTA A Bf g Gil D E F G 4 l 0) A A f[ B B Cc CH D D E = F F G G A 4 | 0 50 “/o 0 A B G D E F G H | 50%. 0 50% A B Cc D E F 5 G H I 50%. 0 50 Vo Fic. 4. Frequency distributions of the orientation of the Ni*t-immobilized specimens of P. caudatum under the influence of a centrifugal force (110G). Shaded columns show the specimens oriented upward. The number in each histogram shows the culture age in day. See the text for the details. and stay in upper half of the tube, and the downward-oriented specimens swim dowanward and stay in lower half of the tube when their cilia beat, the geotaxis index of the non-motile popula- tion of the specimens, Gp, which corresponds to Gt in motile population, can be formulated as: Go Nd—(Nu+Nd)/2 (Nu+Nd)/2_ , where Nd is number of the specimens oriented downward and Nu that oriented upward. —Gp was calculated from the data shown in Figure 4 and plotted against the culture age as shown in Figure 5. Change in —Gt with culture age was also fo) -Gt(e) , -Gp(o) Culture age (days) Fic. 5. Change in the geotaxis index in a population of the specimens of P. caudatum as a function of cul- ture age (day). Open circle; the negative geotaxis index calculated from the data in Fig. 6 (—Gp). Solid circle; negative geotaxis index calculated from actual distribution of the specimens in the assay tube (—Gt). presented in Figure 5 (open circles). Gt in this figure was determined with the specimens from the culture, with which determination of Gp was made. Both —Gp and —Gt were highest in the specimens from 1- or 2-day culture. However, each value of —Gt did not coincide with each corresponding value of —Gp. In the next series of experiments, the specimens in the assay tube was divided into two groups, one presents in upper half and the other in lower half of the tube, after they were kept in the tube for more than 10 min. The specimens in each group was immobilized by Ni**, immersed into the gum arabic-containing solution and subjected to a centrifugal force (110 G) for 30 sec. Then their spacial orientation was determined. As shown in Figure 6, the distribution pattern of the orientation was identical between the two groups. Change in the cell shape with culture age In order to examine relationship between cell shape and geotactic activity, change in cell shape with culture age was determined. Specimen of Paramecium is cylinder-shaped. Thickness of the cylinder is different between anterior and posterior half of the specimen. We, therefore, measured radius of a cross section of the cell cylinder at the levels of both anterior and posterior contractile Geotaxis in Paramecium II. 793 A A B B a Cc Cc S E = F F 5 G G H H 1 1 1 : A pe B wy c 3 > D D E E S 2 ; I 1 (LE eee Eee 0 50°. O 50% A f& A A ef e Cc D D D E E E F F F G 2 G H H 1 & 3 I 4 l 5 A A A B B B Cc (S C D D D E E E F F F G G G H H H I 1 I (le SS Sa Te gee pe ee ag gy 0 5Ovey 0 50% 0 50 %e Fic. 6. Frequency distribution of the orientation of Ni**-immobilized specimens of P. caudatum under the influence of a centrifugal force (110 G). Open column; obtained with the specimens collected from the lower half of the assay tube. Shaded columns; obtained with the specimens collected from the upper half of the assay tube. Each number in each histogram shows the culture age. For further explanation, see the text. vacuoles (r,; at the anterior, r,; at the posterior vacuole). The difference between the squares of the radii (17-1, = Ar’) was used to characterize the cell shape. When the value is negative (positive), the anterior half of the specimen is thinner (thicker) than its posterior half. 4r? was determined in 200 specimens from a single culture. The frequency distribution of Ar’ for different cultures with different culture age is shown in Figure 7. Ar* was negative for most of the specimens from 1-2-day culture. Number of the specimens whose dr? was positive tended to increase with culture age. More than half of the specimen from 5-day culture showed positive Ar’. This indicates that the cell shape changes from its original thinner anterior-shape to thinner pos- terior-shape with culture age. The specimens from 4-day culture in the assay tube were separated into two groups, one present in the upper half and the other in the lower half of the tube after they were kept in the tube for more than 10min. 4r* value was determined with 200 specimens from each group and presented as a histogram (lower parts of Fig. 7). Distribution patterns of the cell shape were almost identical between these two groups. DISCUSSION Three physical models for the mechanism of negative geotaxis in free swimming microorgan- isms have been proposed: 1) the gravity-buoyancy model of Verworn [5], 2) the drag-gravity model of Roberts [7] and 3) the propulsion-gravity model of Winet and Jahn [11]. We determined the orientation of Ni**- immobilized specimens in a solution with a specific density identical with that of the specimen, under 794 K. TANEDA, S. MIYATA AND A. SHIOTA (*/o) 1 2 3 4 -e) 30 20 10 -400 -200 0 -200 0 -200 0 +200 -200 0 +200 -200 O +200 Ar2(ym2) = Ar2(um? ) Ar (him) AraC: )ieeAneeuimen) (*/o) 20 x 10 CV 0 INSES [che = (i -200 0 -200 0 +200 Again.) Nee (ine upper half lower half Fic. 7. Change in the shape of the specimens of P. caudatum. The change is presented as a change in the frequency distribution pattern of the Ar? value. See the lower left inset for the definition of Ar*. Negative value of Ar? (shaded columns) shows that the anterior half of the specimens is thinner than the posterior half. For further explanation, see the text. the influence of centrifugal force (110 G). As shown in Figure 4, number of the upward-oriented specimens, which are heavier in their posterior region, was larger in a population of the specimens showing stronger negative geotaxis. The geotaxis index (Gp) calculated on the assumption that the upward-oriented specimens stay in the upper half of the assay tube, while the downward-oriented specimens in the lower half of the tube, was almost identical with the geotaxis index (Gt) calculated from actual distribution of the specimens in the tube (Fig. 5). These findings are favorable to the gravity-buoyancy model for the negative geotaxis. However, our findings that histogram for the orientation of the immobilized specimens was almost identical between the two groups of the specimens, one obtained from the upper half and the other from the lower half of the assay tube (Fig. 6) indicates that the upward orientation of the specimens due to the gravity-buoyancy torque is not essential factor for the negative geotaxis. We also determined Ar* which characterizes the cell shape. Ar’ is proportional to the anterior and the posterior halves of the specimen due to difference in their sizes. As shown in Figure 7, Ar* changed in close association with change in the geotactic activity of the specimens. This is favor- able to the drag-gravity model for the negative geotaxis. However, the histogram for Ar’ was almost identical between two groups of the speci- mens, one from the upper half and the other lower half of the assay tube (see lower portion of Fig. 7). This finding strongly suggests that the cell shape is not essential factor for the negative geotaxis. It is interesting to note that some specimens from 4-day culture showed sudden change from negative to positive geotaxis or vice versa (Fig. 1). According to the gravity-buoyancy model, such change should be accompanied by a sudden reversal of the portions for the center of gravity Geotaxis in Paramecium II. 795 and for the center of buoyancy in the cell. The most probable cause for such reversal is redistribu- tion of food vacuoles in the cell. However, our observation (unpublished) proved that the food vacuoles were hardly seen in the specimens from 4-day culture. Sudden change in the cell shape is a possible cause for the change in the polarity of the geotaxis. This, however, is not examined yet. The remaining propulsion-gravity model seems to be more reasonable for explanation of the geotactic behavior in the specimens of Para- mecium. Examination of the model will appear in the succeeding paper. ACKNOWLEDGMENT We thanks Professor Y. Naitoh, University of Tsuku- ba, for valuable comments on the manuscript. REFERENCES 1 Jensen, P. (1893) Uber den Geotropismus niederer Organismen. Pfliiger’s Arch. Gesamte Physiol., 53: 428-480. 2 10 11 Davenport, Ch. B. (1897) Experimental Morpholo- gy, Macmillan Co., New York. Kanda, S. (1914) On the geotropism of Paramecium and Spirostomum. Biol. Bull., 26: 1-24. Kuznicki, L. (1963) Reversible immobilization of Paramecium caudatum evoked by nickel ions. Acta Protozool., 6: 301-312. Verworn, M. (1889) Psychophysiologische Protis- tenstudien, G. Gischer Verlag, Jena. Fukui, K. and Asai, H. (1980) The most probable mechanism of the negative geotaxis of Paramecium caudatum. Proc. Japan Acad., Ser. B., 56: 172-177. Roberts, M. (1970) Geotaxis in motile micro- organisms. J. Exp. Biol., 53: 687-699. Naitoh, Y. and Eckert, R. (1972) Electrophysiology of the ciliate Protozoa. In “Experiments in Physiolo- gy and Biochemistry”, Vol. V. Ed. by G.A. Kerkut, Academic Press, London, pp. 17-38. Taneda, K. (1987) Geotactic behavior in Para- mecium caudatum. I. Geotaxis assay in individual specimen. Zool. Sci., 4: 781-788. Bean, B. (1977) Geotactic behavior of Chlamydo- monas. J. Protozool., 24: 394-401. Winet, H. and Jahn, T. L. (1974) Geotaxis in pro- tozoa. I. A propulsion-gravity model for Tetrahyme- na (Ciliata). J. Theor. Biol., 46: 449-465. lot iisenirspe el (fea). wee shit Sean ST £ a co. ee actinenM ity ‘each 1 ito © ina «tee te AO Srerris alk staA, oat tat: vs Sok agg pirortt orale ei v ha rite} \ SNR Tec oot reise RO. Bee 7 * f er : we) wal ; ui i : AY to aa Aleit ea Ky a as 4 : far tht ‘4 LUE ih by ii 1 4s Wii wae) pers ¥ My oy soit ? LS, COB ie CLONE eer: Tiawidngso 2 yucholey odaogtt (OCR SE took bute Morinda”) a | aie meri a ase aqad S ae aes certs pity Sait Jon vr ‘hed ca ae! viov ! “pocimatooig bie Ya Ar Pas: abst it ae 2a Paige A Beye. Yh ea “ea dis asd ws Pista ‘Ot 2 5 (veer) a ; nbons rT ! é ; sina e Gi Vette CIOS) Al RDO STS | | REY 1RV ch Sie doe” jdomitssee tie iaeaite ey nee wiosioaw Pel) .a anal ny ey 1h see a Hoasaynr a hn a 4: ae ea) Mis AT Mi" Fi suevesth: Ao Vt ‘T) A I nak Eph A sar WW 5 H if : steve sks t Or ba tyepet ty Vive 14" peel oe D. ' PAPAL A A acd un Raniigoduatl ee . ah bb Se heidi. ww watt { {\ 1 (eine, >) magi: eet ‘Ware? vce 7 Ni byl ee Apc Ya ¥ ¥ } sity # Pex, ' fais a n . retur y ie 5 | * j y waa o a3 P PLE BENG ea Se oe | : f i ay ni ; ons t apt i sew i . ' ere ee Ee wat" Me penal | i he, by hea LCLieow his ey va iT Cee Le Pee \ Bit i , : i i ( - useby ; Y ey Set Coma ' f i ters or Lye TERE 4 (is ' iY 4; eb Ra) : ii uberiene® aii rae a ne | Dee ro. eciver Ot her Pree Wi uy it mini’ af Lourie 1 Re jie Wiper hall and (omni pomtive pene A I as hs toh: TREE Oe i . iy MR bk tote eat egy! iis at Ane the any mibvaney ne : ire he RF Ya ’ vs, vs ie a ht ; te jah: aves set fi # i pe st Ee et i TURNER in what ye a all ane ital ‘e iy ic seocnivins Que oy (he a teviy tovebe! (dere ly ot! he ‘pone Dane | ZOOLOGICAL SCIENCE 4: 797-801 (1987) © 1987 Zoological Society of Japan The Structure and Innervation of a New Muscle in the Tailfan of the Crayfish, Procambarus clarkii Puitie L. NEWLAND Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan ABSTRACT—The structure and innervation of a newly discovered muscle in the exopodite of the crayfish, Procambarus clarkii, were examined using histochemical and electrophysiological techniques. Based on the histochemical detection of the specific activity of ATPase, the muscle was found to contain only lightly stained fibres typical of the crustacean slow type. Muscle fibres were often divided into separate subunits and had sarcomere lengths of 6-104m. The muscle was innervated by one excitatory axon from the second root of the sixth ganglion, and one inhibitory axon from the third root. Both the excitatory and inhibitory axons produced small junction potentials which exhibited facilitation and summation characteristic of crustacean slow muscles. INTRODUCTION The uropods, the paired terminal appendages of decapod crustaceans, are involved in a great variety of behavioural acts. In recent years considerable attention has focused on the uropods in terms of the muscles and the organization of their innervation [1, 2], their patterns of activation during postural movements [3] and their role in different behaviour patterns [4]. Unlike many crustacean joints that move in a single plane, such as the joints between the basal segments of the antennae [5, 6], the joints between the telson and the protopodite, and the protopodite and exopo- dite are mobile in a number of planes, resulting in complex movements. These movements range from a phasic streamlining of the tailfan during the extension phase of the swimming cycle [7], to slow postural movements made during steering [8]. The work of both Larimer and Kennedy [1, 2] and Takahata et al. [3] on crayfish, provide the most recent descriptions of the underlying muscu- lature producing movements of the uropods. However, this work was restricted in the number of muscles examined in the protopodite and exopodite. Previous experiments (Newland, un- published data) have shown that a new, previously Accepted May 6, 1987 Received February 10, 1987 undescribed muscle was located in the exopodite of the crayfish. This paper describes the structure and physiology of this new muscle. MATERIALS AND METHODS Freshwater crayfish, Procambarus clarkii (Girard), of both sexes (10 to 12 cm in length) were used for experiments. These animals were maintained in large aquaria and were fed reg- ularly. Crayfish abdomen were pinned ventral side up in a small bath containing cooled physiological saline [9]. To expose the muscle, the cuticle around the insertion was cut on three sides and a small patch of cuticle was removed. The adductor muscle was displaced laterally to allow access to the new muscle for recording. The sixth abdominal ganglion was also exposed, and the sensory bun- dles of the second and third roots, and the entire fourth root were cut. A small oil-hook electrode was placed on the motor nerve innervating the new muscle for extracellular recording of motor activity or stimulation. The second and third roots were stimulated using oil-hook electrodes with square wave pulses of 0.01-0.1 ms duration at various frequencies. For intracellular recordings of muscle potentials, glass microelectrodes filled with 3M KCI and of 10-15 m() resistance were used. All signals were amplified and plotted using a signal 798 P. L. NEWLAND processor (Nihon-Kohden Addscope) and X-Y plotter (Rikadenki). The procedure used for the characterization of the uropod muscles for the specific activity of ATPase was based on that of Ogonowski and Lang [10]. In brief, the uropods, telson and sixth abdominal segment were removed and quickly frozen in isopentane with liquid nitrogen. The tissues were then blocked in O. C. T. compound (Miles Scientific Co.) and mounted on the chuck of a cryostat. Serial sections of 25 um were cut and picked up on slides coated with chrome alum- gelatine, and air-dried for 30-60 min before stain- ing. The staining procedure used was as described by Ogonowski et al. [11]. Measurements of the sarcomere lengths were made directly on freshly dissected preparations which were teased apart on a microscope slide. Fibres were viewed under Nomarski optics on an Olympus Vanox micro- scope which was calibrated with an ocular micro- meter. RESULTS General morphology Each of the previous studies on the uropod musculature has adopted a different nomenclature for the uropod muscles. In this study the nomen- clature of Larimer and Kennedy [1] is used. The location of the newly discovered muscle is shown in Figure 1. The muscle, which is relatively small in size (named here the Medial Rotator), originates on a large sclerite between the exopo- dite and the protopodite on the dorsal surface at its posterior margin, and inserts on the ventral surface of the exopodite. In animals of the size range used in these experiments this muscle was about 1.5 mm long and 0.5 mm in diameter. It was composed of about 15 separate fibres (Fig. 2), which were often subdivided into closely connected subunits, typical of crustacean slow muscles [12, 13]. Sarcomere lengths were determined for different regions of the muscle, and were found to be similar in all regions examined. The sarcomere lengths had a mean of 7.26+0.21 um. When stained for myofibrillar ATPase activity, the new muscle showed a uniform light staining Fic. 1. Diagram showing the position of the medial rotator (M. Rot) in the exopodite (EXO) relative to the lateral abductor (L. Abd) and reductor (Red) muscles. The adductor has been omitted for clarity. The new muscle originates on a small sclerite between the exopodite and protopodite (PRO) and inserts on the ventral surface of the exopodite. The endopodite (ENDO) is also shown. Ventral view. Scale bar, 2 mm. Histochemical identification of 2 uropod mus- cles stained for the activity of myofibrillar ATPase. The new muscle (single arrowhead) stains lightly indicating the presence of slow fibres. The adduc- tor (double arrowheads) is more intensely stained, Fic. 2. typical of fast muscle fibres. Horizontal section through the tailfan. Some of the muscle fibres can be seen to be divided into subunits. Scale bar, 0.25 mm. compared to that of the fast fibres of the adductor muscle which showed an intense dark staining (Fig. 2). The activity of myofibrillar ATPase is 2-3 times greater in crustacean fast muscles than in slow muscles [11, 14]. Consequently the fast fibres stain much more intensely. New Crayfish Uropod Muscle 799 Innervation Stimulation of the second motor root of the sixth ganglion close to the muscle, with an increasing intensity, excited firstly an inhibitory axon fol- lowed at a higher intensity by an excitatory axon (Fig. 3). A further increase in the stimulus intensi- 1ImvV 500 msec Fic. 3. Intracellularly recorded responses of the new muscle to electrical stimulation of its motor nerve with an increasing stimulus intensity. An inhibitory response is followed at a higher stimulus intensity by an excitatory response. Stimulus intensity in- creases linearly from left to right during the period indicated by the shaded bar (1-8 V). The begin- ning of the response is marked with an arrowhead. Stimulus frequency, 20 Hz. R2,3 tn 50 msec Fic. 4. Excitatory and inhibitory responses of the new muscle to electrical stimulation of different motor roots of the sixth ganglion. Upper trace, root 2 stimulation at 9.5 V (R2); second trace, root 3 stimulation at 7.5 V (R3); third trace, simultaneous stimulation of both root 2 and root 3 at 9.5 V (R2, 3). Lower trace, stimulus monitor. Stimulation of both roots results in an inhibition of ejps. Marked facilitation and summation are evident for both ejps and ijps. Stimulus frequency, 40 Hz. ty, above that necessary to stimulate the excitatory axon, failed to recruit additional axons. Recording from different fibres of the muscle showed that all had a similar pattern of innervation. Separate stimulation of root 2 and root 3 showed that the excitor was recruited by stimulation of root 2, whereas the inhibitor was recruited by stimulation of root 3 (Fig. 4). Simultaneous stimulation of both roots often resulted in an inhibition of the excitatory junction potentials (Fig. 4). The inhibi- tory axon crossed from the motor branch of root 3 to that of root 2 in an area where both motor branches were in close proximity within the pro- topodite. Resting potentials of the muscle fibres ranged yp 4 40 4 EEE t+ ss mV 500msec Fic. 5. Intracellularly recorded responses of the new muscle to continuous stimulation. A. Response to root 2 stimulation (7 V). B. Response to root 3 stimulation (7 V). Stimulus frequency was changed from 4 Hz to 40 Hz, and back to 4 Hz at the times indicated by the stimulus markers (lower trace of A and B). On returning to 4 Hz, post-tetanic poten- tiation of ejps and ijps was clearly evident. Summation and facilitation can be observed by comparing responses at 4 Hz and 40 Hz. 800 P. L. NEWLAND from —56 to —68mV and had a mean of —64 mV. Single stimuli to root 2 resulted in small excitatory junction potentials of about 1 mV in amplitude. At stimulus frequencies greater than 4 Hz marked facilitation and summation occurred. Maximum facilitation was reached at 40 Hz where individual ejps were about 2 mV in amplitude (Fig. 5A). Summation of these ejps produced mem- brane depolarizations ranging from about 3 mV at 10 Hz, to 14 mV at 40 Hz. Inhibitory junction potentials were, in general, much smaller than ejps (Fig. 5B). The largest ijps recorded at a stimulus frequency of 4 Hz were —0.35mV in amplitude. In most cases little facilitation of these potentials occurred even at frequencies up to 40 Hz. Summation of ijps was observed at stimulus frequencies above 10 Hz, producing membrane hyperpolarizations of about —4mV at 40 Hz. When the stimulus frequency was reduced from a high to a low frequency, marked post-tetanic potentiation of both the ejps and ijps was observed (Fig. 5). This effect was most evident when the frequency during tetanic stimulation was between 20 Hz and 40 Hz. DISCUSSION In terms of structure and physiology, this new muscle exhibits characteristics that are typical of slow or tonic crustacean muscle fibres. It has muscle fibres which are divided into closely con- nected subunits in cross section [12], and long sarcomere lengths which are comparable to those of other tonic muscles [1, 13, 15]. Staining for ATPase activity supports this data showing all fibres to be of the slow type [10, 11]. Typical of uropod slow muscles, the junction potentials re- corded never gave rise to spikes [1]. The uropods move about three axes of rotation: promotion/remotion, flexion/extension and rota- tion. The muscles bringing about these move- ments have therefore been categorised into three groups acting in these three planes [1, 3]. Based on the line of action of this new muscle, it is likely that contraction of the muscle would raise the posterior edge of the exopodite closing it on the endopodite, while the leading edge of the exopodite blade would be brought downward, tending to “cup” or rotate the exopodite blade. Therefore, the muscle has been named the medial rotator. In view of the discovery of this new muscle, it seems possible that the interpretation of the electrical records in previous work may have led to inaccuracies in the identification of the origins of motor axons. For example, stimulation of root 2 near the muscle (Fig. 3) suggests that the inhibi- tory and excitatory axons both exit the sixth ganglion through root 2. However, separate stimulation of root 2 and 3 near the sixth ganglion shows that this is misleading, and that the excita- tory axon actually exits through root 2, while the inhibitory axon exits through root 3 (Fig. 4). The work of Larimer and Kennedy [1] describes a clear separation of the axons in the second and third roots of the sixth abdominal ganglion. The root 2 motor neurones only innervate the closer muscles (the remotors), while the root 3 motor neurones only innervate the opener muscles (the promot- ors). It is not clear from the previous study exactly where the electrical activity was monitored in order to determine the origin of the motor neurones. Without monitoring this activity close to the ganglion and near the target muscle it is not possible to describe with absolute certainty, the origin of the motor neurones of these two roots. It remains to be determined how many axons actual- ly cross; however, from the data presented here, we now know that at least one axon does cross from one root to the other. Therefore, it may be necessary to re-examine the innervation of the uropod muscles which are innervated by the second and third roots in order to clarify their origins. ACKNOWLEDGMENTS I am grateful to Professor M. Hisada for critically reading an early draft of this manuscript and to M. Takahashi for his help with the muscle histochemistry. This work was supported by a fellowship from the Japan Society for the Promotion of Science. REFERENCES 1 Larimer, J. L. and Kennedy, D. (1969) Innervation patterns of fast and slow muscle in the uropods of New Crayfish Uropod Muscle 801 crayfish. J. Exp. Biol., 51: 119-133. Larimer, J. L. and Kennedy, D. (1969) The central nervous control of complex movements in the uropods of crayfish. J. Exp. Biol., 51: 135-150. Takahata, M., Yoshino, M. and Hisada, M. (1985) Neuronal mechanisms underlying crayfish steering behaviour as an equilibrium response. J. Exp. Biol., 114: 599-617. Newland, P. L. (1985) The control of escape be- haviour in the Norway lobster, Nephrops norvegi- cus. Ph. D. Thesis, University of Glasgow, Scotland. Vedel, J.-P. (1980) The antennal motor system of the rock lobster: competitive occurrence of resist- ance and assistance reflex patterns originating from the same proprioceptor. J. Exp. Biol., 87: 1-22. Vedel, J.-P. and Monnier, S. (1983) Reflex reversal resulting from active movements in the antenna of the rock lobster. J. Exp. Biol., 101: 121-133. Webb, P. W. (1979) Mechanics of escape responses in crayfish (Orconectes virilis). J. Exp. Biol., 79: 245-263. Yoshino, M., Takahata, M. and Hisada, M. (1980) Statocyst control of the uropod movements in response to body rolling in crayfish. J. Comp. Physiol., 139: 243-250. 9 10 11 12 13 14 Van Harreveld, A. (1936) A physiological solution for freshwater crustaceans. Proc. Soc. Exp. Biol. Med., 34: 428-432. Ogonowski,M.M. and Lang,F. (1979) Histo- chemical evidence for enzyme differences in crusta- cean fast and slow muscle. J. Exp. Zool., 207: 143-151. Ogonowski, M.M., Lang, F. and Govind, C. K. (1980) Histochemistry of lobster claw-closer muscles during development. J. Exp. Zool., 213: 359-367. Parnas, I. and Atwood, H.L. (1966) Phasic and tonic neuromuscular systems in the abdominal ex- tensor muscles of the crayfish and rock lobster. Comp. Biochem. Physiol., 18: 701-723. Sherman, R. G. and Atwood, H. L. (1971) Struc- ture and neuromuscular physiology of a newly discovered muscle in the legs of the lobster, Homar- us americanus. J. Exp. Biol., 176: 461-474. Lehman, W. and Szent-Gyorgyi, A. G. (1975) Reg- ulation of muscular contraction: Distribution of actin and myosin control in the animal kingdom. J. Gen. Physiol., 66: 1-30. Lang, F., Govind, C. K. and Costello, W. J. (1978) Experimental transformation of muscle fiber prop- erties in lobster. 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Porn ep IN Ds We ee | Date | ib etn: s yim , 6 hs é [ ne J 3 % on a pfin§ 4 oe » P . , : ait PE: oe iat a +) Pie FRje Saceadlal a i oh: ‘tke A a t? ogy ay ; ry i ere? rove 4 - Very Paar Sgr i Ruths Cod MN he Le OL eee A be Ra) AA EGES olin AEP Nig lng fh ut Ler: ait A aM Let ae ey " if wait r fit ; eo SEERA ire) Oi iny ae, OT th eee none ' pe nee re > Pa i Mt) t ; t 4 ' , Vit) & + } ‘) i q a f - i ae ? , : 1 i Mw - 4 } ‘os : fu i x hee ¥ a iti, ib? nl t nal A es ‘ 4 fF yin” f a \ ( an 5 ay i 7 i t OF y j } j ‘ « : ee, i i tare ee ak | 4 Lee Tg ZOOLOGICAL SCIENCE 4: 803-811 (1987) Changes in Blood Volume after Hemorrhage and Injection of Hypertonic Saline in the Conscious Quail, Coturnix coturnix japonica YOSHIO TAKEI and IppEI HATAKEYAMA Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan ABSTRACT—Continuous changes in blood volume after hemorrhage or injection of hypertonic NaCl solution were measured in the conscious quail, Coturnix coturnix japonica. Initially, an intravenous injection of 0.1 ml of 2.5% Evans blue was made into birds, and the rate of disappearance of the dye from the circulation more than 10 hr after injection was estimated by fitting the concentrations in plasma measured prior to the 10hr point to a dual exponential function. The estimated dye concentrations fell within 95% confidence intervals of 3.7-6.9% from the measured values for 4 hr. Then, the change in blood volume was calculated in different birds from changes in the dye concentration in plasma and in the hematocrit, after hemorrhage or injection of hypertonic saline was applied 10 hr after injection of dye. It was found that the response of blood volume to 1 ml of hemorrhage, which amounted to 12.5% of total blood, was quick; blood volume overshot the level prior to hemorrhage within 15 min. Blood volume also responded quickly to an intravenous injection of 0.1 ml of 7% NaCl, which theoretically increases Na concentration of extracellular fluids by 3.6%; blood volume increased immediately after injection, and the maximal increase was attained after 45 © 1987 Zoological Society of Japan min. These changes in blood volume were compatible with the changes in the hematocrit, blood pressure and plasma Na concentration after hemorrhage or injection of hypertonic saline. INTRODUCTION It is generally accepted that volemic regulation of body fluid in terrestrial animals is achieved primarily by drinking (input) and urinary excretion (output). Thus, it is a common practice in the study of volemic regulation to measure con- tinuously those parameters that influence drinking and production of urine after manipulation of hydromineral balance [1]. These parameters are blood volume, plasma osmolality, plasma electro- lyte concentrations, blood pressure, and plasma levels of hormones which are involved in hydro- mineral metabolism. We reported recently that the quail drank in response to hemorrhage or an intravenous injection of hypertonic NaCl solution [2, 3]. Since drinking is regulated primarily by blood volume, plasma osmolality and plasma angiotensin II concentration [4], we attempted to Accepted May 15, 1987 Received April 18, 1987 measure these parameters continuously after hemorrhage and injection of hypertonic saline. We could measure plasma osmolality and plasma angiotensin II concentration continuously, but we could not find a technique suitable for continuous measurement of blood volume in such small animals as quail whose blood volume is less than 10 ml. The present study was undertaken, first, to establish a reliable technique for continuous measurement of blood volume. The technique should be sensitive enough to be applicable to small animals such as the quail. Evans blue was used as a tracer to determine plasma volume, since this dye binds strongly to plasma proteins and, thus, leaves the circulation slowly [5]. Then the technique was applied to measurements of changes in blood volume that follow hemorrhage or injec- tion of hypertonic saline in the conscious quail. The changes in blood pressure and plasma Na concentration were also measured after these procedures to evaluate the reliability of the 804 Y. TAKEI AND I. HATAKEYAMA measurements. MATERIALS AND METHODS Animals Male Japanese quail, Coturnix coturnix japoni- ca, aged 3 weeks, were purchased from a quail farm (Suzukei, Toyohashi) and kept, 5 to a cage (45x45 x90 cm), under a natural photocycle: in August, September and October, in Japan, at 22+1°C. Commercial quail diet containing 150 mEq/kg of Na (Nippon Haigo Shiryo, Yokoha- ma), and tap water were given ad libitum, unless otherwise specified. The birds were acclimated to these conditions for longer than 2 weeks before use, and weighed 108+8 g (mean+SD, n=34) at the time of experiment. Surgery For measurements of blood volume and plasma Na concentration, the quail were anesthetized by an intraperitoneal (ip) injection of Nembutal (40 mg/kg), and a U-shaped polyethylene catheter (o. d.: 0.8mm) was inserted cephalad into the right external jugular vein by 4cm. The tip of the catheter was located at the neck region where blood was constantly circulating. For measure- ments of blood pressure, quail were lightly anes- thetized by an ip injection of urethane (0.3 g/kg), and a polyethylene catheter (o.d.: 0.61 mm) was inserted caudad through the right external jugular vein into the atrium, and another polyethylene catheter (0. d.: ca. 1mm) made from a thicker catheter by heating and pulling, was inserted caudad into the right common carotid artery. Less than 0.05 ml of blood was lost during each surgery. The jugular catheter was filled with saline contain- ing calcium heparin (10 U/ml) when not in use. Experimental protocol Pilot experiment After more than 18hr of postoperative recovery, 0.1 ml of 2.5% Evans blue (Wako Pure Chemical., Tokyo) in 0.9% NaCl was injected in 10 sec through the jugular catheter, and followed by a flush with 0.1 ml of 0.9% NaCl. Dead space in the catheter was approximately 0.03 ml. Blood samples (50 4) were taken just before injection of dye and at 0.25, 0.5, 0.75, 1, 3, 6, 9, 10, 11, 12, 13, 14 and 24hr after the injection. Blood was collected directly into heparinized hematocrit tubes, and the blood that remained in the catheter was reintroduced into the circulation with 0.03 ml of heparinized saline. After measure- ment of the hematocrit, 10 41 of plasma was diluted into 0.5 ml of distilled water in duplicate. Absorbances at 624nm were determined in a spectrophotometer (Hitachi 124) with a micro- cuvette. The linearity of the dilution curve had been confirmed. Plasma collected before injection of dye was used as a plasma blank for spectropho- tometry. Nielsen and Nielsen [6] pointed out considerable variations of the absorbance at 620 nm in the human plasma collected consecutively from the same subject. Thus, they recommended to correct each dyed plasma sample measured at 620 nm with the blank absorbance of the same sample which was calculated from the absorbance at 740 nm. However, we did not make the correction in this experiment, because we meas- ured absorbances of diluted plasma samples at 620 nm which were collected from 3 birds on the time schedule mentioned above without injection of the dye, and found that the absorbance was small and consistent (0.0017 +0.0004, mean+SD, n=42). The rate of disappearance of dye from the circulation more than 10hr after injection was then estimated by fitting the absorbances meas- ured before the 10hr time point to a dual exponential function (A,=ae °'+be~ “'+c), where A, is the absorbance at time t, anda, b,c, @ and £ are constants. A constant (c) was added to the function because the dye remaining in the circulating blood as long as 5 days after injection, as observed in the present study, appeared to be assumable as a constant during the experimental period of 24 hr. The fitting was done with a 3-step regula falsi method which was principally based on the “peeling” method [7]. The outline of the 3-step regula falsi method was: (1) The constant, c, of the dual exponential function was determined by fitting the later set of data before the 10 hr point to a single exponential function with a constant (A,=de ”‘+c), where d and Y are dummy parameters. The later sets of Continuous Measurement of Blood Volume 805 data were those measured 1-10 hr, 3-10 hr, and 6-10 hr after injection of dye. (2) The constants, a and a, were determined by fitting the earlier set of data to a single exponential function (A,—c=ae™ “*'), where c is now constant. The earlier sets of data were those measured 0.25-0.5 hr, 0.25-0.75 hr, 0.25-1 hr, and 0.25-3 hr after injection of dye. (3) The constants, b and £, were determined by fitting all data before the 10 hr point (0.25-10 hr) to a single exponential function (A,—c—ae™ “'= be~ 8 ‘), where a, @ and c are now constant. The fitting to a single exponential function at each step was done by the regula falsi method. The computer program was written in APL and run on an IBM 4341 computer. The program will be supplied on request. Among all combinations of earlier and later sets of data, the best combination was selected with a criterion that the data more than 10 hr after injection of dye could be estimated best, that is, squares sum of relative residuals (estimated value/measured value—1) on 11-24 hr data was minimal. An example that shows a fitness of the estimated absorbances to the measured values is illustrated in Figure la. Change in blood volume after hemorrhage or injection of 7% NaCl Blood samples (50 pl) were collected up to 10hr after injection of dye, according to the time schedule described for the pilot experiment. Immediately after the sampling at 10 hr, hemorrhage (0.5 or 1 ml) was induced (n=4 each) or 0.1 ml of 7% NaCl was injected intravenously (n=4), both of which procedures were followed by injection of 0.03 ml of heparin- ized saline. Samples of blood were taken 0.25, 0.5, 0.75, 1, 2, 3, 4and 14 hr after each procedure. An example that shows a deviation of the absorbances from the estimated values after injection of 7% NaCl is illustrated in Figure 1b. Time controls (n=4) were prepared for the birds that were induced to hemorrhage in which no procedure was applied at 10 hr and blood samples were collected on the same time schedule as described above. As controls for the birds that were injected with 7% NaCl, 4 birds were injected with 0.1 ml of 0.9% NaCl at 10 hr. Water was withheld for 4 hr after each procedure to eliminate the influence by induced water intake on blood volume. Plasma a 0.3 0.2 wo o Absorbance 0.2 0.1 7% NaCl 0 5 10 15 20 24 Time after dye injection (hr) Fic.1. a. An example of the fitness of estimated absorbances of Evans blue (4) to the measured values (@) in a quail, after intravenous injection of the dye. The estimation was made with the best combination data measured prior to the 10 hr point as described in the text. The multiple correlation coefficient was 0.999. b. An example that exhibited a deviation of absorbances, measured after intravenous injection of 0.1ml of 7% NaCl (@), from the estimated values (4) in a quail. The multiple correlation coefficient was 0.962. volume after the treatment (PV’) was calculated by the equation: PV =PV XxA,/A’, where PV is plasma volume before treatment, A, and A’ are absorbances estimated and measured after the treatment, respectively. PV was determined by the equation: PV=5(A,—A, )/(Ajp—A,), where A,, A,, and A, are absorbances of a reference standard, a blank for the reference, and the plasma blank, respectively. Ag was determined by extra- polating the data measured between 0.25 and 1 hr to time 0 with a single exponential function (A,=ae “'+b) with a regula falsi method. The reference standard was prepared by additions of 0.1 ml of the 2.5% dye solution and 0.1 ml of 0.9% NaCl to 5 ml of 1% bovine serum albumin (BSA, Wako Pure Chemical., Tokyo) in 0.9% NaCl. 806 Y. TAKEI AND I. HATAKEYAMA One percent BSA in 0.9% NaCl was used as the blank for the reference. Blood volume was calculated by correction of the plasma volume with hematocrit. The hematocrit was not corrected for trapped plasma. The change in blood volume was also calculated from changes in hematocrit alone (hematocrit method) to allow comparison with the present method employing Evans blue. Measurements of blood pressure and Na concen- tration in plasma_ Changes in blood pressure was monitored following 1ml of hemorrhage, and intravenous injection of 0.1 ml of 0.9 or 7% NaCl in the lightly anesthetized quail. The hemorrhage (n=4) and the injection of NaCl solutions (n=5) were made in different birds. The signal from the transducer was amplified by a carrier amplifier (Type 3126, Yokogawa Electric Works Ltd.) and recorded by a pen recorder (Rectigraph 85, San-ei Instrument Co. Ltd.), while the signal after am- plification was also stored in a data recorder (R-260, TEAC) for further analyses. Concentrations of Na ions in plasma were measured 0, 0.25, 0.5, 0.75, 1, 2, 3, 4 and 14 hr after 0.5 ml of hemorrhage, 1 ml of hemorrhage, or intravenous injection of 0.1 ml of 7% NaCl. Controls were prepared for each procedure as described for the measurement of blood volume. According to the above time schedule, 30 “1 of blood was collected into heparinized hematocrit tubes (Terumo), centrifuged, and 2 ul of plasma was diluted in 4ml of double distilled water in duplicate. The concentration of Na ions was determined with an atomic absorption spec- trophotometer (Hitachi 180-80). Double distilled water collected in the hematocrit tube was diluted as above, and used as a blank for plasma samples. The absorbance of the blank was always less than 0.1% of that of diluted plasma samples. Statistical analyses All results are expressed as means+SD or +95% confidence interval. In order to make clearer the change in blood volume after hemor- rhage or injection of hypertonic saline, the change in each bird was expressed in terms of a ratio to the initial blood volume, and mean of the initial blood volume was given in the text. Since blood volume and concentration of Na ions in plasma at each time point after hemorrhage or injection of hyper- tonic saline are not independent on each other, the data concerning changes with time in blood volume and plasma Na concentration were treated as time-series data and analyzed en bloc by the sign test or Hotelling T-test [8]. Statistical significance and rejection of null hypothesis were achieved when P<0.05. All computations were carried out with an IBM 4341 computer using an APL interpreter. RESULTS Plasma volume in water-replete quail was 4.75+1.03 ml/bird (n=34). Blood volume, cor- rected by the hematocrit (40.7 + 5.9%, n=34), was 8.04+1.56 ml/bird or 7.47+1.47 ml/100 g body weight (n=34). Pilot experiment The concentrations of dye more than 10 hr after injection were estimated best when the 0.25—0.5 hr and 3-10hr data were applied to the dual exponential function as earlier and later sets of data, respectively, as described in Materials and Methods. The mean deviations of the estimated concentrations of dye from the measured values were —0.5, 4.9, 2.9, 10.5 and 34.7% after 1, 2, 3, 4, and 14hr of estimation, respectively. The time-series analysis by the Hotelling test showed that the values thus estimated were biased signif- icantly towards values greater than the measured values. Therefore, the estimated values were corrected by the deviation at each time point for calculations of the changes in blood volume, in the following experiments. The estimated values thus corrected fell within 95% confidence intervals of +3.7, +5.6, +6.9, +6.6 and +16.0% from the measured values after 1, 2, 3, 4 and 14hr of estimation, respectively. Changes in blood volume Blood volume returned to the level prior to hemorrhage between 1 and 2 hr after withdrawal of 0.5 ml of blood (6.2% of total blood volume), while recovery from hemorrhage was quicker (within 15min) after larger volume (1 ml) of hemorrhage (Fig. 2a, b). The time-series analysis Continuous Measurement of Blood Volume Change in blood volume (ratio to time 0) Time after treatment (hr) Fic. 2. Time course of change in blood volume after (a) 0.5 ml of hemorrhage, (b) 1 ml of hemorrhage, and (c) intravenous injection of 0.1 ml of 7% NaCl, calculated from changes in the concentration of Evans blue in plasma and in the hematocrit in the quail (n=4 each). Changes in blood volume were expressed in terms of ratios to the values before treatments. Average blood volumes before treat- ment were (a) 7.24+1.08 ml, (b) 8.26+0.51 ml, and (c) 7.77+0.32 ml. The changes after hemor- rhages and injection of 7% NaCl were corrected by mean changes in time controls and 0.9% NaCl- injected controls, respectively. Values are means + SD. showed that the level of blood volume for 14 hr after hemorrhage was greater than the level just after hemorrhage, but was not different from the level prior to hemorrhage in both groups subjected to 0.5 and 1 ml of hemorrhage. There was no significant change in blood volume in time con- trols. Blood volume increased immediately after in- travenous injection of 0.1 ml of 7% NaCl, and the maximal increase of 12.5% was achieved after 45 min (Fig. 2c). The increase became smaller there- after but the level was still greater than the level prior to injection after 14 hr. The increase for 14 hr after injection was statistically significant. In controls injected with 0.1 ml of 0.9% NaCl, there 807 was a transitory increase in blood volume due to the volume of saline injected, but the blood volume returned to the level prior to injection within 1 hr. Hematocrit method for determination of changes in blood volume The pattern of changes in blood volume after hemorrhage determined by the hematocrit alone was similar to that determined by the present method that employs Evans blue (Fig. 3a, b). However, the initial increase appears to be greater by the hematocrit method, and blood volume overshot the previous level within 15 min after both 0.5 and 1 ml of hemorrhage (Fig. 3a). Blood volume appeared scarcely to increase during the 1 hr period after intravenous injection of 0.1 ml of 147 a 12 So 10 = — os Ss 0 ek z = 12 = 10 5 S 08 2 0 Ss 14 | aD @ = S 10 6S 08 0 01234 14 Time after treatment (hr) Fic. 3. Time course of change in blood volume after (a) 0.5 ml of hemorrhage, (b) 1 ml of hemorrhage, and (c) intravenous injection of 7% NaCl, calcu- lated from the change in hematocrit alone, in the quail (n=4 each). The data were obtained from the same birds as in Fig.2. Changes in blood volume after hemorrhages and injection of 7% NaCl were expressed in terms of ratios to the values before treatments, and corrected by the changes in corresponding controls as detailed in Fig. 2. Values are means+SD. 808 Y. TAKEI AND I. HATAKEYAMA 7% NaCl, when the change was determined by the hematocrit method (Fig. 3c). Blood volume in- creased thereafter and the maximal increase was 10% 14 hr after injection. Change in blood pressure The mean blood pressure of the quail lightly anesthetized with urethane was 117.8+13.8mm Hg (n=14). Blood pressure decreased by 40.0+9.9% (n=4) after 1 ml of hemorrhage, but the level was restored in 11.6+4.0 min. Blood pressure increased by 35.3+3.0% (n=5) after intravenous injection of 0.1 ml of 7% NaCl and returned to the level prior to injection in 6.8+2.3 min. In 4 of 5 cases, blood pressure decreased further below the pre-injection level thereafter. In controls injected with 0.9% NaCl, the increase in blood pressure was 13.0+2.8% (n=5), which disappeared in 1.5+0.5 min. 725.4 14 Time after treatment (hr) Fic. 4. Time course of change in plasma Na concentra- tion after (a) 0.5 ml of hemorrhage (n=5), (b) 1 ml of hemorrhage (n=5), and (c) intravenous injec- tion of 0.1 ml of 7% NaCl (n=6) in the quail. Changes in Na concentration after hemorrhage and injection of 7% NaCl was corrected by the mean changes in time controls and 0.9% NaCl-injected controls, respectively. Values are means+SD. Change in concentration of Na ions in plasma The concentration of Na ions in plasma in intact birds was 139.4+4.7mM (n=17). Plasma Na concentrations tended to increase after both 0.5 and 1 ml of hemorrhage (Fig. 4a, b). The time- series analysis showed that the increase for 14 hr was Statistically significant in both groups. The levels of Na ions returned to normal 45 min after intravenous injection of 0.1 ml of 7% NaCl, which theoretically increases Na concentration in ex- tracellular fluids by 3.6%. The level decreased further below the pre-injection level thereafter (Fig. 4c). Thus, the time course of change in plasma Na concentration for 14 hr was not signif- icantly different from the normal level after injection of 7% NaCl. The levels of Na ions in plasma did not change in time controls for hemor- rhage, and in controls injected with 0.9% NaCl. DISCUSSION Two techniques have, thus far, been established for continuous measurement of blood volume. The first method consists of the injection of a known amount of °!Cr-tagged red blood cells into the circulation, with a continuous monitoring of radioactivity in an extracorporeal arterio-venous shunt loop, and has been tested in anesthetized dogs [9, 10] and conscious rats [11]. This technique enables continuous monitoring of blood volume in its true sense, but, due to a considerable volume of blood required for external circulation, it cannot be applied to the quail which has only 8 ml of blood. The other technique follows the change in blood volume by means of the changes in hemato- crit and concentration of hemoglobin [12]. This technique scarcely disturbs homeostasis of the animal and, thus, is suitable for use in the human in whom experimental manipulations should be avoided [13]. However, accurate measurement is impossible if red blood cells are released from the storage sites during the experiment. This techni- que has been applied to the sheep fetus, in which release of red blood cells is not significant [14]. In adult mammals, however, the spleen is known to act as a blood reservoir and, even in splenecto- mized dogs, red blood cells are released into the Continuous Measurement of Blood Volume 809 circulation after injection of vasoactive drugs [15]. In the present study, we attempted to monitor the change in blood volume by measuring both plasma volume and hematocrit at the same time. Evans blue was used as a tracer for measurement of plasma volume, because it has been shown to bind strongly to plasma proteins and scarcely to escape from the vascular space [5]. In the present study, however, Evans blue seems to disappear rather quickly from the circulation as illustrated in Figure 1. This is partly because we injected a considerable amount of dye (2.5 mg/bird) to ensure high absorbance 10 hr after injection of dye (0.060 +0.002 after 1/51 dilution, n=20), and thus dyes unbound to plasma proteins might escape from the circulation during the initial phase after injection. Our preliminary experiment using polyacrylamide gel electrophoresis showed, however, that all the dyes bound to plasma proteins within 15min after injection of this amount of dye into the quail (Takei and Ihara, unpublished observation). Thus, the quick dis- appearance of dye appears to be due to a high metabolic rate in birds. We adopted a dual exponential function to simulate the disappearance of dye from circulation, because semi-log plot of the disappearance curve resulted in a diagonal line which signifies an involvement of two exponential terms, and because two major routes of excretion of the dye have been reported in mammals, the reticulo-endothelial system and the liver [5]. In fact, fitting all the data measured during the 24 hr period to the dual exponential function invariably produced multiple correlation coefficients of more than 0.998 in the pilot experiment of this study. Furthermore, spleens and livers of the quail were found to be stained deep blue at post-experimental autopsy. It seems that >'Cr-tagged red blood cells might be a better tracer for monitoring the change in blood volume, because the decay of the tracer should be slower and, thus, the estimation of future decay could be made with a greater accura- cy than in the case of Evans blue. However, in many institutions like ours, the use of radioactive materials in in vivo studies are strictly limited because of the lack of facilities to dispose of experimental animals contaminated with radioac- tive materials. On the other hand, the present technique could be utilized in the laboratories equipped only with a spectrophotometer and a microcomputer. Thus, the present technique has an advantage to be utilized in almost any labo- ratories. It has been shown that the estimation of the dye concentration could be made within the range of 6.9% from the measured values for 4 hr with more than 95% confidence, while hemorrhage and injection of 7% NaCl caused much greater changes in the dye concentration. In view of this fact, this technique seems to be reliable. For the accurate measurement, however, the rate of disappearance of dye from the circulation should not be modified by hemorrhage or injection of hypertonic saline. Thus, we will discuss below the reliability of the present measurement in relation to this problem by comparing the change in blood volume with changes in hematocrit, blood pressure and plasma Na concentration after hemorrhage and injection of hypertonic saline. It is rather surprising that blood volume over- shot the previous level within 15 min after 1 ml of hemorrhage. This overshot was also observed when the change in blood volume was assessed by changes in hematocrit alone. Stallone and Braun [16] examined the validity of hematocrit as an indicator for the change in blood volume in the domestic fowl, and found that the change in blood volume measured by hematocrit alone after isosmotic volume expansion or hemorrhage was similar to the change measured by >!Cr-labeled red blood cells. Furthermore, the present study showed that blood pressure returned to the normal level within 15 min after 1 ml of hemorrhage. We also observed that, 15 min after hemorrhage, blood flooded from the jugular catheter more vigorously than before hemorrhage, indicating that venous pressure was elevated due to an increased expansion of volume in this low pressure side of the circulation. These observations support the quick restoration of blood volume after hemor- rhage. In other avian species, it is shown that blood volume restored quickly after 10 and 20% of hemorrhage in the domestic fowl [16], because the maximal decrease in hematocrit was completed 5 min after each hemorrhage. In the pigeon, 810 Y. TAKEI AND I. HATAKEYAMA Kaufman and Peters [17] showed that plasma volume was restored within 4 hr after hemorrhage when the change was measured by the change in hematocrit. Blood volume increased immediately after injec- tion of 7% NaCl, and the maximal increase was 12.5% after 45min. Ruch and Hughes [18] also reported that in a few species of birds, the volume of extracellular fluid increased immediately after an intravenous injection of hypertonic NaCl solu- tion. The increase in Na concentration in ex- tracellular fluid should be 3.56% after injection of 0.1 ml of 7% NaCl; extracellular Na concentration is 139.4mM (present study) and extracellular space is approximately 23% of body weight in birds [19]. If injected excess Na ions were diluted only by practically Na-free cellular fluid and all the cellular water entered the vascular space, and if no Na ions were excreted during that time, the increase in blood volume should be 10.2%. Thus, the maximal increase in blood volume in the present study is more than the maximal increase expected theoretically. One interpretation of this result is that the excretion of the dye was stimu- lated after injection of hypertonic saline due to increases in volume and pressure of blood, which results in a false increase in blood volume. However, blood pressure rather decreased more than 7 min after injection of 7% NaCl as shown in the present study. Thus, another interpretation is that decreased blood pressure stimulated an influx of interstitial fluid into the vascular space. This interpretation also explains the sustained increase in blood volume for 14hr after injection of the hypertonic saline, although the data at 14 hr are not so reliable as those by 4 hr after injection. On the other hand, the maximal increase in blood volume was attained 45 min after injection of 7% NaCl, which coincides well with the restoration of plasma Na concentration to normal that occurs 45 min after injection. The blood volume scarcely increased for 1 hr after injection of 7% NaCl when the change was measured by hematocrit alone. This result may be due to a release of red blood cells after the hyperosmotic volume expansion. The present study showed that, when an acute reduction of blood volume was induced in the quail, the compensation of blood volume exceeded the previous reduction. The degree and rate of the compensation were greater as the reduction was greater. It is also shown that, when an acute increase in plasma Na ions was induced in the quail, the increase in blood volume was more than that required to dilute the increased Na ions to the previous level. These excess responses to exoge- nous acute alterations seem to occur often in many biological systems, when the system possesses a mechanism to respond quickly to the alterations. For instance, Drischel [20] reported that, when glucose was injected into the circulation, blood glucose level rather decreased after a brief increase for a few minutes in the rat. These phenomena are generally termed ‘rebound phenomenon’ [21], or ‘overcompensation’ in the cybernetic terms. It is likely that the responses of blood volume to hemorrhage and injection of hypertonic saline in the quail of the present study may exemplify these phenomena. ACKNOWLEDGMENTS The authors express their appreciation to Dr. Toshiro Sato, Department of Internal Medicine, Kitasato Uni- versity School of Medicine, and to Dr. Tetsuya Hirano, Ocean Research Institute, University of Tokyo, for their critical reading of the manuscript. We also thank Ms. Junko Okubo and Sanae Nishida for technical assistance. This investigation was supported in part by a Grant-in- Aid from the Ministry of Education, Science and Culture, Japan (574307). REFERENCES 1 Guyton, A.C., Hall,J.E., Manning, R.D., Jr., Norman, R. A., Jr., and DeClue, J. W. (1978) A systems analysis of volume regulation. In “Osmotic and Volume Regulation”. Ed. by C. B. Jorgensen and E. Skadhauge, Munksgaard, Copenhagen, pp. 283-294. 2 Kobayashi, H. and Takei, Y. (1982) Mechanisms for induction of drinking with special reference to angiotensin II. Comp. Biochem. Physiol., 71A: 485-494. 3 Takei, Y., Uemura, H. and Kobayashi, H. (1985) Angiotensin and hydromineral balance: With special reference to induction of drinking behavior. In “Current Trends in Comparative Endocrinology”. Ed. by B. Lofts and W.N. Holmes, Hong Kong Univ. Press, Hong Kong, pp. 933-936. 10 11 12 13 Continuous Measurement of Blood Volume Fitzsimons, J. T. (1972) Thirst. Physiol. Rev., 52: 468-561. Gregersen, M. I. and Rawson, R. A. (1959) Blood volume. Physiol. Rev., 39: 307-342. Nielsen, M.H. and Nielsen, N.C. (1962) Spec- trophotometric determination of Evans blue dye in plasma with individual correction for blank density by a modified Gaeblers method. Scan. J. Clin. Lab. Invest., 14: 605-617. Mancini, P. and Pilo, A. (1970) A computer pro- gram for multiexponential fitting by the peeling method. Comput. Biomed. Res., 3: 1-14. Hotelling, H. (1936) Relations between two sets of variables. Biometrika, 28: 321-377. Leonard, J.I. and Abbrecht,P.H. (1974) A method for continuously monitoring blood volume. J. Appl. Physiol., 36: 506-508. Tanaka, Y., Morimoto, T., Miki, K., Nose, H. and Miyazaki, M. (1981) On-line control of circulating blood volume. Jpn. J. Physiol., 31: 427-431. Nose, H., Morita, M., Yawata, T. and Morimoto, T. (1986) Continuous determinantion of blood volume on conscious rats during water and food intake. Jpn. J. Physiol., 36: 215-218. Dill, D. B. and Costill, D. L. (1974) Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration. J. Appl. Physiol., 37: 247-248. Hubbard, R. W., Matthew, W.T., Horstein, D., Francesconi, R., Mager,M. and Sawka,M.N. 14 15 16 17 18 19 20 21 811 (1984) Albumin-induced plasma volume expansion: Diurnal and temperature effects. J. Appl. Physiol., 56: 1361-1368. Brace, R. A. (1983) Blood volume and its measure- ment in the chronically catheterized sheep fetus. Am. J. Physiol., 244: H487-H494. Baker, C.H. (1965) Blood reservoirs in the splenectomized dog. Am. J. Physiol., 208: 485-491. Stallone, J. N. and Braun, E. J. (1986) Osmotic and volemic regulation of plasma arginine vasotocin in conscious domestic fowl. Am. J. Physiol., 250: R644-R657. Kaufman,S. and Peters,G. (1980) Regulatory drinking in the pigeon, Columba livia. Am. J. Physiol., 239: R219-R22S. Ruch, F.E., Jr. and Hughes,M.R. (1975) The effect of hypertonic sodium chloride injection on body water distribution in ducks (Anas platyrhyn- chos), gulls (Larus glauchscens) and roosters (Gulls domesticus). Comp. Biochem. Physiol., 52A: 21-28. Skadhauge, E. (1981) Osmoregulation in Birds, Springer Verlag, Berlin, Heidelberg and New York, p25: Drischel, H. (1956) Blutzukerregelung. In “Re- gelungsvorgange in der Biologie”. Ed. by H. Mittelstaedt, Verlag R. Oldenborg, Miinchen, pp. 60-75. Sherrington, C. S. (1939) Selected Writings of Sir Charles S. Sherrington. Ed. by D. Denny-Brown, Hamish Hamilton, London. iueliiaes suitliok setad Ley Ranies aR ‘hynga wins paedasared Ae lact OR ae: yt heer agi sini ranged — (F801) A My soe + fiiusi a 2a ah SB ote Tat ot iowa st? pe aeaiage ae teh -238 "hoe" sans Pied, OBGb hahianadah isp Bea cine Gl ei): &) O)OMICROM Unein i) bries ig dee coh eit Glenedaas se ib) ode. Wy lanAoylie baleen momen UD)), oF e's ws Mi ar tie act: PAF we i" axe DlteS oh; P rig % oa 2 :? yang, +e PL aa RA. f, ; J A (7 Ny a Te ‘ aR AOS ) Aciseg ag "hi yockn if; RS Pa eee ay eae iMAC UMN cae Alc ame. Abe eat ‘a oe bio fee neiaatels Lethe" woot: Jot Aelia. in nin Ret cape oh arith sae Ee p20, niet tie, r Whe B20 208, Nahe HAM Poa, a ahve Ler it rm ri rayne ch iO, ) (us DAIS, Posie ; iy a | “ue ee a Saukitude "ey oP Rat ohay fh se eri rsa ilig? | } Te eda k elvan dG ie sn Le | mp endy eee teas Abel est) “hitacboaiet (ue hie aoa ah Re. bic detdkty jemand hab Pio AROYEROMG E23, : in. bork WMA, ee ae 1h Gh he panies Ay, rm ' H! 2 F nf ie’ ime ity RS ALS Ae Ue tas AAS _ cw Tei agit afar} orion (PECL) 2 7) noranthere Pt ) r ‘, +0) 7! Be Vit s) HOE re a Pe | ij Ton i $rt pat wen > BORGER TURE eer ah F 7 hae ; - ar { : $ i Se h ; ; +2 j i $4 ‘kia i Se | i 4h 7 q fh Sey 4 c : aa ay Lideet.% iwi if i ay 3) ky A ) { ¥ val ; foes { ry a hy \ } poy” if ‘ dieiernait Ate FESS Brrr ig 2 Rota BAP Groene ‘Sor mstay Sinie.brot : ‘oy Koheyaati ; dre (veenyh iad i stan in pled coe ni 46 ps eayoon Hf ae aM } no af fone (ap inde a: ayers ha ene! siamese: ui he, ra ‘nae ndbineltsctiadity, (he rae he HRI REN, rig ibe A Ak é ad | aah Teh eiety: if ; 3 +)? ee a W/ i ntiire al rent Dae hik weonw aati enya KiCaple at , a i in nina (#903) Vo beh Xs 842 S.-I. ABE AND S. ASAKURA ized. A small area containing the marked cells was cut out, stripped off the plastic dishes, and mounted on supporting blocks. Ultra-thin sections were made with a Porter-Blum microtome MT-1, stained with aqueous uranyl acetate followed by lead citrate, and examined under a JEM 100C electron microscope. RESULTS Differentiation of primary spermatocytes Primary spermatocytes in metaphase (Fig. la) completed the first division within a couple of hours (Fig. 1b) and formed pairs of secondary spermatocytes. During interphase II, the nuclei appeared homogeneous (Fig. 1c). The secondary spermatocytes proceeded through metaphase II (Fig. 1d) and telophase II (Fig. le) and formed quadruplets of round spermatids (Figs. 1f and 2). Soon, a flagellum developed in each round sper- matid (Fig. 3). Small vacuoles appeared in the cytoplasm (Fig. 3) and became larger and fewer. Finally, after a couple of days, a round transparent acrosome, attached over a considerable area of the nuclear envelope, formed in each spermatid (Figs. lg and 4). The diameter of the acrosome was almost the same as that of the nucleus. Some spermatids disappeared in a few days but most survived. After about a week, the acrosome began to condense (Figs. 1h and 5) and the cells assumed an elongated shape. Then the acrosome complete- ly condensed (Fig. li, j). Flagella attained the length of 20-40 4m after one week’s culture and did not increase after an additional week. A round nucleus was located in one side of the cell. Electron microscopic observations revealed an interesting behavior of the mitochondria in sper- matids. Just after the second division, small round mitochondria were scattered throughout the cyto- plasm (Fig. 2). Within a day after the second division, some mitochondria assembled around the nucleus (Fig. 3). When the large acrosome was formed, most of the round mitochondria attached to the nuclear envelope (Fig. 4). During acrosome condensation (Fig.5) and spermatid elongation, the mitochondria remained attached to the nuclear envelope. Phase contrast microscopic observation revealed a dark line along the posterior half contour of the nuclear envelope (Fig. 1j). The dark line should represent the cluster of round mitochondria attached to the posterior part of the nuclear envelope. In the cytoplasm of the elongat- ing spermatid, membraneous structures and some microtubules were observed. After first meiotic division, cell fusion between paired secondary spermatocytes frequently occur- red. An average of 16% of primary spermatocytes (149 cells) in three experiments fused after first meiotic division. Likewise, fusion among sperma- tids frequently occurred following the second meiotic division. Thirty percent of secondary spermatocytes (189 cells) which had not fused after the first division fused after the second division. The fused spermatids differentiated normally ex- cept that a single large acrosome formed in a fused celle Efficiency of differentiation The fate of 138 primary spermatocytes in metaphase from three experiments is summarized in Figure6. More than 90% of the primary spermatocytes completed the first and second divisions and differentiated into round spermatids which formed flagella and acrosomes. About 70% of the initial primary spermatocytes formed sper- matids with condensing acrosomes and about 60% advanced to elongated spermatids. Thus, dif- ferentiation progressed at a very high efficiency. Differentiation at a very high efficiency could be due to some factor(s) which might be secreted from Sertoli cells in culture. In order to exclude the possible effect of Sertoli cells on the dif- ferentiation of spermatocytes or spermatids, Serto- li cells were removed from cultures as described in Materials and Methods. Before removal, the percentage of the Sertoli cells per inoculum was about 2% (1X10° Sertoli cells per inoculum of Fic. 2. Electron microscopic photograph of a round spermatid a couple of hours after the second meiotic division. Bar indicates 1 ~m. Arrows indicate round mitochondria through Fig. 2—Fig. 5. Fic. 3. flagellum. Fused spermatids. About 20 hr after the second meiotic division. Some small vacuoles appeared. FI, In vitro Spermatogenesis in Xenopus laevis 843 844 S.-I. ABE AND S. ASAKURA Fic. 4. A round spermatid with a large acrosome. Ac, acrosome; Nu, nucleus. Bar indicates 1 um. Fic. 5. A round spermatid with a condensed acrosome. In vitro Spermatogenesis in Xenopus laevis 845 100 50 number cell PC 2SC 4tids fl Ac° Ac‘ elongate (mI) max tids 138 cells Fic. 6. The percentage of differentiated cells resulting from primary spermatocytes in metaphase. PC, primary spermatocytes in metaphase; 2SC, pairs of secondary spermatocytes; 4tids, quadruplets of round spermatids; fl, spermatids with flagella; Ac” max, spermatids each with a large acrosome; Ac“, spermatids with condensed acrosomes; elongate tids, elongated spermatids. In this graph, a pair of secondary spermatocytes and a quadruplet of sper- matids derived from a primary spermatocyte were counted as one cell. 5X 10* cells). After removal, the number of Sertoli cells was lower than 10 per dish. Even when very few Sertoli cells were present, differentiation proceeded at almost the same efficiency compared to those cultures that contained large numbers of Sertoli cells. Thus, the differentiation of sperma- tids in vitro does not require substantial numbers of Sertoli cells. Time schedule of differentiation The initially marked primary spermatocytes TABLE 1. Period Average time +S.D. were observed at constant intervals and the time required for the progression of cells from one stage to another was obtained. From the primary spermatocyte to the formation of the acrosome in spermatids the changes were rapid and observa- tions were made every 1-3hr. Following the formation of the acrosome, the changes were very slow and the cells were observed every 10 hr. The period between telophase I and telophase II extended about a day (Table 1). Interphase II lasted about 18 hr. Within 3 hr following telophase II, a short motile flagellum (about 1 um) de- veloped in each round spermatid. Within about 1.3 days after telophase II the complete formation of the acrosome occurred. About 7-8 days were required for the completion of the acrosome condensation and the elongation of the sperma- tids. DISCUSSION The fate of individual primary spermatocytes of Xenopus laevis that attached to pLL-coated cul- ture dishes was observed with phase contrast microscopy. We obtained direct evidence that each spermatocyte completed two meiotic divi- sions and the resultant quadruple round sperma- tids formed flagella and acrosomes. The acro- somes condensed, the cells elongated and the nuclei were situated on one side. Nuclear elonga- tion, however, did not occur even after a pro- longed culture period. Our results are similar to those of Risley who used different medium and cultured the cells in suspension [5]. We also observed by electron microscopy that Time schedule of spermatogenesis in Xenopus laevis at 22°C in vitro No. of cells telophase I~telophase II interphase II telophase II ~ acrosome formation telophase II~ acrosome condensation telophase II ~ elongate spermatids examined 22.2- 8.6 ht 42 cells Witsoe Sets 39 SIRS 9S 36 (1.3+0.4 days) 135.1+18.3 (5.6+0.8) ae 185.2+22.4 73 7.7 40:9) 846 S.-I. ABE AND S. ASAKURA round mitochondria in acrosomal spermatids attached to the posterior part of the nuclear envelope. Clearly, this phenomenon is the pri- mary step toward mitochondrial assembly in the middle piece of mature sperm. What is the mechanism to assemble the mitochondria in the region close to the nuclear envelope?: Do micro- tubules transport them to the nuclear envelope? Or does the nuclear envelope attract the mitochon- dria by exposing some substances on the nuclear envelope? It needs a further study to answer these questions. The possibility that Sertoli cells promote the differentiation of spermatocytes by secreting some factors into culture media was examined by reduc- ing the presence of Sertoli cells to less than 10 per culture. Nevertheless, differentiation proceeded at an efficiency similar to that in cultures contain- ing high numbers of Sertoli cells. This result suggests that Sertoli cells are not required in vitro for spermatogenesis from the stage of primary spermatocytes in metaphase to elongated sperma- tids. The function of Sertoli cells in spermatogene- sis of Xenopus laevis remains to be elucidated. The efficiency of differentiation was very high. More than 90% of the primary spermatocytes developed into spermatids with flagella and acro- somes. This efficiency of differentiation in Xeno- pus is comparable to that obtained in primary spermatocytes of the newt [7]. The time schedule of Xenopus spermatogenesis in vitro was obtained at 22°C. According to Kalt [8] who studied Xenopus spermatogenesis in vivo, the most rapidly maturing cells in the testis spend one day in meiotic division and 12 days in spermiogenesis at 18°C. The time schedule of our cells in vitro is comparable to that in vivo. In our current studies with Xenopus, we used pLL to attach cells to the culture dishes. Various molecular weights of pLL were tested for their effect on viability and differentiation of cells; pLL with molecular weight lower than 27,000 had no inhibitory effect on meiotic divisions and dif- ferentiation of spermatids. But, pLL with high molecular weights (47,000, 90,000 and 120,000) was toxic to the cells. It should be noted that cell fusion frequently occurred between secondary spermatocytes and between spermatids when cells were cultured in pLL-coated dishes. However, cell fusion rarely occurred when the cells were cultured in suspen- sion in the dishes uncoated with pLL. Thus, cell fusion appears to be aided by pLL. Polycation polyarginine also induces fusion between sea urchin eggs [9]. In a fused spermatid differentia- tion proceeded normally except for the acrosome formation; even when a cell contained 2 or 4 nuclei, a single acrosome larger than normal was formed. How such a single acrosome is formed in a fused cell is now under investigation. Previously, we showed in the newt that single primary spermatocytes completed two meiotic divisions and differentiated into round spermatids that formed flagella, acrosomes, rings and neck- pieces [1]. Thus, we have shown that L-15 medium supports meiotic divisions and early-mid- spermiogenesis in vitro in both the newt and Xenopus laevis of which mature sperms are entire- ly different in size and shape from each other. One of the differences observed in sperma- togenesis in vitro between Xenopus and the newt is that the flagellar length in Xenopus is shorter (20-40 um) than that in the newt (100-400 um) [1]. Another difference is that the acrosome in the Xenopus spermatid is as large as the nucleus and easily observed by phase contrast microscopy, whereas the acrosome in the newt is small and not easily observed. Also, in the Xenopus spermatid mitochondria attached to the posterior part of the nuclear envelope when the acrosome was formed. However, in the newt acrosomal spermatids, mitochondria were scattered throughout the cyto- plasm. These results correctly reproduce the difference in in vivo spermatogenesis between both species [10-16]. Since the culture of dissociated spermatogenic cells in Xenopus and the newt has been refined, future studies on the differences between newt and Xenopus spermatogenesis in vitro at the cellular and molecular levels should provide insight into the regulatory mechanisms governing the flagellar formation, acrosome formation, and the assem- blage of mitochondria in the middle piece of the mature sperm. In vitro Spermatogenesis in Xenopus laevis 847 ACKNOWLEDGMENTS We thank Professor Marie A. DiBerardino (The Medical College of Pennsylvania) for editing this manu- script. This work was supported by a Research Grant from the Ministry of Education, Science and Culture of Japan (no. 60540467) and grants from the Ito Science Foundation and Naito Science Foundation to S. A. REFERENCES 1 Abé,S.-I. (1981) Meiosis of primary spermatocytes and early spermiogenesis in the resultant spermatids in newt, Cynops pyrrhogaster, in vitro. Differentia- tion, 20: 65-70. 2 Abé, S.-I. and Uno, S. (1984) Nuclear elongation of dissociated newt spermatids in vitro and _ their shortening by antimicrotubule agents. Exp. Cell Res., 154: 243-255. 3 Abé,S.-I. (1987) Differentiation of spermatogenic cells from vertebrates in vitro. Int. Rev. Cytol., 109: 159-209. 4 Risley, M. S. and Eckhardt, R. A. (1979) Evidence for the continuation of meiosis and spermiogenesis in in vitro cultures of spermatogenic cells from Xenopus laevis (1). J. Exp. Zool., 207: 513-520. 5 Risley, M. S. (1983) Spermatogenic cell differentia- tion in vitro. Gamete Res., 4: 331-346. 6 Wallace, R. A., Jared, D.W., Dumont,J.N. and Sega, M. W. (1973) Protein incorporation by iso- lated amphibian oocytes. III. Optimum incubation conditions. J. Exp. Zool., 184: 321-334. 7 Nishikawa, A. and Abé,S.-I. (1983) Progression throughout all stages of meiosis from the early 10 11 12 13 14 iI5) 16 prophase of newt primary spermatocytes in vitro. Dev. Growth Differ., 25: 323-331. Kalt, M. R. (1976) Morphology and kinetics of spermatogenesis in Xenopus laevis. J. Exp. Zool., 195: 393-408. Bennett, J. and Mazia, D. (1981) Interspecific fu- sion of sea urchin eggs. Surface events and cytoplas- mic mixing. Exp. Cell Res., 131: 197-207. Kalt, M. R. (1973) Ultrastructural observations on the germ line of Xenopus laevis. Z. Zellforsch.., 138: 41-62. Reed, S. C. and Stanley, H. P. (1972) Fine structure of spermatogenesis in the South African clawed toad Xenopus laevis Daudin. J. Ultrastruct. Res., 41: 277-295. Meves, F. (1897) Ueber Structur und Histogenese der Samenfaden von Salamandra maculosa. Arch. Mikrosk. Anat. Entwickl., 50: 110-141. Barker, K.R. and Biesele,J.J. (1967) Sper- mateleosis of a salamander Amphiuma tridactylum Cuvier. A correlated light and electron microscope study. Cellule, 57: 91-118. Picheral, B. (1972) Les elements cytoplasmiques au cours de la spermiogenese du Triton Pleurodeles waltlii Michah. I. La genese de l’acrosome. Z. Zellforsch., 131: 347-370. Picheral, B. (1972) Les elements cytoplasmiques au cours de la spermiogenese du Triton Pleurodeles waltlii Michah. II. La formation du cou et l’evolu- tion des organites cytoplasmiques non integres dans le spermatozoide. Z. Zellforsch., 131: 371-398. Picheral, B. (1972) Les elements cytoplasmiques au cours de la spermiogenese du Triton Pleurodeles waltli Michah. III. L’evolution des formations caudales. Z. Zellforsch., 131: 399-416. ae oe Ay REFORM RENEE, teint seojctoiad “nes sae ee an 10. iowrs af A ti vgey bits faa (op as ae SC sn ahigcll ,) 4 ; Rye 4 ee et eee fe acd Y RUE pati { ns ae Saison | eS seen Widbembelte SRG FE nal alteve mht easton nets dost he in fyi eos ea Mo FRSA is ond Ate} Wit: mien BiiAr a: ou), MITA Ca aad ime nae sul het i aan AOS 3 2 OT, ntoe ye to wih ai ge he #4 pRee Y { ' Ne pape bt EPEC ap PT Pate ans hy tye ane MCLE a RUT RRR a fa hig ie IMs CPA RSIS ANE BAY ri See ag ae e lesvenlisktels eu tom feiiewy soruently L: niberetl divs) tad, aivpaaaga tas year ovhatdie : SY Chat Dd Si pel se detierer ties | RES by aE yaa 1 ety i udaseae ie GOAL) Y= BS) a falban ' Hepat ocinor TORE. NOY whbale AEE yah ‘om ; i Ae adls’ Wo; ce fides PLT Oe Tai ‘a ThA. 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Wat iealte’s PRS wed 423 euneicite ae ine Pah je ik Ai ais ey tf 5 AM has eae) en aie mertiee inthis. samt i a ye f ¥ - g i ay i 0 j ! H — a i yee ee ee} sd ‘ ry b suc? e ‘ t J { iu Pyar st i ove mk us : +" . is ' 2 ! MiGd ug 2H on te fe ie +s Dalen. ; Fah J Behe tae ee bi ar i q , He 7% Lil ipey be 3 ‘ = ,! 4 F ) y fed pa Pe es j 1 OE Ae ; mh OF et ¢ p sx HOLEC Pee Piet Tri e ZOOLOGICAL SCIENCE 4: 849-854 (1987) Plasma Thyroid Hormone Levels in Metamorphosing Larvae and Adults of a Salamander, Hynobius nigrescens SHINTARO SUZUKI Department of Comparative Endocrinology, Institute of Endocrinology, Gunma University, Maebashi 371, Japan ABSTRACT—Plasma thyroxine (T,) and 3, 5, 3’-triiodothyronine (T;) were measured by radioimmu- noassay (RIA) in metamorphosing larvae, metamorphosed animals, and adults of a salamander, Hynobius nigrescens. At the onset of metamorphosis the plasma T, was at an undetectable level. As metamorphosis proceeded, the plasma T, gradually increased and reached its highest level (1.96 ng/ ml) at the metamorphic climax, accompanied with striking regression of a dorsal fin and sudden reduction in the size of gills. Thereafter, the plasma T, decreased towards the end of metamorphosis. The plasma T; of these metamorphosing larvae was detected only at the metamorphic climax. In the animals tested two weeks after metamorphosis, the plasma Ty, level increased again. In the adults which were collected before spawning, the plasma T, level was 0.34-5.77 ng/ml, although the plasma T, was undetectable after spawning. Plasma T3 was undetectable in any of the adults collected. These results from metamorphosing larvae resemble those reported in anuran tadpoles. However, the data from metamorphosed animals and adults suggest that there is a positive correlation between thyroid © 1987 Zoological Society of Japan activity and molting and the spawning behavior in the aquatic habitat. INTRODUCTION Thyroid hormones are indispensable for amphi- bian metamorphosis and it has been suggested that a gradual rise in the circulating thyroid hormone level is necessary for anuran metamorphosis [1-4]. Recently, it has been reported that the plasma thyroid hormone level rose sharply at the onset of metamorphic climax and fell towards the end of metamorphosis [5-10]. In urodeles, in which the metamorphic events seem to be much less striking than in anurans, the data available thus far are fragmentary [11, 12] and the plasma thyroid hormone levels have not been elucidated in rela- tion to distinct metamorphic stages. In the present communication, we report plasma thyroid hormone levels in the metamorphosing larvae of the Japanese salamander, and also refer to the plasma thyroid hormone levels both in the metamorphosed animals and in the adults before and after spawning to define the role of thyroid hormones. Accepted June 2, 1987 Received March 9, 1987 MATERIALS AND METHODS Animals The eggs of the Japanese salamander, Hynobius nigrescens Stejneger, were collected from a pond in a mountain region in Gunma Prefecture. After hatching, the larvae were kept at 20+2°C in well water and were fed with a living Tubifix twice a week. The developmental stages were determined from the normal table (sketches) reported by Usui and Hamasaki [13] (UH stages 62 to 66), and two stages (stages 67 to 68) by the time of completion of metamorphosis were added to the UH stages. Larvae were anesthetized with MS222 (tricaine methanesulfonate, Sankyo Co., Tokyo), and blood samples were pooled into heparinized capillaries after cutting the ventral aorta. After centrifugation the plasma was stored at —60°C for RIA. The animals kept for two weeks after metamorphosis were also used for measurement of thyroid hormones. The body length and weight of these animals were 4.6—5.0 cm and 0.7-1.0 g, respectively. Adult salamanders with a body weight ranging 7.4-14.1g were collected from the same pond during the breeding season in March. These adults collected before 850 S. SUZUKI spawning were kept in the laboratory for two days and blood samples were taken for the measure- ment of thyroid hormones. Some of the blood samples from the remainder were also obtained at one day after spontaneous spawning in the labora- tory. Some adults were collected from the same pond after spawning. Blood samples were col- lected from these larvae and adults between 11: 00 a.m. and 2:00 p.m. Radioimmunoassay Plasma T, and T3 were measured by appropriate RIA methods [14, 15], as described previously [9]. [!*°I] Ty (1250 uCi/ xg) and [!*I] T3 (1200 »Ci/ pg) were purchased from New England Nuclear, U.S.A. Antisera for T, and T3 were prepared by immunizing rabbits with their conjugates with bovine serum albumin, and used at a final dilution of 1:3000 and 1: 2500, respectively. The cross reactivities of these anti- sera were 0.1% (T4 antiserum to T3) and 0.14% (T3 antiserum to T,). For duplicate RIA deter- mination, T, and T3 in 100 «l plasma were ex- tracted with 1 ml of ethanol [6]. The minimum detectable level was estimated to be 0.2 ng/ml for T, and 0.15 ng/ml for T3. RESULTS Identification of developmental stages in metamor- phosing larvae The developmental stages of metamorphosing TABLE 1. larvae and some characteristics are shown in Table 1, with corresponding stages of Ambystoma macu- latum [16]. At UH stage 62, slight shrinkage of the dorsal fin and gill platelets was observed. Howev- er, the body length and body weight of the larvae continued to increase with the growth of the forelimbs (UH stages 63 to 64). At UH stage 65, the regression of the dorsal fin reached to halfway between the forelimbs and hindlimbs, and con- spicuous shrinkages of gill platelets were observed. After this stage, the operculum began to fuse to head, with rapid regression of dorsal fin and gills. At UH stage 66, a striking puffing of eyelids and the formation of square-jaws were observed in these larvae. Their gills changed into nubbins, and the regression of the dorsal fin reached the joints of the hindlimbs. At stage 67, the head shape changed into that of the adult due to rapid regression of the hyobranchial skeleton. Complete fusion of the operculum and the first molting were observed in the larvae. Thereafter, small pieces of tail fin disappeared and many white spots emerged on the dark brown skin of the larvae (stage 68). Within a few days or a week, metamorphosed animals began to feed again. After the first molting at stage 67, regular molting with a cycle of 2.4 days was observed for at least two months. The body length and weight of these metamorphosing larvae increased to the UH stage 65. However, a rapid decrease in body weight was observed at the end of metamorphosis (Fig. 1(B)). Developmental stages and some external characteristics of metamorphosing larvae of Hynobius nigrescens, and corresponding stages of Ambystoma maculatum Hynobius Ambystoma Developmental Some external characteristics Developmental stage stage 62 Onset of shrinkages of dorsal fin and gill platelets; puffing of eyelids 56 64 Shrinkages continuing; striking wrinkles and undulating tail fin 65 Regression of dorsal fin reaching to halfway between forelimbs and hindlimbs 66 Regression of dorsal fin as far back as hindlimbs; small gill pieces Sd) existing 67 Disappearance of gill pieces; complete fusion of opercular fold; 58 change in head shape due to rapid regression of hyobranchial skeleton; small pieces of tail fin existing; 1st molting of entire skin 68 Gular fold almost smoothing out; disappearance of tail fin; sudden 59 increase in number of white spots on the dark skin (ng/ml) TA Body length (cm) o Plasma T, and T3; Levels in Hynobius 851 2 (A) 14 1 ee 6) 62 64 66 68 Developmental stage (B) 5 1.5 4 o = ioe © 5 4 10 > iS a Developmental stage Plasma T, and T; levels in metamorphosing larvae and metamorphosed animals Plasma T, and T; levels in both metamorphosing larvae and animals kept two weeks after meta- morphosis are shown in Table 2. The plasma T, was undetectable at the onset of metamorphosis (UH stage 62). As metamorphosis proceeds, its plasma T, gradually increased (UH stage 64, 0.23 ng/ml) and reached its highest level (UH stage 66, 1.96 ng/ml) at the metamorphic climax when remarkable regression of gills and dorsal fin is observed. Thereafter, the T, values decreased towards the end of metamorphosis. In the animals which were kept for two weeks after metamor- phosis, the plasma T, value was 1.32 ng/ml. On the other hand, the plasma T3 level was undetectable at the early stages of metamorphosis (UH stages 62, 64 and 65). At the metamorphic climax (UH stage 66) the plasma T; value was 0.54 ng/ml. Thereafter, the plasma T3 decreased and reached an undetectable level at stage 68 (Fig. 1(A)). In the animals two weeks after metamor- phosis, the plasma T3 was at an undetectable level. Fic. 1. Plasma T, and T3 levels (A), and changes in body length and weight (B) during metamorphosis of Hynobius nigrescens. Symbols (A) in Fig. 1 (A) show undetectable levels of T, and T3. Each point in Fig. 1 (B) shows the mean value +standard error (n=10). TaBLE2. Plasma T, and T; levels in Hynobius nigrescens at various developmental stages Developmental stage Number of animals 62 23 64 50 65 27 66 39 67 DA 68 15 Metamorphosed 29 animals*** * Not detectable, T,<0.2 ng/ml ** Not detectable, T3<0.15 ng/ml Iv T3 (ng/ml) 0.23 = 0.50 = 1.96 0.54 1.44 0.11 0.71 i 1252 = *** Animals kept for two weeks after metamorphosis 852 TABLE 3. after spawning S. SUZUKI Plasma T, and T; levels in the adult salamanders, Hynobius nigrescens before and Body length (cm) Animal number N @) Pad 16.2 II) 14.3 2 14.0 14.0 Animals before spawning 16.0 bei 14.2 15.74 16.0 123 Animals after spawning \o co ~ Nn PWN — i=) 11 12 Sesser es SS) oe oe os * Not detectable, T,<0.2 ng/ml ** Not detectable, T;<0.15 ng/ml Plasma Tq and T; levels in the adult salamanders Plasma T, and T3 in the adults were measured before and after spawning. As shown in Table 3, in all adults collected before spawning, the plasma T, values changed greatly from 0.34 to 5.77 ng/ml. However, after spawning the plasma T, was at an undetectable level. T, value was also undetectable in adults collected from the same pond after spawning. In all adults tested at the same time, the plasma T3 was undetectable both before and after spawning. No significant sex differences in the plasma levels of T, and T3 were found. DISCUSSION Until now, plasma thyroid hormones in meta- morphosing larvae of several urodeles have been measured by RIA [11, 12]. Plasma Ty, values in metamorphosing larvae of Ambystoma gracile (30 ng/ml) were much higher than those in both non-metamorphosing larvae and metamorphosed animals (below 10 ng/ml) [11]. Larras-Regard et al. [12] classified the metamorphosing larvae of Ambystoma tigrinum into three groups (beginning, middle, and end of metamorphosis) according to the metamorphic stage. They found a significant Body WA T; weight (g) (ng/ml) 10.0 0.34 a 9.6 a) fT) 9.6 1.24 Woe 11.6 1.87 —** 10.3 JS 13.0 2.14 we 7.6 = a WD = a 10.9 = 12 a —** 14.1 = 7.4 —* = elevation of plasma T, levels only during meta- morphosis, although T, values fluctuated consider- ably in different animals. Plasma Ty, values reached the highest level at the end of metamor- phosis (1-15 ng/ml). However, T3 levels were much higher at the middle than at the end of metamorphosis (0.98-1.95 ng/ml). In particular, they observed high plasma T3, although plasma T, was undetectable in several metamorphosing animals. The present results obtained in the metamorphosing larvae of Hynobius clearly showed that a slight increase in plasma T, is found at the early stages of metamorphosis and plasma T, reached its highest level at the metamorphic climax, accompanied with striking regression of the dorsal fin and gills. Thereafter, the plasma T, level fell towards the end of metamorphosis. The circulating T3 level in metamorphosing animals was very low and detected only at climactic stages. These changes in the plasma T, and T3 during metamorphosis of the salamander Hynobius re- semble those found in anuran tadpoles [5-10]. It seems that a gradual rise in plasma thyroid hormone levels is required for early metamor- phosis, and a decrease is observed at the end of metamorphosis even in urodeles. It was reported that molting in urodeles is Plasma T, and T3 Levels in Hynobius 853 influenced by thyroid function and the frequency of molting is determined by the level of thyroid activity [17-19]. In the present study it was shown that the level of plasma T, in metamorphosed animals which were continuing a cyclic molting was much higher than that in larvae which had just completed metamorphosis (stage 68). This result suggests that the higher plasma Ty, level is a prerequisite to frequent molting in metamor- phosed animals. Larras-Regard et al. [12] reported the plasma T, and T3 levels in adult Ambystoma tigrinum. In seven of these animals, plasma T, and T; levels were below the limits of detection (T,, below 0.5 ng/ml; T3, below 0.05 ng/ml). In one animal the plasma T, level was quite elevated (5.2 ng/ml), but plasma T3 was not detected. In neotens of the same species, Norris et al. [20] found circannual changes in plasma Ty, levels. The plasma T, was at a much higher level (5.4—5.5 ng/ml) from Novem- ber to March (prespawning) and decreased to its minimum level (2.2+0.31 ng/ml) from June to September (postspawning). They emphasized antagonistic roles in thyroid function and repro- ductive events such as gonadal development in these animals. In the present study it was shown that plasma T, values for adult Hynobius in prespawning were much higher than those in postspawning. There was no significant change in the amounts of total serum proteins among these adults (unpublished data, mean+standard error: prespawning, 19.3+0.4mg/ml; postspawning, 18.9+2.6). From the results of Norris et al. [20] and our results, it appears likely that the thyroid function of the adult salamanders is more active in prespawning. In newly metamorphosed Ambystoma tigrinum, Duvall and Norris [21] found that plasma T, levels are significantly higher in animals displaying spon- taneous land preference than in those displaying water preference. It is assumed therefore that the higher thyroid activity found in Hynobius kept in the laboratory for two weeks after metamorphosis may also reflect their land preference. On the other hand, the adult Hynobius migrates to water only during the breeding season and returns to the terrestrial life after laying its eggs. Therefore, in these animals it seems that the role of thyroid hormone should be considered in controlling changes in habitat. Recently, Moriya [22] found that prolactin-treated salamanders, Hynobius re- tardatus, sink to the bottom of the water, although the control salamanders that do not receive prolac- tin float near the surface. Moreover, Moriya and Dent [23] reported the effect of prolactin and T, on habitat choice, the specific gravity of the whole body and osmotic pressure of serum in the newt, Notophthalmus viridescens. They showed that the animals move from water to land, and both specific gravity and serum osmolality are decreased when they are immersed in a solution containing Ty. As already mentioned above, we found a conspicuous difference in plasma T, between the prespawning and the postspawning Hynobius, both of which are staying in the water. It seems likely that thyroid activity is in some fashion correlated with spawn- ing behavior in the aquatic habitat. ACKNOWLEDGMENTS I express my gratitude to Professor M. Suzuki of our Institute, for his valuable suggestions during this work. Thanks are also due to Mr. T. Kakegawa and Mr. T. Narita, for their technical assistance. REFERENCES 1 Etkin, W. (1935) The mechanisms of anuran meta- morphosis. I. Thyroxine concentration and the metamorphic pattern. J. Exp. Zool., 71: 317-340. 2 Etkin, W. (1968) Hormonal control of amphibian metamorphosis. In “Metamorphosis”. Ed. by W. Etkin and L. I. Gilbert, North-Holland Publ. Co., Amsterdam, pp. 313-348. 3 Just, J. J. (1972) Protein-bound iodine and protein concentration in plasma and pericardial fluid of metamorphosing anuran tadpoles. Physiol. Zool., 45: 143-152. 4 Dodd, M. H. I. and Dodd, J. M. (1976) The biology of metamorphosis. In “Physiology of the Amphi- bia”, Vol. 3. Ed by B. Lofts, Academic Press, New York and London, pp. 467-599. 5 Leloup, J. and Buscaglia, M. (1977) La triiodothy- ronine, hormone de la metamorphose des amphi- biens. C. R. Acad. Sci., Ser. D, 284: 2261-2263. 6 Miyauchi, H., LaRochelle, F.T., Jr., Suzuki, M., Freeman, M. and Frieden, E. (1977) Studies on thyroid hormones and their binding in bullfrog tadpole plasma during metamorphosis. Gen. Comp. Endocrinol., 33: 254-266. 10 11 12 13 14 15 854 Regard, E., Taurog, A. and Nakashima, T. (1978) Plasma thyroxine and triiodothyronine levels in spontaneously metamorphosing Rana catesbeiana tadpoles and in adult anuran amphibia. Endocrinol- ogy, 102: 674-684. Mondou, P. M. and Kaltenbach, J.C. (1979) Thy- roxine concentrations in blood serum and pericar- dial fluid of metamorphosing tapoles and of adult frogs. Gen. Comp. Endocrinol., 39: 343-349. Suzuki, S. and Suzuki, M. (1981) Changes in thy- roidal and plasma iodine compounds during and after metamorphosis of the bullfrog, Rana cates- beiana. Gen. Comp. Endocrinol., 45: 74-81. Weil, M. R. (1986) Changes in plasma thyroxine levels during and after spontaneous metamorphosis in a natural population of the green frog, Rana clamitans. Gen. Comp. Endocrinol., 62: 8-12. Eagleson,G.W. and McKeown,B.A. (1978) Changes in thyroid activity of Ambystoma gracile (Baird) during different larval, transforming, and postmetamorphic phases. Can. J. Zool., 56: 1377-1381. Larras-Regard, E., Taurog,A. and _ Dorris, M. (1981) Plasma T, and T; levels in Ambystoma tigrinum at various stages of metamorphosis. Gen. Comp. Endocrinol., 43: 443-450. Usui, M. and Hamasaki, M. (1939) Tafeln zur Entwicklungsgeschichte von Hynobius nigrescens Stejneger. Zool. Mag., 51: 192-206 (In Japanese). Larsen, P.R. (1972) Direct immunoassay of triiodothyronine in human serum. J. Clin. Invest., 51: 1939-1949. Larsen, P. R., Dockalova, J., Sipula, D. and Wu, F. M. (1973) Immunoassay of thyroxine in unextracted S. SUZUKI 16 17 18 i) 20 21 (as ep) human serum. J. Clin. Endocrinol. Metab., 37: 177-182. Ballard, W. W. (1972) Characterization of develop- mental stages. Part VI. Salamander. In “Biological Data Book”, Vol. 1. Ed. by P. L. Altman and D. S. Dittmer, Fed. Am. Soc. Exp. Biol., U.S. A., pp. 185-189. Jorgensen, C. B. and Larsen, L.O. (1960) Hor- monal control of moulting in amphibians. Nature, 185: 244-245. Clark, N. B. and Kaltenbach, J.C. (1961) Direct action of thyroxine on skin of the adult newt. Gen. Comp. Endocrinol., 1: 513-518. Larsen, L.O. (1976) Physiology of molting. In “Physiology of the Amphibia”, Vol. 3. Ed. by B. Lofts, Academic Press, New York and London, pp. 53-100. Norris, D. O., Duvall, D., Greendale, K. and Gern, W.A. (1977) Thyroid function in pre- and post- spawning neotenic tiger salamanders (Ambystoma tigrinum). Gen. Comp. Endocrinol., 33: 512-517. Duvall, D. and Norris, D. O. (1980) Stimulation of terrestrial substrate preferences and locomotor activity in newly transformed tiger salamanders (Ambystoma tigrinum) by exogenous or endogenous thyroxine. Anim. Behav., 28: 116-123. Moriya, T. (1982) Prolactin induces increase in the specific gravity of salamander, Hynobius retardatus, that raises adaptability to water. J. Exp. Zool., 223: 83-88. Moriya, T. and Dent, J. N. (1986) Hormonal in- teraction in the mechanism of migratory movement in the newt, Notophthalmus viridescens. Zool. Sci., 3: 669-676. ZOOLOGICAL SCIENCE 4: 855-860 (1987) © 1987 Zoological Society of Japan Proliferation of Prolactin Cells in the Rat: Effects of Estrogen and Bromocryptine SUMIO TAKAHASHI and SEIICHIRO KAWASHIMA Zoological Institute, Faculty of Science, Hiroshima University, Naka-ku, Hiroshima 730, Japan ABSTRACT—The mitosis of prolactin (PRL) cells was immunohistochemically identified in colchi- cine-treated rats. The administration of estrogen increased the mitotic activity of PRL cells and serum PRL levels in male rats, while bromocryptine, a dopamine agonist, blocked the estrogen-mediated increase in mitotic activity and PRL levels. In female rats, bromocryptine treatment in the early afternoon of 2nd day of diestrus and proestrous day, which should block the increase in serum PRL level in the evening of proestrous day, decreased the mitotic activity of PRL cells at estrus, which was very high in non-treated females. These results suggest that the proliferation of PRL cells was closely associated with PRL release from the pituitary gland. INTRODUCTION Mitotic prolactin (PRL) cells were immunohis- tochemically and electron microscopically iden- tified in intact rats [1, 2]. The mitotic activity of PRL cells varied with the estrous cycle, and the mitotic index at estrus was higher than those at other stages of the cycle [3]. Estrogen treatment stimulated and ovariectomy decreased the mitotic activity of PRL cells [3, 4]. Estrogen is a stimulatory agent on PRL release. In the course of estrogenic stimulation, the syn- thesis of PRL-messenger RNA (mRNA) began to increase 12 hr after estrogen treatment and further increased over the next 48hr [5]. Pituitary PRL concentration and DNA synthesis also rose at 30 hr after estrogen treatment [6]. Earlier study found that the number of mitotic cells was in- creased during the second day after estrogen treatment, although the cell types undergoing mitosis were not identified in this study [7]. Chronic estrogen treatment induced an increase in the number of immunohistochemically identified PRL cells [8-10]. Yamamoto et al. [9] recently found the increase in PRL mRNA content per PRL cell as assessed by the in situ hybridization in Accepted May 9, 1987 Received April 14, 1987 chronic estrogen-treated rats. However, little has been known about the time-related changes of the mitosis of PRL cells and serum PRL levels after estrogen treatment, and the first aim of the present study was to solve this problem. Dopamine agonists, such as ergocryptine and bromocryptine, inhibited the syntheses of PRL mRNA and PRL, and the release of PRL [11, 12]. Bromocryptine also inhibited DNA synthesis and mitosis in the pituitary cells in estrogen-treated rats [13, 14]. In this conjunction, the second aim of the present study was to examine the effects of dopamine agonist, bromocryptine (CB-154), on the mitosis of PRL cells in estrogen-treated male rats and intact female rats exhibiting normal estrous cycles to clarify the relationship between PRL secretion and mitosis. The mitotic PRL cells were observed immunohistochemically in colchi- cine-treated rats throughout the present study. MATERIALS AND METHODS Animals Adult male and female rats (about 60 days of age) of the Wistar/Tw strain were used. They were housed in a temperature-controlled room with a 12-hr light (06: 00-18:00) and 12-hr dark cycle, and given rat chow (Clea Japan Inc., Tokyo) 856 S. TAKAHASHI AND S. KAWASHIMA and water ad libitum. In female rats vaginal smears were recorded daily for at least two weeks before the experiments. Estrogen treatment Intact male rats were given a single sub- cutaneous injection of 50 wg estradiol-178 (Ep, Sigma, St. Louis) dissolved in 0.1 ml sesame oil. This dose was chosen from the data of our previous experiments [4]. These rats were killed at 0, 3, 6, 24, 36, 48, 72 or 96 hr after the treatment for the observations of the mitotic PRL cells. Rats given sesame oil only served as controls (0, 48 and 96 hr). For serial blood samplings, other rats were similarly given E> or oil, and blood samples were collected from the jugular vein under light ether anesthesia on the same schedule as for mitosis observations. Bromocryptine treatment Intact male rats were given a single sub- cutaneous injection of E,, and 24 or 42 hr later they were subcutaneously given bromocryptine (CB-154, Sandoz, Ltd., Basel) at a dose of 4 mg/kg. Bromocryptine was dissolved in a minimal amount of 100% ethanol and diluted with 0.9% saline to the final concentration of 2 mg/ml. Final ethanol concentration did not exceed 1%. Vehi- cle-treated rats served as controls. All rats were killed 48 hr after E, treatment. About half of them were used for the observation of the mitosis, and the remaining animals were used for the measure- ment of serum PRL levels. Intact female rats were given bromocryptine at a dose of 4 mg/kg on 2nd day of diestrus (15: 00 hr), proestrous day (15:00 hr) or estrous day (10:00 hr). Vehicle-treated rats served as controls. All rats were killed in the afternoon of estrous day (16: 00 hr). Immunohistochemistry and counting of mitotic PRL cells Colchicine (Wako Pure Chemical Industries, Ltd., Osaka) was injected intraperitoneally at a dose of 1mg/kg. Three hours later, between 16:00 and 18:00, the rats were killed by decapita- tion. The pituitary glands were quickly removed and fixed in Bouin’s fluid. Horizontal paraffin sections 6 um thick were cut. Immunohistochemi- cal staining was performed according to PAP method of Sternberger et al. [15], using rabbit antiserum to rat prolactin (NIADDK-anti-rat pro- lactin S-7). Immunospecificity of the antiserum to rat PRL was checked in a previous report [3]. Peroxidase activity was demonstrated by the reac- tion with 3, 3’-diaminobenzidine tetrahydrochlor- ide. After the immunohistochemical staining, sections were counterstained with hematoxylin. Sections near the horizontal medial plane were selected for histometry, because in a preliminary study the value from the horizontal medial plane represents the mean value of PRL cell population of three different levels of horizontal planes. The total number of anterior pituitary cells in colchi- cine-arrested metaphase and the number of im- munoreactive mitotic cells (mitotic PRL cells) were counted. In the sections used for the differential mitotic cell counting, the area of anterior pituitary glands was measured using a tablet digitizer MG 200-1 (Mutoh Kogyo, Tokyo). The mitotic index is expressed as the number of mitoses per mm’ of section. The sectional area of 1 mm? contained about 10,000 cells in 60-day-old rats. Radioimmunoassay of PRL Blood samples were collected under light ether anesthesia from the jugular vein or the decapitated trunks. PRL levels were measured using the NIADDK RIA kit. The data were expressed as ng/ml of NIADDK-rat-prolactin RP-2. Statistics Statistical analyses of differences were per- formed with Student’s t test. RESULTS Mitotic index of PRL cells and serum PRL levels in estrogen-treated male rats E, significantly increased the mitotic indices of PRL cells and the total anterior pituitary cells 36 hr after the injection, and the significantly high mitotic activity lasted until 96 hr (Fig. 1). At 6, 36, 48, 72 and 96 hr after the E, injection the mitotic Proliferation of Prolactin Cells 857 25 20 (mitoses/mm’) a x< © 10 Uv = = e ss 0 036 24 «36 2=— 48 72 96 Time after Estradiol-176 treatment (hr) Fic. 1. Mitotic index of prolactin cells (@) and total pituitary cells (©) after estradiol-17 (50 ug) injec- tion. The mitotic index in vehicle-treated controls was described in the text. The numbers on each point depict the numbers of rats, and the bars indicate the standard errors of means. index of total cells including PRL cells was significantly greater than that at the time of injection (P<0.05, at 6hr; P<0.01 at 36, 48, 72 and 96 hr), and that of PRL cells was higher at 36, 48, 72 and 96 hr than that at the time of injection (P<0.01, in all comparisons). In vehicle-treated controls the mitotic indices of PRL cells and total cells (n=the number of rats) were 1.4+0.3 (PRL cells) and 4.0+0.8 (total cells) (n=5) at Ohr, 0.6+0.1 and 2.6+0.5 (n=5S) at 48 hr, and 1.5+0.3 and 4.0+0.3 (n=6) at 96hr, respectively. E, significantly increased serum PRL levels 36 hr after the injection and maintained the elevated PRL levels until 96 hr (Fig. 2). In vehicle-treated controls, on the other hand, PRL levels tended to decrease during the successive blood samplings, and the significant differences in serum PRL levels between estrogen-treated rats and vehicle-treated rats were observed at 36, 48, 72 and 96hr (P<0.001, at 36, 48 and 72 hr; P<0.05, at 96 hr). 40 30 (ng/ml) Prolactin 036 24 36 48 72 96 Time after Estradiol-178 treatment (hr) Fic. 2. Serum prolactin concentration in male rats given estradiol-17 (50 ug, 7 rats, @) or sesame oil (6 rats, O). The value at 0 hr is the composite data of estradiol-17f-treated rats and vehicle-treated rats (n=13). The bars depict the standard errors of means. Effect of bromocryptine on the mitotic index of PRL cells and serum PRL levels in estrogen-treated rats Bromocryptine injection at 24 hr after E, treat- ment blocked the E,-induced increase in the mitotic index of PRL cells, while bromocryptine injection at 42 hr after E, was not effective on this parameter (Table 1). Mitotic indices of cells other than PRL cells were not influenced by bromocryp- tine injection. Serum PRL levels significantly decreased when bromocryptine was injected either 24 or 42 hr after E, treatment. Effect of bromocryptine on the mitosis of PRL cells in intact female rats Mitotic index of PRL cells was observed at 16:00 hr of estrous day, which has proved to be maximal during the estrous cycle [3]. Bromocryp- tine treatment on 2nd day of diestrus (15: 00 hr) or proestrous day (15:00 hr) significantly decreased 858 S. TAKAHASHI AND S. KAWASHIMA TABLE 1. estrogen-treated male rats Effect of bromocryptine (CB-154) on the mitosis of PRL cells and serum PRL level in Time at Treatment CB-154 treatment (hr) PRL cell Control 24) 12.1+2.9 CB-154 24 3.9+0.6* Control 42) 10.9+1.7 CB-154 42. 12.9+4.4 a: Mean+S.E.M., 1): CB-154 or vehicle was injected 24 or 42 hr after E, injection. treatment. Mitotic index (mitoses/mm7) Serum PRL level Other cells (ng/ml) 13.942.3 (7)? 80.3+16.4 (8) Mea (G) Miles ILS) 13.142.2 (5) 78.651 eaten 8.4+1.9 (5) 1 dense a) b: The number in parentheses indicates the number of rats. All animals were killed 48 hr after E, Difference from matched controls: *P<0.05, **P<0.001. TABLE 2. Effect of bromocryptine (CB-154) on the mitosis of PRL cells in intact female rats Mitotic index (mitoses/mm7) at estrus Treatment pay ot meet treatment rats Control 2nd day of 5 CB-154 diestrus 5 Control Proestrous 5 CB-154 day 5 Control Estrous 6 CB-154 day 6 PRL cell Other cells 58.2+9.5? 13.4+3.5 bofsh ate) foe 3:62-100" 29.7+4.0 16.7+2.9 1.9+0.2** 325 Oras 31.0+2.2 19.3+3.2 32520) 18, 7223:5 a: Mean+S.E.M. Difference from matched controls: the mitotic indices of PRL cells and other pituitary cells (Table 2). On the other hand, bromocryptine treatment at estrus 6 hr before autopsy failed to affect the mitotic indices of PRL cells and other cells. DISCUSSION Mitotic PRL cells were immunohistochemically shown in the present study. The result in the present study indicates that the proliferation of PRL cells is closely associated with PRL release from the pituitary gland. Davies et al. [13], Lloyd et al. [14] and Jahn et al. [16] previously presented the evidence that PRL release was accompanied by the increases in DNA synthesis and the number of mitotic cells in the pituitary glands. However, the P0055) oe 0/008 cell types of the mitotic cells were not verified in their reports. Immunohistochemical observations after colchicine treatment in the present study showed that the increase in the mitotic activity of total pituitary cells in estrogen-treated rats could be explained partly by the increase in the mitotic activity of PRL cells. The sharp increase in the mitotic activity of PRL cells at estrus in female rats [3] was associated with the elevation in serum PRL levels in the afternoon of proestrous day (proestrous PRL “surge”) as shown in Neill et al. [17]. In the present study, bromocryptine treatment in intact female rats in the early afternoon of 2nd day of diestrus and the morning of proestrous day inhibited the increase in the mitotic activity of PRL cells along with that of other cell types at estrus. The same dose of Proliferation of Prolactin Cells 859 bromocryptine as used in female rats effectively blocked the estrogen-induced PRL release 24 hr later in male rats (the present study) and also in estrogen-treated female rats [11]. Therefore, the present bromocryptine treatment in female rats at 15: 00 hr of 2nd day of diestrus could be effective in blocking the proestrous PRL surge in the following day. In estrogen-treated male rats, bromocryptine decreased the mitotic activity of PRL cells as shown in female rats, but did not affect the proliferation of other cell types in male rats. The difference between sexes may be ex- plained if some of the immunonegative cells in intact female rats are PRL cells. However, the verification of this view should await future study. The proestrous PRL “surge” is ovarian estrogen and hypothalamic dopamine dependent [17, 18]. Plasma estrogen began to rise from the afternoon of 2nd day of diestrus and reached a peak in the afternoon of proestrous day, and plasma PRL level started to rise 3 hr after the estrogen peak [19]. Similarly, the increase in the mitotic activity of PRL cells at estrus was ovary dependent, because ovariectomy on 2nd day of diestrus or proestrous day blocked this increase [3]. It is well known that dopamine is the physiological PRL-inhibitory hor- mone [18]. The dopamine concentration in the portal blood is lower at proestrus compared with that at other states of the estrous cycle [20]. This low dopamine level on proestrous day is another factor of the proestrous PRL “surge” and the elevation of the mitotic activity of PRL cells. Thus, the PRL secretion and proliferation of PRL cells are regulated by the two factors, estrogenic stimulation and dopaminergic inhibition. The changes in the mitotic activity of total pituitary cells after estrogen treatment were in good agreement with the earlier report by Hunt [7]. The significant increase in the mitotic activity of total pituitary cells and PRL cells at 36 hr after estrogen treatment in male rats may be regarded as a mimic of the increase in the mitotic activity of PRL cells in intact female rats at estrus. However, the effect of estrogen treatment on serum PRL levels and the mitotic activity of PRL cells con- tinued until 96 hr later. The decrease in serum PRL levels in vehicle-treated controls may be due to the procedure of sequential blood sampling, although the significant difference in serum PRL levels between estrogen-treated rats and vehicle- treated rats was apparent during the course of samplings after 36 hr. In female rats, the mitotic activity of PRL cells, and estrogen levels rapidly decreases after estrus [3, 19], and these changes were associated with the fluctuation in serum PRL levels. Based on the fact that the decrease in the mitotic activity of PRL cells and the decrease in estrogen and PRL levels are all well correlated, the long-lasting effects of E, on male rats in the present study might indicate that the injected dose was large enough to maintain the high mitotic activity and PRL level for longer period. Bromocryptine failed to affect the mitotic activ- ity of PRL cells if given 6hr before autopsy in estrogen-treated male or intact female rats. Simi- larly, ovariectomy on the morning of estrous day was without effect on the mitotic activity of PRL cells 6 hr later [3]. Such ineffectiveness suggests that the mitotic process had already been in progress at the time of these treatments. Estrogen may stimulate the proliferation of pituitary cells other than PRL cells, because estrogen increased the mitotic activities of pitui- tary cells which were immunohistochemically negative to anti-rat PRL serum. The possibility is not ruled out that the mechanism of cell prolifera- tion, which is dependent on serum estrogen levels but independent of serum levels of PRL, may exist in the pituitary cell population. ACKNOWLEDGMENTS We wish to thank Drs. W. Weidmann and H. Friedli, Sandoz Ltd., Basel for providing bromocryptine. We also wish to thank Dr. S. Raiti, and National Hormone and Pituitary Program, NIADDK for the supply of PRL RIA kit. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1 Sato, S. (1980) Postnatal development, sexual dif- ference and sexual cyclic variation of prolactin cells in rats: special reference to the topographic affinity to a gonadotroph. Endocrinol. Jpn., 27: 573-583. 2 Shirasawa, N. and Yoshimura, F. (1982) Immuno- histochemical and electron microscopical studies of 10 11 12 860 mitotic adenohypophysial cells in different ages of rats. Anat. Embryol., 165: 51-61. Takahashi, S., Okazaki, K. and Kawashima, S. (1984) Mitotic activity of prolactin cells in the pituitary glands of male and female rats of different ages. Cell Tissue Res., 235: 497-502. Takahashi, S. and Kawashima, S. (1986) Mitotic potency of prolactin cells in the pituitary gland in rats. In “Pars Distalis of the Pituitary Gland— Structure, Function and Regulation”. Ed. by F. Yoshimura and A. Gorbman, Elsevier Science Publishers B. V., Amsterdam, pp. 497-501. Seo, H., Refetoff,S., Vassart,G. and Brocas, H. (1979) Comparison of primary and secondary stim- ulation of male rats by estradiol in terms of prolactin synthesis and mRNA accumulation in the pituitary. Proc. Natl. Acad. Sci. USA, 76: 824-828. Jacobi, J., Lloyd, H. M. and Meares, J. D. (1977) Onset of oestrogen-induced prolactin secretion and DNA synthesis by the rat pituitary gland. J. Endocrinol., 72: 35-39. Hunt, T. E. (1947) Mitotic activity in the anterior hypophysis of ovariectomized rats after injection of estrogen. Anat. Rec., 97: 127-137. Takahashi, S. and Kawashima, S. (1981) Respon- siveness to estrogen of pituitary glands and prolactin cells in gonadectomized male and female rats. Annot. Zool. Japon., 54: 73-84. Yamamoto, N., Seo, H., Suganuma,N., Matsui, N., Nakane, T., Kuwayama, A. and Kageyama, N. (1986) Effects of estrogen on prolactin mRNA in the rat pituitary. Analysis by in situ hybridization and immunohistochemistry. Neuoendocrinology, 42: 494-497. Perez, R. L., Machiavelli,G. A., Romano, M. I. and Burdman, J. A. (1986) Prolactin release, oes- trogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats. J. Endocrinol., 108: 399-403. Brooks, C. L. and Welsch, C. W. (1974) Reduction of serum prolactin in rats by 2 ergot alkaloids and 2 ergoline derivatives: a comparison. Proc. Soc. Exp. Biol. Med., 146: 863-867. Maurer, R. A. (1980) Dopaminergic inhibition of prolactin synthesis and prolactin messenger RNA 13 14 15 16 ty) 18 19 20 S. TAKAHASHI AND S. KAWASHIMA accumulation in cultured pituitary cells. J. Biol. Chem., 255: 8092-8097. Davies, C., Jacobi, J., Lloyd, H. M. and Meares, J. D. (1974) DNA synthesis and the secretion of prolactin and growth hormone by the pituitary gland of the male rat: effects of diethylstilboestrol and 2-bromo-ergocryptine methanesulphonate. J. En- docrinol., 61: 411-417. Lloyd, H. M., Meares, J.D. and Jacobi, J. (1975) Effects of oestrogen and bromocryptine on in vivo secretion and mitosis in prolactin cells. Nature, 255: 497-498. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J. and Meyer, H. G. (1970) The unlabeled antibody enzyme method of immunohistochemistry. Prepara- tion and properties of soluble antigen-antibody complex (Horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spiro- chetes. J. Histochem. Cytochem., 18: 315-333. Jahn, G. A., Machiavelli,G.A., Kalbermann, L. E., Szijan,I., Alonso,G.E. and Burdman, J. A. (1982) Relationships among release of prolactin, synthesis of DNA and growth of the anterior pituitary gland of the rat: effects of oestrogen and sulpiride. J. Endocrinol., 94: 1-10. Neill, J.D., Freeman,M.E. and _ Tillson,S. A. (1971) Control of the proestrous surge of prolactin and luteinizing hormone secretion by estrogens in the rat. Endocrinology, 89: 1448-1453. MacLeod, R. M. (1976) Regulation of prolactin secretion. In “Frontiers in Neuroendocrinology, Vol. 4”. Ed. by L. Martini and W. F. Ganong, Raven Press, New York, pp. 169-194. Smith, M.S., Freeman,M.E. and Neill, J.D. (1975) The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology, 96: 219-226. Ben-Jonathan, N., Oliver, C., Weiner, H. J., Mical, R.S. and Porter,J.C. (1977) Dopamine in hypophysial portal plasma of the rat during the estrous cycle and throughout pregnancy. Endocri- nology, 100: 452-458. ZOOLOGICAL SCIENCE 4: 861-865 (1987) © 1987 Zoological Society of Japan Effects of Two Juvenile Hormone Analogs (R-20458, RO203600) and Three Juvenile Hormones (JH 1, JH 2, JH 3) on the External Morphology and Length of the Spiculum Copulatus (SC) in the Male German Cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) CHRISTINE M. WHEELER and AYODHYA P. GUPTA Department of Entomology and Economic Zoology, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, N. J. 08903, U.S.A. ABSTRACT—The spiculum copulatus (SC) of adult male Blattella germanica (Dictyoptera: Blattelli- dae) is a robust, pollex-shaped cuticular process characterized by an irregularly sculptured exterior, no membranous articulation and numerous sensilla trichoidea. It is crucial for copulation to occur. The insect growth regulator (R-20458, RO203600, and Juvenile Hormones 1, 2 and 3)-treated SCs showed various degrees of distortion to the exterior surface and a significant reduction in length for all treatments. INTRODUCTION Various sensory structures, important in receiv- ing tactile and/or chemical information during the mating behavior of cockroaches, have been in- vestigated in one or both sexes of Periplaneta americana [1-3], Blattella germanica [4], Leucophaea maderae [5], and Diploptera punctata [6]. Another unusual sensory structure, first discovered by Sreng [7] and later independently reported and designated as the spiculum copulatus (SC) by Ramaswamy et al. [8], plays a major role in the mating behavior of male B. germanica. During the stereotyped mating sequence of males, the tergal glands, located beneath the modified 7th and 8th abdominal terga, release an attractant concurrently with male wing-raising; the female mounts the male and ingests these attractants [4, 8, 9]. While feeding, the female stimulates this sensory structure (140-200 wm long) which is located between the anterior and posterior pairs of tergal gland orifices on the 7th abdominal tergum, and is invested with numerous bifurcate and trifurcate sensilla. Prodding of the SC with an Accepted May 27, 1987 Received March 30, 1987 insect pin caused arching and extension of the abdomen in 87% of all males tested [8], whereas heat cauterization of this structure resulted in failure of 68.3% of the males to elicit this behavior after female feeding; it was concluded from this that the SC is crucial for copulation to occur. Reductions in the number of sensilla on the antennae and/or maxillary and labial palps of juvenile hormone-treated male L. maderae [10], P. americana [11] or female B. germanica [12] have been reported; in these cases, mating was pre- vented. No one has reported the juvenile hormone effects on the SC in male B. germanica; a structure critical for copulation. We describe the normal morphology of the SC and report the effects of two juvenile hormone analogs (JHAs) (R-20458, RO203600) and three juvenile hormones (JH 1, JH 2, JH 3) on the external morphology and length of the SC. MATERIALS AND METHODS German cockroaches were selected from a laboratory colony maintained at 25°C under a photoperiod of 12 hr per day and reared on Purina laboratory chow and water. Thirty (1 to 3-day-old) 6th (last) instar males were treated topically with 862 C. M. WHEELER AND A. P. GuPpTA 0.2 wg/insect of R-20458 (6, 7-epoxy-1-(p- ethylphenoxy)-3, 7-dimethyl-trans-2-octene) (2 pl), 2.5 ug /insect RO203600 (6, 7-epoxy-3-methyl- 7-ethyl-1-(3, 4-methylenedioxyphenoxy)-2-octene) (5 wl), 2 ug/insect Juvenile Hormone 1 (JH 1) (methyl-10, 11-epoxy-7-ethyl-3, 11-diemthyl-2, 6- tridecadienoate, cis/trans mixture) (2 wl), 2 ng /insect Juvenile Hormone 2 (JH 2) (2 pl), 7.5 pg/insect Juvenile Hormone 3 (JH 3) (6 wl) or 2, 5 or 6 wl of acetone, respectively (controls). After adult emergence, 7th abdominal terga from both treated and control groups were excised and processed for light and scanning electron micros- copy [12]. Changes in spur morphology were noted and the lengths of 30 individual SCs for each treatment were determined using an ocular micro- meter calibrated by means of a 2mm reference stage micrometer. Comparisons were made be- tween the treated and control groups with respect to SC length (Analysis of Variance (ANOVA); Duncan). RESULTS Based on our present SEM studies and earlier work [8], the SC is a robust, pollex-shaped cuticular process, which is rigidly connected to the integument that separates the left and right ante- rior and posterior pairs of tergal gland orifices on the 7th abdominal tergum. The SC is character- ized by an irregularly sculptured exterior (Fig. 1A-C); it lacks a membranous articulation and its width varies throughout its length (approximately 60 «m at the base and 4 um at the apex). With the help of SEM studies, and the morphological [12] and nomenclatural [2] descriptions, we have char- acterized the sensilla on the SC as sensilla trichoidea. To simplify the morphological descrip- tions, we have divided the SC into three regions: the apical (Fig. 1A), central (Fig. 1B), and basal (Fig. 1C) regions. The narrow distal area of the apical region (approximately 20 ~m long) tapers to a blunt rotundate apex (Fig. 1A) and lacks sensil- la; a wider proximal area (approximately 33 ~m long) contains a few sensilla (Fig. 1A). The central region, narrower than the proximal area of the apical region and 33 um long, has several nodules and numerous sensilla trichoidea (Fig. 1B) on its Fic. 1. Montage showing full length of SC. 1,500. A. Apical area showing blunt, rotundate apex (up- per white arrow) and wide proximal area (lower white arrow). B. Central region with nodules (white arrow). C. Basal region. Spiculum Copulatus in Male German Cockroach 863 surface. The broad basal region (55 um long) is continuous with the cuticle (Fig. 1C). All five treatments resulted in various degrees of distortion in SCs, rendering the three SC regions indistinguishable from one another (Fig. 2A-F); JHA-treated SCs showed the greatest distortion (Fig. 2B—C). Since the analogs are not the natural compound in the insect and they are not easily metabolized, their effects may be more potent. The irregularly sculptured surface of normal SCs was replaced by a relatively smooth exterior in the stump-like, R-20458-treated (Fig. 2B) and pyri- form, RO203600-treated SCs (Fig. 2C). One hun- dred percent of all males with JH-induced abnor- malities had distorted, pyramiform SCs with com- pressed, blunt apices bent to one side, giving a slightly geniculate appearance to the distal end of the SC (Fig. 2D-F). Unlike normal SCs, the JHA- and JH-treated SCs were broad at the base and progressively narrowed toward the apices, with JHA-treated SCs having obtuse, severed apices. TaBLE1. Effect of JHAs and JHs on spiculum copulatus length of male B. germanica Spiculum length (4m) Treatment (Mean -+S.D.) Acetone Control 144.7+19.4 a R-20458 Siialae ee) |) RO203600 SeOse De) © JH 1 Hl Wo © JH 2 lise Aa (Gl JH 3 94.2+ 80d Numbers followed by the same letter are not significantly different at the p=0.05 level (ANOVA; Duncan). From the measurements reported in Table 1 and the scanning electron micrographs (Fig. 2A-F), there is a significant reduction in SC length for all treatments (R-—20458, RO203600 and JH 1, 2 and 3), compared with the control. JH 1-treated SCs were significantly shorter than JH 2-treated SCs (ANOVA; Duncan). The JH 1 titer in the nymph is higher than JH 2, which may explain the significant difference in the lengths of the JH 1- and 2-treated SCs. DISCUSSION Application of two juvenile hormone analogs (R-20458, RO203600) and three juvenile hor- mones (JH 1, 2 and 3) to 6th (last) instar male B. germanica results in a 35 to 65% reduction in the length of the SC (Table 1). R-20458 was the most potent with the SC reduced by 65%. Apparently, JH-induced changes in the length or morphology of the SC does not interrupt the mating sequence of B. germanica males, as long as the neural mechanism to the structure remains intact. In another study, we found also that JH-treated males showed a reduction in sensilla number on the 7th terga, the tergum involved in mating, without interruption in the mating sequence. The male may not require a certain quantity of specific sensilla types for mating; their mating sequence requires contact between the sexes and pheromone perception is short range so that successful stimuli reception may occur with only a few sensory receptors. Previous reports [8] have shown that B. germa- nica males, whose SCs were heat cauterized, reinitiated precopulatory behaviors, but were un- receptive to copulation and thus not able to arch and extend the abdomen. Although no elec- trophysiological recordings were done, they sug- gest that once the female tergal feeding begins, the subsequent mating behaviors are released by mechanoreceptive or tactile stimuli; the SC and its sensilla may be an innervated mechanoreceptor. That the SC may function as an innervated mechanoreceptor is further substantiated by a report on the behavioral responses of abdominal vibration receptors (AVRs) in P. americana males, which are involved in detecting cuticular vibrations [13]. It was found that the P. americana AVRs respond to mouthpart movements on the cuticle and suggested that in those cockroaches, which require female tergal feeding in the courtship sequence, AVRs may be present and could func- tion as female position detectors. It is possible that the SC, a cuticular extension containing a concen- trated group of mechanoreceptive sensilla, is functioning like an AVR, capable of detecting the correct position of the female during courtship. 864 C. M. WHEELER AND A. P. GupTA Spiculum Copulatus in Male German Cockroach 865 ACKNOWLEDGMENT This report is the New Jersey Agricultural Experiment Station Publication No. 08125-—21-86 supported by state and U.S. Hatch funds and Rutgers Research Council. REFERENCES Schafer, R. J. (1973) Postembryonic development in the antenna of the cockroach, Leucophaea mader- ae: Growth, regeneration and development of the adult pattern of sense organs. J. Exp. Zool., 183: 353-364. Schafer, R. and Sanchez, T. V. (1973) Antennal sensory system of the cockroach, Periplaneta amer- icana: Postembryonic development and morphology of the sense organs. J. Comp. Neurol., 149: 335-354. Schaller, D. (1978) Antennal sensory system of Periplaneta americana: Distribution and frequency of morphologic types of sensilla and their sex- specific changes during postembryonic develop- ment. Cell Tissue Res., 191: 121-139. Roth, L. M. and Willis, E.R. (1952) A study of cockroach behavior. Am. Middl. Nat., 47: 66-129. Engelmann, F. (1960) Hormonal control of mating behavior in an insect. Experientia, 16: 69-70. Stay, B.and Roth, L. M. (1956) The reproductive behavior of Diploptera punctata (Blattaria: Diplo- pteridae). Proc. 10th Int. Congr. Entomol., 2: 547-552. Fic. 2. SEM micrographs of normal and treated SCs. 7 10 11 12 13 Sreng, L. (1976) Les glandes tergales du male de Blattella germanica L. (Insecte Dictyoptere). Ultra- structure, development, chimie de la secretion. These de troisieme Cycle, Univ. Dijon, France. Ramaswamy, S.B., Gupta, A. P. and Fowler, H. G. (1980) External ultrastructure and function of the “spiculum copulatus” (SC) of the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae). J. Exp. Zool., 214: 287-292. Roth, L. M. (1969) The evolution of the male tergal glands in the Blattaria. Ann. Entomol. Soc. Am., 62: 176-208. Schafter, R. J. (1977) The nature and development of sex attractant specificity in cockroaches of the genus Periplaneta. III. Normal intra- and _ inter- specific behavioral responses and responses of in- sects treated with juvenile hormone. J. Exp. Zool., 199: 73-84. Schafter, R. and Sanchez, T. V., (1974) Juvenile hormone inhibits differentiation of olfactory sense organs during postembryonic development of cock- roaches. J. Insect Physiol., 20: 965-974. Ramaswamy, S. B. and Gupta, A. P. (1981) Sensil- la of the antennae and the labial and maxillary palps of Blattella germanica (L.) (Dictyoptera: Blattelli- dae): Their classification and distribution. J. Mor- phol., 168: 269-279. Florentine, G. J. (1968) Response characteristics and probable behavioral roles for abdominal vibra- tion receptors of some cockroaches. J. Insect Physiol., 14: 1577-1588. x600. A. Normal SC. Band C. R-20458-treated and RO203600-treated SCs showing smooth exteriors (lower white and black arrows) and obtuse apices (upper white and black arrows). D and E. JH 1- and JH 2-treated SCs showing the distorted surfaces (lower black and white arrows) and geniculate distal ends with compressed apices (upper black and white arrows). F. JH 3-treated SC with distorted surface (lower black arrow) and geniculate distal end with compressed, hook- shaped apex (upper black arrow). “ 4 7 ! eee rh aney ake secon rife) abs ip Ayes eee eh — ae BRAG dell aay ZOOLOGICAL SCIENCE 4: 867-873 (1987) Sex Difference in the Early Histopathological Changes of the Kidney in Wistar/Tw Rats WIN WIN YEE and SEIICHIRO KAWASHIMA Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima 730, Japan ABSTRACT— Early signs of histopathological changes of the kidney were detected at 3 months of age in male rats and at 9 months of age in female rats of the Wistar/Tw strain. Criteria for early lesions include the enlargement of Bowman’s capsule space and glomerular capillary walls, slight mesangial thickening and the formation of few casts in the renal tubules. Enlargement of Bowman’s capsule space in male rats was 1.6 times of that in female rats at 3, 6 and 9 months of age. Moreover, in male rats 50% of glomeruli were affected at 3 months of age and about 90% at 13 months. In contrast, in female rats the percentage of affected glomeruli was only about 40% at 9months of age. The enlargement of Bowman’s capsule space and the incidence of affected glomeruli were closely related with age and sex of rats. However, there were no significant changes in water metabolism up to 13 months of age. Degenerated glomeruli counted about 20% of the total glomeruli in male rats at 13 months of age, but those were very few in female rats. Thus, the marked changes of renal function in male rats at more advanced ages may be related to the high percentage of glomerular degeneration. © 1987 Zoological Society of Japan INTRODUCTION Kobayashi and Kawashima [1, 2] and Kawashi- ma and Kobayashi [3] reported that polydipsia and polyuria, with the decrease in urinary electrolyte concentration, occurred in male rats of the inbred Wistar/Tw strain at the age of about 17 months. However, polydipsia and polyuria occurred sig- nificantly later in female rats than in males [4]. These senile changes in water and electrolyte metabolism are the consequences of retardation in renal function, because spontaneous kidney dis- ease is common in rats of this substrain as well as in old rats maintained in other laboratories [5-11]. In male Wistar/Tw rats frequent occurrence of de- generated glomeruli, enlargement of Bowman’s capsule space and marked increase in hyaline cast in the renal tubules were the features at the age of 17-18 months, while the histopathological changes were less pronounced in female rats of the same age ([12] and their unpublished observations). Furthermore, Kawashima et al. [13] suggested that definite sex difference in water metabolism may be Accepted May 23, 1987 Received March 26, 1987 due to the difference in the onset of degenerative changes of the kidney. The aim of the present study is to know the early signs of histopathological changes of the kidney and whether there are sex differences at younger ages. MATERIALS AND METHODS Male and female rats of the Wistar/Tw strain raised in this laboratory were used in the present study. They were housed in a _ temperature- controlled room at a 12-hr light (6: 00-18: 00) and 12-hr dark cycle, with free access to laboratory chow (CA-1, Clea Japan Inc.) and tap water. At 1, 3, 6, 9 and 13 months of age, five male and five female rats each were used for the measurements of water intake and urinary volume. For the measurement, rats were kept individual for 3 days in aluminum metabolic cages in which the urine could be collected without contamination with feces. After the measurement the animals were killed by decapitation. The kidneys, after weighing, were cut into several frontal pieces. Superior, medial and inferior parts of the kidney were fixed 868 WIN WIN YEE AND S. KAWASHIMA in Bouin’s fluid and embedded in paraplast. Frontal sections were cut at 8 wm thickness and divided into two series. A series of sections were stained with hematoxylin and eosin, and the other series, with periodic acid Schiff (PAS). Histoquan- titative studies were carried out only on the medial parts of the kidney. The space of Bowman’s capsules in sections cut at the plane of renal corpuscle including the vascular and urinary poles was measured with the aid of an image analyzer connected with a computer (Mutoh Industry Ltd., Tokyo). In each kidney the total number of glomeruli and the number of affected glomeruli were counted in a total of 18 sections at different levels (each section shows more than 150 glomeru- li). The affected glomeruli were expressed as percentage as applied by Elema and Arends [14]. The affected glomeruli showed various degrees of lesions from sclerosis to complete degeneration. The severity of lesions was increased as a function of age. Progressive glomerulonephrosis (PGN) was used as the general term for the age-related kidney lesions in the present study. Initial signs of PGN Fic. 1. a. Normal. b. Grade 1. tubules with some casts (arrows). c. Grade 2. Dilated tubules with casts (arrows). d. Grade 3. tubules are filled with casts. Degenerated glomeruli associated with enlargement of Bowman’s capsule space. Grades for histopathological changes of the kidney of Wistar/Tw rats. PAS preparations. Scale: 0.5 mm. Glomeruli are compact and tubules are devoid of PAS-positive casts. Irregularly thickened Bowman’s capsule space with thickened glomerular capillary wall and Most of the e. Grade 4. Adhesion of glomerular tufts to Bowman’s capsule, dilated tubules and tubules filled with large amount of casts. Sex Difference of the Kidney 869 were based on criteria described in several papers [6, 15-17] and PGN was arbitrarily graded from 1 to 4. The grading system was generally based on Coleman et al. [16]. However, as their criteria for early pathological changes did not fit well to our materials, we have rated the changes with some modifications as follows. Grade 1: The earliest lesion. Presence of irregularly thickened Bowman’s capsule space with thickened glomerular capillary wall and slight mesangial thickening in some glomeruli. A few casts were present in Henle’s loop preferentially in the region at the cortico-medullary junction (Fig. la). The casts were strongly PAS-positive, and the kidneys without any PAS-positive cast were clas- sified as normal. Grade 2: Presence of scattered dilated tubules lined by flattened epithelium with hyaline casts particularly at the cortico-medullary junction (Fig.1b). Grade 3: Presence of large casts throughout the renal tubular system, the atrophy of capillary tufts and the degeneration of some glomeruli. Enlargement of most of the Bowman’s capsule space (Fig. Ic). Grade 4: In addition to exaggerated Grade 3 lesions, the adhesion of glomerular tufts to Bow- man’s capsules with wide-spread glomerulosclero- sis (Fig. 1d). Water intake (ml/1009.b.w./ day) 1 3 6 3 13 Months Urinary volume (ml/100g.b.w./day) Bowman’s capsule space and the number of affected glomeruli were measured in all kidneys. The statistical analyses between two groups of rats were performed by Kruskal-Wallis and Mann- Whitney U tests. The relationship between the weight of the kidney and the Bowman’s capsule space was analyzed by coefficients of rank correla- tion. RESULTS Daily water intake and urinary volume Water intake was the greatest at 3 months of age in both sexes than at other ages (Fig. 2). After 3 months water intake decreased in female rats, while in male rats water intake was almost constant until 9 months of age and it slightly decreased at 13 months. The urinary volume decreasd steadily as a function of age in females after 3 months of age. However, in male rats daily urinary volume was almost constant between 3 and 9 months of age but decreased at 13 months (Fig. 2) (3 vs. 13 months, P<0.005). Incidence glomerulonephrosis (PGN) In male rats the initial lesions (Grade 1) were first detected at 3 months of age (Table 1) and of progressive Months Fic. 2. Age-related changes in daily water intake and urinary volume of Wistar/Tw rats of both sexes. Vertical bars indicate standard errors of means. Each point depicts the mean of 5 rats. 870 WIN WIN YEE AND S. KAWASHIMA TABLE 1. rats at various ages Incidence of progressive glomerulonephrosis (PNG) in male and female Wistar/Tw Sex Age No. of Incidence mules) ats Normal Grade 1 Grade 2 Grade 3 Grade 4 Male 1 5 5 — pity bus are 3 5 fi 4 — —- sine 6 5 — = 5 ain 9 5 — ahi arch 3 13 5 — bate ee ie 5 Female 1 5 5 — == or, = 3 5 5 — = ss pit 6 5 4 1 = Ms BL 9 5 — 5 aaa ies pe? 13 5) — ace 5 es. hay definite lesions of Grade 2 developed at 6 months of age. Grade 4 lesions were observed in two out of five male rats at 9 months and all rats at 13 months of age (Table 1). In contrast, in female rats the kidney was affected only slightly until 6 months of age. Initial lesions were observed at 9 months. The definite lesions of Grade 2 were encountered at 13 months of age (Table 1). Thus, male rats showed earlier the development of PGN after 3 months than female rats. Figure 3 illustrates the incidences of affected glomeruli and degenerated glomeruli in the kidney of rats at various ages. In male rats about 50% of glomeruli were affected at 3 months of age and about 90% at 13months. The incidence of affected glomeruli significantly increased only at 9 months in female rats to about 40%. The increase in percentage of degenerated glomeruli occurred at 9 months in male rats (about 20%). However, in female rats degenerated glomeruli were very few even at 13 months (about 4%) (Fig. 3). Age-related changes in Bowman’s capsule space The increase in Bowman’s capsule space is another striking feature of age-related changes of the kidney. Figure 4 shows that the enlargement of Bowman’s capsule was progressive during 1 to 9 months of age in male rats and during 1 to 13 months in females. At 1 month of age there was no sex difference. Bowman’s capsule space of 3- month-old male rats was almost the same as that of 9-month-old female rats, indicating the rate of enlargement was greater in male than female rats. Bowman’s capsule space in male rats was 1.6 times of that in female rats at 3, 6 and 9 months of age [_] affected glomeruli,male Au affected glomeruli,female a degenerated glomeruli 100 Affected and degenerated glomeruli(%) Months Fic. 3. Age-related increase in mean percentage of affected glomeruli and degenerated glomeruli in total number of glomeruli in 18 sections from me- dial part of the kidney in each rat (total counts of more than 2700 glomeruli). Open columns: percen- tage of affected glomeruli in male rats, hatched columns: percentage of affected glomeruli in female rats, solid columns: percentage of degenerated glomeruli in both sexes. Vertical bars indicate standard errors of means. Sex Difference of the Kidney 871 16 (]Male {i} Female 2 Bowman's capsule space (10 um) 3 1 3 6 9 13 Months Fic. 4. Age-related changes in the mean maximum space of Bowman’s capsules. Vertical bars indicate standard errors of means. Each column shows the mean of 5 rats. y = 0.2696+0.00021 x Fr =0.97 Absolute kidney weight(g) Bowman's capsule space(10 pm’) Relationships between the weight of kidney and the space of Bowman’s capsule regardless of ages (months; M) in male and female rats. Rank correlation (r) is statistically significant in both sexes of rats. Fic. 5. (Fig. 4). Figure 5 shows the coefficients of rank correla- tion between the weight of the kidney and Bow- man’s capsule space regardless of ages in both sexes. Rank correlation was highly significant at r=0.97 in males and r=0.93 in females for fitted regression lines. These results show that the increase in the weight of kidney is correlated with the space of Bowman’s capsule. DISCUSSION The frequency of PGN was observed by light microscopy in Wistar/Tw rats until 13 months of age in both sexes. The onset of pathological lesions was characterized by histological and histo- quantitative criteria. Male rats showed the de- velopment of lesions as early as at the age of 3 months and definite lesions developed at 6 months. In contrast, in female rats lesions de- veloped 6-7 months later than in male rats. However, water intake and urinary output were conceivably within the range of normal functional variation until 13 months of age in both sexes, as has been reported previously [3, 4]. According to Hackbarth and Harrison [18] the kidney is en- dowed with a great compensatory ability, allowing y= 0.3697+ 0.00021X r=0-93 ? Absolute kidney weight (g) Bowman's capsule space( 10 m) 872 WIN WIN YEE AND S. KAWASHIMA it to maintain near normal function even in chronic kidney disease or with extensive histological changes. Thus, the absence of any marked phys- iological abnormalities may be the characteristics of compensatory ability of the kidney of rats younger than 13 months of age in Wistar/Tw strain. Moreover, our Wistar/Tw strain male rats showing enlargement of Bowman’s capsule space with marked degenerated glomeruli about 70% at 17-18 months were accompanied by polydipsia and polyuria. These changes in water metabolism may indicate the marked changes in renal function due to degeneration of glomeruli. The present study showed the possible rela- tionships among the incidence of PGN, affected and degenerated glomeruli, the weight of kidney, Bowman’s capsule space and the age and sex of animals. Elema et al. [19], Guttman and Kohn [20], Striker et al. [21] and Berg [6] reported that the glomerular lesion precedes tubular lesion. The present results were consistent with the findings of these workers, because we found that the primary change began in the glomerulus. There were many divergent views upon the enlargement of renal corpuscles. Using the same substrain as ours, Kobayashi and Kawashima [12] suggested that the increase in diameter of renal corpuscles at 17-18 months of age was due to the enlargement of Bowman’s capsule per se and not to the glomerular enlargement. Bauer and Rosenberg [22] inter- preted that the glomerular enlargement was a reflection of the dilatation of glomerular capilla- ries. These results altogether may indicate that the enlargement of renal corpuscle is probably due firstly to the dilatation of capillaries, and secondly to the repletion with casts. In addition, in the present study the observed enlargement of Bow- man’s capsule space together with marked tubular dilatation filled with casts of various sizes probably induced the increase in kidney weight as age advanced. Based on histological observations Gray et al. [17] reported that Sprague-Dawley female rats showed the onset of disease during their second year of life. In their study Brown Norway (BN/Bi) rats were in an initial disease state at about 30 months of age in both sexes, and Wistar (WAG/ Rij) male rats attained a similar state at 13-15 months of age, approximately 8 months earlier than female rats. In our Wistar/Tw male rats the initial lesions occurred at least 10 months earlier in both sexes than the Wistar rats used by other investigators. From the present results together with the previous findings we may conclude that the pres- ence of sex difference is evident in the process of PGN in Wistar/Tw rats and that the early lesions are usually followed by eventual development of polydipsia and polyuria at the age of about 17 and 24 months in male and female Wistar/Tw rats, respectively. Therefore, the development of poly- dipsia and polyuria may be pathological changes of the kidney. ACKNOWLEDGMENTS The authors wish to thank Dr. T. Machida for his helpful discussion. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1 Kobayashi, Y. and S. Kawashima (1980) Polydipsia and polyuria in aged male rats of the Wistar/Tw strain. Proc. Japan Acad., Ser. B., 56: 643-648. 2 Kobayashi, Y. and S. Kawashima (1984) Age- related changes in the water and electrolyte metab- olism in male rats of the Wistar/Tw strain. Exp. Gerontol., 19: 107-113. 3 Kawashima,S. and Y. Kobayashi (1982) Mor- phometric study of the hypothalamo-neuro- hypophyseal system in aged rats. J. Sci. Hiroshima Univ., Ser. B., Div. 1, 30: 229-242. 4 Kobayashi, Y. and S. Kawashima (1982) Sex differ- ence in water metabolism during aging and life span in rats of the Wistar/Tw strain. J. Sci. Hiroshima Univ., Ser. B, Div. 1, 30: 243-248. 5 Foley, W. A., D.C. L. Jones, G. K. Osborn and D. J. Kimeldorf (1964) A renal lesion associated with diuresis in the aging Sprague-Dawley rat. Lab. Invest., 13: 439-450. 6 Berg, B.N. (1965) Spontaneous nephrosis with proteinuria, hyperglobulinemia and hypercholester- olemia in the rat. Proc. Soc. Exp. Biol. Med., 119: 417-420. 7 Berg,B.N. (1976) Pathology and aging. In “Hypothalamus, Pituitary and Aging”. Ed. by A. V. Everitt and J. A. Burgess, C.C. Thomas Publ., Illionis, pp. 43-67. 10 11 12 ie. 14 15 Sex Difference of the Kidney Sworn, M.J. and M. Fox (1972) Donor kidney selection for transplantation. Br. J. Urol., 44: 377-383. Christian, J. J. (1976) Anterior pituitary in relation to renal disease. In “Hypothalamus, Pituitary and Aging.” Ed. by A. V. Everitt and J. A. Burgess, C. C. Thomas Publ, Illinois, pp. 297-332. Goldman, R. (1977) Aging of the excretory system: Kidney and bladder. In “Handbook of the Biology of Aging.” Ed. by C. E. Finch and L. Hayflick, Van Nostrand Reinhold, New York, pp. 409-431. Goyal, V. K. and P. C. Chatterjee (1980) Changes with age in mouse kidney. Exp. Gerontol., 15: 151-160. Kobayashi, Y. and S. Kawashima (1983) Histologi- cal changes in the kidney of aged rats of the Wistar/Tw strain showing polydipsia and polyuria. osc. Enoshima’ Univ., Ser. B., Div. 1, 31: 149-154. Kawashima, S., K. Kawamoto and Y. Kobayashi (1986) Aging of the Hypothalamo-neuro- hypophysial system and water metabolism in rats. Zool. Sci., 3: 227-244. Elema,J.D. and A. Arends (1975) Focal and segmental glomerular hyalinosis and sclerosis in the rat. Lab. Invest., 33: 554-561. Bras, G. and M. H. Ross (1964) Kidney disease and nutrition in the rats. Toxicol. Appl. Pharmacol., 6: 16 17 18 19 20 za 22 873 247-262. Coleman, G. L., S. W. Barthold, G. W. Osbaldis- ton, S. J. Foster and A. M.Jonas (1977) Pathologi- cal changes during aging in barrier-reared Fischer 344 male rats. J. Gerontol., 32: 258-278. Gray, J. E., M. J. van Zwieten and C. F. Hollander (1982) Early light microscopic changes of chronic progressive nephrosis in several strains of aging laboratory rats. J. Gerontol., 37: 142-150. Hackbarth, H. and D. E. Harrison (1982) Changes with age in renal function and morphology in CS7BL/6, CBA/HT6 and B6CBAF, mice. J. Gerontol., 37: 540-547. Elema, J. D., J. Koudstaal, H. B. Lamberts and A. Arends (1971) Spontaneous glomerulosclerosis in the rat. Arch. Pathol., 91: 418-425. Guttman, P. H. and H. I. Kohn (1960) Progressive intercapillary glomerulosclerosis in the mouse, rat and chinese hamster, associated with aging and X-ray exposure. Am. J. Pathol., 37: 293-307. Striker, G. E., R. B. Nagle, P. W. Kohnen and E. A. Smuckler (1969) Response to unilateral nephrec- tomy in old rats. Arch. Pathol., 87: 439-442. Bauer, W. C. and B. F. Rosenberg (1960) A quan- titative study of glomerular enlargement in children with tetralogy of Fallot. Am. J. Pathol., 37: 695-712. ay EAD. Veet. Py CU VTA He ne er it iris aN ae parker Bea 34 neg? ik ie anh hee etal he Sa noe a failapath E peraey pa y Lect hem | ii eihingahy ARwaoTeT Frit awh enFY a W pele. te catante; leraed Wi ee per ewieneigage 4 . 1 aie SE RE bo OLIRRBOR IE ce rt du 2 fii J fies WARE 3 ra sie a} titacaenbh « ry ys vat ah nod "DAR nant GAS, of 4 riley i ere iy te a a8 tia ay Hel Raine HAM View Ape Une ateniamn vi gpy | toh Meet tee eae Sea. Be “P et Leh HORE NT INNS ey Pag: cited) ehnsir.° on SEES a data tga te eae flip. avicnayy et. ODN. by oaeyh A et irs, Heth mene Age hy Rare Fle Lr ong i nix nigel ae Cra P| sities oy bs su te é Wwapl: es UN Bato WR” PESO a” alsin ROA ia T" hice nies PON RE RS ean eal tlh ry al Rea ae Aaa ya ig Wy 20h. ae as he net ele oon | parts oF Derrcunaes Hh iY A eerie Adss: MA Sates AF 12 leet iyady wig}. belo me tee a , tp, ae a Ae < ME oA PAE i ait Iasi, valk vse! ta ty A Hy 142 ovis uy Wael) | 2 at Neg ryt a sete “eae 0 rie oe te “ates ma” ant et itn iy Pa } i 00, te ee i rit ah oe ; 1 eee ae ear Sie 74 D Tey ; ‘ . 1 , ) 94 " 4 i ! r* ’ j " _ ‘i y 9 , } v ") bo A *\ & ‘ " j i i] ? ty 4 e 5 Lig é : < iy 1 4 yh 2 ik Pi} ot . ee +e ari! xt " ole a5 i ee art aes VAL He \ _ ‘ L ‘ " a 3 Reo Mu Rie ty eS i . re 1 tire oa Peo , tena peeve af CTR BERR Sith Ui Ca toed ail? dertienoties lt * finn nei siuupahe Ye i nae) tbe (ee et ‘open “eaary ani anemedltie yor eda nium hiraelinees. “aE v0 q 8 7 iskenrutio 4 ¥ tine vetoes GT on- Ceriladroeny ht awit eos (tt wining rot then hell Wie a wad) hed! Rue ts Lynd vc tly i Ga ¢t oa Sal Orr ee. Tyr ¥ ol wy ij Ra riba it ry ay f +7 ‘Vins ip.t Wy 9 ai. iP Ps 7 DDS) } ‘BAG: : 4 .y SON, : / . hs ey me ae we Pay oP a hi 4evith ZOOLOGICAL SCIENCE 4: 875-880 (1987) Ultrastructural Observations of the Developing Basophilic Granulocytes in the Loach Kidney Kriyoto ISHIZEKI Department of Oral Anatomy, School of Dentistry, Iwate Medical University, Morioka 020, Japan ABSTRACT—The developing basophils in the intertubular space of the loach kidney were examined electron microscopically. These basophils could be classified into early, intermediate, and mature stages based on ultastructural appearance. The majority of the cells identified as in the early stage had a developing rough endoplasmic reticulum and Golgi apparatus, and several immature granules that consisted only of a fine granular matrix. Among these cells, typically large lymphoid cells that contained a nucleus with a prominent nucleolus, many ribosomes, a few large mitochondria, and occasionally vesicles containing flocculent material were considered to be the most immature cells of the basophil series. Intermediate cells contained well-developed Golgi apparatus, rough endoplasmic reticulum, and numerous immature and mature electron-dense homogeneous granules, and they were similar morphologically to the myelocytes of the mammalian basophils. In the intertubular tissues, cells with an eccentric oval nucleus and numerous electron-dense homogeneous granules ultrastructur- ally resembled the mature basophils found in the peripheral blood. In conclusion, the loach has basophils clearly distinguishable from other granulocytes and the ultrastructural features of its granules © 1987 Zoological Society of Japan closely resemble those of higher vertebrates. INTRODUCTION In higher vertebrates, basophils are common blood cells. However, in lower vertebrates such as teleost fishes, basophils have been found in some, but not in other families. Some light microscopic [1, 2], histochemical [3] and electron microscopic [4, 5] studies failed to find basophils in several teleost fishes, but several studies [6-10] revealed the presence of basophils in the blood of some teleost fishes. Lester and Desser [11] demonstrat- ed electron microscopically the blood basophils of the white sucker (Catostomus commersoni) which contained large, round, membrane-bound electron-dense granules consisting of fine granular material. Catton [12], Kamishiro [13] and Bielek [14] reported that these basophils are produced in the kidney of some teleost fishes. However, detailed ultrastructural studies of the developing basophils in teleost fishes have not been reported. In the present study, developing basophils in the Accepted May 6, 1987 Received March 4, 1987 intertubular space of the loach kidney were ex- amined by electron microscopy, to clarify their morphological characteristics. MATERIALS AND METHODS Commercially supplied adult loaches, Misgurnus anguillicaudatus (5-10 g in weight, 10-15 cm in length) were used. For electron microscopy, specimens of the kid- ney and whole blood were centrifuged and the resulting pellets were fixed in cold 2.5% glutaral- dehyde buffered in sodium cacodylate (pH 7.4) for 1 hr, and then postfixed in 1% OsO, for | hr at 4°C. The fixed materials were then dehydrated by an ethanol series and finally embedded in Epon 812. The specimens were sectioned on an LKB- ultratome using a diamond knife. Ultrathin sec- tions were stained with uranyl acetate and lead citrate, and examined with a JEM-100B electron microscope at 80kV. Blood smear preparations obtained from decapitated animals were stained with May-Griinwald Giemsa solution and were used for light microscopic observations. 876 K. ISHIZEKI RESULTS Loach blood contained three kinds of granulo- cytes: basophils, heterophils and eosinophils. However, basophils were easily distinguished, because they differed clearly from the other granulocytes in staining features, hemopoietic sites, and fine structural features of specific gra- nules as previously reported [15]. Light microscopically, the basophil was not found so frequently in smear preparation as other granulocytes. Almost all the basophils observed were Oval or round, and contained a large nucleus and granules which were stained dark blue (Fig. la). The ultrastructure of such basophils (Fig. 1b) in the peripheral blood exhibited an eccentric oval nucleus with condensed heterochromatin and many electron-dense homogeneous granules (Fig. Ic). A few rough endoplasmic reticulum, mitochondria and immature granules with a fine granular matrix were also recognizable. In the present observations, the basophils showing these morphological features were identified as mature basophils of the loach. In the intertubular space of the kidney, various kinds of developing basophils could be seen. Among these cells, the most immature cells were of large lymphoid-shaped and had an oval nucleus with a high percentage of euchromatin and a prominent nucleolus. A few large mitochondria ‘gi Fic. 1. a: Light micrograph of the mature basophil in the blood smear preparation. A large oval nucleus and granules stained dark blue can be seen. x 1,500. b: Oval nucleus (N) with condensed heterochromatin, many large round homogeneous granules, a few mitochondria (MT), rough endoplasmic reticulum (rER) and immature granules containing a fine granular matrix (arrowhead) can be seen in the cytoplasm. 20,400. c: High magnification of mature granule that consists of an electron-dense homogeneous matrix. 46,000. Ultrastructure of the Loach Basophils 877 and many ribosomes were distributed throughout the cytoplasm, but other organelles were unde- veloped (Fig. 2a). Occasionally, the cytoplasm contained a few vesicles that were partially filled with flocculent material (Fig. 2a, inset). In addi- tion, more developed basophils were distinguished by an increased amount of heterochromatin at the nucleus rim, developing Golgi apparatus and rough endoplasmic reticulum (Fig. 2b). The imm- ature granules (0.3-0.6 ~m in diameter) were spherical and consisted of an_ electron-inter- mediated fine granular matrix (Fig. 2b, inset). Furthermore the cells which were identified as in the intermediate stage (Fig. 3a, b) were relatively large and were characterized by a well-developed Golgi apparatus and an increased number of electron-dense homogeneous granules (0.8-1.1 ym in diameter). Eccentric nuclei had more condensed heterochromatin than those of the early stage cells and were invaginated slightly near the Golgi apparatus, which consisted of several cis- ternae, enclosed the progranules arising from the ends of the outer cisternae (Fig. 3b, arrow). The other characteristics at this stage were mingling of immature and mature granules. Immature gran- ules were similar to those seen in the early stage cell, and mature granules showed an electron- dense homogeneous matrix. In the intertubular tissue, cells similar in ultra- structural appearance to those of mature basophils in the peripheral blood were observed (Fig. 4). This type of cell had an eccentric condensed nucleus and many mature dense granules, but other organelles tended to decrease. Mature granules had neither precise crystalline cores nor myelin figures, such as those observed in amphib- ian [16-18] or human basophils [19]. Thus, in the present observation, these cells were classified as A few large mitochondria (MT) and many ribosomes of the polysome type are present. Note that the vesicle contains flocculent material (frame). 13,800. Inset: High magnification of area shown in the frame. x 40,000. b: An early stage of a basophil having a large oval nucleus (N), prominent nucleolus (NC), rough endoplasmic reticulum (rER), mitochondria (MT) and developing Golgi apparatus (GA). number of immature granules that consist of fine moderately-dense granular matrices. Immature granule seen at an early stage at a high magnification. Note the small <11,000. Inset: x 23,400. 878 K. ISHIZEKI Fic. 3. a: A basophil at the intermediate stage. Many immature granules and mature granules, well-developed Golgi apparatus (GA) around the centriole and eccentric nucleus (N) with moderately clumped heterochroma- tin characterized the cell of this stage. Arrowhead shows immature granule with a fine granular matrix. 11,000. b: High magnification of portion of the Golgi apparatus (GA) shown in (a). Immature granules can be seen at the end of the Golgi cisterna (arrow) and in the surrounding area (arrowhead). Note the Ultrastructure of the Loach Basophils 879 mature basophils. DISCUSSION In the teleost fishes, basophils have been re- ported in only a few species [6-11]. They derive from the intertubular space of the kidney [12-14]. In the loach kidney, there were many maturing basophils, which relates to the hemopoietic nature of this tissue. Generally, the morphological change of mammalian developing basophils is similar to that of eosinophils or neutrophils [20-22], and mature basophils possess a lobulated nucleus [23-25]. However, in the loach, such segmented basophils, though matured cells, were not found light- microscopically: it was therefore difficult to direct- ly apply mammalian criteria to the development of loach basophils. Therefore, in the present study, basophils were classified into three stages of maturation by ultrastructural appearance. The cytoplasmic granule, as seen in large lym- phoid-cells (Fig. 2a), was similar to the immature granules of the basophil series. Also, this type of granule resembled the progranulocyte (myelo- blast) of the amphibian (Plethodon glutinosus; [17]) and rabbit basophilic myelocytes [23]. Heter- ophils as well as basophils also arose from the intertubular space of the kidney, the two cell types were morphologically distinct because heterophils contained granules with more electron-dense material than those of basophils even though progranules. Therefore, the cells of this type were identified as the most immature basophils of the loach. Also, among the basophils classified as in the early stage, cells containing a few granules with a fine granular matrix alone were also seen, but by the appearance of rough endoplasmic reticulum and heterochromatin, which is condensed in the nuclear periphery, they seemed to be more de- veloped than the cell shown in Figure 2a. The mammalian basophilic myelocytes and metamyelo- cytes are characterized by a centrally located large heterophilic granules with more electron-dense crystalloid structures. nucleus, well-developed Golgi apparatus and rough endoplasmic reticulum, and two or three types of cytoplasmic granules [20-23]. Such cells seemed to be morphologically similar to the cell types that were classified as being at the intermedi- ate stage in this study. In mammalian basophils such as in humans [19, 22, 24], rabbits [23], minks [26] and in amphibian basophils [18, 27], two types of granules, particu- late and homogeneous matrix, have been observed. Furthermore, Lester and Desser [11] reported that the basophils in the peripheral blood of the white sucker (Catostomus commersoni) possess two types of granules with large, round membrane-bound granules: some were uniformly electron-dense, while others were in a granular matrix. Both granules resembled ultrastructurally the immature or mature granules of the loach basophils, but generally granules with a fine granular matrix have been described in mature basophils [18, 24, 27]. Although the stage of appearance of these granules varies among animal species, the structure of such granules might reflect the phylogenetic characteristic of the basophils. In the present investigation, basophil granules occurred as condensed vacuoles containing floccu- lent material at the end of the Golgi cisternae, and progressed to a fine granular matrix and were finally transformed into electron-dense homogeneous granules, which were numerous in near-mature or mature basophils. Thus, the large homogeneous granules were a mature form of fine granular matrices and consequently, the loach basophils consist of one population of granules. Although it is unknown whether the granule contents of the basophils vary with the species, if teleost basophils have similar granules, the present findings would be important for classifying basophils. Further study is needed to examine whether this cell is functionally analogous to basophils of the higher vertebrates. HP, heterophil, N, nucleus, MT, mitochondria, Co, centriole, rER, rough endoplasmic reticulum. 29,500. Fic. 4. Mature basophil in the kidney showing condensed nucleus (N), many electron-dense mature granules, a few rough endoplasmic reticulum (rER) and mitochondria (MT). One large immature granule is indicated by the arrowhead. This type of cell is similar to the mature basophils in the peripheral blood. X 12,300. 880 ACKNOWLEDGMENT The author wish to thank Prof. T. Nawa for critical reading of the manuscript. 10 ii 12 13 14 REFERENCES Chlebeck, A. and Phillips, G. L. (1969) Hemato- logical study of two buffalofishes, Ictiobus cyp- rinellus and I. bubalus (Catostomideae). J. Fish. Res. Bd. Canada, 26: 2881-2886. Conroy, D. A. (1972) Studies on the haematology of the atlantic salmon (Salmo salar L.). Symp. Zool. Soc. Lond., 30: 101-127. Barber,D.L. and Westermann, J.E.M. (1975) Morphological and histochemical studies on a PAS- positive granular leukocyte in blood and connective tissue of Catostomus commersonnii Lacépéde (Tele- ostei: Pisces). Am. J. Anat., 142: 205-220. Cannon, M.S., Mollenhauer, H. H., Eurel, T. E., Lewis, D.H., Cannon, A.M. and Tompkins, C. (1980) An ultrastructural study of the leukocytes of the channel catfish, Jctalurus punctatus. J. Mor- phol., 164: 1-23. Savage, A.G. (1983) The ultrastructure of the blood cells of the pike Esox lucius L. J. Morphol., 178: 187-208. Duthie, E. S. (1938) The origin, development and function of the blood cells in certain marine teleosts. part 1. Morphology. J. Anat., 73: 396-414. Weinreb, E. L. (1963) Studies on the fine structure of teleost blood cells. 1. Peripheral blood. Anat. Rec., 147: 219-238. Watson, L. J., Shechmeister, I. L. and Jackson, L. L. (1963) The hematology of goldfish, Carassius auratus. Cytologia, 28: 118-130. Srivastava, A. K. (1968) Studies on the hematology of certain freshwater teleosts. IV. Leucocytes. Anat. Anz. Bd., 123: 520-533. Williams, R. W. and Warner, M. C. (1976) Some observations on the stained blood cellular elements of channel catfish, Jctalurus punctatus. J, Fish Biol., 9: 491-497. Lester, R. J.G. and Desser,S.S. (1975) Ultra- structural observations on the granulocytic leuco- cytes of the teleost Catostomus commersoni. Can. J. Zool., 53: 1648-1657. Catton, W. T. (1951) Blood cell formation in certain teleost fishes. Blood, 6: 39-60. Kamishiro, K. (1955) Untersuchungen tber die Leukozyten der Fishe (Pisces). Kyoto daigaku iga- kubu daini kaibou ronbunshu, 3: 1-43. (In Japanese, Abstract in Germany). Bielek, K. (1981) Developmental stages and local- ization of peroxidatic activity in the leucocytes of K. ISHIZEKI 15 16 C7) 18 19 20 21 OD 23 24 25 26 27 three teleost species (Cyprinus carpio L.; Tinca tinca L.; Salmo gairdneri Richardson). Cell Tissue Res., 220: 163-180. Ishizeki, K.. Nawa,T., Tachibana, T., Sakakura, Y. and lida, S. (1984) Hemopietic sites and develop- ment of eosinophil granulocytes in the loach, Mis- gurunus anguillicaudatus. Cell Tissue Res., 235: 419-426. Tooze,J. and Davies,H.G. (1968) Light and electron microscopic observations on the spleen and the splenic leukocytes of the newt Triturus cristatus. Am. J. Anat., 123: 521-555. Curtis,S.K., Cowden, R.R. and Nagel, J. W. (1979) Ultrastructure of the bone marrow of the salamander Plethodon glutinosus (Caudata: Pletho- dontidae). J. Morphol., 159: 151-184. Ishizeki, K. (1982) The cytodifferentiation in the basophilic granulocytes of the newt (Triturus pyr- rhogaster) in organ culture. J. Iwate Med. Assoc., 34: 551-567. (In Japanese, Abstract in English). Hastie, B. and Chir, B. (1974) A study of the ultrastructure of human basophil leukocytes. Lab. Invest., 31: 223-231. Bessis, M. (1973) The granulocytic series. In “Liv- ing Blood Cells and their Ultrastructure”. Springer- Verlag, Berlin, Heidelberg, New York, pp. 285-355. Cawley, J.C. and Hayhoe, F.G.J. (1973) The basophil. In “Ultrastructure of Haemic Cells: A Cytological Atlas of Normal and Leukaemic Blood and Bone Marrow”. W.B. Saunders Company Ltd., London, Philadelphia, & Toronto, pp. 24-29. Rhodin, J. A.G. (1974) Blood and lymph. In “Histology: A Text and Atlas”. Oxford University Press, New York, London & Toronto, pp. 93-138. Wetzel, B. K., Horn, R. G. and Spicer, S. S. (1967) Fine structural studies on the development of heterophil, eosinophil, and basophil granulocytes in rabbits. Lab. Invest., 16: 349-382. Zucker-Franklin, D. (1967) Electron microscopic study of human basophils. Blood, 29: 878-890. Dvorak, A. M., Galli, S. T., Morgan, E., Galli, A. S., Hammond,E. and Dvorak,H.F. (1981) Anaphylatic degranulation of guinea pig basophilic leukocytes. I. Fusion of granule membranes and cytoplasmic vesicles: formation and resolution of degranulation sacs. Lab. Invest., 44: 174-190. Davis, W.C., Spicer,S.S., Greene,W.B. and Padgett, G. A. (1971) Ultrastructure of cells in bone marrow and peripheral blood of normal mink and mink with the homologue of the Chediak-Higashi trait of humans. II. Cytoplasmic granules in eosin- ophils, basophils, mononuclear cells and platelets. Am. J. Pathol., 63: 411-431. Campbell, F. R. (1970) Ultrastructure of the bone marrow of the frog. Am. J. Anat., 129: 329-356. ZOOLOGICAL SCIENCE 4: 881-888 (1987) Body Color and the Preference for Background Color of the Siamese Fighting Fish, Betta splendens TSUNEO MoriyA and YoOKO MIYASHITA Department of Biology, Sapporo Medical College, Sapporo, 060, Japan ABSTRACT—The preference of the fish, Betta splendens, for black or white background color was observed using an experimental tank which was divided into white and black halves. This fish has originally a greater preference for black than for white. When fish were kept for one week in the tank with a black background, this preference for black was enhanced, while keeping fish in a white tank increased the time the fish spent in the white area. These results show that the environmental color affects the background color preference. Next, we examined the relationship between preference for background color and fish body color. The difference of preference for background color in the female and male was examined because they exhibit a pronounced difference in their body colors, the male being darker than the female when they are exposed to the same background. Males exhibited a greater preference for black than females did. We also compared the preference for background color of reserpinized fish, which had darker bodies than that of control fish (reserpine-untreated). Reserpinized fish preferred a black background more than control fish did. Throughout the observation, there was no significant difference between the number of appearances of control and reserpinized fish in the white area of the experimental tank. However, the average time control fish spent in the white area at each appearance was always longer © 1987 Zoological Society of Japan than that of the reserpinized fish. INTRODUCTION Some species of lower vertebrates change their body color according to the background color. This is regarded as camouflage against predators and such changes are called “protective color changes” [1]. In addition to being camouflage, the body color and color patterns have been consid- ered to serve for communication [2-5]. The Siamese fighting fish, Betta splendens, demon- strates unique color changes on various occasions, such as antagonistic and reproductive displays [6]. When two males are housed together in a tank, they fight each other, opening their opercula and spreading the tail fin, and the body color becomes darker. The fish beaten in the fight becomes pale and two longitudinal black lines appear on its body sides [6]. Since the loser can avoid attack by its opponent after changing its body color, it is considered that the body color functions as a Accepted May 1, 1987 Received January 22, 1987 communicatory signal. Thus, it is plausible to assume that animals are aware of their own body color. If this is the case, animals might be able to move positively toward an appropriate environ- ment suitable for their hue. Kohda and Watanabe [7] reported that the cross-striped fish, Oyanirami Coreoperca kawamebari, prefers a_ vertically, rather than a horizontally, striped background. They suggested that the cross-stripes, appearing vertically when the fish is swimming or resting, could function as a cryptic pattern among vertical plants in the aquatic habitat. Brown and Thompson [8] demonstrated that fish adapted to black background choose black more frequently than fish adapted to white background in eight species of fish. The above mentioned papers [7, 8] reported that fish select some background area corresponding to their body color and/or color pattern; however, it has not yet been clarified whether they perceive their own body color or some hormonal factors act on the brain, causing them to choose a suitable background. The formation and change of body color of 882 T. MorryA AND Y. MIYASHITA animals, including teleost fish, are controlled by neural or/and hormonal regulatory systems [9]. It is possible that certain nervous or hormonal conditions which regulate the skin color have some behavioral effects [10]. Karplus and Samuel [11] demonstrated that MSH injected into white- adapted Xenopus tadpoles induced a significant shift in their preference toward black background, and they speculated that the effect of MSH was mediated by its action on the brain rather than its skin darkening action. In the present study, in order to expand our knowledge of the relationship between the prefer- ence for background color and body color, we have tried to learn the fundamental preference for background color in the Siamese fighting fish, Betta splendens. We also examined whether the exposure of fish to a black or white background affects their background color preference. In the third part of this study, we analyzed whether the body color of the fish affects its background color preference or not. MATERIALS AND METHODS Adult Siamese fighting fish, Betta splendens, hatched in our laboratory were maintained singly in vessels. For the adaptation to background color, the fish were kept in a white or black plastic tank 43 x43 x20 cm deep made of opaque plastic plates for one week under a 15hr-light and 9 hr-dark cycle. The water in each tank was adjusted to 10 cm in depth and maintained at 25°C. The fish were individually identified and designated by the letters A-S and a-l. After the adaptation to white or black background color, single fish was transfer- red to an experimental tank 43x43 x20 cm deep divided into white and black halves and their preference for background color was observed. The movement of the fish in each tank was monitored with a video camera (Olympus VX-304) for Shr from 10 AM to 3 PM under conditions of continuous illumination. For the purpose of analysis, the time fish stayed in the white area and the number of appearances in the white area from the black area for 30-min periods was checked from video tapes using a quick visual searching system. In the treatment with reserpine, an inducer of body darkening in fish [12], half the number of fish which had been kept in a white tank for one week were immersed in water containing 3x10~°M reserpine (Daiichi Seiyaku Co. Ltd., Tokyo) for 20 hr in a dark room. As the control, the other half were kept in water containing no reserpine. Reserpine treatment caused a darkening effect on the body color even with a white background. This effect continued for more than one week in the white tank (details shown in Miyashita [12]). In contrast, the control fish became pale in a white background. The data were tested statistically using Student’s t-test, the Cochran-Cox test, the paired t-test and the Kruskal-Wallis test [13]. For these statistical tests (except for the Kruskal-Wallis test), we employed the computer programs prepared by Ishii et al. [14]. RESULTS AND DISCUSSION Betta splendens, especially females, rapidly changed their body colors according to the back- ground color. When the fish were transferred into a black tank from a white one, their body colors became dark, and when they were returned to the white tank, the body color became pale. This change occurred within two minutes after transfer and the altered body color was maintained as long as the fish stayed in the same tank. First, we examined the influence of previous exposure to a black or a white background on the background color preference. Female fish were kept in either a black or a white tank for one week (black or white adaptation). Then they were subjected to a choice between white and black environments. The time the fish spent in the white area and the number of appearances in the white area from the black one are shown in Table 1. In both groups, the number of appearances in the white area decreased with the observation time (r(70) = —0.595, p<0.001 and r(70)=—0.562, p<0.001 for the black-adapted and the white- adapted group, respectively). There was no signif- icant difference between the two groups in total number of appearances (mean+S.E. were 47.0+10.8 (n=7) and 52.9+7.5 (n=7) for the TABLE l. Background-color Preference in Fighting Fish black environments Time (hr)/Fish Group 1_ Black-adapted fish 883 The background color preference of the female Betta splendens after adaptation to white or A B C D E F G 0.0-0.5 253) a les Go)» SUIS) 58( 4) 274( 8) 219(12) 289(15) 0.5-1.0 345( 6) 104( 3) 221(11) 0( 0) 449(10) 532(12) 469(10) 1.0-1.5 LOZ (3a 225-3) 211 (13) 0( 0) 284(13) 526(10) 227( 9) 1.5-2.0 SP) (eS) @ PVA oa) eSB CG) 47( 2) 168(10) 242( 7) 434(14) 2.0-2.5 22 (3) NOCD) 302-4) SOD) ns ACD) eq ws 50S) ww SLO) 2.5-3.0 GONG). wuts) 2632) OG0) e1037(C5) OGO) 5 225.8) 3.0-3.5 88 ( 2) ONO) Stilts) DW) Te) em OARS) Se TE (ES) 3.5-4.0 30 ( 1) 0( 0) 86 ( 3) 0( 0) TUES) Vee S)) SBC G) 4.0-4.5 COCO) 0s) 22) OG) a 3082) 2G) 204) (GO) 4.5-5.0 0( 0) CSIC) G2) 0( 0) 9513) 36( 1) 104( 4) Total 1034 (24) 1064(24) 2038(67) 159( 8) 2061(69) 2256(52) 2709 (85) Time (hr)/Fish a a 2 ae ‘au af i 0.0-0.5 328169) — ai101\13) 4( 1) 160( 9) 347( 9) 488( 6) 650( 9) 0.5-1.0 535 (20) 707 (12) OC O) pae239iES)) SECS) sere 2001S) FP a1648(14) 1.0-1.5 516( 7) 825 (10) SCA) 392.2) 462) (10) Sr S39iKG 9) etS3 G9) 1.5-2.0 1639 (10) 694( 8) 6D) Ci) pa 71S) ee G61: sk) LOE) ee S916) 2.0-2.5 2659) par OIOGa)ery 121'( 3) 900 '( S)irnfa374-(<5) 5 252041G4) al866) 2.5-3.0 951( 4) 435( 6) 23\( 3) 9 9rl220i( 2) 64( 8) 150( 7) 806( 4) 3.0-3.5 TAT (2) ierny-215 © 5) 7 C DireS860iGS)gi5Z251 Ghd 334) 6 6516) 3.5-4.0 232i i 1360813) AC) 27312) 3220'S) 27 ES) e200) 4.0-4.5 46( 1) 254( 2) 152) ae 60313) 118 (4) CGO) e523) 4.5-5.0 340 ( 2) (AGH 1033) 6031G1) 427 (8) 121) P2284, 3) Total 5599 (67) 4961(67) 414(16) 6027(36) 2935(66) 1904(53) 4434 (65) The time the fish stayed in the white area is expressed in seconds. The total times and total numbers of appearances for 5 hr were All the individual values for time and number of white area is shown in parentheses. analyzed statistically according to Student’s t-test. appearances in 30-min periods were analyzed according to the Kruskal-Wallis test. evaluation. black-adapted and the white-adapted group, re- spectively). In the black-adapted fish, the time spent in the white area for 30-min periods gradually decreased with the passage of time (r(70) = —0.448, p<0.001). In contrast, in the white-adapted fish, no changes in the time spent in the white area were observed. The mean total time the fish spent in the white area during the entire observation period (5 hr) was 1617 +336 sec (n=7) for black-adapted fish and 3753+783 sec (n=7) for white-adapted fish and there was a significant difference between them (t=2.507, p<0.05). Even in the white-adapted fish, the time The number of appearances in the See text for statistical was only 20% of the entire observation period. These results show that this fish strongly prefers a black background to a white background. Their preference for background color seems to be similar to that of the blunt-nosed minnow and doughbelly, which show a smaller degree of modification of background choice as a result of residence in an area with a black or a white background, as reported by Brown and Thompson [8]. Even within the same group, there were statisti- cally significant differences among individuals for the time (H,,,(7)=30.6) and the number of 884 T. MorryA AND Y. MIYASHITA appearances (H,o,(7)=31.3) in black-adapted fish, as well as in white-adapted fish, for which the respective figures were H,.,(7)=32.7 and Heor (7) =22.8, p<0.001. Each fish might have a unique character as far as its color preference is concerned. In order to eliminate the individual variations and to confirm the above observation, we ex- amined the preference for background color for each fish after alternately keeping it in a white or black tank for one week using 19 females. As shown in Figure 1, when the fish were kept in a black background for one week, they showed a strong preference for black, and when the same fish were kept in a white background for the same period, the time the fish stayed in the white area increased, except for four fish, L, O, R and S. The change in background preference according to the pre-adaptation was statistically significant (t=3.972, p<0.001, paired t-test). These results strongly suggest that the tone condition of the environment in which the fish had stayed affected later preference for background color. However, it is possible that the long-term exposure to a white or black background brings 100 A Creek | wON td min. in white area Time about some difference in the hormonal and/or neural conditions as well as in the body color and that one or both of these differences could affect the background color preference. In order to confirm whether the body color is related to background color preference or not, we examined the background preference of the female and male fish. This was done because their body hues are distinguishably different even while in the same background; the male is always darker than the female in a white background. After adapta- tion to a white background for one week, back- ground preference was observed. As shown in Figure 2, males exhibited a greater preference for the black background than females. The above observations suggest that differences in the body color of fish might directly reflect the preference for background color; darker fish prefer to stay longer in the black background than pale fish. However, we can’t disregard the possibility that this difference in the choice of background color is due to sexual differences. Therefore, we tried to compare the differences in the preference of fish having different hues in the same environmental color using the same sex. Individuals Fic. 1. splendens. The influence of previous exposure to a black or white background on the color preference of the fish, B. Nineteen female fish (A-S) were used. White columns show the time that the fish stayed in the white area for 5 hr after white adaptation. Black columns show the time that the same fish stayed in the white area after black-adaptation. The difference in time for each fish was statistically significant (p<0.001, paired t-test). in white area Time oO min. 60 30 Time in white area for each 30 min period —— o min Male Background-color Preference in Fighting Fish 885 Female NAS SSS Oy so Ose 0 y l l AY INNS NEE For this purpose, the fish were treated with reserpine. Throughout the observation period, reserpine- treated fish (black fish) always showed a strong preference for black compared to the control fish and no change in staying time was observed in either group (Fig. 3). As shown in Table 2, in both the male or female groups, as well as in the mixed male-female group, there was a significant differ- ence between the control and reserpinized groups. The difference in preference between females and males was also observed in the control group (Table 2). This result confirmed a previous ex- perimental series which showed a difference in the Fic. 2. The difference between preference for back- ground color of the female and male of B. splen- dens. Columns show the time that fish stayed in the white area throughout the observation time (5 hr). Bars show the standard errors. There is a signifi- cant difference in time between males (n=12) and females (n=16) (p<0.01, Cochran-Cox test). ’ AV ALLL ne aa atta i se SO Un * SSS BSS [QGSSSSSS NANG: Fic. 3. The difference between preference for background color of reserpinized and control fish in B. splendens. Columns show the time that the fish stayed in the white area for each 30-min period. White columns; control group (consisted of seven males and eight females). Hatched columns; reserpinized group (consisted of six males and ten females). Bars show the standard errors. 886 T. Moriya AND Y. MIYASHITA preference for background color between the female and the male (Fig. 2). However, this difference disappeared in the reserpinized groups (Table 2), indicating that the difference of prefer- ence for black or white is mainly due to the difference of body hue rather than the sexual difference. The number of appearances of both the control and reserpinized fish in the white area decreased in correlation with the observation time (r(150) = —0.516, p<0.001 for the control group; 1(160) = —0.327, p<0.001 for the reserpinized group) (Fig. 4). There was no significant differ- ence between the two groups (mean number of appearances+S.E. was 40.4+3.6 (n=15) and 45.9+3.6 (n=16) for the control and reserpinized groups, respectively). This indicates that the movements of the fish to the white area from the black area were not affected by reserpine. The changes in duration time for each appear- ance in the white area throughout the entire observation time (5 hr) are shown in Figure 5. In the control fish, the duration in the white area gradually increased from 40 sec to 160 sec with the passage of time. This result suggests that control fish become used to staying in the white area after transfer to the experimental tank. In contrast, the time the reserpinized fish spent in the white area was almost constant (between 30 and 40 sec) throughout the observation period. These results show that the reserpinized fish move into the white area from the black area as frequently as the control fish do, but that they return to the black area more quickly than the control fish. Interest- ingly, the time the reserpinized fish spent in the white area seems to correspond to the time it would have taken to change the body color from black to white had they not received reserpine. This suggests that they might be aware of their unchanged body color and try to move into black background color. The information is lacking about how they recognize their own body color. We can only speculate that they see their body color or that they are affected by some physiological changes regulating the integumental color, such as the elevation of the MSH level in the plasma. It has been reported that, in the amphibian tadpole (Xenopus laevis), the content of MSH in the blood significantly increased after the adaptation to a black background, and decreased after the adapta- tion to a white one [15]. Karplus and Samuel [11] reported that the Xenopus tadpole kept in a white background showed a significant preference for white, and that injection of MSH induced an increase in their preference for black. They assumed that MSH acts on the brain and induces a preference for black. The increase of MSH content in the plasma is also known in black- adapted trout [16, 17] and eel [18]. In this study, we kept fish in a black or white background for one week before they were used in an experimental TABLE 2. The difference in preference for background color between reserpinized and control group B. splendens Duration time in white area for 30 min* Group Male plus female Male Female *: expressed in sec (mean+S.E.) Control 332--20i(n—50)s 269+26 (n= 70)? 388 +28 (n= 80)° Reserpinized 216+ 13 (n=160)* 190+16 (n= 60)" 231+18 (n=100)° These data were obtained from the same experimental series shown in Figure 3. All the values for time the fish spent in the white area for 30-min periods were analyzed. Difference between a and a’, p<0.001, t=4.96 (t-test). Difference between b and b’, p<0.05 (Cochran-Cox test). Difference between c and c’, p<0.01, t=4.73 (t-test). Difference between b and c, p<0.01, t=3.11 (t-test). No significant difference between b’ and c’, t=1.71 (t-test). Background-color Preference in Fighting Fish 887 oe feet diel | Talli Number of appearances in the white area time Observation Fic. 4. The change in number of appearances of the fish, B. splendens, in the white area for 30-min periods throughout the observation time. White circles show the mean number of appear- ances for control fish. Black circles show the mean number of appearances for reserpinized fish. Bars show the standard errors. series. During this period, the level of MSH in the plasma might have changed. We observed that black-adapted fish showed a slightly stronger preference for black compared with the reserpi- nized fish (data not shown). We can’t exclude the possibility that, in the black-adapted fish, in- creased endogenous MSH caused their preference for black. However, in the reserpine-treatment expriments, two groups of fish (control and res- erpinized fish) were both kept in a white back- ground for the same period (one week). Thus, we can assume that the level of MSH in reserpinized fish might be almost the same as that in control fish. In spite of having the same level of MSH, reserpinized fish preferred to stay in a black background more than control fish did. This datum gave us the impression that the body color affected background color preference. In the lower vertebrates, protective color change e ae E U el i) = ie) wo Qa a. i je) ved S iia | 5 lho | | 0 | ol 2s wo 2 | T | tL 3 ghee 2 Io | | x Tees oe Oe a Peale ve RET Fe eae ee eo JL 3 or 9 jee fi S oid Fypeagc (a RES ages fi pats E = OD 10 15°20 25°50 8 5 40 45 40) 25 5: i) UO ss) 20 AS 60) S540) 5 50 hr Observation time Fic.5. The changes in time the fish, B. splendens, spent in the white area for each appearance in the white area. White circles show the mean time for control fish. Black circles show the mean time for reserpinized fish. Bars show standard errors. The line (y=16.4x+48.2) for the control group was obtained by a least square calculation with a cor- relation coefficient of r(150)=0.304, (p<0.001). is very important for the protection against pre- dators. However, it is not the only method. Animals can also move to a better background color to hide, as it appears to be the case in Siamese fighting fish, Betta splendens. REFERENCES 1 Brown, F. A., Jr. (1961) Chromatophores and color change. In “Comparative Animal Physiology”. Ed. by C.L. Prosser, Saunders Co., Philadelphia & London, pp. 915-950. 2 Kohda, Y. (1983) The effects of color patterns on aggressive behaviors of a freshwater serranid fish, Coreoperca kawamebari. Zool. Mag., 92: 356-360. 3 Kohda, Y. and Watanabe, M. (1982) Agonistic behavior and color pattern in a Japanese freshwater serranid fish, Coreoperca kawamebari. Zool. Mag., 91: 61-69. 10 11 888 Kohda, Y. and Watanabe, M. (1982) Relationship of color pattern to dominance order in a freshwater serranid fish, Coreoperca kawamebari. Zool. Mag., 91: 149-145. Kohda, Y. and Watanabe, M. (1983) Reproductive behavior and color pattern in a freshwater serranid fish, Coreoperca kawamebari. Zool. Mag., 92: 207-215. Robertson, G. M. and Sale, P. F. (1975) Sexual discrimination in the Siamese fighting fish (Betta splendens Regan). Behavior, 54: 1-25. Kohda, Y. and Watanabe, M. (1986) Preference for vertical-striped backgrounds by the Oyanirami Coreoperca kawamebari, a freshwater serranid. Ethology, 72: 185-190. Brown, F. A., Jr. and Tompson, D. H. (1937) Melanin dispersion and choice of background in fishes, with special reference to Ericymba buccata. Copeia, 3: 172-181. Fujii, R. and Oshima, N. (1986) Control of chroma- tophore movements in teleost fishes. Zool. Sci., 3: 13-47. Kastin, A. A., Sandman,C.A., Stratton, L. O., Schally, A. V. and Miller, L. H. (1975) Behavioral and electrographic changes in rat and man after MSH. Prog. Brain Res., 42: 143-150. Karplus, I. and Samuel, D. (1978) The effect of exposure to black background and to MSH on black-white background preference in the amphi- bian Xenopus laevis (Daudin). Horm. Behav., 11: 12 13 14 15 16 17 18 T. MortyA AND Y. MIYASHITA 151-159. Miyashita, Y. (1985) Melanophore responses and color change in female Siamese fighting fish (Betta splendens)—A brief note. J. Lib. Arts Sci., Sap- poro Med. Coll., 26: 35-39. Siegel, S. (1956) Nonparametric Statistics for Be- havioral Sciences, McGraw-Hill, New York. Ishii, S., Kohno, S., Wakabayashi, K., Wada, M. and Kubokawa, K. (1983) N88-BASIC Jitsuyou Puroguramu Shyu (N88-BASIC Programs for Biologist), Vol. 2, Baifukan, Tokyo. Wilson, J.F. and Morgan,M.A. (1979) Alpha melanotrophin-like substances in the pituitary and plasma of Xenopus laevis in relation to color change responses. Gen. Comp. Endocrinol., 38: 172-182. Bowley, T. J., Rance, T. A. and Baker, B. I. (1983) Measurement of immunoreactive a-melanocyte- stimulating hormone in the blood of rainbow trout kept under various conditions. J. Endocrinol., 97: 267-275. Rodrigues, K. T. and Sumpter, J. P. (1984) Effects of background adaptation on the pituitary and plasma _ concentrations of some _ pro-opio- melanocortin-related peptides in the rainbow trout (Salmo gairdneri). J. Endocrinol., 101: 277-284. Baker, B. I., Wilson, J. F. and Bowley, T. J. (1984) Changes in pituitary and plasma levels of MSH in teleosts during physiological color change. Gen. Comp. Endocrinol., 55: 142-149. ZOOLOGICAL SCIENCE 4: 889-896 (1987) © 1987 Zoological Society of Japan Interspecific Differences in Some Courtship Behavioral Properties among the Four Species Belonging to the Drosophila auraria Complex! Yuzuru Ocuma, Haruo Kurokawa, Sumio M. AKaAr’, HISASHI TAMAKI and Jiro KAJITA Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki 305, Japan ABSTRACT—The four species comprising the Drosophila auraria complex were examined for their sexual activities, with regard to the forms of consecutive behavior exhibited during courtship. The mating frequencies and mating indices of the four species showed no diurnal changes, though under a dim red light, no insemination was observed during an initial 30-min observation period. The forms of courtship behavior observed on video tape recordings were analyzed for six parameters, i.e., time to initiation of courtship, total time of courtship, number of courtship bouts, length of courtship bout, number of tappings, and number of wing displays. Significant species differences were found in some courtship parameters. INTRODUCTION A large number of observations of courtship behavior have been performed using many Dro- sophila species including D. auraria [1]. One of the objectives of these investigations was to assign specific values to the behavioral elements in a courtship sequence which could be used as taxo- nomic characteristic for clarifying phylogenetic relationships. The D. auraria complex consists of four sibling species, i.e., D. auraria, D. biauraria and D. triauraria, which are widely distributed in Japan and Korea, and D. quadraria, which has been recorded only in Taiwan. Although interspecific crosses are successful and can produce hybrid offspring in the laboratory, each species retains a completely or almost completely discrete gene pool even in a sympatric area in the natural population [2-4]. It is thus concluded that some form of ethological isolating mechanism has be- come well developed, being strong enough to Accepted May 12, 1987 Received April 4, 1987 " Special Research Project on Instinct, University of Tsukuba. * Present address: Yamanashi Gakuin Junior College, Kofu, Yamanashi 400, Japan. separate the gene pool of one species from those of the others. The major aim of our consecutive works is to elucidate the role of male courtship behavior, which constitutes the behavioral basis of sexual isolation. Grossfield [5, 6] reported that mating success in the D. auraria complex was highly dependent on light. This fact suggests that a visual stimulus may be indispensable for partner recognition in this group. The diurnal rhythmicity of courtship be- havior in D. auraria has also been investigated by Hardeland [7]. The present paper describes interspecific differ- ences in some properties of courtship behavior among the four species belonging to the D. auraria complex. MATERIALS AND METHODS Species used Species used in this experiment were D. auraria (A541, Tsukuba), D. biauraria (B16, Tokyo), D. triauraria (1544, Tsukuba) and D. quadraria (Q, Taiwan, Texas stock No. 3075.1). These stocks had been maintained in mass laboratory cultures for several years at the time of initiation of the study. 890 Y. Ocuma, H. Kurokawa et al. Rearing conditions Flies were raised and aged in the standard Drosophila medium (sucrose—-dried yeast-corn meal) seeded with live yeast at 25+0.5°C and 60% R. H. under an artificial light (200-300 lux) and dark cycle (LD 14:10), the light period being between 5:00 and 19:00. Virgin flies emerging within 8 hr were sexed under CO, anesthesia, and aged in groups of 30 males or 20 females in food vials for a given period as described in Results. For observation of behavior using a video record- ing, a single male stored in a vial was employed. Sexual maturity For the record of mating success which was employed for measuring the degree of sexual maturity according to the time after eclosion and the diurnal change in sexual activity, two kinds of measurement were obtained from mass mating test. Ten females were placed with 15 males in each mating vial (¢3x10cm), and 20 replicates were run for each test. Both male and female flies were removed from the vial with an aspirator as soon as they had paired. The number of matings in every 5-min interval was scored during a 30-min observation period, and then a mating index was calculated using the formula proposed by Spiess et al. [8]. Mating frequency was measured according to the proportion of females mated within 30 min. In order to estimate the time taken to attain sexual maturity after eclosion, mating frequencies were examined for flies which had been aged for 1, 3, 4, 6 and 12 days, respectively. Diurnal changes To study diurnal changes in mating activity, both mating frequencies (%) and mating indices were measured either every two hours during the light period or every three hours during the dark period. The dark condition was furnished by a dim red light. Percentage of insemination under dark conditions Ten 4-day-old females were placed with fifteen 4-day-old males in each mating vial under the illumination conditions shown in Table 2. The prepared vials were left for 2, 12, 24 and 48 hr, respectively. After each of the given time periods had passed, the females were dissected and their ventral receptacles were examined for sperm. Observation of courtship behavior In order to analyze the parameters of courtship behavior, each of 20 single pairs was recorded for a 30-min period using a video recorder set (camera, Sony SLD-325; video recorder, PVM-171; video- recorder monitor, ACV 1150D) at 10 magnifica- tion. Afterward, the forms of behavior were analyzed by replaying the tapes. The observation chamber measured 15 mm in diameter and 5 mm in depth. The observation and the recording were carried out from 9:00 to 12:00 a.m. Parameters of courtship behavior There are basically three male courtship be- havior elements, i.e., orientation/following, wing display and attempted copulation. All of these forms of behavior were observed and noted via a video monitor. The parameters determined from the playbacks were as follows: time to initiation of courtship, number of courtship bouts in a 30-min observation period, length of courtship bout, total time of consecutive courtship behavior, number of tappings, number of wing displays per 10-min courtship period, latency to copulation and dura- tion of copulation. The time to initiation of courtship meant a measure of the time taken for the initiation of the first form of male courtship behavior after introducing the fly into the observa- tion chamber. The calculation of the number of tappings or wing displays per 10 min was done as follows: if the total courtship time is 2 min with 30 episodes of tapping and 5 episodes of wing display in a 30-min observation period, we multiply them by five. Therefore the figures for the two para- meters become 150 and 25, respecitvely. RESULTS The time taken for flies to attain sexual maturity can be estimated by observing the daily changes in their mating frequency (Fig. 1). There was no significant difference among the four species in their mating frequencies at the 4 days. The values reached a maximum at 4 to 6 days after eclosion. Behaviors in the D. auraria Complex 891 MATING FREQUENCY 5 10 DAYS AFTER ECLOSION Fic. 1. Changes in sexual activity (percentage of in- semination) with age. Ten females and 15 males in a vial were tested for a 30-min observation peiod. A: D. auraria, B. D. biauraria, T: D. triauraria, Q: D. quadraria Accordingly, observation of courtship behavior was carried out using 4- to 6-day-old flies in the subsequent experiment. Diurnal changes in the mating frequency and the mating indices are summarized in Table 1. No mating was observed within the first 30-min observation period under the dim red light during the dark phase. Neither following nor tapping was observed under this condition. The male fly began to actively court the female under a low light intensity of 2-3 lux. No particular change in the diurnal activity of mating was revealed for any of the species. D. auraria showed 70% mating frequencies on average while D. biauraria, D. triauraria and D. quadraria showed higher levels. This tendency could also be seen in the mating index, that is, the mating indices of D. auraria ranged from 70.5 to 87.2, while those of the others ranged from 104.1 to 137.4. Thus the latter three species seemed to be sexually more active than D. auraria during the light period. The results of the insemination test under dark conditions are shown in Table 2. There was a slight difference in light dependency among the four species. Under dark conditions the per- centage of insemination in D. auraria increased gradually with time and reached 89.7% at 12 hr after the beginning of the test. However, D. biauraria showed a much lower value and D. triauraria and D. quadraria showed values in- termediate between those of D. auraria and D. biauraria. The sequence of male courtship behavior in the D. auraria complex fundamentally coincided with that described by Spieth [1]. The general features of this behavior are as follows: After the male fly has been introduced into the observation chamber, he moves around for a while, and then orients toward a female. He follows and taps her with his fore leg and extends one wing at 90 degrees and vibrates it. A receptive female becomes to move slowly after a short time and the male can make contact with her, after which he attempts copula- tion, extending one wing to 90 degrees following copulation. During copulation, the male rubs the female’s abdomen with his mid legs and vibrates one of his wings. Toward the end of the copulation period, the female spins and kicks the male. The male rubs her more frequently and vibrates his wing in concert with her movements, then he withdraws and dismounts. The frequency of each courtship element was found to be clearly different among the four species (Table 3). The results of the analyses of courtship behavior are shown in Table 4. The mean latency time to copulation and the duration of copulation are shown in Table5. The six parameters obtained could be used to build up pictures of courtship for each species by Mann- Whitney U-test [9] (Tables 6-1, 6-2 and 6-3). The species characteristics of male courtship behavior in the D. auraria complex are summa- rized as follows: 1. D. auraria Males spent a very long time from being introduced into the observation cham- ber to initiation of courtship (=time to initiation of courtship) (Tables 4 and 6-1). On the contrary, the total time of courtship (56.9 sec) was much shorter, and the number of courtship bouts (2.8 sec) was lower than the other three species (Tables 892 Y. Ocuma, H. Kurokawa et al. TABLE 1. Diurnal changes in mating frequency, mating index with the standard error of the mean (S.E.M.) and its coefficient of variation (CV) of the 4 species of the D. auraria complex under a light period of the diurnal cycle using 4-day-old flies Mating index Mating Run frequency CV Observation Mating frequency Mating index Run (%) with cise © 0 (%) withsEM D. auraria D. triauraria 19 70.0 WM ssyae 555) Sel 6:00 20 90.5 ae 30) 10.3 19 TEL 78.4+6.4 S5m/ 8:00 19 88.9 125.6+4.8 16.6 19 70.0 74.5+6.2 36.4 10:00 20 88.5 134.3+5.6 18.5 20 76.0 75.9+4.5 26.7 12:00 19 83.1 116.4+5.8 Alli 20 76.0 T7.9£6.3 835/20) 14:00 17 84.7 129.3+4.5 14.3 20 74.5 76.7+6.3 36.9 16:00 20 76.5 OS \azs)3) Dod 18 ed 87.2+6.2 30.0 18:00 17 90.0 IDC ae Ss) 17.4 135 TSsf3 Ve 2achS 34.2 Mean 132 86.0 ae Il 1.3 D. biauraria D. quadraria 19 76.8 114.8+5.4 20.7 6:00 82.6 132 SsOe2 20.3 19 Se) 104.64+3.8 16.0 8:00 20 76.5 118.4+4.4 16.7 20 82.5 22S a2 50 18.4 10:00 20 WS IS Y.5)42 5.4! 18.1 20 76.5 104.1+5.7 24.4 12:00 19 13 117.2+6.4 23.6 20 77.0 MISSES). 25 14:00 20 76.0 120.4+7.7 28.4 20 80.5 115.7+4.8 18.6 16:00 20 76.0 127.4+5.7 22 20 82.0 111 9326-2 S29) 20 84.0 134.0+5.7 18.9 WIA ss) 2,72 PPB) i7) 138 78.0 126.1+2.4 22.0 The group comparisons showing a significant difference (P<0.001, by t-test) in mating indices are marked with a brace (} TABLE 2. D. auraria D. biauraria D. triauraria D. quadraria ). Percentage of inseminated females under constant light (LL), constant dark (DD) and a normal diurnal cycle (LD) 100 100 100 50 2hr (%) LL DD 95.0 58.0 TO» OD) 84.0 44.0 99.0 20.0 y 36.0 116.8 33.0 90.3 12 hr (%) 24 hr (%) , 48 hr (%) ‘ SERIE ESE EIS DE ee Ze pee) CR Ee, 1DID) Avi Dim spp SPT IEID)T eID ID) 4 98.0 89.7 ns. 100 960° ns’ 100 100m wes 950 4.0" "162.0" 92.0) 60.0" 26.3" 100" O20 ane 95:0 42.0 62:7 93.0520" 401 910) STG Ue OeE 980° 62.0 181 98.0 65.0) 159° 99.0 100eernte All tests were performed in vials containing 10 femals and 15 males. * All chi-squared values were calculated with the Yates’ correction [9] and are significant (P=0.001). n.s. means not significant. 4 and 6-2). tion time was extended to 60 min. Attempted copulation was not observed in the first 30-min observation period (Table 3), but a few were found when the observa- 2. D. biauraria Many of the males showed wing display (85.0%) and tapping (75.0%) (Table 3). The mean numbers of tappings and wing displays per 10 min were 41.0 and 15.6, respective- ly, each of which was significantly higher than the others (Table 6-3). The mean time of initiation to TABLE 3. Behaviors in the D. auraria Complex the 30-min observation period D. auraria D. biauraria D. triauraria D. quadraria 893 Frequency (%) of males showing courtship behavior and each courtship element in Wing Attempted : Run Courtship Tapping feplay eoniiation Copulation 40 60.0 30.0 3:0 0 0 20 95.0 75.0 85.0 30.0 20.0 20 100 75.0 30.0 0 70.0 20 100 60.0 5.0, 50.0 40.0 TABLE 4. Comparison of parameters of courtship behavior in the males Run D. auraria 40 D. biauraria 20 D. triauraria 20 D. quadraria 20 TABLE 5. Time of Total Number of Length of Mean number per initiation of time of courtship courtship 10min courtship period courtship courtship bouts bout (sec+S.E.M) (sec+S.E.M) with S.E.M (sec+S.E.M) Tapping Wing Display D46:4 83:05 950.9215 1p 2.82205 26562-1087 2228.8 105-0: (ADEE LD SAO) +855) 12-04-2260 60.9416.4 41.0+9.2 15262 329 WS 8ce4729 903M E1379 §4-24-087)) 1233, 4a rlesm O92 S16 4.6+1.0 TOMSee29- SSSI =eailiyagn O:4cborS D9 sae 09 OeEOR! One OF! species in the 30-min observation period D. biauraria D. triauraria Comparison of latency to copulation and duration of copulation among the three N Latency (sec+S.E.M.) Duration (sec+S.E.M.) 4 1105.3+232.4<——, S21 S=E2S.9 14 n99:1460 | 35/9 431356 8 Us\i eae CRS 289.1+ 8.6 D. quadraria * Significant (P=0.01) by Mann-Whitney U-test. courtship (71.2 sec) was significantly shorter than those of D. auraria and D. quadraria (Tables 4 and 6-1). The male courted the female with short gaps and short lengths of courship bouts in the 30-min observation period (Tables 4 and 6-2). Although the male courted the female frequently, the percentage of pairs copulated (20.0%) was low in the 30-min observation period (Table 3). 3. D. triauraria A very long length of the courtship bout (233.4 sec) revealed that the male of this species courted the female with a long period of orientation and following with only few episodes of tapping. This characteristic feature was comprehensible by the fact that the total time of courting (903.1 sec) was about half the time of the observation period (Tables 4 and 6-1). After the long time of courtship, most of the males were able to attain the copulation (70.0%) (Table 3) with a few episodes of tapping and wing display. 4. D. quadraria The males courted the females frequently almost without any episodes of wing display and tapping (Tables3 and 4), and only half the number of males showed attempted copulation. The time to initiation of courtship of this species was 161.3 sec, a value intermediate between those of D. auraria and D. biauraria. The characteristic feature of courtship behavior in this species was that the males showed only a few episodes of tapping and wing display despite the long courtship bout. 894 Y.Ocuma, H. Kurokawa et al. TABLE 6-1. Results of Mann-Whitney U-test for courtship parameters, time to initiation of courtship and total time of courtship biauraria triauraria quadraria U=20 U=41 WRG p<0.0001 p<0.0001 p<0.0001 U=53 Wi=18525 U=100 p<0.0001 not sig. 0.01—-/a. Seta Ja inserted at about mid-distance along length of pteromorph. Seta /m at the level of seta Ja. Mutual distance of setae /m wide, twice as long as distance /Ja-lm. The mutual distance of setae /p 0.8 times as long as that of setae /m and almost equal to distance c-la. Seta hz just behind the level of seta Jp. Distance between /p—h; almost equal to that between /a—lp. Distance h3;—h>z almost equal to distance h—h;, and a little longer than mutual distance of setae hy. Distance ps3—ps2 about equal to distance ps2—ps), and about twice as long as mutual distance of setae DS}. Four pairs of areae porosae round, indistinct; Aa, A; and A> near setae /a, Ip and h>, respective- ly, while A; at the middle level between setae ps, and h;. Five pairs of lyrifissures short; lyrifissure ia just near Ah. Legs Setation: trochanters 1-1-2-1, femora 5-5-3-2, genua 3(1)-3(1)-1(1)-2, tibiae 4(2)- 4(1)-3(1)-3(1), tarsi 18(2)-—15(2)-15-12. Tarsi I and II monodactylous. Tarsi III and IV hetero- tridactylous; side claws very thin, curving only slightly. An acute ventral spur on each of genua I, II and femur II. Lamelliform ventral mdge on femora III and IV as well as trochanters III and IV. Remarkable setae are as follows. Tarsi: all setae fc swollen at basal half. Tibiae: v” III and IV dully pointed, thicker and shorter than v’ III and IV, respectively. Genua: /” I and II very thick and dully pointed, v” I and II minute. Femora: bv” I and v’ I thin, ev’ IV minute. Trochanters: v’ IV minute, while setae on other legs barbed, thin and very long. Type-series Holotype (NSMT-Ac 9794 in spir- it) and 7 paratopotypes: Ishinden-Kozubeta, Tsu, Mie, May 4, 1986, collected and extracted by N. Ohkubo from dead leaves that are cast at the shore of a pond. All specimens will be deposited in the collection of the National Science Museum (Nat. Hist.), Tokyo. Remarks The type species Z. furcatus has been collected from Europe frequently. Though the information on its morphology is not sufficient, the illustrations that were given by Warburton and Pearce [2], Willmann [8], Schweizer [9] and Shaldybina [6] indicate some specific characters: 1) long cuspis, 2) fairly thick base of cuspis, 3) barbation on sensillus (except in [8]), and 4) relatively pointed tip of sensillus (except in [8]). The body length (500 «m [2] and 440 um [8]) is greater than that of the present new species. Argentine species Z. argentinensis is characterized by 1) very long cuspis, 2) presence of hairs on sensillus, 3) large areae porosae, 4) more posterior position of setae ps3, 5) long notogastral setae, 6) more setae on tibia II and 7) longer and shorter setae on leg II. Its body length 500 um. Two species were recorded from Chile: Z. cristatus (Hammer [7]) and Z. spinosus (Hammer [7]). The former species is different from the new species in 1) long cuspis, 2) short lamellar seta, 3) distinct areae porosae on notogaster, 4) anterior position of genital seta g>, 5) lateral position of adanal seta ad; and 6) thick lateral claws of leg IV. The body length 580 um. The latter species is characterized by 1) anteriorly tapering cuspis, 2) widely sepa- rated lamellae and 3) anterior position of genital sete g> and g3. The body length 380 um. DISCUSSION The genus Zetomimus Hull was not popular at first and its type species was rather known as a member of Ceratozetes until Balogh [10] accepted the genus as a well isolated taxon. Later, Shaldybi- na [11] constructed new system of Ceratozetoidea where she gave a family level taxon Zetomimidae to the genus. Her taxon was adopted in the identification keys of Balogh [4]. During the present study, however, the author notices that Zetomimus fairly resembles Ceratozetes. Behan- Pelletier [12] presented fine diagnosis of Cerato- zetes. Examining his description, one can find that the present new species has almost all features of Ceratozetes as to adult. The genus Zetomimus is distinguishable only by some additional features as mentioned after. The family Ceratozetidae con- tains many genera which have little resemblance to Ceratozetes. The author, therefore, is doubtful about the family level separation of the two genera. The genus Zetomimus is distinguished from Ceratozetes as follows: 1) legs I and II monodactyle and legs III and IV tridactyle, 2) notogaster 902 N. OHKUBO relatively wide, 3) lamella and cuspis relatively narrow, 4) sensillar head club-shaped, 5) area porosa A; situated just before seta /p, 6) median part of rostral tip not excavated. The next features have a possibility to become generic characters: 1) organ Ah extremely developed, 2) the border between sdm and psdm of bothridium only slightly curved, and 3) the presense of membranous secretion under pteromorphs and near dorso- sejugalis. ACKNOWLEDGMENT The author wishes to thank Dr. J. Aoki, Yokohama National University, for reading the manuscript. REFERENCES 1 Hull, J.E. (1916) Terrestrial Acari of the Tyne Province. Trans. Nat. Hist. Soc. Northumberland, 4: 381-410. 2 Warburton, C. and Pearce, N. D. F. (1905) On new and rare British mites of the family Oribatidae. Proc. Zool. Soc. London, 2: 564-569, 2 pls. 3 Hull, J.E. (1915) Acari from bird’s nests, with description of a new species. Naturalist, 707: 10 11 iw 398-399. Balogh, J. (1972) The Oribatid Genera of the World. Budapest, pp. 1-188, 71 pls. Hammer, M. (1958) Investigations of the oribatid fauna of the Andes Mountains. I. The Argentine and Bolivia. Biol. Skr. Dan. Vid. Selsk., 10: 1-129, 34 pls. Shaldybina, E. S. (1975) Ceratozetoidea. In “A Key to Soil-Inhabiting Mites (Sarcoptiforms)”. Ed. by M. S. Gilyalov, Moscow, p. 491. (in Russian) Hammer, M. (1962) Investigations of the oribatid fauna of the Andes Mountains. III. Chile. Biol. Skr. Dan. Vid. Selsk., 13: 1-95, 30 pls. Willmann, C. (1931) Moosmilben oder Oribatiden (Cryptostigmata). Tierw. Deutschl., 22: 79-200. Schweizer, J. (1956) Die Landmilben des schweizer- ischen Nationalparkes. 3e partie: Sarcoptiformes. Ergeb. wiss. Unters. shweiz. Nationalparkes, 34: 215-377. Balogh, J. (1965) A synopsis of the world oribatid (Acari) genera. Acta Zool. Hung., 11: 5-99. Shaldybina, E.S. (1966) Postembrionic develop- ment of horned mites of superfamily Ceratozetoidea Balogh, 1961 and their system. In “1st Conference of Acarology. Abstracts”. Nauka, pp. 225-226. (in Russian) Behan-Pelletier, V.M. (1984) Ceratozetes (Acari: Ceratozetidae) of Canada and Alaska. Can. En- tomol., 116: 1449-1517. ZOOLOGICAL SCIENCE 4: 903-911 (1987) © 1987 Zoological Society of Japan Redescription of Villa myrmeleonostena (Insecta, Diptera, Bombyliidae), A Parasitoid of Ant Lion in Japan Kintaro Basa, AxirA Nacatomi!, Hisako NAGATOMI? and NEAL L. EvENHUIS>° Kurokawa-mura, Kitakambara-gun, Niigata Pref. 959-28, ‘Entomological Laboratory, Faculty of Agriculture, Kagoshima University, Kagoshima 890, * Biological Laboratory, Kagoshima Women’s College, Hayato 899-51, Japan, and * Department of Entomology, B. P. Bishop Museum, Honolulu, Hawaii 96817, U.S.A. ABSTRACT — Villa myrmeleonostena (Baba) comb. n. from Japan is redescribed and illustrated. The larva of this species is parasitic upon the ant lion. INTRODUCTION Baba [1] gave the biological notes upon a bee fly species from Japan, whose larva parasitizes the ant+ lion. Later, Baba [2] named this species Argyr- omoeba (?) myrmeleonostena. In this paper, Villa myrmeleonostena (Baba, 1953) comb. n. is rede- scribed and illustrated and its biology is introduced according to Baba [1, 2]. Genus Villa Lioy Villa Lioy, 1864, Atti Ist. Veneto Sci. ser. 3, Vol. 9: 732. Type species: Anthrax concinnus Meigen, 1820 [=abbadon (Fabricius, 1794)]. By designation of Cogquillett (1910). Anthrax, authors, not Scopoli. For diagnosis of Villa, see Engel [3], Hesse [4], Oldroyd [5], Hull [6], Hall [7] and Greathead [8]. This genus contains numerous species and is present in all of the continents (e.g., Bowden [9, 10]; Hull [6]; Painter and Painter [11]). Oldroyd [5] wrote, “Clear-winged species of Villa are among the easiest Bombyliidae to recog- nize generically, but the most difficult to identify specifically.” Accepted May 23, 1987 Received April 18, 1987 ‘ To whom reprints should be requested. Villa myrmeleonostena (Baba) comb. n. (Figs. 1-11) [Japanese name: Arijigoku-tsuriabu] Argyromoeba (?) myrmeleonostena Baba, 1953, Arijigo- ku no Seibutsushi [=Biology of the Ant Lion], p. 69. Type locality: Niigata Prefecture, Honshu. According to Hall [7], “palpus usually about half length of proboscis or more. Thoracic bristles well-developed. Pulvilli present or absent” in the North American Villa and its related genera. In V. myrmeleonostena from Japan, the thoracic and scutellar bristles are lacking, the palpus is small and inconspicuous and difficult to recognize, and the pulvilli are absent. According to the Key to the Palaearctic species by Engel [3], V. myrmeleonostena falls into the couplet 7 and is similar to V. albida Becker, 1916 from Hungary (#), but may easily be distin- guished from the latter by having the following characters: bristles on mesonotum lacking and abdominal terga 4-6 with black erect hairs at sides. Male Head (Figs. 1-5): Dark brown to black, and more or less pale gray pollinose; frons, face (except clypeus), and antennal segments 1-2 with black, trim, rather stout hairs; postocular rim (including cheek), ocellar triangle and proboscis with short, black haris which are recumbent on postocular rim; posterior border of postocular rim covered with white, trim hairs directed posteriorly, which become black on lower part; frons, face 904 K. Basa, A. NAGATOMI et al. Fics. 1-5. Villa myrmeleonostena, male. 1-4. Head, anterior, dorsal (ocellar triangle is kept horizontal), facial and lateral views. 5. Antenna, dorso-inner view (based on 2nd specimen). (except clypeus), and postocular rim with small, pale (or pale brown) scales; half width of head from a direct frontal view 0.9 times distance from antenna to median ocellus and 1.0-1.1 times width of frons at antennae; ocellar triangle 0.8—1.0 times as wide as long; space between antennae 2.0-2.4 times width of ocellar triangle; width of frons at median ocellus 0.16-0.18 times distance from antenna to median ocellus, 0.18—0.20 times width of frons at antenna, 0.4—0.5 times narrowest part of face, and 1.3-1.5 times width of ocellar triangle; distance from lower margin of clypeus to antenna 1.2-1.3 times that from antenna to median ocellus; clypeus 0.3-0.4 times as wide as long and 5.5-12.0 times as wide as narrowest part of parafacials; antenna 0.5 times as long as distance from antenna to median ocellus and 2.8-3.3 times as long as width of frons at median ocellus; when measured along inner surface, antennal segment 3 (including short apical spine) 1.9-2.1 times as long as and 0.6 times as thick as segment 1, and 3.5—-5.0 times as long as and 0.8 times as thick as segment 2; in segment 3, basal wide part 0.4—0.6 times as long as apical narrow part (including spine); proboscis (along ventral surface) 0.8-0.9 times as long as clypeus; data based on 5 specimens. Thorax (Fig. 6): Dark brown to black, and more or less pale gray pollinose; mesonotum, scutellum, \ | Fic. 6. Thorax and abdomen of Villa myrmeleonoste- na, male, dorsal view. Redescription of Villa myrmeleonostena 905 meta-, and upper part of meso-, and anteroupper part of pteropleura covered with long, white haris which are trim, vertically erect, and transversely running on anterior part of mesonotum; pro-, lower part of meso-, sterno- (except bare large anterolower part), and pteropleura (before meta- pleura) with black hairs which are long, dense, trim, and rather stout on propleura and running transversely above fore coxae; hypo- and lower part of pteropleura bare; halter creamy white or creamy pale brown, and partly tinged with dark brown. Wing (Figs. 7-8): Hyaline; veins brown to dark brown; subcostal cell more or less brown fumose; supernumerary crossvein sometimes present con- necting Rj,3 and Ry, (see Fig. 8). Legs (Figs. 9-11): Dark brown to black; coxa and femur more or less pale gray pollinose and with black hairs; femur also covered with small, pale (or pale brown) scales; fore tibia at dorsal surface and mid and hind tibiae throughout sur- faces clothed with spicules; pulvilli absent; relative lengths of segments (excluding coxa and trochan- ter) of fore leg 66 (63-69): 67 (66-69): 20 (18-21): 9(8-10): 7 (6-7): 5 (5): 11 (11-12), of mid leg 75 (73-77): 80 (77-82): 24 (23-26): 9 (8-9): 7 (6-7): 5 (5-6): 11 (11), of hind leg 100: 108 (104-111): 37 (45-38)= 15 (3-19): » 11 (10-13): 7 (7-8): 13 (13-14) and in hind leg, relative thickness of femur, tibia, and tarsal segments 1-3, 10 (9-11): 7 (7): 4 (4-5): 3 (3-4): 3 (3-4); (N=4). Fic. 7. Wing of Villa myrmeleonostena, male. Abdomen (Fig. 6): Dark brown to black, and more or less pale gray pollinose; dorsum with white hairs which are dense on the sides and become chiefly or partly black on sides of terga 4-6; tergum 2 (except sides) with a large semi- circular apical band which is covered with black scales; terga 3-7 (except sides) with white scales which become black along posterior borders (ex- cept that of tergum 7) and along mid lines; venter with black hairs which become white on sides of sternum 2; venter also with black scales which become pale (or pale brown) on sides. Length: Body 6.4-8.8 mm (N=8); wing 7.0-8.8 mm (N=6); hind femur 2.1-2.4 mm (N=4). Female Similar to male except as follows. Head: Space between antennae 1.8-2.0 times width of ocellar triangle; width of frons at median ocellus 0.25—0.29 times distance from antenna to Fics. 8-11. Villa myrmeleonostena, male. 8. Part of wing (showing abnormal crossvein connecting R;,3 and R,). 9. Fore femur, tibia and tarsus, posterior view. 10. Hind coxa, femur, tibia and basitarsus, anterior view. 11. Hind tarsal segments 2-5, ventral view. 906 K. Baba, A. NAGATOoMI et al. median ocellus, 0.30-—0.33 times width of frons at antennae, 0.6-0.7 times narrowest part of face, and 1.8—2.0 times width of ocellar triangle; anten- na 1.9-2.3 times as long as width of frons at median ocellus; when measured along inner sur- face, antennal segment 3 (including apical short spine) 1.7-1.8 times as long as and 0.7-0.8 times as thick as segment 1, and 4.0-5.5 times as long as and 0.8-0.9 times as thick as segment 2; data based on 5 specimens. Thorax: Mesonotum (except sides and anterior border) and scutellum (except posterior border) covered with white scales and clothed with fine, erect black hairs which become partly white on scutellum and posterior border of mesonotum. Wing: As in male. Legs: Relative lengths of segments of fore leg 66 (65-67): 67 (65-69): 20 (19-20): 8 (8): 7 (6-7): 6 (5-6): 12 (12), of mid leg 76 (76): 78 (74-82): 27 (24-31): 9 (8-9): 7 (6-7): 6 (5-6): 11 (10-11), of hind leg 100: 107 (106-108): 38 (34-41): 13 (13-14): 10 (9-11): 8 (7-8): 13 (12-15) and in hind leg, relative thickness of femur, tibia, and tarsal segments 1-3, 12 (11-12): 7 (7-8): 5 (5): 4 (4): 4 (3-4); (N=3). Abdomen: As in male. Length: Body 7.4-8.6 mm (N=8); wing 7.2-8.6 mm (N=8); hind femur 2.1-2.3 mm (N=3). Distribution Japan (Honshu). Specimens examined (10/7 %, 92 $): Niigata Pref.: 20%, 12, Kinoto, 18 & 29. vii. 1948, K. Baba; 1%, Araihama, 19. vii. 1959, K. Baba; ALS, 32 2, Senami, 4-14. vii. 1985, K. Baba; 20h, S$, Senami, 4-24. vii. 1986, K. Baba; 1%, Senami, 28. viii. 1986, K. Baba. There is a single male specimen from Yakushi- ma (Kagoshima Pref.) (Kurio, 27. vii. 1967, A. Nagatomi). Its size is as follows: body 10.2 mm; wing 9.8 mm; hind femur 2.75 mm. No significant difference is found between this specimen (from Yakushima) and myrmeleonostena (from Niigata Pref.). The male genitalia are not yet examined, however. Fics. 12-17. 15. Dorsal view. 16. Lateral view. Mouthparts of Villa myrmeleonostena, female. 12. Lateral view. 13. Ventral view. 14. Lateral view. 17. Theca, ventral view. CP, cibarial pump; H, hypopharynx; LC, lacinia; LE, labellum; LR, labrum; P, palpus; TH, theca; TO, torma. Redescription of Villa myrmeleonostena 907 Mouthparts of Villa myrmeleonostena (Figs. 12-17) Nagatomi and Soroida [12] described and illus- trated the female mouthparts in 3 genera (Anthrax, Bombylius and Ligyra) and 3 species of Bombyliidae. The structure of the mouthparts in V. myr- meleonostena is given below, basedon2/ 4,17. Differing from the 3 genera and 3 species (? 2) above as follows: Hypopharynx not longer than labrum and mid-distal interlabellar process lacking. Male Labrum longer than wide, and much shorter (0.4-0.5 times) than cibarial pump; dorsal part of labrum willow leaf-like, narrower apically, and with basal lesser half desclerotized; ventral part of labrum pipe-like, concave dorsally, paral- lel-sided, with apical margin nearly transverse, and with a V-shaped pale spot near apex. Hypopharynx thin dorsoventrally, shorter (0.9 times) than labrum, longer than wide, widest behind middle (or roughly parallel-sided), and desclerotized at basal part. Cibarial pump wider (1.7-2.0 times) than labrum, wider (2.8-3.3 times) than breadth between dorsal cornua, much longer (3.7—4.8 times) than wide, widest before middle, and gradually narrower posteriorly. Torma long and its base situated before (i. e. near anterior end) middle of cibarial pump; a pair of more sclerotized lateral lines not fused at posterior ends. Palpus elliptic, small and inconspicuous, 1- segmented, and longer than wide. Lacinia about as long as labrum. Labellum shorter (0.7 times) than cibarial pump, longer (1.7-1.9 times) than theca, and with 2 longitudinal darker spots. Length: Labrum 0.40-0.43 mm; cibarial pump 0.93-1.03 mm. Female (Figs. 12-17) No significant difference is found between sexes. Length: Labrum 0.38 mm; cibarial pump 0.89 mm. Specimens dissected: 2{%, Senami, Niigata Fics. 18-22. Male genitalia of Villa myrmeleonostena. 18. Dorsal view. 19. Ventral view. 20. Lateral view. 21. Ventral view. 22. Lateral view. AA, anterior bar of aedeagus; ADP, aedeagal dorso-anterior plate; B, basistyle; BDP, basistylar dorso-inner anterior process; D, dististyle; DB, dorsal bridge; DP, dorsal plate; VL, ventral lobe; VP, ventral plate. 908 K. Basa, A. NAGATOMI et al. Pref., 10 & 24. vii. 1986, K. Baba; 12, Senami, Niigata Pref., 14. vi. 1985, K. Baba. Genitalia of Villa myrmeleonostena (Figs. 18-32) Hull [6] and Theodor [13] described and illus- trated the male genitalia in many genera and species of the family Bombyliidae. The following descriptions of the genitalia of V. myrmeleonostena contain the familial and generic characters. It is not possible to extract the specific characters, because only one species is examined. The term of each part of the male genitalia is the same as that in Nagatomi [14] on the lower Brachycera. Male genitalia (Figs. 18-25) Basistyle neither thickened nor folded into dorsal piece, and com- posed only of ventral piece, but one narrow dorsal band present before dististyle; fused basistyles (from ventral view) widened at anterior part, longer than wide, gently concave at anterior margin and deeply so at posterior margin; basistyle (except anterior part) with ventral hairs; basistylar dorso-inner anterior process is strip-like, and a cove formed by this process and basistyle is narrow. Dististyle short in relation to basistyle and composed of outer wide and inner narrow pro- cesses, having hairs. The so-called aedeagus composed of the dorsal and ventral plates, dorsal bridge, anterior bar of aedeagus, and aedeagal dorso-anterior plate (in- cluding a pair of diverging processes). Dorsal plate longer than wide; distal part of dorsal plate Fics. 23-25. Male genitalia of Villa myrmeleonostena. 23 cercus; S10, sternum 10; T9, tergum 9. flattened dorsoventrally, wider anteriorly, and longer than distal part of ventral plate (which is probably composed of ventral plate and a part of dorsal plate). Ventral plate pointed and forming a tube at apex, and its basal part composed of a pair of long diverging processes. Anterior bar of aedeagus flattened laterally and wider and round- ed anteriorly (=toward base of abdomen). Tergum 9 widely concave at anterior margin, straight at posterior margin, with anterolateral angle pointed ventrally, and with dorsal long hairs which are absent on anterior part (whose middle area is large). Cercus triangular in shape from dorsal view, rounded at posterior margin from lateral view, and widely bordered with membranous area covered with hairs. There is no distinctly sclerotized sternum 10. Specimens dissected (3/4): 1%, Kinoto, Niigata Pref., 18. vii. 1948, K. Baba; 2/3, Senami, Niigata Pref., 10 & 24. vii. 1986, K. Baba. Female genitalia (Figs. 26-32) Tergum 7 and sternum 7 much wider than long and the former slightly longer and slightly wider than the latter. Tergum 8 and tergum 9+ 10 much narrower than the preceding sgment. Each side of tergum 8 and tergum 9+ 10 developed into a ventral piece. Each of sternum 10 and cercus divided into a pair of sclerites which are flattened laterally. Sternum 8 flattened dorsoventrally, somewhat narrower than tergum 8, much wider than long, with anterior and lateral margins forming a semicircular line, and with anterior part desclerotized. . Dorsal view. 24. Ventral view. 25. Lateral view. C, Redescription of Villa myrmeleonostena 909 / i y fi i NS ik Ws LR ex \) Fics. 26-32. Female genitalia of Villa myrmeleonostena. 26. Ventral view. 27. Dorsal view. 28. Lateral view. 29-30. Genital fork, dorsal and ventral views. 31. Sternum 8, dorsal view. 32. Ventral view. C, cercus; GF, genital fork; MAS, midanterior stick in tergum 8; S7, sternum 7; S8, sternum 8; S10, sternum 10; 17, tergum 7; T8, tergum 8; T9+10, tergum 9+ 10. Dorsal piece of tergum 8 trapezoidal, wider anteriorly, much wider than long, with a con- spicuous midanterior stick, and with long soft hairs at posterior margin. Ventral fold of tergum 8 widely bordered with membrane which is covered with long soft hairs; this membrane developed into a process at middle and absent on posterior margin of ventral fold except outer angle; posterior part of ventral fold haired. Dorsal piece of tergum 9+ 10 wider than long, rectangular, and with an antero- lateral sclerite + membrane (the membrane may be connected with postero-outer part of ventral piece); posterolateral part of tergum 9+10 with a vertical row of rod-like long spines, 10 or so in number; tergum 9+10 covered with long soft hairs. Inner border of sternum 10 darker and with soft hairs. Cercus membranous and haired. Genital fork longer than wide, somewhat pen- tagonal, and with posterolateral long margins and midanterior short line sclerotized; as a part of genital fork, there is a U-shaped ventral sclerite. Specimens dissected (322): 12, Senami, Niigata Pref., 14. vii. 1985, K. Baba; 12 , Senami, Niigata Pref., 24. vii. 1986, K. Baba; 12, Senami, Niigata Pref., 19. vii. 1986, K. Baba. Biology of Villa myrmeleonostena (Figs. 33-36) For biology and immature stages of the family Bombyliidae, see Clausen [15], Hull [6] and du Merle [16]. Ant-lion parasites in the family Bombyliidae have been recorded from the New World genus Dipalta Osten Sacken and the Old World genus Micomitra Bowden. Anthrax confluensis Roberts, and anomalous representative of this ubiquitous genus from Australia have also been recorded as parasitic on immatures of Myrmeleontidae. A. confluensis may belong in a new genus, but detailed phylogenetic study will have to be under- taken first to confirm this. For further details on the life history of Micomitra stupida (Rossi) 910 K. Baba, A. NAGATOMI et al. parasitic on Myrmeleontidae, see Steffan [17]. Baba [1, 2] gave an article on the biology of V. myrmeleonostena. An abstract of this article is given below. The habitat The bee fly in question was col- lected on sand dune of the beaches in northern part of Niigata Prefecture and that at Akashi in Hyogo Prefecture. The investigation was made at the former. The adult The parent fly appears in July and August. It visits the flower, e.g. Fagara schinifolia Engl. (=inuzansho). The copulation was seen on the flower. The fly hovers over sand dune under a burning sun, gradually descends and quietly alights on the sand, but takes flight at once and hovers again. The hosts The parasite larva is found exclusive- ly within the cocoon of the ant lion, that is, Grocus bore (Tjeder) (=kuro-ko-usuba-kagero). There were 191 examined cocoons of G. bore, of which 97 were parasitized. Whereas in Distoleon con- tubernalis (MacLachlan) (=ko-kasuri-usuba- kager6), out of 45 cocoons examined, only 1 was parasitized. The data above were obtained during 3 years from 1933 to 1935, at Kinoto, Niigata Prefecture. In 1985-86, at Senami, Niigata Prefec- ture, there were 88 examined cocoons of G. bore, of which 17 were parasitized. The egg, Ist instar larva, and 2nd instar larva Fics. 33-36. Villa myrmeleonostena.(after Baba [1]). 33. The youngest larva on host pupal surface. 34. Full-grown larva. 35. Pupa. 36. Pupal head. (within the host body) The stages above are not found. However, it is certain that the 1st instar larva (=planidium) enters into the host larval body. The molting time into the 2nd instar is unknown. The larva must spend a long time within the host body, waiting for the host pupation. The larva upon the host pupal surface The youngest parasite larva found in the host cocoon is filiform and 4-5 mm long (Fig. 33). It crawls out from the host at the pupation time. When new host pupal thorax appears from the host larval cuticle, half or over half of the parasite body is usually seen already. It remains unknown whether the parasite larva at this time is the 2nd instar or the 3rd. It is always located at the venter of the host pupa between middle and hind legs and winds itself semicircularly round the host body. It becomes full-grown after 7-10 days (Fig. 34), passes a prepupal stage for 4-6 days upon the host body which is shrank completely, and then pu- pates. The pupa The parasite pupa (Figs. 35 and 36) bores a hole through the host cocoon and reaches to the ground. The head and thorax appear out of the sand and emergence from the pupa takes place there. The pupal period is somewhat more than 10 days. The habitat and life history of hosts Grocus bore (Tjeder) (=kuro-ko-usuba-kager6): The larva of this species is common at sand dune of the beaches, but is also seen at that of the rivers and lakes. It makes a pitfall. The larva, devoted whole 1 to 3 years to mature, cocoons and pupates from late June to early August. In August, a number of new, ant lion pitfalls are seen. Distoleon contubernalis (MacLachlan) (=ko- kasuri-usuba-kagerd): This species may not be regular as the host. The larva is seen both at seasides and at hilly places. At seasides, it lives in stationary sand dune under young pine trees. It does not make a pitfall. ACKNOWLEDGMENTS We wish to express our sincere thanks to Dr. Kanetosi Kusigemati (Kagoshima University) and Dr. Hiroshi Shima (Kyushu University) for their aid in various ways. Redescription of Villa myrmeleonostena 911 REFERENCES Baba, K. (1936) Studies of the ant lion, IV. On the bee fly parasitic upon the pupa of the ant lion. Shizen Kenkyu (=Nature Study), 6: 16-23. (In Japanese) Baba, K. (1953) Arijigoku no _ Seibutsushi (=Biology of the Ant Lion). Essa Entomol. Soc., Niigata, 107 pp. (In Japanese) Engel, E. O. (1932-37) Bombyliidae. 25. In “Die Fliegen der palaearktischen Region”. Ed. by E. Lindner, Vol. 4, Pt. 3. E. Schweizerbart’sche Ver- lagsbuchhandlung, Stuttgart, pp. 1-619. Hesse, A. J. (1956) A revision of the Bombyliidae (Diptera) of southern Africa. Part II; Part III. Ann. S. Afr. Mus., 35: 1-464, 465-972. Oldroyd, H. (1969) Diptera Brachycera. Section (a). Tabanoidea and Asiloidea. Handbooks for the identification of British Insects, Vol.9, Part 4. Royal Entomol. Soc. of London, 132 pp. Hull, F. M. (1973) Bee flies of the world: the genera of the family Bombyliidae. Smithsonian Institution Press, Washington, D. C. 687 pp. Hall, J.C. (1981) Bombyliidae. In “Manual of Nearctic Diptera”. Ed. by J. E. McAlpine, B. V. Peterson, G. E. Shewell, H. J. Teskey, J. R. Vock- eroth and D.M. Wood, Res. Br., Agric. Can. Monogr., 27(1): 589-602. Greathead, D. J. (1981) The Villa group of genera in Africa and Eurasia with a review of the genera comprising Thyridanthrax sensu Bezzi 1924 (Dip- tera: Bombyliidae). J. Nat. Hist., 15: 309-326. Bowden, J. (1975) Family Bombyliidae. In “A 10 11 WA 13 14 15 16 i, Catalog of the Diptera of the Oriental Region”. Ed. by M. D. Delfinado and D. E. Hardy, Vol. II. The University Press of Hawaii, Honolulu, pp. 165-184. Bowden, J. (1980) Family Bombyliidae. In “Cata- logue of the Diptera of the Afrotropical Region”. Ed. by R.W. Crosskey, British Museum (Nat. Hist.), London, pp. 381-430. Painter, R.H. and Painter, E.M. (1965) Family Bombyliidae. In “A Catalog of the Diptera of America North of Mexico”. Ed. by A. Stome, C. W. Sabrosky, W. W. Wirth, R.H. Foote and J.R. Coulson, U. S. Dept. of Agriculture, Washington, D. C., pp. 407-446. Nagatomi, A. and Soroida, K. (1985) The structure of the mouthparts of the orthorrhaphous Brachycera (Diptera) with special reference to blood-sucking. Beitr. Entomol., 35: 263-368. Theodor, O (1983) The Genitalia of Bombyliidae (Diptera). The Israel Academy of Sciences and Humanities, Jerusalem, 275 pp. Nagatomi, A. (1984) Male genitalia of the lower Brachycera (Diptera). Beitr. Entomol., 34: 99-157. Clausen, C. P. (1940) Entomophagous insects. McGraw-Hill Book Co., New York, 688 pp. du Merle, P. (1975) Les hoétes et les stades pré- imaginaux des Diptéres Bombyliidae: revue bibliog- raphique annotée. Bull. Sect. région. Quest Paldearctique (Organisation Internationale de Lutte Biologique). 1975 (4): 1-289. Steffan, J. R. (1967) Exoprosopa stupida (Rossi) parasite de fourmilions dans l’ancien monde (Dipt., Bombyliidae). L’entomologiste (Paris), 23: 78-79. cgniaot Wa sit mee i : nil his ee he SHIGE pai a it oe “eH} (04 at Miatonalta ; e ri _ nt Tidus DME AEE k Ne _ RA Helge dito, Seng sat to a . et cveehell Sather deeo tT ANicRe yal aad ce 91 ey 4 Rae1),. = este a to Biol? AX als het We iss ya AY i an ie aateatiae afte ay ae sl, OOS tag ge int. Hod Jina WOW Valea: pnb ea ag te Be) fyeGl mens aeeaArD 2004 Ft at: i mek le th Wate ane SEniRE Toll RY a Aevlsahanewe Sky reiahane he. at @ 122 eeoRr euedant aadrea nalLe SAH unllean Net a le ML p-boold Of doagtatsy lal oa it ine. 4 aia j Bat at SARA ORME hie Spel ae tha Shiels Moe hes (ater NASR eee itr. C3 cio RE Damion? I VaobsoA isevel ott sqneaay: . Th, Boon RL eshte. artiguarrn, 4 ‘auMinarmghhs; a oe RG AE aH Tassels oR (HBR ED, As rec eH abd i | a nf ag domigig a b ue ee (graeel). Payee, | aS RO AUT iagntt ghana a fOBeL i ae 2 poeak > " rea fr wins a Bae aH sa wor how ‘a tt if wits} ak ; tov a (evel) 4 SHAE tay" a welvchetih POY “Aabachaandtn Bn Sight i barter At ny Tove nein afte oMigtoy (eee ionptga Wi Sit ah ART AY Shah patiemenee suite LNBs 4b) Aa Carat ne (our Ria L Matas enioiuitt iat 60 isis as toy “Bb ShP ya me. Tete lige is) mloncpirya Sagrengons Patil y shew note | BERG RTSE eS (aie) 33) AOS TY Bet: Mien? wth err Oarien ce ey ty if re te a yl oo EEE Giant Cet ile y Mien ack ive ik Fh vrpnaig | Yh, ‘., haute” mit Serpe m ities ists { WR, wy iny ae ite 4 AG; " eo SALAM MAE, epee fut ; ven SE nett) i Hheaat Appt § hig ea | Rout area BOMBS | sregland, ue 1h ake \ 164 oe ka a ea ‘Hebd neyise SMe EUSA Tb em Fiomudivart reopensa diem a x nich 9 epee ia aah). v i aniglAoM. a’, Le, Mi hae, + a hae Vote Wr ee way" Seiad fa i te ‘ § ae | 04 07 vy iy : Bie if GS Why Ty eoir rg lie aid) # bie! jek wands ooh PSE ONE 22h eelE ta } “bball ct ae ee ZOOLOGICAL SCIENCE 4: 913-918 (1987) © 1987 Zoological Society of Japan Proposal of New Terminology for the Morphology of Nauplius Y (Crustacea: Maxillopoda: Facetotecta), with Provisional Designation of Four Naupliar Types from Japan TATSUNORI ITO Seto Marine Biological Laboratory, Kyoto University, Shirahama, Wakayama 649-22, Japan ABSTRACT—New terminology for the morphology of first stage nauplius y larvae, mainly for the plates on their cephalic shield, is proposed. The proposed terminology aids in the identification of plates which vary in place and/or shape among naupliar types, and simplifies the description of nauplius y larvae. Four nauplius y larvae from Japan which are cited to explain the terminology are provisionally designated as separate types, symbolized as VIII-a, IX, X, and XI, the last being for the nauplius which was formerly described as a larva of Hansenocaris pacifica Ito. INTRODUCTION The nauplius y type IV larvae originally de- scribed by Hansen [1] and later reviewed by Schram [2] are characterized by the sculpture made of many clear “plates” on the cephalic shield. Many undescribed nauplii which are more or less similar to type IV in cephalic-shield sculpture are present in Tanabe Bay on the Pacific coast of Japan, where three types of nauplius y larvae have already been reported [3]. During the past three years I have raised many different nauplii into “cypris y” stages in the laboratory (for an abstract of the first successful case see [4]). The results of these rearing experiments should be taxonomically useful because the current tax- onomy within the Facetotecta is based only upon the cypris y stage and remains tentative [5]. However, the lack of adequate terminology to describe cephalic-shield plates of different shapes and different arrangements poses an unexpected problem. Steuer [6] tried to identify plates of his material with their counterparts in Hansen’s type IV larva by labeling them alphanumerically, and Schram [2] named plates in an advanced manner (see Fig. 1A). Although Schram’s terminology, Accepted May 21, 1987 Received April 22, 1987 which was based upon the smallest form of his type IV larvae, seemed to be usable for my material, its application to various nauplius y larvae in Japan has often been very difficult even for larvae at the same stage (first naupliar stage) as his smallest form. Therefore, I have had to modify Schram’s terminology to make it more universal. This improved terminology for first stage larvae, which is described in the present paper, will facilitate our forthcoming descriptive works on nauplius y larvae. Provisional designation of naupliar types Three of the four nauplii cited in the present paper (Fig. 2) are selected from undescribed forms found in Tanabe Bay on the Pacific coast of Japan. They are provisionally named as types VIII-a, IX, and X, though they will be fully described in forthcoming papers. The other one cited in this paper is called type XI; this nauplius was formerly described by It6 [3] as a nauplius of Hansenocaris pacifica It6, 1985. Closer examination has re- vealed that this form is not the larva of H. pacifica but belongs to a separate species, the cypris y stage and the naupliar development of which are to be described elsewhere. Comments on larval stages and general morphology To judge from my successful rearing experi- 914 T. I76 Fic. 1. (B) for cephalic-shield plates. Diagrams showing relationship between Schram’s terminology (A) and new terminology The diagram A was redrawn from Schram [2] with permission. Abbreviations— A: axial; B: brim; C: crescentic; E: elongate; F: frontal; I: intercalary; M: marginal; O: occipital; P: polygonal; S: superlateral; W: window. ments, the number of naupliar stages of face- totectans is typically five. The four naupliar types from Japan cited in the present paper represent the first naupliar stages of four separate species. It is apparent from certain characteristics in the struc- ture of the cephalic shield that the original type IV larva described by Hansen [1] is also the first naupliar stage of a separate species. Characteris- tics available for identification of each stage will be dealt with elsewhere. In the present paper the term “cephalic shield” is used to denote a particular dorso-anterior part of integument of the nauplius y larvae. The remain- der of the integument is not yet named; I would like to call it “faciotruncal” integument. The border between these two parts is occasionally unclear, especially in a larva at an intermolt phase, so one might doubt the necessity for discriminating the cephalic shield from the faciotruncal integu- ment. However, they are actually separate units of the naupliar integument. After a molting, a nauplius y larva often leaves two partial exuviae; the dome-shaped one with no appendages is the cephalic shield and the other one with appendages is the faciotruncal integument. When a nauplius starts to molt, the cephalic shield splits from the faciotruncal integument except along its rear edge and is pushed up by the emerging body which exits through this anteriorly facing gap. The connection between these two parts of the cast integument is weak and they easily detach from each other; the true contour of the cephalic shield can be clearly determined by examining such a cast one. TERMINOLOGY As can be seen in Figure 2 (especially A and BE), the cephalic-shield plates principally form almost concentric circles interrupted by a midlongitudinal belt of plates. This latter belt is separated into two parts by a single special plate called “window” (abbreviated as “W”; in the following description, parenthesized symbols represent abbreviated forms of plate names); the plates which form the anterior belt are called “frontal” plates and num- bered toward the front (F-1 to F-4), and the plates which form the posterior belt are called “occipital” plates and are numbered toward the Terminology for Nauplius Y 915 Fic. 2. Diagrams showing cephalic-shield plates of four naupliar types from Japan and Hansen’s type IV larva. A: Type X; B: Type IX; C: Type VIII-a; D: Type XI; E: Hansen’s type IV (window is not illustrated in the original figure by Hansen). For abbreviations see Fig. 1. rear (O-1 to O-7). These frontal, window, and occipital plates may all be called “axial” plates (the reason is mentioned later). The following explanation refers to the plates (or plate groups) of only one half of the cephalic shield, because the plate arrangement is symmet- rical about the axials. The plates of all non-axial plate groups are numbered toward the rear. Two plates which are placed alongside some of the anterior occipitals are called “crescentic” plates (C-1 and C-2). Two plates which are placed antero-lateral to the crescentics are called “elongate” plates (E-1 and E-2). Three plates which are placed antero-lateral to the elongates are called “intercalary” plates (I-1 to I-3). Six plates which border the outer sides of the interca- laries, E-2, C-2, and a few posterior occipitals are called “polygonal” plates (P-1 to P-6). Seven plates which border the outer sides of the polygo- nals are called “marginal” plates (M-1 to M-7). 916 T. IT6 There is a triangular plate which wedges itself between M-2 and M-3; it is called the “superlater- al” plate (S). The externalmost part of the cephalic shield except for its rear edge (the rear edge of O-7) is called “brim” (B) as a whole (Fig. D©): FURTHER EXPLANATION AND DISCUSSION Schram’s “axial” plates (Al to Al1; in Schram’s terminology, an abbreviated plate name is repre- sented by a combination of a capital letter and a number with no hyphen between them) accord with the longitudinal belt of frontal, window, and occipital plates of my terminology (cf. Fig. 1A and B). The reason for dividing this simple belt of plates is given below. With a light microscope we usually observe the cephalic shield which is mounted horizontally on a slide glass in dorsal view, not in frontal (anterior) view. To observe a cast cephalic shield in frontal view is actually impossible. This practical condi- tion causes a difficulty in the observation of anterior plates. In certain nauplii the frontal region is rounded and in normal preparations is situated almost parallel to the line of sight. In such specimens, anterior axial plates appear to be closely appressed and accurate observations of their borders are difficult. This problem is more severe at later developmental stages because their axial plates are changed into numerous smaller plates by division. If Schram’s terminology, in which the anteriormost plate is labeled the first axial (A1), were applied to nauplii whose first axial could not be surely ascertained, no axial plate could be confidently labeled, even an easily observable one on the top of the cephalic shield. My system avoids this difficulty because it starts from an easily identifiable plate, namely the window, which is placed almost on the top of the cephalic shield. As already reported by Ito [3], in two types of nauplius y from Tanabe Bay the window is a special plate above the nauplius eye; it is characterized by its smoothness (though partially filled with a mesh-like texture in certain nauplii), whereas other plates are usually not smooth, but of mesh-like texture. Although the window is usually detectable in the first stage nauplius y larvae as shown in Figure 2, it is possible that we meet unusual nauplii in which the window is not detected. For describing such unusual nauplii the term “axial” in the sense of Schram would be still available and I think it should be reserved. It is also useful as a general term for a context in which there is no necessity to distinguish frontal, window, or occipital plates; in fact, I have already used it in this sense in former paragraphs. Hansen [1] did not indicate the window in his type IV nauplius, but there is little doubt that it had a window because other similar nauplii have the window (Schram’s larva 2 from Akeroya [2]; the nauplius illustrated by Steuer [6] based upon the material from Adriatic Sea). In Figure 2E, I indicate a possible situation of its window with reference to Schram’s larva 2 from Aker6ya. Schram clearly illustrates the window in his type IV larvae (especially in his Fig. 3E), and also mentions that “a circular platelete is present inside the original axial plate No.5.” The circular platelet (misspelled as “platelete”) in his sense accords with the window. However, the window is not always a platelet embraced by A5 (0-1 in my terminology). The window of the type X nauplius is placed medially just between F-1 and O-1 (Fig. 2A), and it would be difficult to say that the window belongs to O-1. In the type IX nauplius (Fig. 2B), O-1 is subdivided with a faint ridge and the window is wedged deeply between the halves. In contrast, the window of the type XI nauplius does not appear to wedge itself deeply into the O-1, which is also subdivided as in type IX. The window of the type VIII-a nauplius is encircled with a single large plate, which seems to be a fusion of F-1 and O-1. Such a fused plate encircling the window can be called a “circum- window” plate. Of the “crescentic” plates, C-—1 is the same as Schram’s single “crescentic” plate. However, I call a plate posterior to it the second crescentic (C-2), while Schram calls it “P7”, namely the seventh polygonal. The main reason for considering Schram’s P7 a “crescentic” plate is that the P7 has a tendency to fuse with the crescentic (cf. Fig. 2A, B, C). Another reason can be understood best in connection with the other polygonals sensu Terminology for Nauplius Y 917 Schram, which are dealt with later. The first elongate plate (E-1) accords with the homonymic plate (E1) in the sense of Schram, but E-2 is called P5 in Schram’s terminology. As can be seen in types IX and XI nauplii (Fig. 2B, D), E1 and P5 sensu Schram are fused with each other; hence, I treat P5 as an “elongate” plate. Similarly, P2, E2 and P3 have a tendency to fuse (cf. Figs. 1 and 2) and are regarded as members of the same plate-group. As there is no available name for them in Schram’s terminology, the new term “intercalary” is introduced. Among the intercalary plates of the type XI nauplius, I-2 and I-3 seem to form a unit isolated from I-1 (Fig. 2D). Similar arrangements are also seen in the other nauplii shown in Figure 2; for example, in the type IX nauplius (Fig. 2B), I-2 and I-3 are broadly connected with each other, while the connection between I-1 and I-2 is very narrow. In the intercalary plates, type X and Hansen’s type IV nauplii are similar to type IX. In the type VIII-a nauplius, the three intercalary plates are fused with each other, but I-1 is still discernible from the others because the fused plate has a neck which indicates the border between I-1 and I-2. My concept of the polygonal plates differs greatly from Schram’s. Schram’s polygonal plates occur as two groups separated by a radial row of three “elongate” plates (Fig. 1A). In types VIII—a and XI nauplii, there is no such radial row of plates; hence, elongate plates in the sense of Schram would be meaningless for these nauplii. Schram numbered the plates within the posterior group of polygonals outwards as he supposed transversal rows. However, such numbering is impossible for types IX, VIII-a and XI nauplii. I find it much more useful to recognize a row of plates which originates from P1 (the same as P-1) and extends posteriorly; thus, Schram’s P2, P3, PS, and P7 are abandoned and, instead, a continuous row of six polygonal plates (P—1 to P—6) is formed. In the type VIII-a, five polygonal plates (P-2 to P-6) are almost fused with each other, which demonstrates the usefulness of this grouping. The marginal (M-1 to M-7) and superlateral plates are the same as the homonymic ones in Schram’s terminology. The brim is also the same as the homonymic part in Schram’s terminology, though I do not yet recognize any first stage nauplius which has plates within the brim (Fig. 2 and [3]). The brim is usually not seen in dorsal view, but in the type VIII—a larva it is clearly seen even in dorsal view, except for an anterior portion (Fig. 2C). The type XI nauplius has a peculiar single plate which has two “arms” extending posteriorly along- side the window, O-1, O-2, and O-3 plates (Fig. 2D). There is no doubt that this plate is made by fusion of F-1 and a pair of C-1 plates. A similar plate with two “arms” is present also in the type VIlI-a nauplius (Fig. 2C), but it is made of F-2 fused with the E-1 and E-2 plates on each side. Among the four nauplii from Tanabe Bay shown in Figure 2, the one closest to Hansen’s type IV nauplius in the sculpture of the cephalic shield is the type X nauplius, although the proportions of its cephalic shield are greatly different. In contrast to these two similar nauplii, whose cephalic-shield plates may be regarded as the model of the proposed terminology, the other nauplii show various degrees of modification, mainly due to fusion and slight translocation of plates as already mentioned. However, it is important to acknowl- edge that their modified plates can readily be identified using the proposed terminology. These nauplii may be assumed to be closely related compared with other nauplii whose cephalic-shield sculpture can not be described using this terminol- ogy, like the type I nauplii described by Hansen [1] and Schram [7] or the Pacific type I described by Itd [5]. I expect that such similarity and dissimilar- ity in the fundamental structure of the cephalic- shield sculpture will be reflected in the taxonomy within the Facetotecta in the future. The proposed terminology, which has been elaborated here specifically for application to the first naupliar stage, is also available for later naupliar stages to a certain extent, although it must be supplemented by other terms. An adaptation of this terminology to later stages will be demon- strated in a forthcoming paper which is in prepara- tion. 918 T. [76 ACKNOWLEDGMENT I would like to express my sincere thanks to Dr. Mark J. Grygier for reviewing the manuscript. This study is supported in part by the Grant-in-Aid for Scientific Research, No. 62540567, from the Ministry of Educa- 4 tion, Science and Culture, Japan. REFERENCES 1 Hansen, H. J. (1899) Die Cladoceren und Cirripe- dien der Plankton-Expedition. Ergebnisse der Plank- ton-Expedition der Humboldt-Stifung, 2 (G.d): 1-58, 7 pls. I-IV. 2 Schram, T. A. (1972) Further records of nauplius y type IV Hansen from Scandinavian waters. Sarsia, 50: 1-24. It6, T. (1986) Three types of “nauplius y” (Maxillo- poda: Facetotecta) from the North Pacific. Publ. Seto Mar. Biol. Lab., 31: 63-73. It6, T. (1984) Nauplius y and cypris y (problematic crustacean larvae) from Japan. Zool. Sci., 1: 1000. It6, T. (1986) A new species of “cypris y” (Crustacea: Maxillopoda) from the North Pacific. Publ. Seto Mar. Biol. Lab., 31: 333-339. Steuer, A. (1905) Uber eine neue Cirripedien larve aus dem Golfe von Triest. Arb. Zool. Inst. Univ. Wien, 15: 113-118. Schram, T. A. (1970) On the enigmatical larva nauplius y type I Hansen. Sarsia, 45: 53-68. ZOOLOGICAL SCIENCE 4: 919-927 (1987) © 1987 Zoological Society of Japan Rediscovery of Heptacarpus jordani (Rathbun) with Notes on Morphological Variations (Decapoda, Caridea, Hippolytidae) KeEN-IcH1 HAYASHI and TosHiRo CuIBA! Department of Aquaculture and Biology, Shimonoseki University of Fisheries, Shimonoseki, Yamaguchi 759-65, and 'Kesennuma Municipal Office, Kesennuma, Miyagi 988, Japan ABSTRACT—Heptacarpus jordani (Rathbun) is rediscovered from northern Japan. Based on samples collected during one year from Zostera belts, morphological variations and population growth are examined. A part of the key to the Asian species of the genus is revised. INTRODUCTION During the survey of the shore fauna in Kesen- numa City, Miyagi Prefecture, northern Japan, a considerable number of specimens representing a single species of the hippolytid genus Heptacarpus were collected. They belonged to the species group with an epipod on the first three pereopods, and resembled H. jordani (Rathbun), reported originally from Hokkaido in 1902 [1], and subse- quently from Sagami Bay in 1914 [2], but there- after no material has been collected [3]. Variations in some important characters of these specimens, however, made the specific identification rather difficult. Thus, three specimens were sent to the Smithsonian Institution, where the holotype was deposited. Direct comparison revealed some dis- crepancies between them. In order to examine the morphological variability, we collected this species monthly for one year from Hajikami-akedo, near the mouth of Kesennuma Bay. The examination of many specimens proved that the present mate- rial represented the rediscovery of H. jordani after 70 or 80 years and demonstrated that this species showed considerable morphological variations. Hayashi [3] presented a key to the Asian species of the genus Heptacarpus, but it is necessary to revise that of the species group with an epipod on the first three pereopods. Accepted May 22, 1987 Received September 26, 1986 MATERIALS AND METHODS Material was collected from three localities at Kesennuma City, Miyagi Prefecture by one of the authors (TC). The monthly collection was made from littoral Zostera belts at Hajikami-akedo, just outside of the mouth of the Kesennuma Bay during October, 1983 to September, 1984. The scoop net used there had a 60 cm X60 cm mouth frame and covered with fine net of 6mm mesh. The intertidal vegetation at a depth of about 0.5- 1.0 m at low tide was swept over an area of about 200 m X50 m with this net by hand. Small collec- tions were added from two other localities both facing the Kesennuma Bay: at Tajiri, Oshima Island, where specimens were collected from Sargassum belts by another small hand net, and at Nagaiso-hama, the shrimps were obtained from growing cages of the abalone juveniles hanging in the area. After preservation in 10% neutral formalin, the samples collected prior to October, 1983 were examined morphologically in detail and the other samples were used for studies of morphological variation and of population growth including fecundity, breeding season and recruitment. The specimen size was represented by the carapace length (cl), from the orbital margin to the posterior end of the carapace. Three specimens (18, 22) were deposited in the collection of the United States National Museum of Natural His- tory (USNM), and 14 specimens (11%, 1 ovig. ¢, 920 K.-I. HAYASHI AND T. CHIBA 2’) in the Shimonoseki University of Fisheries (SUF). The specimens with the abbreviations in parentheses are deposited in the respective institu- tion. The remaining material without abbreviation is in the private collection of the author (TC). Hajikami-akedo, Apr. 28, 1971-12 (SUF); Mar. 6, 1982-68 (SUF); June 27, 1983- ¢ (USNM), 4% (SUF), 1 ovig. 2 (SUF), 1? (USNM), 22 (SUF); Oct. 5, 1983-179, 419; Nov. 19, 1983 - 60% , 232; Dec. 19, 1983 - 439, 4 ovig. 2, 207; Jan. 20, 1984 - 219, 11 ovig. $, 49; Feb. 15, 1984 -12¢,5ovig. 2,69; Mar. 15, 1984 - 13%, 4 ovig. 2,29; May 4, 1984-29, 4 ovig. 7, 12; May 19, 1984-62, 3 ovig. $; June 16, 1984-124, 2 ovig. 2, 42; July 28, 1984-59, 22, 13 juv.; Aug. 25, 1984-29, 462, 15 juv.; Sept. 22, 1984-10¢, 554, 6 juv. Tajiri, Sept. 5, 1982 - 212 (USNM); Dec. 12, 1983 - 210,12. Nagaiso-hama, Jan. 14, 1984-1¢,14. RESULTS Heptacarpus jordani (Rathbun) (Figs. 1-4) Fic. 1. Spirontocaris jordani Rathbun, 1902, p. 44, fig. 17 [1]. Spirontocaris jordani: Balss, 1914, p. 44 [2]. Heptacarpus jordani: Holthuis, 1947, p. 12 [4]. Heptacarpus jordani: Hayashi, 1979, p. 25 [3]. Diagnosis: Moderate size species (Fig. 1). Females larger than males. Rostrum shorter than carapace, with 5-9 teeth on dorsal margin and 1 or 2 teeth on ventral margin; posterior 1 or 2 dorsal teeth usually situated on carapace (Fig. 2a); ven- tral teeth situated near rostral apex. Carapace smooth, always with antennal spine. Pterygosto- mial spine usually present in females and present or absent in males (Fig. 2b). Abdomen smooth. Pleuron of fourth somite usually not pointed, but that of fifth somite always pointed (Fig. 2c). Telson with 3-6, usually 4, pairs of spines. Posterior margin pointed at middle, flanked by 2, or rarely 3, pairs of spines and 1, or rarely 2, pairs of plumose setae (Fig. 2d). These spines and setae sometimes arranged asymmettri- cally. First segment of antennular peduncle with 2-6, usually 4, spinules on distal margin. Large spine present on second and third segments (Fig. 2a). Mouthparts typical of the genus (Fig. 3). First pereopod rather elongated in mature males. ) oS Zs Heptacarpus jordani (Rathbun). Male, 5.0mm in carapace length. Scale represents 2.0 mm. 921 Rediscovery of Heptacarpus jordani ws > ~S (Cm 3 (7 ( "rae Fic. 2. Body parts of Heptacarpus jordani (Rathbun). a-c, female, 7.3mm in carapace length (cl); d, female, 7.6mm in cl; e, f, female, 8.9mm in cl; g, h, male, 6.0mm in cl. a, anterior part of body in dorsal view, left eye and setation of right antennal scale removed; b, anterior part of carapace in lateral view; c, third to sixth abdominal somites; d, tail fan, setation of uropods omitted; e, first pereopod; f, distal four segments of third pereopod; g, first pleopod; h, endopod of second pleopod. Scales for a-f represent 2.0 mm and scale for g, h represents 1.0 mm. Merus usually with a subterminal spine in both sexes (Fig. 2e). Meri of last three pereopods with 1-3 spines, usually 2 or 3 spines on third (Fig. 2f), 1 or 2 spines on fourth and single spine on fifth pereopod. Endopod of male first pleopod elongated, distal part slightly broadened with some retinacula (Fig. 2g). Appendix masculina on male second pleopod shorter than appendix interna, with many long stiff setae (Fig. 2h). The live individuals are unicolored, without stripes or bands, but rather variable from light- brown to dark-brown or dark green. In general the color merges into the habitats, from which they were collected, and is lighter in males than in females. The antennal flagella are always light 922 K.-I. HAYASHI AND T. CHIBA \ Y if la J E = Fic. 3. Mouthparts of Heptacarpus jordani (Rathbun). Male, 4.3 mm in carapace length. a, mandible; b, first maxilla; c, second maxilla; d, first maxilliped; e, second maxilliped; f, third maxilliped. Scales represent 1.0 mm. TABLE 1. Variations in rostral formulae of Heptacarpus jordani (Rathbun) Rostral formula No. of specimens (Comalivonimal) Male (%) Female (%) Juvenile (%) 6/0 0 1( 0.4) 0 10/1 C0) 0 0 9/1 (024) 15 ( 6.5) 0 8/1 96 (45.3) 100 (43.3) 2( 5.9) 7/1 99 (46.7) 85 (36.8) 14 (41.2) 6/1 9( 4.2) 18 ( 7.8) 15 (44.1) 5/1 0 0 3( 8.8) 9/2 0 1( 0.4) 0 8/2 2( 0.9) 7 ( 3.0) 0 We 0 8) (Jigs) 0 6/2 0 1( 0.4) 0 Total 212 231 34 Rediscovery of Heptacarpus jordani brown and the distal parts of the posterior pereopods are somewhat lighter than the other parts of body. Variations: A total of 495 specimens, 217 males, 244 females including 34 ovigerous females and 34 juveniles, were examined for morphologi- cal variations of some important characters. They showed considerable variations and differed partly from the holotype. The carapace length of the largest specimens TABLE 2. 923 was 6.2 mm in male and 10.2 mm in female. The ovigerous females were 6.1-8.6 mm in cl. The variations in the rostral formulae are shown in Table 1. The combination of 7 or 8 dorsal with 1 ventral teeth was common in both sexes, more than 90% in males and 80% in females, but the juveniles had commonly 6 or 7 dorsal teeth. Appearance of spines on the pterygostomial angle varied with sexes (Table 2). The juveniles were all provided with a spine on both sides. The females were also provided with the spine in most Presence or absence of a spine on pterygostomial angle, fourth abdominal pleuron and merus of first pereopod of Heptacarpus jordani (Rathbun) Pterygostomial angle Fourth abdominal pleuron No. of specimens Merus of first pereopod Male Female Juv. Male Female Juv. Male Female Juv. (%) (%) (%) (%) (%) (%) (%) (%) (%) Present on 76 208 34 2 18 1 202 234 30 both sides C520) (8523) ew (100) (OLS) i (723) ee (239) (94.4) (95.9) (100) Present on 21 32 10 21 6 S) 9 one side 9a) (13h) CAN eS) vey) G42) (assy) Absent on 120 4 205 206 Zi 3 1 either side (S58) eG JES) (94.5) (84.1) (79.4) (1.4) (0.4) Total Zale, 244 34 ZAG 245 34 214 244 30 TABLE 3. Variations of dorsal spines on telson and marginal spinules on first antennular segment of Heptacarpus jordani (Rathbun) Spines or spinules 6-6 6-5 6-4 5-5 5-4 5-3 4-4 4-3 Me) 33 40 3-1 De) Telson Male Female 0 2 3 4 25 24 1 0 150 185 9 Oe, 1 1 fe 3 1 0 No. of specimens Total (%) 20.5) 7( 1.6) 49 (11.2) 1( 0.2) 335 (76.5) 36 ( 8.2) 2 (0.5) 5( 1.1) G02) Antennular peduncle Male Female 1 DD 3 4 23 19 60 61 4 6 93 87 26 46 1 0 5) Si/ 1 12 0 1 0 3 Total (%) 3( 0.6) 7( 1.4) 42 ( 8.5) 121 (24.4) 10 ( 2.0) 180 (36.4) 72 (14.5) 1( 0.2) 42 ( 8.5) 13 ( 2.6) 1( 0.2) 3( 0.6) SE In asymmetrical number of spines or spinules, the arrangement does not precisely represent the side of the actual specimen. 924 K.-I. HAYASHI AND T. CHIBA specimens, and it was restricted to one side only in 32 specimens (13.1%) or entirely absent on both sides in 4 specimens (1.6%). On the other hand, the males may have a tendency to lose this spine with growth. More than half of the males ex- amined had no spine on either side. Nearly 16% of males less than 3.9 mm in cl had no spine, but this ratio increased to 42% at 4.0-4.4mm, 62% at 4.5-4.9 mm and finally 76% in those larger than 5.0 mm in cl. The pleural spine on the fourth abdominal somite was rather variable (Table 2), but usually absent in most specimens (88%) that showed no sexual dimorphism. In most specimens of both sexes, the merus of the first pereopod was provided with a single subterminal spine on both sides in more than 95%. A few, only 3 males and 1 female, had no such spine on either side (Table 2). The spines on the telson, usually 4 pairs, were rather variable from 2 to 6 spines, which were usually paired or sometimes asymmetrically arranged (Table 3). The first antennular peduncle was provided with 1 to 6 marginal spinules on one side, and usually with 4 spinules but 3 or 5 spinules were also observed in considerable ratio (Table 3). The outer spines on the meri of the last three pereopods were | to 4, usually 2 or 3, on third, 1 to 3, usually 1 or 2, on fourth and usually 1 spine on fifth pereopod. Biology: The carapace length composition of H. jordani collected monthly from Hajikami- akedo during October, 1983 to September, 1984 is shown in Figure4. The ovigerous females appeared first in December and the ratio of them to the adult females was 18%. This value rapidly increased to more than 70% in January and the high percentage continued to May, except Febru- ary. The breeding season ended in June. The new recruits, 1.9-2.6 mm in cl, appeared from July to September. The male is distinguished by the differentiation of the appendix masculina when they grew up to 2.7 mm in cl. Sexual size differences already appeared in September, though not large. After then animals gradually grew up and the differences became conspicuous. esq Juvenile 20? Nov. 19 (N=83) ge : Le 15 (N23) {es 15 (N=19) R (N=7) stay 19 (N=9) | June 16 (N=18) A of DUC gue 4 INn5ROL GUAT ANO NENG NNAO CARAPACE LENGTH (mm) Fic. 4. Carapace length composition of Heptacarpus jordani (Rathbun) collected from Zostera belts at Hajikami-akedo, Kesennuma City during the period from October 1983 to September 1984. The mean carapace length attained 6.0mm in females and 4.5mm in males in November. The males attained about 5.0 mm in January, while the females continued to grow until February, in which they were as large as 8.0 mm. The largest male was 6.2mm in cl and nearly the same size as the smallest ovigerous female. From December to early May the sexual size differences attained 2.6 to 3.0 mm. In late May the larger animals of both sexes disappeared and the mean size value de- creased considerably. The males recovered the value in June or July, when the females, however, were nearly 1.0mm smaller than those in early Rediscovery of Heptacarpus jordani 925 May. The female-male ratio varied from month to month and the male dominated from November to June, except February and early May. Based on 11 ovigerous females (6.1-8.2 mm in cl), 6 collected in January, 1 in May and 4 in December, number of eggs and egg size were examined. The total number of eggs of these females was counted, and 10 eggs selected at random from each ovigerous female were meas- ured the long and short diameters with the micrometer. Two specimens less than 7.0 mm in cl bore only 350 and 653 eggs, respectively. The remaining 9 specimens, more than 7.4 mm in cl, bore 866-1111 (mean 991) eggs. The log of egg numbers shows a significant linear correlation with the log of the carapace length of the female (p<0.05). The fitted regression equation is given by E=5.4049L7°°” (r=0.68), where E is number of eggs and L is the carapace length of the mother shrimp in mm. The short diameter was 0.58-0.72 (mean 0.64+0.03) mm, and became slightly larger in eggs incubated in large number. The long diameter was 0.70-0.98 (mean 0.83+0.06) mm, and became larger in the advanced developmental stages, though the short diameter did not change during developmental stages. Other shrimps collected together with H. jorda- ni were the following eight species; Heptacarpus futilirostris, H. grebnitzkii, Eualus leptognathus, E. sinensis, Spirontocaris ochotensis, Pandalus prensor, Crangon affinis, and Metacrangon angus- ticauda. H. jordani and H. grebnitzkii were the dominant shrimps in littoral vegetations at the present research fields. DISCUSSION Dr. Fenner A. Chace, Jr. of the Smithsonian Institution examined the holotype and compared it with a part of the present material. The holotype is a large female, 9.2 mm in cl, and shows differences in two important characters from most specimens of the present material. The meral spine on the first pereopod has been thought to be one of the key characters in the genus Heptacarpus and used to separate two related species from each other, such as absent in H. futilirostris, while present in H. rectirostris [5|. This character, however, varies slightly in the present species as stated previously. More than 95% of the specimens have a single such spine, but the holotype has no spine on either side as in 4 specimens of the present material. The pleural spine on the fourth abdominal somite is present on both sides in the holotype, though nearly 90% of the samples have no spine. Similar differences were shown in the case of H. commensalis {3}. With regard to these two points, no specimens showing the combination of characters noted in the holotype were found among nearly 500 specimens examined. The holotype, therefore, seems to be a rather aberrant form. There are no other distinct discrepancies between the holotype and the pres- ent specimens. The breeding season of this species was mainly in the winter and spring, like the most Japanese members of this genus [6-10] and the related genus Eualus [11], and H. pictus from the East Pacific Ocean [12]. Judging from the breeding season and the carapace length distribution, the life span of this species is thought to be 12 months at Kesennuma area. In comparison with other species the ovigerous females were rather scarce, which may indicate that spawning or releasing larvae takes place in deeper waters. Two peaks of the ovigerous season, which were observed in H. pandaloides at Ishinomaki Bay (Kurata [6] as H. propugnatrix) were not apparent in the present species. H. jordani bears an epipod on all maxillipeds and the first three pereopods. This character is shared with five Asian species: H. commensalis, H. futilirostris, H. grebnitzkii, H. minutus and H. rectirostris. The most striking feature, by which H. jordani is separated from five other species, is the presence of 3-5, usually 4, marginal spinules on the first antennular segment. A single spinule is present in H. commensalis, H. futilirostris and H. rectirostris and 2 spinules in H. grebnitzkii, though the spination is uncertain in H. minutus. Of these six species the rostral formulae differ markedly in H. minutus with many ventral teeth, and the arrangement differs in H. grebnitzkii by having dorsal and ventral teeth restricted in the central or proximal part. H. jordani is similar to 926 K.-I. HAYASHI AND T. CHIBA the three other species in number and arrangement of the teeth. In H. futilirostris males are much larger than females but in H. commensalis, H. grebnitzkii and H. rectirostris females are larger than males like the present species. The sexual size difference of H. minutus is uncertain, because it has been known from the male holotype only. H. commen- salis is much smaller than the other species, only 3.3 mm in cl of the ovigerous female. In view of the morphological variations of H. Jordani, it is necessary to partly revise the key to the Asian species of the genus. The following key to the Asian Heptacarpus species with an epipod on all maxillipeds and the first three pereopods is offered to modify the couples after couplet 5 of the key adopted by Hayashi [3]. 5 Rostrum longer than carapace, with 6 teeth on dorsal margin and 7 teeth on ventral margin. First pereopod without meral spine. Pleuron of fourth abdominal somite without spine. AOR TAL ETI), Se H. minutus (Yokoya) 5 Rostrum with fewer than 4 teeth on ventral MAE OTe NE. coed Re ETE 6 6 Rostrum longer than or as long as carapace. 2-4 teeth present on central part of ventral margin. First antennular peduncle with 2 spinules. First pereopod with meral spine. Pleuron of fourth abdominal somite without spine. Males smaller than females. ie augue ae cones Stree H. grebnitzkii (Stimpson) 6 Ventral rostral teeth present distally. ....... 7 7 First antennular peduncle with 3-5 marginal spinules. Rostrum shorter than carapace, with 7-8, rarely 6 or 9, teeth on dorsal margin and 1 or 2 teeth on ventral margin. First pereopod usually with meral spine, but rarely absent. Pleuron of fourth abdominal somite usually without spine. Males smaller than females. SER PEA icant be CURNNGE H. jordani (Rathbun) 7 First antennular peduncle with single marginal spinule eee A Pe a ee OE RRs 8 8 First pereopod with meral spine. Pleuron of fourth abdominal somite with spine. Rostrum with 5-6 teeth on dorsal margin and 3-4 teeth on ventral margin. Males smaller than females. POLIS da, AERIS ae ae H. rectirostris (Stimpson) 8 First pereopod without meral spine. ........ 9 9 Body large. Rostrum shorter than carapace. Mature males larger than females with elon- gated third maxilliped and strengthened first pereopod. Free living, usually in coastal weed Beltstece 8 ose et a. H. futilirostris (Bate) 9 Body small. Rostrum as long as or longer than carapace. Mature males smaller than females and any thoracic appendages neither elongated nor strengthened. Usually commensal with littoral coelenterates. OES, a AUER Saat H. commensalis Hayashi ACKNOWLEDGMENTS We wish to express our gratitude to Dr. Fenner A. Chace, Jr., of the Smithsonian Institution for examining the holotype of Spirontocaris jordani Rathbun and comparing it with a part of our material, as well as critical reading of the manuscript. We are indebted to Mr. H. Onodera of the Hashikami Fisheries Cooperative Asso- ciation for collecting the shrimps at Nagaiso-hama. REFERENCES 1 Rathbun, M. J. (1902) Japanese stalk-eyed crusta- ceans. Proc. U.S. Nat. Mus., 26: 23-55. 2 Balss, H. (1914) Ostasiatische Decapoden. II. Die Natantia und Reptantia. Abh. Bayer Akad. Wiss., Suppl. 2 (10): 1-101, pl. 1. 3. Hayashi, K. (1979) Studies on hippolytid shrimps from Japan—VII. The genus Heptacarpus Holmes. J. Shimonoseki Univ. Fish., 28: 11-32. 4 Holthuis, L. B. (1947) The Decapoda of the Siboga Expedition. Part IX. The Hippolytidae and Rhyn- chocinetidae collected by the Siboga and Snellius Expeditions with remarks on other species. Siboga Exped. Monogr., 39a°: 1-100. 5 Miyake,S. and Hayashi, K. (1968) Studies on hippolytid shrimps from Japan—IV. Two allied species, Heptacarpus rectirostris (Stimpson) and H. futilirostris (Bate), from Japan. J. Fac. Agr. Kyushu Univ., 14: 432-447. 6 Kurata, H. (1963) Ecology of shrimps on eel-grass bed. I. Spirontocaris propugnatrix. Bull. Hokkaido Reg. Fish. Res. Lab., 26: 81-85. 7 Kurata, H. (1968) Larvae of Decapoda Natantia of Arasaki, Sagami Bay—II. Heptacarpus futilirostris (Bate) (Hippolytidae). Bull. Tokai Reg. Fish. Res. Lab., 55: 253-258. 8 Kurata, H. (1968) Larvae of Decapoda Natantia of Arasaki, Sagami Bay—III. Heptacarpus geniculatus (Stimpson) (Hippolytidae). Bull. Tokai Reg. Fish. 10 Rediscovery of Heptacarpus jordani Res. Lab., 56: 137-142. Yamashita, K. and Hayashi, K. (1979) Larvae of Decapoda Macrura in the vicinity of Miyazima, the Seto Inland Sea, I. Heptacarpus _ rectirostris (Caridea, Hippolytidae). Proc. Jap. Soc. Syst. = Zool. 7245-51. Yamashita, K. and Hayashi, K. (1980) Larvae of Decapoda Macrura in the vicinity of Miyazima, the Seto Inland Sea, II. Heptacarpus pandaloides (Stimpson) and H. geniculatus (Stimpson) (Caridea, iI 12 927 Hippolytidae). 16-23. Oya, F. and Oka, K. (1985) Growth and breeding ecology of the hippolytid shrimp Eualus sinensis (Xi) Zool Scien 251257263. Bauer, R. T. (1976) Mating behavior and sperma- tophore transfer in the shrimp Heptacarpus pictus (Stimpson) (Decapoda: Caridea: Hippolytidae). J. Nat. Hist., 10: 415-440. Proc. Jap. Soc. Syst. Zool., 19: i cae ue wane ee caine gir! point Llwiflvwentlr (RID: Snes aga hai aeas’. tag anti errant att he )yaelaoy pal . ni), tag AVES Wee A Sa om RE a bbe oly eelod ‘ait, (Oren 7 sith rat ~ ah a! 4% ciematia "ont ih a ate th maene Stare (mers ; eb Gag INRHEPS Loe ay (abontapeye® Hy FOE EY vrs, Fo Ae tenahe th ORL BY Sevres: ehyalbtiqna candy : Vik ; ALLS hi Huey t f ti thy (rt! aw gf B) Cane ae) t" re oH EYRE OmUDR eA : ti " asker: VOT h ' Ley oth 7%, vign ete nt hie Ke at: en Gruath { MOR PCeS! hy i aes , #4 \ i oe ee ' 3) (eh ea hy ri 4 } ig woe Ly Fee iso | a if ar i, i pe e) 7 ‘ 5. © é he FL) i} P ; i Lae He rey ; : t ts wy ‘Gy in Bid Lr, 4 ‘ i i i), y al 1 F 1) " * F fy ’ 1 BA , 6) ie Ba } ." i f TL ' } : va ee " ¥ eae Toleu Tite 2 #\ caw An wb h Sere fe : Le | hoes : $ ‘ y he at ] ey ‘ bike Leal MM | n f i ' ian i sh ae ‘ ve te ty I if ae rads ei 4 ue i + tae rm! i 4 " bi ; fy H g i = r ‘ " Hf { jis vi i y : if thy < ie oar o Me h , i ; ry ¢ " i at i wi ‘ t r ay j i p a ZOOLOGICAL SCIENCE 4: 929-931 (1987) [COMMUNICATION] © 1987 Zoological Society of Japan In Vitro Synthesis of Connectin in an Extract of Chicken Embryo Muscles ATSUSHI ASAKURA!*, YoICHI NABESHIMA~ and KoscaK MARUYAMA2”” ‘Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo 170, and *Department of Biology, Faculty of Science, Chiba University, Chiba 260, Japan ABSTRACT — Incorporation of *°S-methionine into con- nectin during protein synthesis in an extract from chicken embryo muscles was detected by autoradiography after immunoprecipitation with anti-connectin antibodies fol- lowed by SDS gel electrophoresis. Any radioactivity of peptides smaller than connectin but larger than actin was not detected with anti-connectin antibodies reacted samples. On the other hand, incorporation of radioactive methionine into nebulin was not observed. INTRODUCTION Connectin (also called titin) is a very long, elastic filamentous protein of striated muscle and its MW is estimated as huge as 2.8 million from SDS gel electrophoresis mobility (cf. review [1]). Whether connectin is comprised of a single peptide or not has as yet remained uncertain. In the present study, it was attempted to observe in vitro synthesis of connectin by the incorporation of °S-methionine in a chick embryo muscle extract. Autoradiography of anti-connectin antibodies reacted sample revealed a faintly radioactive band corresponding to connectin. MATERIALS AND METHODS Breast muscles from five chick embryos incu- bated for 15 days were gently homogenized in an Accepted June 16, 1987 Received June 3, 1987 > To whom reprint requests should be addressed. equal volume of cold salt solution containing 0.25 M KCl, 10mM MgCl, 6mM 2-mercaptoethanol and 10mM_ Tris-HCl buffer, pH7.4. The homogenate was centrifuged for 7 min at 7,000 xg, and the supernatant was subjected to Sephadex G-25 column (1 x 15 cm), using the salt solution as elutant. Amino acid free protein-rich fractions including polysomes were collected for protein synthesis assay [2]. A reaction mixture, 0.2 ml, containing muscle extract, 0.15M KCl, 10mM MgCl, 6mM 2- mercaptoethanol, 0.25mM GTP, 2mM ATP, 4 mM creatine phosphate, 10 yg creatine kinase, 40 YM amino acid mixture (minus Met), 200 #Ci 35§-Met (NEN Research Products) and 10mM Tris-HCl, pH 7.4, was incubated for 1 hr at 37°C [2]. After incubation, 40 1 of the reaction mixture was treated with 20 ul of affinity chromatographed anti-@-connectin antibodies [3], anti-nebulin [3] and also non immune IgG for 1.5hr at room temperature followed by the incubation with 20 ul of Affi-Gel Protein A (Bio Rad) for 1.5 hr at room temperature [4]. The precipitate was washed 5 times with a solution of 0.5 M NaCl and 20 mM Tris-HCl buffer, pH7.5. The precipitate was added with an SDS solution (final concentrations, 5% SDS, 20mM DTT, 5mM EDTA, and 50 mM Tris-HCl, pH 6.7) and heated for 2 min at 100°C. A portion of the total reaction mixture and also a piece of adult chicken breast muscle, were also treated with SDS. SDS gel electrophoresis was carried out according to Laemmli [5] using 2.5% 930 A. ASAKURA, Y. NABESHIMA AND K. MARUYAMA polyacrylamide for stacking gel and 4.5% polyac- rylamide for separation gel. The gel stained with Coomassie Brilliant Blue was incubated with a scintilating agent EN°NANCE (NEW Research Products) for 30 min, washed with distilled water and dried. Fluorography was carried out for 7 days at —80°C. RESULTS AND DISCUSSION Figure 1 shows autoradiograms of a chicken embryo muscle extract incubated with radioactive methionine. In the whole mixture, strong radioac- tivity was recognized at the top of the gel, Fic. 1. Incorporation of *°S-methionine into connectin synthesized in vitro in chicken embryo muscle ex- tract. For conditions and method of detection, see text. (a) Coomassie Brilliant Blue stained whole sam- ple of adult chicken breast muscle. C, connectin; N, nebulin; M, myosin heavy chain; A, actin. (b) Coomassie Brilliant Blue stained whole sam- ple of embryo muscle extract after incubation. (c) Autoradiogram of the whole sample. (d) Autoradiogram of the anti-nebulin antibodies precipitated fraction. (e) Autoradiogram of the anti-connectin anti- bodies precipitated fraction. (f) Autoradiogram of the non-immune IgG pre- cipitated fraction. unknown band lower than nebulin, myosin, and other smaller proteins (Fig. 1c). In the Coomassie Brilliant Blue stained gel, bands corresponding to connectin and nebulin were very faint, if any (Fig. 1b). A strong radioactive band near nebulin (Fig. lc) was significantly lower than the nebulin band (Fig. la). Furthermore, anti-nebulin antibodies precipitated sample did not show any radioactivity at the nebulin band (Fig. 1d). On the other hand, in the anti-connectin antibodies precipitated sam- ple, faint but significant radioactivity was recog- nized in the connectin band (Fig. le). This radioactive band was not detected with anti- nebulin antibodies (Fig. 1d) and non immune IgG (Fig. 1f) precipitated samples. It is to be noted that strong radioactivities detected in the lower fronts of Figure ld, e, f, might be due to contami- nant proteins of low molecular weight, e.g. actin, that could not be washed out. A trial to prepare mRNA for connectin from chick neonatal muscles was unsuccessful. This was very probably due to breakdown of very long mRNA during preparation procedures. There- fore, an embryo muscle extract containing poly- somes for connectin was prepared and its in vitro translation was demonstrated in the present study. However, the extent of radioactive methionine incorporation was very low. This is not surprising because of breakdown of the mRNA even during a gentle homogenization of muscle tissues. It is to be mentioned that there were not any radioactive peptides smaller than connectin but larger than actin (Fig. le). This suggests that a large peptide of connectin is synthesized in vivo [cf. 6]. At present the largest mRNA encoding a muscle protein is that of Duchenne dystrophy gene and its size is 14 kb corresponding to a protein of MW of half a million [7]. Cloning of connectin gene will give a final answer to the nature of this gigantic protein. REFERENCES 1 Maruyama, K. (1986) Int. Rev. Cytol., 104: 81-114. 2 Heywood,S.M., Dowben,R.M. and Rich, A. (1967) Proc. Natl. Acad. Sci., 57: 1002-1009. 3 Sugita, H., Nonaka, I., Itoh, Y., Asakura, A., Hu, D.H., Kimura, S. and Maruyama, K. (1987) Proc. Connectin Synthesis in Chick Embryo Extract 931 Jap. Acad., 63B: 107-110. 6 Yoshidomi,H., Ohashi,K. and Maruyama, K. 4 Olliver,C.L. and Boyed,C.D. (1984) Methods (1985) Biomed. Res., 6: 207-212. Mol. Biol., 2: 157-160. 7 Monaco,A.P., Neve,R.L., Colletti-Feeper, C., 5 Laemmli, U.K. (1970) J. Biol. Chem., 244: Bertelson, C. J.. Kurnit,D.M. and Kunkel, L. M. 4406-4412. (1986) Nature, 223: 646-650. a i ' ahh ee i ii. a“ sok a spatien tied ah pints ne UY Mba (DUD wodtiase sir chy. Be Wine ah pels goes. Sadan bat SURE fi ‘cigars oF ae “Bin EERE fF | ti PO va Lop etelgaN een tn we ing Se THE Oeaicaie VERB? MP AGEL) boc pe hg WR | fi 4 Wee, Caer ae y (it ‘ 2 a F citys iM SAND DESCUNSHIN moth ‘ i t ‘ i; trie 135% i it 3 q ti eat cc” a! sh.b8 i 4 Pa 4 wv 1/ { r i] t P 4 , Pn ‘ >) a ‘ : tals ‘ s Ae ites J ® “i d a % a ~ 5 os 1.” 1 ' i eh) Ah 3 , +” on { { Fe P a0 a 4 2 La LS | i 5 v5 ad bee 7 ui} tm i eh j iy ere itl ‘ ry ‘ : i, et Beary P é tg wey Oe aa - Fes yer’ rite u 7a E ears inte ; ‘ “3 y . : a n4ud | y yf 4 At eae or poet ty ek i ei ; ; t Bren ay ae a * ab A an OTE TA i) bu * ' faire ‘ ‘ oor { a é hy i RL OR ‘ i i : S F Feo ir a ee ; f Pee ke 1 cn ee he gd i} Liens pw ‘ “ . - 2 4 e : a re . » i 1 Wi © -) saa OW le An A Se ely oy | i sho ! staan brand ear 7 Aue! ois The) tend ee ‘pay ia) Pay, Sta ite CO Pw iT Pe Tet A. Cr a pes Weer IM LS ae a ee alias ne ah lemma wil 1 a “" (ey Was biglisi ie ty 2 bio Heh Piet or aa os mn a ee fia sip or . PRG wea a ' ‘ ATA AE MESO Cr: 5 Ore ssh eat CPyel¥ |: Sette he! : eos ie = \ i i“ Ba’ i yo ®, 4 ‘ : i a 4 ueei@ 5 Tres oP i cae { iin f npiyi “ Vii ry I * % tpi Lun on. . £ v7 6G if Published by 7, (4 the Japanese Society of Developmental Biologists Papers in Vol. 29, No. 5. (October 1987) 40. REVIEW: H.Suimapa: DNA Replication and Its Regulation in Cleavage Embryos. 41. S. Mryata and H. K. Kimara: Effects of Endopeptidase Inhibitors on Gastrulation of Xenopus eggs. 42. K.Ousumi: The Periodic Changes in Microvilli Density in Activated Xenopus Eggs That Correspond to the Cleavage Cycle. 43. J.-F. Riou, De Li Sui, T. DARRIBERE, J.-C. BoucAauT and J. CHARLEMAGNE: Expression of Three Gastrula Cell Surface Glycoproteins During Embryonic and Larval Development in the Amphibian Pleurodeles waltlii. 44. M. Ozawa, S. YONEZAWA, M. Sato, H. UEHARA, E. Sato and T. Muramatsu: Three Groups of Teratocar- cinoma Antigens Co-Expressed in the Visceral Endoderm are Located in Mutually Exclusive Sites in the Adult Kidney. 45. M. TANAKA, H. ASAHINA, N. YAMADA, M. Osumi, A. WEDA and K. ISHIHARA: Pattern and Time Course of Cleavages in Early Development of the Ovoviviparous Pond Snail, Sinotaia quadratus historica. 46. C.K. Newrn, C. ARNAL, R. A. GOULART and M.H. Nanna: Founder Cell Differentiation and Acrasin Production in an Aggregateless Mutant of Polysphondylium violaceum. 47. W.R. Ecxperc and A. G. CArroLi: Evidence for Involvement of Protein Kinase C in Germinal Vesicle Breakdown in Chaetopterus. 48. T. Takamatsu and S. Funta: Growth of Notochord and Formation of Cranial and Mesencephalic Flexures in Chicken Embryo. 49. K.Dan: Studies on Unequal Cleavage in Sea Urchins. III. Micromere Formation under Compression. 50. S.Komazaki: A Yolk-Granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster. 51. K.K.-K. Oxupa, E. Kuwano, M. Eto and O. YAmasuita: Inhibitory Action of an Imidazole Compound on Ecdysone Synthesis in Prothoracic Glands of the Silkworm, Bombyx mori. 52. B. ALBERS: Competence as the Main Factor Determining the Size of the Neural Plate. 53. M.LasMAN: Encystment-Inducing Factor from Cultures of Acanthamoeba palestinensis. Development, Growth and Differentiation (ISSN 0012-1592) is published bimonthly by The Japanese Society of Developmental Biologists, Department of Biology, School of Education, Waseda University, Tokyo 160, Japan. 1987: Volume 29. Annual subscription U.S. $110.00 including air speed delivery except Japan. Application to mail at second class postage rate is pending at Jamaica, NY 11431, U.S.A. Outside Japan: Send subscription orders and notices of change of address to Academic Press, Inc., Journal Subscription Fulfillment Department, 6277 Sea Harbor Drive, Orlando, FL 32887, U.S. A. Send notices of change of address at least 6-8 weeks in advance. 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There is no need for a bulky stand. * Hydraulic remote control ensures totally vibration-free operation. * 3-D movements achieved with a single joystick. Micromanipulators Microelectrode pullers Stereotaxic instruments NARISHIGE SCIENTIFIC INSTRUMENT \2ek® / LABORATORY CO., LTD. 4-9-28, Kasuya, Setagaya-ku, Tokyo 157 JAPAN Telephone: 03-308-8233 Telex: NARISHG J27781 (Contents continued from back cover) Oguma, Y., H. Kurokawa, S.M. Akai, H. Tamaki and J. Kayjita: Interspecific differ- ences in some courtship behavioral prop- erties among the four species belonging to the Drosophila auraria complex Taxonomy Ohkubo, N:: (Acari: Oribatei) from Japan Baba, K., A. Nagatomi, H. Nagatomi and N. L. Evenhuis: meleonostena (Insecta, A new species of Zetomimus Redescription of Villa myr- Diptera, Bomby- liidae), a parasitoid of ant lion in Japan ito: E.: morphology of nauplius y (Crustacea: Maxil- lopoda: Facetotecta), with provisional de- signation of four naupliar types from Japan Proposal of new terminology for the Hayashi, K.-I. and T. Chiba: Rediscovery of Heptacarpus jordani (Rathbun) with notes on morphological variations (Decapoda, Caridea, Hippolytidae) ZOOLOGICAL SCIENCE VOLUME 4 NUMBER 5 OCTOBER 1987 CONTENTS REVIEWS Asakura, A., Y.Nabeshima and_é CK. Terakado, K.: Fine structure of ascidian Maruyama: In vitro synthesis of connectin Smooth. muscle oi2257 ae eee (Dil in an extract of chicken embryo muscles Keller, R.: Cell rearrangement in morpho- (COMMUNICATION) 9) °F 2-0 eee eee 929 PENCSIS | a5). 0.05 Lh ee ee ere eee ee 763 ; Developmental Biology ORIGINAL PAPERS Abé, S.-I. and S. Asakura: Meiotic divisions Ay sicloey and early-mid-spermiogenesis from cultured : Taneda, K.: Geotactic behavior in Para- primary spermatocytes: of ene OE SOO I. Geotaxis assay of in- Endocrinology dividual Specimen? <<. 5,285.J.cgesecaceee 781 Suzuki, S.: Plasma thyroid hormone levels in Taneda, K.: Geotactic behavior in Para- mecium caudatum. II. Geotaxis assay in a population of the specimens Newland, P. L.: tion of a new muscle in the tailfan of the The structure and innerva- crayfish, Procambarus clarkii Takei, Y. and I. Hatakeyama: Changes in blood volume after hemorrhage and injec- tion of hypertonic saline in the conscious quail, Coturnix coturnix japonica Arii, N., K. Namai, F. Gomi and T. Naka- zawa: Cryoprotection of medaka embryos during development Hidaka, T. and T. Miyahara: inhibitory neuromuscular transmission in fish Excitatory and FEGMMUSCLE§ 2 521.5 2h eet aeerenc eee 819 Cell Biology Sawai, T: Surface movement in the region of the cleavage furrow of amphibian eggs ...825 Biochemistry Takahashi, S. and K. Maruyama: Activity changes in myosin ATPase during meta- MOnphosis Of sinuithily | eee ee 833 INDEXED IN: Current Contents/LS and AB & ES, Science Citation Index, ISI Online Database, CABS Database metamorphosing larvae and adults of a sala- mander, Hynobius nigrescens ............ 849 Takahashi, S. and S. Kawashima: Prolifera- tion of prolactin cells in the rat: Effects of estrogen and bromocryptine ............. 855 Wheeler, C.M. and A.P. Gupta: Effects of two juvenile hormone analogs (R-20458, RO203600) and three juvenile hormones (JH1, JH2, JH3) on the external morphology and length of the spiculum copulatus (SC) in the male German cockroach, Blattella ger- manica (L.) (Dictyoptera: Blattellidae) ..861 Morphology Win Win Yee and S. Kawashima: Sex differ- ence in the early histopathological changes of the kidney in Wistar/Tw rats Ishizeki, K.: Ultrastructural observations of the developing basophilic granulocytes in the loach kidney Behavior Biology Moriya, T. and Y. Miyashita: Body color and the preference for background color of the Siamese fighting fish, Betta splendens_ ....881 (Contents continued on inside back cover) Issued on October 15 Printed by Daigaku Printing Co., Ltd., Hiroshima, Japan OOLOGICAL >CIENCE An Internatio nal Jou ZOOLOGICAL SCIENCE The official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Hideshi Kobayashi (Tokyo) Toshin-building, Hongo 2-27-2, Bunkyo-ku, Managing Editor: — er Tokyo 113, Japan. Tel. 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ZOOLOGICAL SCIENCE is distributed free of charge to the members, both domestic and foreign, of the Zoological Society of Japan. To non-member subscribers within Japan, it is distributed by Business Center for Academic Societies Japan, 6-16—3 Hongo, Bunkyo-ku, Tokyo 113. Subscriptions outside Japan should be ordered from the sole agent, VNU Science Press BV, Europalaan 93, 3526 KP Utrecht, (postal address; P.O. Box 2093, 3500 GB Utrecht), The Netherlands. Subscription rates will be provided on request to these agents. New subscriptions and renewals begin with the first issue of the current volume. All rights reserved. No part of this publication may be reproduced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1987, The Zoological Society of Japan Publication of Zoological Science has been supported in part by a Grant-in-Aid for Scientific Publication from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 4: 933-944 (1987) © 1987 Zoological Society of Japan REVIEW Rectal Gland and Crypts of Lieberkiihn ;” Is There a Phylogenetic Basis for Functional Similarity? CHRISTOPHER A. LORETZ ee ae Sie, POM\THSON An JUL 2¢ 1988 LIBRARIES Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, U.S.A. INTRODUCTION Clinicians are surely familiar with the debilitat- ing and potentially fatal effects of intestinal malab- sorption, especially secretory diarrhea. Secretory diarrhea can result from a variety of causes, all of which produce active fluid secretion to override the fluid absorption which normally occurs. The ultimate cause may be exogenous like enterotoxins (e.g., cholera toxin) of bacterial origin, or endoge- nous, such as hormone-secreting tumors (€.g., vasoactive intestinal peptide-(VIP-)oma in pan- creatic cholera syndrome) or choleric enteropathy where, because of insufficient enterohepatic recycling, bile salts pass into the colon [1-6]. In many such cases, these agents act through the same intracellular mediators, for example, causing an elevation of cyclic AMP and the subsequent stimulation of active Cl~ secretion [1-6]. The secretory flux of Cl” (and Na™) draws water into the intestinal lumen, thereby producing the diarrhea. The loss of this fluid brings about the profound dehydration requiring clinical care. Physiologists appreciate the complexity of the simultaneous absorptive and secretory processes at the intestinal epithelium which contribute in their balance to the net overall Na* and Cl” fluxes. In recent years, considerable attention and ex- perimental ingenuity have been directed toward localizing and understanding these transport pro- cesses and their control. The list of hormonal and Received August 1, 1987 other mediators implicated in the regulation of gastrointestinal function is extensive [3,7]. Many of these agents have multiple actions, including effects on the absorptive and secretory compo- nents of ion and water transport, intestinal vascu- lar flow, and general intestinal smooth muscle tone (e.g., [3]). For the physiologist studying ion transport, the simultaneous operation of absorptive and secre- tory mechanisms can confound even baseline transport studies on untreated tissues. Animal models with simpler transport schemes, perhaps involving only absorptive or secretory compo- nents, are useful in the study of particular aspects of intestinal ion transport and its control. Although the intestinal epithelium is simple in the histological sense, its mixed cell composition and folding to produce villi and crypts seem more reflective of its multifunctional role in the organ- ism [8, 9]. The intestinal epithelium of mammals (and most vertebrate classes) is arranged in a series of folds which involve the lamina propria (referred to as villi), or additionally the muscularis mucosae and submucosa (referred to as the plicae circulares or rings of Kerckring); the latter may, in fact, have a villous covering. There are also glandular pits penetrating into the underlying lamina propria of the mucosa, or even into the submucosa. There may occur submucosal glands (Brunner’s glands), restricted in their distribution to the duodenum, as well as mucosal glands (crypts of Lieberkthn) which are found throughout the small intestine and colon. The villi and crypts are distinct in their 934 C. A. LORETZ histology. In addition to the abundant absorptive cells and somewhat less abundant mucus cells common to most regions of intestinal epithelium, the crypts also contain Paneth cells and enterochromaffin/argentaffin cells. The Paneth cells, with their apical secretory granules, secrete lysozyme which serves a protective role through its ability to digest the cell coat of potentially harmful intestinal bacteria [8]. The enterochromaffin cells are endocrine cells of the gastroenteropancreatic series which can be individually identified by immunocytochemical staining [7]. In mammals, the crypts are also the site of active mitosis to regenerate the epithelial cells lost following their normal migration from the crypts to the villus tips. The segregation of the epithelium into villar and crypt regions with these subtleties of cell distribution invites speculation regarding the dis- tribution of function. REGIONAL SEGREGATION OF NaCl TRANSPORT FUNCTION With respect to Na* and Cl transport, the villar and crypt regions have been characterized functionally based on several lines of experimental evidence. Osmotic shock to damage the villar portion of the jejunum did not diminish the secretory response to cholera toxin, suggesting a secretory function for crypt epithelium [10]. Cholera toxin exposure on the luminal side inhibits NaCl absorption very rapidly but net secretion is stimulated only after a longer delay, consistent with diffusion of toxin into the crypts [11]. Welsh etal. [12], using a visual technique, showed for the rabbit colon that the crypts are the site of active fluid secretion. When colonic tissue covered with paraffin oil was stimulated with PGE,, the sec- reted fluid appeared as droplets only over the crypt duct openings. Weiser and Quill [13] reported greater increases in adenylate cyclase activity in isolated villus cells compared with crypt cells following exposure to cholera toxin; although these experimenters conclude correctly that the major adenylate cyclase response is at the villus, this finding does not address directly the issue of the location of ion transport systems. Field and coworkers [14] reported the intriguing finding that cyclic AMP reduced net Na* and Cl” absorption by the winter flounder (Pseudopleuronectes amer- icanus) intestine but did not stimulate the active Cl~ secretion characteristic of mammalian smail intestine and colon. Similar data have been reported for two additional species, the flouider (Platichthys flesus ; [15, 16]) and the goby (Gillich- thys mirabilis; [17, 18]). Field et al. [14] related these data to the well documented absence in fish intestine of crypts of Lieberkiihn ({19]; Figs. 1A, B and 2), consistent with the notion that active secretion originates in the crypts. The absence of crypts, and also of active fluid secretion, in fish intestine is physiologically signif- icant. Active Cl secretion along the length of the fish intestine would be maladaptive in both seawa- ter and fresh water. Seawater-adapted teleosts make good their diffusive losses of water to the external environment by ingesting seawater. Absorption of Na* and Cl” from the lumen reduces the osmolality of the luminal fluid, which drives water absorption. Low circulating levels of the fresh water-adapting hormone prolactin, which normally reduces intestinal water permeability, allow greater coupling between NaCl and water absorption [20, 21]. Following this absorption of seawater, the organism relies on another set of epithelial organs (gills, opercular membrane and skin), spatially separated from the intestine, to actively excrete Na* and Cl, leaving the fish with a net gain of free water. Active secretion of Cl” by the intestine, to reduce the osmotic gradient, would be counterproductive to the overall goal of fluid absorption. Fluid secretion is probably not required to generate an aqueous milieu for chemi- cal and enzymatic digestion (as might be argued for terrestrial vertebrates with a relatively dry diet) since drinking in seawater-adapted teleosts can be quite substantial [21-23]. Secretion of mucus, for the protection of the intestinal epithelium from digestive enzymes and to lubricate the flow of digesta, and of HCO 3 [24] and K* [25, 26], for acid-base and ionic regulation, do occur. In fresh water-adapted fishes, where the drink- ing rate is low and intestinal water permeability is low due to the action of prolactin, NaCl secretion would clearly be detrimental. Reduced coupling of water absorption to NaCl absorption limits intes- Rectal Gland and Crypts 935 Wd ctattige gs on Ne patel” Ones Fic. 1. Light photomicrographs of intestinal histology. (A): Goby (Gillichthys mirabilis) posterior intestine. There are no mucosal or other glands and the muscularis externa is very poorly developed. (65) (B): Bowfin (Amia calva) posterior intestine. The epithelium is highly folded but no mucosal glands are apparent; the muscularis externa is highly developed and extends well below the field of view. (60) (C): Cat ileum. Both villi extending into the lumen and crypts of Lieberktthn extending downward to the muscularis mucosae can be distinguished in this section. (65) (D): Cat duodenum. In addition to villi and crypts, Brunner’s glands are visible in the submucosa (lower right). (60) (E): Cat colon. Crypts form the major portion of colonic mucosa with the villi reduced to the horizontal layer of surface cells. (135) 936 C. A. LoRETZ tinal water gain while the kidney and urinary bladder produce a copious, hypotonic urine. Although teleost fish lack crypts of Lieberkuhn and the associated intestinal active fluid secretion, they do possess a number of other salt excretory organs: branchial “chloride cells”, opercular mem- brane and skin, rectal salt gland and dendritic organ. The inability of teleost fish intestine to actively secrete NaCl and the simultaneous pres- ence of other secretory structures stimulated my interest in the possible phylogenetic and functional relationships of one of the latter, secretory, organs. In particular, similarities between the rectal gland and the mammalian crypts of Lieber- kiihn will be addressed in this paper and one possible interpretation of the data will be pre- sented. The discussion will be largely synthetic, making use of anatomical and physiological data. RECTAL GLAND STRUCTURE AND HOMOLOGY The argument for homology of the crypts of Lieberkthn and the rectal gland is based on morphological considerations. A morphological series, aS an approximation to the intermediate stages of a phylogenetic or evolutionary series, can be constructed using the anatomies of extant species. Many of these data are old and careful reading of the original reports reveal that the general notion of homology, while not fully de- veloped, is certainly not new. The morphologies of the various organs will be summarized herein and a morphological series constructed which includes the mammals. An abundant literature exists on the various morphologies of the fish rectal gland, which has gone by a variety of names since its earliest anatomical recognition by Severini in 1645 (in [27, 28]); rectal gland synonymies include: rectal gland caecum postanal gland bursa cloacae caecal gland or appendage appendix digitiformis appendix vermiformis superanal gland nodular gland The first report of the glandular nature of the organ was in 1852 when Leydig described well vascularized clusters of ducts in macerated speci- mens; he compared the glandular structure of the rectal gland with that of Brunner’s glands (in [27, 28]). As noted below for the coelacanth, there has also been some confusion in identity and nomen- clature. Probably the best known rectal gland is that of elasmobranchs such as the dogfish shark (Squalus acanthias ; [27-32]; Fig. 2). The dogfish rectal gland is a digitiform dorsal diverticulum of the postvalvular intestine supported by dorsal mesorectum. Although a number of functions have been attributed to the rectal gland since its first description (including accessory reproductive structure, urinary bladder, renal accessory excre- tory structure, hormone secretion, and blood function; cf. [27,28]), its function as a NaCl excretory organ, emptying its effluent into the terminal intestine via the rectal gland duct is now well established (e.g., [33-35]). In addition to active NaCl secretion by the gland measured directly, the secretory cells of the ducts bear all the hallmarks of ion-secreting epithelial cells, includ- ing abundant mitochondria and a well-developed tubulovesicular system and associated cisternae continuous with basolateral membrane invagina- tions. The Nat-K*-ATPase has been localized using histochemistry to this series of basolateral membranes [32, 36], and is responsible for gener- ating the electrochemical gradient for Na~ into the cell which drives the active secretion. Arterial supply to the gland is from the rectal gland artery (synonymous with the posterior mesenteric artery and caecal artery, [27-29, 37, 38]), a branch from the dorsal aorta. Three courses of flow are possible for blood passing toward the rectal gland in the rectal gland artery. The rectal gland artery, upon reaching the gland, supplies a series of circumferential arteries and then continues along the dorsal surface of the rectal gland to beyond the junction of the gland with the intestine, where it joins the circulation of the postvalvular intestine. Blood delivered to the circumferential arteries can follow two routes. Blood can pass to a network of superficial venules and then via a pair of larger veins through the Rectal Gland and Crypts 9577 NO GLANDS AGNATHS TEEEOSTIS MAMMALS INTRAMURAL RACEMOUS GLANDS DIGITIFORM RECTAL GLAND SARCOPTERYGIANS CHONDRICHTHYEANS LOBED RECTAL GLAND SOME CHONDRICHTHYEANS CHIMAERAS Fic. 2. Morphological series illustrating the variety of mucosal morphologies discussed in the text. The series of intestinal morphologies shows the gradual transition (counterclockwise in the figure) from mammalian crypts to the elasmobranch-type rectal gland. In these drawings, submucosal, muscularis externa and serosal layers are depicted for simplicity as single lines. dorsal mesentery to the posterior cardinal vein system [37]. Alternatively, blood can be delivered to sinusoids in the deeper, secretory regions of the gland. This blood passes through capillaries sur- rounding the secretory ducts and is collected centrally in the rectal gland vein which passes into the intestine as the dorsal intestinal vein, a tributary of the hepatic portal vein. Clearly, there may be shunting of blood from the rectal gland artery to the vessels of the posterior intestine, bypassing the gland altogether. Satchell [38] has reported that subambient pressures in the dorsal intestinal vein produced by cyclic vasoconstriction of the rectal gland sinusoids may actually cause retrograde flow to the rectal gland of blood in the intestinal veins. He further proposed that the advantage of such flow would be the delivery of NaCl-rich blood (resulting from intestinal NaCl absorption) directly to the gland for secretion, before delivery of that blood to the general circulation. The coelacanth (Latimeria chalumnae) postanal gland (=rectal gland or nodular gland) has a structure and vascular connections similar to those of the Squalus elasmobranch type ([39, 40]. Although its salt secretory function has not been directly measured, the ultrastructural appearance of the parenchymal cells and high levels of Na*- K*—ATPase in the gland support such a role [39- 41]. 938 C. A. LORETZ The lungfish (Protopterus aethiopicus and P. dolloi) cloacal gland (=rectal gland) is a descrete encapsulated gland which is located posterior to the urogenital sinus as opposed to the anterior position of other rectal glands [42]. There also occurs in lungfish another structure, the cloacal caecum (=urogenital sinus) whose placement is more typical of the elasmobranch rectal gland. In the earlier literature, the relationship of this cloacal caecum to the rectal gland of elasmo- branchs was unknown. Its placement was noted to be similar to that of the elasmobranch rectal gland although a different (nonglandular) internal struc- ture was acknowledged [43]. Description of the vascular connections has not been published. Ultrastructurally, the secretory cells of cloacal gland tubules exhibit the typical abundant mitochondria and basolateral cisternae seen in the secretory cells of other rectal glands. Interestingly, a cloacal gland is found only in the lungfish genus Protopterus. This is the only genus of lungfish which estivates for any length of time; Lagios and McCosker [42] hypothesize a role for the gland in osmoregulation during estivation based on this correlation. In some elasmobranchs, such as Hexanchus and Heptanchus, the rectal gland is a postvalvular intestinal diverticulum of the Squalus type but consisting of 2-4 discrete lobes with separate lumina (Fig. 2); vascular supply and drainage are via the caecal artery and dorsal intestinal vein, respectively [27]. In the chimaeras, there is no extramural rectal gland but, instead, a series of 9-12 or more intramural racemous glands located either dorso- laterally (Chimaera monstrosa; Fig. 2) or circum- ferentially (Hydrolagus colliei). These glands are located in the gut wall immediately posterior to the last plicum of spiral valve and drain into the postvalvular intestine via separate ducts; these ducts enter at the bases of intestinal folds [27, 31, 42, 44]. Arterial supply is via the caecal artery and venous drainage presumably, although not de- scribed, via the intestinal veins. The organ is arranged as lobulated clusters of grape-like glands drained by tubular ducts. Both glands and ducts are formed of a simple cuboidal/columnar epithe- lium. The cells of the parenchyma are mitochon- dria rich, show the typical basolateral cisternae, and are rich in Na*-K*—ATPase activity as expected for an ion-secreting structure. As in some sharks, and reminiscent of crypts, particular- ly in the colon, the ducts contain numerous mucus cells [31]. Extramural rectal glands and intramural glands of the chimaeroid type are lacking in tetrapod vertbrates which, instead, possess numerous mucosal glands (crypts of Lieberkthn; Fig. 1C, D, E) or their apparent equivalents, the various cell nests and tubular glands of amphibians and rep- tiles, respectively [8, 19, 45,46]. There are, in addition, submucosal secretory glands in the duodenum (Brunner’s glands; Fig. 1D). The intestines of teleosts and lamprey possess neither crypts of Lieberkthn nor rectal glands of the types described above [14, 17, 19, 47-50]. In- stead, the middle and posterior intestinal segments are simple in organization. They are lined by a simple epithelium folded to various extents, pro- ducing villi (Fig. 1A, B and 2). The submucosal and muscularis externa layers are more or less developed according to species ([14, 17]; Fig. 1A, B). This cryptless epithelium is capable of per- forming many of the functions of mammalian intestine including the absorption of NaCl and water and the secretion of HCO3, K* and mucus (e.g., [17, 22-26, 47]). One function associated with the mammalian crypts of Lieberkihn is the proliferation from stem cells of epithelial cells to replace those lost at the villus [9]. In some teleost species, cell proliferation occurs at the bases of intestinal folds [48, 49] whereas in other species [50], and in the lamprey [51], proliferation occurs throughout the epithe- lium, with each generative cell supplying surround- ing areas. Therefore, cell proliferation is not universally associated with crypts. As an aside, the localization of proliferative cells and the functional segregation of absorptive and secretory processes in mammalian intestine suggest the interesting notion that the functional role of an epithelial cell perhaps changes as the cell matures and migrates toward the villus tip. Two major evolutionary questions arise with regard to the relationships between the crypts and the rectal gland in its various forms. First, are the Rectal Gland and Crypts rectal gland and crypts of Lieberktthn homologous structures? And second, what are the phylogenetic relationships of the various intestinal morpholo- gies? Both of these questions are difficult to answer, of course, because the fossil record does not yield anatomical details of the sort required. Given only the anatomy of extant species, then, there are several possible interpretations of the data. These are: (1) the presence of intestinal salt-secreting structures (crypts and rectal glands) is primitive with respect to the vertebrates, and some groups, notably the teleost fishes, have secondarily lost them, (2) the various intestinal excretory structures in several groups were derived independently from ancestors with the teleost-type intestinal morphology and have subsequently con- verged in appearance, or (3) crypts and rectal glands are advanced specializations shared by relatively closely related taxonomic groups. With respect to the first question, a morpholo- gical series illustrates possible intermediate mor- phological stages in the evolution of these various organs (Fig. 2). No phylogenetic scheme is implied in Figure 2, only the logical arrangement of in- termediate morphological stages from the digiti- form Squalus-type to the crypt-like intramural type. The mammalian crypts of Lieberkthn (an example of tetrapod mucosal glands) are included as an extention of the fish series since it appears to represent an extreme case of multiple intramural glands of the chimaeroid type with circumferential distribution. The assignment of the aglandular teleost and agnathan intestines to an intermediate position in this continuous series (circular, as it is diagrammed) between the crypt-like glands and the digitiform-type gland reflects the earlier argu- ment of the unsure evolutionary origin of this condition, i.e., being an aglandular primitive condition or an advanced condition where secre- tory glands were secondarily lost. The taxonomic distribution of intestinal salt- secreting glands is presented in Figure 3. Various rectal gland phylogenies have been proposed but, according to Forey [39], no single phylogeny can be supported despite the accepted homology of the different rectal gland types. Considering the broad distribution of secretory glands in their various forms and the claims of Fange and Fugelli [31] and \O Oo Ne) (Sharks) Actinopterygii (Rayfins) Myxinoidea (Hagfishes) Petromyzontia (Lampreys) Holocephali (Chimaeras) Dipnoti (Lungfish) Actinistia (Coelacanths) Choanata (Tetrapods) e Elasmobranchii Crossopterygi Sarcopterygi Fic. 3. Phylogeny of the major groups of vertebrates (adapted from [52, 53]). Filled circles indicate those groups with intestinal secretory gland (rectal glands) or tissues (crypts of Lieberkthn). Lagios and Stasko-Concannon [44] that the in- tramural glands are primitive with respect to the extramural rectal gland, the most parsimonious scheme might include the appearance of crypts in early gnathostomes with their subsequent develop- ment into extramural glands in some groups (sharks, lungfish and coelacanth) and their sec- ondary loss in rayfinned fishes. Remarkably, Pang et al. [52] earlier described the identical taxonomic distribution for ureotelism in vertebrates. This parallelism might suggest that ureotelism and intestinal secretory glands are both shared charac- ters associated with the evolution of a particular osmoregulatory strategy (as opposed to each being derived independently in serveral groups). Strengthening this argument is the fact that both ureotelism and rectal gland secretion are adap- tations to the marine environment. Urea retention is diminished in elasmobranches acclimated to brackish water (cf. [54]) and the rectal glands in sharks adapted to fresh water and in freshwater stingrays, although normal in anatomical position, are extremely small [55, 56]. In addition to the similarity of vascular connec- tions, the ontogeny of the elasmobranch rectal gland supports the proposed homology. Early reports [27, 28] describe the developmental origin of the rectal gland as an outgrowth from the dorsal intestinal wall. The gland originates as a single outpocketing to give rise to the rectal gland stalk and duct; subsequent repeated evaginations from this anlage produce the secretory ducts of the parenchyma [28]. The dorsal placement of both adult elasmobranch glands and intramural glands 940 C. A. LorRETz of the Chimaera-type reflect this ontogeny. Crofts [27] and Fange and Fugelli [31] claim the chi- maeroid type to be primitive with respect to the elasmobranch digitiform gland based on its less defined structure; the intramural embryonic origin might substantiate their claim. Lagios and Stasko- Concannon [44] concur with regard to the “primi- tive” status of intramural glands and analogize the transformation into a compact extramural rectal gland with the evolutionary development of the endocrine pancreas from diffusely-arranged en- docrine cells in the gut wall. Ultrastructural similarity also lends support to the proposed homology. Mitochondria-rich cells characterize the glandular epithelium of the rectal glands of elasmobranchs, the coelacanth, the chimaeras, and the absorptive cells of the crypts of Lieberkiihn [9, 29, 30, 32, 40, 42, 44]. Although a similar ultrastructural appearance is consistent with the proposed homology, this is merely sup- portive considering the widespread occurrence of mitochondria-rich cells in transporting epithelia of diverse ontogenetic origin (such as fish skin and opercular membrane from ectoderm; [57-60]). Separate from the issue of rectal gland-crypt homology presented here, Crofts [27] considered vertebrate intestinal caeca (where present) to be homologs of the rectal gland; she based this argument on gross anatomical similarity and the alleged presence in the rectal gland of simple lymphoid tissue similar to that seen in caeca. Crofts [27], in her detailed description, states that the lymphoid tissue is commonly found in the associated mesentery and is sometimes pressed against the rectal gland. Some anatomists (e.g., Morgera in [27]) included the mammalian appen- dix in the homology as well. Hoskins [28], Bulger [29], Fange and Fugelli [31], and others (cf. [27]), however, do not find lymphoid tissue in the substance of the rectal gland, thus weakening the caecal homology argument. PHYSIOLOGICAL AND ENDOCRINOLOGICAL SIMILARITIES BETWEEN RECTAL GLAND AND CRYPTS Physiological and endocrinological studies on intestine and rectal gland demonstrating functional similarity are supported by the proposed homolo- gy. The intestine of mammals, the best studied of tetrapods, has several electrolyte transport func- tions. Two of these are relevant here: (1) NaCl absorption and (2) Cl™ secretion. Current concen- sus places the Na* and Cl~ absorptive function in the villus epithelium and the Cl~ secretory func- tion in the crypt epithelium [1, 12]. The intestine of at least three species of teleost fish possesses only the absorptive transport component; in two flounder species and the goby, cyclic AMP did not stimulate active Cl~ secretion as it does in mam- mals [14-18]. Non-teleost fishes have received little attention; the presence of a valvular intestine (spiral valve) in a number of these forms presents morphological complexities to in vitro study, and a consequent paucity of data. Should the proposed homology be correct and if an intramural secretory organ (such as the chimaeroid type rectal gland or the crypts of Lieberkiihn) represents the primitive condition as some argue, then it can be concluded that the salt secretory function in the intestine of some fishes has been removed to an extramural gland, and in other fishes has been lost altogether. In teleosts, the abundant branchial and epidermal salt-secreting chloride cells may functionally re- place the crypts or rectal gland in the role of salt secretion. Since ion transport processes of the valvular intestine of elasmobranchs which lack crypts, or of other species with rectal glands, have not been reported, the expected absence of secre- tory capability of this tissue cannot be addressed at present. The advantage for fishes of removing NaCl secretion to an extramural site is clear. In teleosts, by transferring the secretory role to chloride cells of the skin, opercular and branchial regions, the problem alluded to earlier of simultaneous absorp- tion and secretion across the intestine is relieved. The intestine could maintain a relatively high permeability to water (in the seawater-adapted fish, for example) facilitating absorption, while the chloride cell-containing epithelia could maintain a relatively low permeability to water limiting os- motically-driven water efflux. Another effective strategy (in the sharks, for example) is the use of an extramural rectal gland to produce a concen- Rectal Gland and Crypts 941 SRC= GOR. CE IRIE —mammal crypt cell — fish rectal gland trated NaCl solution which is delivered to the posterior-most segment of intestine where it might be rapidly voided, thereby limiting the time available for osmotic water loss from the organism across intestinal tissues. Judging from the concen- tration of secreted fluid (ca. 500 mM NaCl; [33- 35]), the water permeability of the rectal gland ducts is reasonably low. The intramural glands of holocephalans, which drain into the intestine in a more crypt-like manner would be less efficient in NaCl removal although the placement of their ducts in the postvalvular segment of intestine is surely adaptive compared with the broad distribu- tion of crypts in tetrapods. In the context of ion transport, functional similarity of the homologous crypt and rectal gland epithelia is supported by the currently accepted models for transport [1,2,34,35]. A simple diagram of the cellular model for active Cl™ secretion, including the major relevant features is shown in Figure 4. Secretion depends ultimately on the basolateral Na*-K*-ATPase to maintain a low intracellular activity of Na*. The system operates as a two-step mechanism involving the apical (luminal) and basolateral (blood-side) cell membranes. Coupled transport driven by the electrochemical gradient for Na* brings Na~* and Cl” into the cell. Cl”, accumulated above elec- trochemical equilibrium, exits in a conductive manner down its gradient into the lumen as secreted Cl~. Accumulated Na‘ is recycled across the basolateral membrane by the Na‘t-K*t- ATPase. Under open circuit conditions, as would be the case in vivo, macroscopic electroneutrality is maintained by the diffusion of Na* through the cation-selective paracellular pathway from blood side to the lumen in response to the lumen- negative electrical potential difference established by Cl™ exit. This general model for Cl” secretion applies not only to rectal gland and crypt cells but also to secretory cells of the teleost skin and opercular membrane and mammalian tracheal epithelium [57, 61]. In contrast to Cl~ secretion, there is a greater diversity of transport mechanisms for the NaCl- absorptive component of intestinal ion transport. There are abundant reviews of these processes available for the interested reader [1, 2, 62, 63]. Fic. 4. Cellular model for active Cl~ secretion by mammalian crypt cells and fish rectal gland. LU indicates the luminal side of the epithelium and BL the blood side. The basolateral membrane Na*-— K*-ATPase maintains a low intracellular Na‘ activity which drives the coupled entry of Na* and Cl~. Cl” exits by a conductive mechanism across the luminal membrane. Na* and K* recycle across the basolateral cell membrane. Under open-circuit conditions, Na* diffuses through the paracellular pathway to the luminal side. (Adapted from [1, 2, 34-36, 85]). An identical mechanism is proposed for Cl~ secretion by chloride cells in fish skin and opercular membrane [59, 60]. Functional similarity between the rectal gland and crypts of Lieberktihn extends also to endo- crine mechanisms of control of ion transport. Active secretion of fluid by both rectal glands and intestinal crypts is stimulated by cyclic AMP. Moreover, in both systems, chemical messengers which act via cyclic AMP produce similar effects. For example, VIP stimulates both tissues to secrete; in both systems, VIP is thought to act via an increase in cyclic AMP [64-69]. Shuttleworth and Thorndyke [70] report that in sharks the natural agonist may not be VIP but, instead, the newly discovered peptide “rectin”. Similarly, somatostatin inhibits VIP-induced secretion in 942 C. A. LoretTz some mammalian intestinal segments and in shark rectal gland [66, 67, 71-73]. Although details of the schemes have yet to be elucidated fully, intracellular Ca** acting as an intracellular mes- senger stimulates secretion by both mammalian intestine and shark rectal gland; this stimulatory effect of Ca*t may be dependent on other intracellular messengers [74-77]. The cryptless teleost intestine bears some func- tional resemblance to the absorptive portion of mammalian intestine located at the villus [1], specifically with respect to control by hormonal and intracellular mediators like Ca?+. Nat and Cl~ absorption by some mammalian intestinal segments is stimulated by somatostatin [71, 72, 78]; urotensin II, a naturally-occurring teleost analog of somatostatin [79] stimulates Na* and Cl~ absorption across teleost intestine [47, 80, 81]. Regulation by the calcium messenger system of coupled NaCl absorption in the goby is similar to the general scheme proposed for coupled NaCl- absorptive systems in mammalian intestine [74, 75, 82]; elevated intracellular Ca** activity inhibits absorption (acting through calmodulin) whereas depressed Ca’ activity stimulates. One contribution of studies on fish tissues can be stated at present with respect to the regulatory effect of somatostatin on NaCl transport. As noted above, somatostatin reduces VIP-induced ion and fluid secretion by the mammalian intestine [71-73]; this effect could result from either a direct effect to block the secretory process of the crypts or an offsetting stimulation of absorption at the villi. The ability of somatostatin to inhibit VIP- induced secretion in rectal gland [67] and of urotensin II to stimulate basal absorption in teleost intestine [81] suggest the dual actions of somato- statin, antisecretory and absorptive, in reducing net intestinal secretion in mammals; several years ago, this question of dual action was unresolved [1]. DO RAYFINNED FISHES REALLY LACK INTESTINAL SECRETORY GLANDS? A special note must be made of a little known accessory secretory structure in teleost fishes. Marine species of the catfish family Plotosidae possess a dendritic organ located caudal to the urinary papilla [83, 84]. The dendritic organ is a highly folded, external epithelial organ; apparent secretory cells are located in short crypts or acini opening to the surface of the tissue leaflets. In the plotosid Cnidoglanis macrocephalus, ligation of the dendritic organ produces an increase in plasma sodium concentration, indicating a salt-excretory role for the organ [84]. A salt excretory function and the location of the organ near the anus invite speculation as to possible homology with the rectal gland. The salt-secreting cells of the dendritic organ are similar ultrastructurally to branchial chloride cells, elasmobranch rectal gland cells and avian nasal salt gland cells [83] reinforcing the suggestion of a role in salt secretion. The place- ment of the glands supports an ectodermal, rather than endodermal, origin, however. It appears, therefore, that teleosts (at least those examined to date) do not possess an intestinal NaCl secretory apparatus. CONCLUSION More study is needed to validate further the use of piscine models in intestinal transport research. Particularly, detailed studies on the biophysical mechanisms, as well as schemes for cellular and endocrine regulation involving larger numbers of putative messengers, are desirable. With further refinement, the teleost intestine may emerge as a model system for mammalian villar transport, with its coupled Na*-Cl~ or Na*-K*-2 Cl” absorp- tion (where present) and K* secretion. The shark rectal gland has already been acknowledged as a model for Cl” secretion not only by intestine, but also by sweat gland and salivary gland [85]. The teleost intestine and elasmobranch rectal gland can be models for the NaCl absorptive and Cl~ secretory components, respectively, of mammalian intestine. As presented here, the basis for such use can, in addition to functional similarity, also include their phylogenetic relationship. At the very least, though, results of studies on one system can provide useful insight into other systems and aid in the framing of interesting and informative hypotheses for future testing. Rectal Gland and Crypts ACKNOWLEDGMENTS Great thanks are due to Dr. J. W. Crim for collecting intestinal tissues from Amia and to Professor H. A. Bern for his encouragement and enthusiasm. 10 11 2 13 14 55 16 REFERENCES Field, M. (1981) In “Physiology of the Gastrointes- tinal Tract”. Ed. by L. R. Johnson, Raven Press, New York, pp. 963-982. Binder, H. J. (1981) In “Physiology of the Gastroin- testinal Tract”. Ed. by L. R. Johnson, Raven Press, New York, pp. 1003-1019. Walsh, J. H. (1981) In “Physiology of the Gastroin- testinal Tract”. Ed. by L. R. Johnson, Raven Press, New York, pp. 59-144. Ruskone, A., Rene, E., Chayvialle, J. A., Bonin, N., Pignal, F., Kremer, M., Bonfils, S. and Ram- baud, J. C. (1982) Digestive Dis. Sci., 5: 459-466. Freel, R. W., Hatch, M., Earnest, D. L. and Gold- ner, A. M. (1983) Am. J. Physiol., 245: G808- G815. Westergaard, H. and Dietschy, J. M. (1986) In “Physiology of Membrane Disorders”, 2nd edition. Ed. by T. E. Andreoli, J. F. Hoffman, D. D. Fanes- til and S. G. Schultz, Plenum Publ. Co., New York, pp. 873-885. Vigna, S. R. (1986) In “Vertebrate Endocrinology: Fundamentals and Biomedical Implicaions”. Ed. by P.K.T. Pang and M.P. Schreibman, Academic Press, New York, pp. 261-278. Bloom, W. and Fawcett, D. W. (1986) A Textbook of Histology, 11th edition, W.B. Saunders Co., Philadelphia. Trier, J. S. and Madara, J. L. (1981) In “Physiology of the Gastrointestinal Tract”. Ed. by L. R. John- son, Raven Press, New York, pp. 925-961. Roggin, G. M., Banwell, J.G., Yardley, J. H. and Hendrix, T. R. (1972) Gastroenterology, 63: 981- 989. deJonge, H.R. (1975) Biochim. Biophys. Acta, 381: 128-143. Welsh, M. J., Smith, P. L., Fromm, M. and Frizzell, R. A. (1982) Science, 218: 1219-1221. Weiser, M. M. and Quill, H. (1975) Gastroenterol- ogy, 69: 479-4872. Field, M., Smith, P. L. and Bolton, J. E. (1980) J. Membr. Biol., 55: 157-163. MacKay, W. C. and Lahlou, B. (1980) In “Epithe- lial Transport in the Lower Vertebrates”. Ed. by B. Lahlou, Cambridge Univ. Press, Cambridge, pp. 151-162. MacKay, W. C., Lahlou, B. and Porthe-Nibelle, J. ily) 18 19 20 724 22 23) 24 25 26 Zi} 28 29 30 Sil 32 33 34 35 36 Si) 38 39 40 4] 42 43 44 943 (1978) C. R. Acad. Sci. Paris, 287: 1239-1242. Loretz,C. A. (1983) Comp. Biochem. Physiol., 75A: 205-210. Mooney, S.M. and Loretz,C. A. (1987) Comp. Biochem. Physiol., 86A : 367-372. Andrew, W. and Hickman, C. P. (1974) Histology of the Vertebrates, C. V. Mosby Co., St. Louis. Loretz, C. A. and Bern, H. A. (1982) Neuroendo- crinology, 35: 292-304. Potts, W. [. W.,., Foster. M.A.” Rudy,.P.P.. and Howells, G. P. (1967) J. Exp. Biol., 47: 461-470. Smith, H. W. (1930) Am. J. Physiol., 93: 480-505. Smith, H. W. (1932) Q. Rev. Biol., 7: 1-26. Dixon, J. M. and Loretz,C. A. (1986) J. Comp. Physiol. B, 156: 803-811. Frizzell, R. A., Halm, D. R., Musch, M. W., Stew- art, C. P. and Field, M. (1984) Am. J. Physiol., 246: F946-F951. Ando, M. and Utida, S. (1986) Zool. Sci., 3: 605- 612. Crofts, D. R. (1925) Proc. Zool. Soc. Lond., 1925: 101-188. Hoskins, E. R. (1917) J. Morphol., 28: 329-367. Bulger, R. E. (1963) Anat. Rec., 147: 95-107. Bulger, R. E. (1965) Anat. Rec., 151: 589-608. Fange, R. and Fugelli, K. (1963) Sarsia, 10: 27-34. Van Lennep, E. W. (1968) J. Ultrastr. Res., 25: 94-108. Burger, J. W. and Hess, W. N. (1960) Science, 131: 670-671. Epstein, F. H.; Stoff,J. S: and Silva, P. (1983) J. Exp. Biol., 106: 25-41. Epstein, F. H., Stoff,J.S. and Silva, P. (1984) In “Chloride Transport Coupling in Biological Mem- branes and Epithelia”. Ed. by G. A. Gerencser, Elsevier Sci. Publ, B. V., pp. 347-357. Eveloff, J., Karnaky, K. J., Jr., Silva, P., Epstein, F. H. and Kinter, W. B. (1979) J. Cell Biol., 83: 16-32. Kent, B. and Olson, K. R. (1982) Am. J. Physiol., 243: R296-R303. Satchell, G. H. (1986) New Zealand J. Zool., 13: 101-105. Forey, P. L. (1980) Proc. R. Soc. Lond. B, 208: 369-384. Lemire, M. and Lagios, M. (1979) Acta Anat., 104: 1-15. Griffith, R. W. and Burdick, C. J. (1976) Comp. Biochem. Physiol., 54B: 557-559. Lagios, M. D. and McCosker, J. E. (1977) Copeia, 1977: 176-178. Parker, W. N. (1892) Trans. R. Irish Acad. Dublin, 30: 109-230. Lagios,M.D. and Stasko-Concannon,S. (1979) 45 46 47 48 49 50 ot 52 53 54 55 56 57 58 59 60 61 62 63 64 944 Cell Tissue Res., 198: 287-294. Patten, S. F., Jr. (1960) Exp. Cell Res., 20: 638- 641. Andrew, W. (1963) Ann. N. Y. Acad. Sci., 106: 502-517. Loretz,C. A., Howard,M.E. and Siegel, A. J. (1985) Am. J. Physiol., 249: G284-G293. Gas, N. and Noaillac-Depeyre, J. (1974) C. R. Acad. Sci. Paris, 279: 1085-1088. Stroband,H.W.J. and Debets,F.M.H. (1978) Cell Tissue Res., 187: 181-200. Trier, J.S. and Moxey, P.C. (1980) Cell Tissue Res., 206: 379-385. Youson, J. H. and Langille, R. M. (1981) Can. J. Zool., 59: 2341-2349. Pang, P.K.T., Griffiths, R. W. (1977) Am. Zool., 17: 365-377. Schaeffer, B. and Williams, M. (1977) Am. Zool., 17: 293-302. Prosser, C.L. (1973) In “Comparative Animal Physiology”, 3rd edition. Ed. by C. L. Prosser, W. B. Saunders Co., Philadelphia, pp. 1-78. Oguri, M. (1964) Science, 144: 1151-1152. Gerst, J. W. and Thorson, T.B. (1977) Comp. Biochem. Physiol., 56A: 87-93. Marshall, W. S. and Nishioka, R. S. (1980) J. Exp. Zool., 214: 147-188. Fosket@au. KS opsden- © Dy “urner,, is, Machen, T.E. and Bern, H.A. (1981) J. Exp. Biol., 93: 209-224. Foskett, J. K., Bern, H.A., Machen, T.E. and Conner, M. (1983) J. Exp. Biol., 106: 255-281. Degnan, K. J. (1984) In “Chloride Transport Cou- pling in Biological Membranes and Epithelia”. Ed. by G. A. Gerencser, Elsevier Sci. Publ. B. V., pp. 359-391. Welsh, M. J. (1986) In “Physiology of Membrane Disorders”, 2nd edition. Ed. by T. E. Andreoli, J. F. Hoffman, D.D. Fanestil and S.G. Schultz, Plenum Publ. Co., New York, pp. 751-766. Schultz, S.G. (1981) In “Physiology of the Gas- trointestinal Tract”. Ed. by L. R. Johnson, Raven Press, New York, pp. 983-989. Powell, D. W. (1986) In “Physiology of Membrane Disorders”, 2nd edition. Ed. by T. E. Andreoli, J. F. Hoffman, D.D. Fanestil and S.G. Schultz, Plenum Publ. Co., New York, pp. 751-766. Schwartz, C. H., Kimberg, D. V., Sheerin, H. E., Field, M. and Said, S. I. (1974) J. Clin. Invest., 54: 536-544. and Atz, J. W. 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 C. A. LORETZ Waldman, D. B., Gardner, J. D., Zfass, A. M. and Makhlouf, G.M. (1977) Gastroenterology, 73: 518-523. Carter, R.F., Bitar, K.N., Zfass, A.M. Makhlouf, G.M. (1978) Gastroenterology, 74: 726-730. Stoff, J.S., Rosa, R., Hallac, R., Silva, P. and Epstein, F. H. (1979) Am. J. Physiol., 237: F138- F144. Krejs, G.J., Browne, R. and Raskin, P. (1980) Gastroenterology, 78: 26-31. Davis, G. R., Santa Ana, C. A., Morawski, S. G. and Fordtran, J. S. (1981) J. Clin. Invest., 67 : 1687- 1694. Shuttleworth, T. J. and Thorndyke, M. C. (1984) Science, 225: 319-321. Dharmsathaphorn, K., Binder, H. J. and Dobbins, J. W. (1980) Gastroenterology, 78: 1559-1565. Dharmsathaphorn, K., Racusen, L. and Dobbins, J. W. (1980) J. Clin. Invest., 66: 813-820. Dharmsathaphorn, K., Sherwin, R. S. and Dobbins, J. W. (1980) Gastroenterology, 78: 1554-1558. Donowitz, M. (1983) Am. J. Physiol., 245: G165- GI7T- Donowitz, M. and Welsh, M. J. (1986) Ann. Rev. Physiol., 48: 135-150. Forrest, J. N., Jr., Dornbusch, J. N., Ross, B. and Murdaugh, A. (1987) Bull. Mt. Desert Is. Biol. Lab., 18: 10-13. Shuttleworth, T. J. (1983) Am. J. Physiol., 245: R894-R900. Guandalini, S., Kachur, J. F., Smith, P. L., Miller, R. J. and Field, M. (1980) Am. J. Physiol., 238: G67-G74. Pearson, D., Shively, J. E., Clark, B. R., Gesch- wind, I. I., Barkley, M., Nishioka, R. S. and Bern, H. A. (1980) Proc. Natl. Acad. Sci. USA, 77: 5021- 5024. Mainoya, J. R. and Bern, H. A. (1982) Gen. Comp. Endocrinol., 47: 54-58. Loretz, C. A., Freel, R. W. and Bern, H. A. (1983) Gen. Comp. Endocrinol., 52: 198-206. Loretz, C. A. (1987) J. Exp. Zool., in press. van Lennep, E. W. and Lanzing, W. J. R. (1967) J. Ultrastr. Res., 18: 333-344. Kowarsky, J. (1973) Comp. Biochem. Physiol., 46A : 477-486. Epstein, F. H. (1979) Yale J. Biol. Med., 52: 517- S23 ZOOLOGICAL SCIENCE 4: 945-954 (1987) © 1987 Zoological Society of Japan REVIEW Insect Circadian Activity with Special Reference to the Localization of the Pacemaker YOSHIHIKO CHIBA and KENJI TOMIOKA Environmental Biology Laboratory, Biological Institute, Yamaguchi University, Yamaguchi, 753 Japan Circadian rhythm, an endogenous biological rhythm with a period approximating the length of a day is ubiquitous among the physiological organ- ization of eukaryotes, forming the basis of daily rhythm in animal life. Characteristic features of this rhythm are, on one hand, persistence in a constant environment, where at least light intensi- ty and temperature are kept as constant as possible and, on the other hand, entrainability to environ- mental (particularly light) cycles of an appropriate length (in general, not much deviated from 24 hr). The internal mechanism has been hypothesized as including, at the very least, a pacemaker (primary generator of temporal information) and photore- ceptors which disperse photic information toward the pecemaker for entrainment. If a tissue includes the pacemaker, then the following criteria should be met. 1) Removal or disconnection of the tissue from the rest of the body should cause the overt rhythm under study to disappear or to become quite atypical. 2) The tissue itself should show circadian rhythm in complete isolation from the rest of the body. 3) Either the phase or the period of the overt rhythm should be transplanted by transplanting the tissue to an individual deprived of rhythmicity. It would, of course, be ideal if all these criteria are satisfied, but difficulties arising mainly from technical prob- lems do not always allow us to investigate the latter two criteria completely. Pacemaker tissues which have so far been demonstrated to meet all or at least two of the Received May 30, 1987 criteria —1) plus one other— are the suprachias- matic nuclei of mammals [1,2], pineal body of birds [3, 4], and eyes or parieto-visceral ganglion of molluscs [5-7]. Studies on the function of the pacemaker at the cellular level have also begun in some animals [8]. In insects where the candidate pacemaker tissue was reported earlier than in any other animals [9], essential progress has occurred only recently. The purpose of this paper is to review the studies on the localization of pacemakers controlling the circad- ian timing of insect behavior or locomotion, in order to augment previous pertinent reviews [10- 1D); THE OPTIC LOBE Disconnection of the optic lobes from the midbrain Nishiitsutsuji-Uwo and Pittendrigh [9] first re- ported that the bilaterally paired optic lobes possibly house some crucial structure (driving oscillator) underlying the circadian locomotor rhythm of the cockroach, Leucophaea maderae. This was based on experimental results meeting the criterion that the overt circadian rhythm free-ran under a 24 hr cycle of light and darkness (LD) after bilateral optic nerve severance, but disappeared when the optic tracts were cut to isolate the optic lobe neurally from the midbrain (cerebral lobes). Each optic lobe has three neuropiles (synaptic regions), the lamina, medulla, and lobula (Fig. 1). Electrical cauterization experiments yielded re- 946 Y. CHIBA AND K. ToMIOKA L.maderae G. bimaculatus Fic. 1. Schematic drawings of cricket (A) and cock- roach (B) brains illustrating the optic nerves (on), optic tract (ot), optic stalk (os) and the region of neuropiles, lamina (la), medulla (me), and lobula (lo). The optic lobes, each including these neuro- piles, lies bilaterally between the midbrain (mb) and the compound eyes. sults suggesting that the cell bodies area adjacent to the second optic chiasma and to the lobula play a particularly important role [13]. Similarly, it has been expressed that the medulla plus the lobula system may be crucial but that the most distal synaptic region, the lamina, is not necessary for the manifestation of rhythm. Rather, it functions as a pathway conveying photic information from the compound eyes to the postulated pacemaker located somewhere in the deeper part [14]. The indispensability of the optic lobe was also demonstrated in crickets (Teleogryllus commodus [15], and Gryllus bimaculatus {16]). The latter has a medulla and lobula which are connected by a long optic stalk in contrast to the cockroach which has the two neuropiles located close together (Fig. 1). In the optic lobe ablation experiment, the optic stalk was cut at its distal end near the medulla, leaving the greater part of the stalk plus the whole lobula attached to the midbrain. However, the bilateral ablation caused the rhythm to disappear or sometimes to become quite atypical (Fig. 2). Thus, whether the view of Sokolove [13] or 49 — ate i ee 1- = SS Se — oe —————————— . SSeS — ane == ——— eae DSSS SS ————— a re Seen Se Se Sa eee ee ae 20- (a Se See ae SS ————— a Se SSS See a re Se ee SSS ee Se ——— ee ee —————— Se 40 = SSS ee ee ee = ee 1C ie hss! | ! 1- = See ee Sarees es ——————— == = —- =< = —— ——a = =a Ss ee SS ee ——— Sa a Se ere ee Se eee ee a ee = ee 20 ae = a re Sa ee ee ae eee a TB a er en eoeniee tn eee ee ae re ee Se ee pe ee ae SE en pipe 40- Se ey Sa (ap ame ee St pee een a eee a Soe Time of day Fic. 2. Activity records of three crickets. A: Intact cricket showing entrained rhythm reversing itself from diurnal to nocturnal 4 to 5 days after imaginal molt. B: Cricket with optic nerves severed bilaterally, showing free-running rhythm. Note the occurrence of rhythm reversal. C: When optic lobes were bilaterally ablated, circadian rhythmicity dis- appeared. Crickets were 8th instar nymphs at time of operation (arrow)— one instar before adult. Roberts [14] applies to the cricket deserves careful investigation. The third insect which, to be rhythmic, may require the optic lobe is the beetle, Carabus problemsticus; bilateral optic lobe ablation re- sulted in the disappearance of the rhythm [17]. Transplantation of the optic lobe If the optic lobes, disconnected from the mid- brain by cutting the optic tracts, are left in situ, the locomotor rhythm reappears in a few weeks. In addition, the rhythm returned in a cockroach Insect Circadian Pacemaker 947 which had lost rhythmicity through optic lobe ablation, when the optic lobe was transplanted from another individual [18]. The transplanted lobe had reestablished structural connections with the midbrain of the host. The free-running period of the restored rhythm was similar to the period of the donor animal’s rhythm before surgery. Page [19] also succeeded in replacing one of the bilaterally paired lobes with a lobe transplanted DD DAY !106- from another individual having a different free- running period. The recipient’s activity showed the two rhythmic components (Fig. 3); one may be controlled by the recipient’s original lobe and the other by the donor’s. There is no other explana- tion for these data except that the oscillation regulating locomotor activity must originate in the optic lobes. T22 DONOR (ROL) T26 HOST (LOL) SS SSS SSS SS 12M 12M | 12M 12M TIME (CST) Fic. 3. Activity record of an animal with a unilateral optic lobe transplant [19]. The host animal had been raised from birth in LD 13:13 (T26) and the donor had been raised in LD 11:11 (122). The right optic lobe (ROL) of the donor was transplanted. The host retained its left optic lobe (LOL). The record begins 106 days after transplantation and 33 days after section of the host optic tract; the record is ’triple-plotted’, and shows 72 hr across. Two clear components to the activity rhythm appear to be free-running independently. An increase in the activity level is evident when the two components come into phase, and activity is nearly eliminated when the components are in anti-phase [19]. (Courtesy of Dr. Terry Page) 948 Y. CHIBA AND K. TOMIOKA Circadian nervous activity of optic lobe efferents In the cricket, G. bimaculatus, lamina-medulla tissue ablation causes the circadian locomotor rhythm to disappear [16]. The “lamina-medulla- compound eye” system (LMCS) exhibited clear circadian rhythms in the multiple unit activities recorded extracellularly from the cut end of the optic stalk with a suction electrode under constant conditions. In LL, all specimens exhibited a rhythm with a peak discharge frequency in the subjective night and with a free-running period longer than 24hr. However, in specimens kept under DD or in those with the optic nerve severed, two types of circadian patterns were observed: one type showed a “diurnal” increase and the other a Multiple unit activity (x10~*) 12 18 24 06 12 Time of day Fic. 4. Circadian rhythms of the lamina-medulla effer- ents after both optic stalk and optic nerve transec- tion under constant light. The multiple unit activity showed either diurnal (A) or nocturnal (B) in- creases. In both cases, both the cerebral lobe and subesophageal ganglion were excised. The opera- tion was performed at 10:00 on day 1. The record should be read from top to bottom [16]. “nocturnal”. Removal of the cerebral lobe and/or subesophageal ganglion, which minimizes the pos- sibility of hormonal control over the system being measured, did not affect these rhythms (Fig. 4). Even though humoral controls were not excluded completely, the results may be regarded as fairly conclusive evidence that the “lamina-medulla’- complex contains a neural mechanism, i.e. a circadian pacemaker [20]. Isolation of the pace- maker tissue from both neural and humoral factors is necessary to make the evidence more conclusive, as is already being tried in the cockroach [21]. Circadian rhythm of the amplitude of the electroretinogram (ERG) Studies on ERG amplitude rhythm also suggest that the optic lobe contains a circadian pacemaker. In the beetle, Blaps gigas, ERG rhythms of the bilateral compound eyes free-ran under DD with specific circadian periods and thereby desynchro- nized with each other, indicating that bilateral pacemakers function independently [22]. The ERG rhythm disappeared after lobula ablation in the beetle, Anthia sexguttata [23]. The cricket, G. bimaculatus, also showed circad- ian ERG rhythm under DD peaking in the subjective night. The rhythm persisted in the LMCS isolated from the lobula and the midbrain (Fig.5). A 24hr LD cycle given unilaterally entrained the rhythm of the ipsilateral LMCS isolated from the lobula and the midbrain, but did not entrain the contralateral normal LMCS kept in DD. Consequently, the two eyes were desynchro- nized, implying that the LMCS constitutes a complete circadian system composed of a receptor (photoreceptor), pacemaker and effector (photo- receptor), and that the bilaterally paired systems can function independently of each other [24, 25]. The question of whether the locomotion and ERG share a common pacemaker was answered to some extent by studies with the cockroach. Bisec- tion of the optic lobe distal to the lobula abolished the ERG rhythm of the cockroach, L. maderae, suggesting that the pacemaker is probably located near the lobula, as in the case of the locomotor rhythm [26]. Insect Circadian Pacemaker 949 100 y : ERG Amplitude (%MAX.) 12 12 12 12 198 20208 -16 12 12 12 12 12 Time of day Fic. 5. ERG amplitude rhythms in a cricket after bilateral severance of the optic stalk. O: right eye, @: left eye. Record was taken in DD. Taus of right and left eyes were 23.2 and 23.4 hr, respectively. Data partly missing on days 2 and 3 due to technical failure [24]. Bilaterally paired pacemaker If the bilateral pacemakers with their intrinsic periods work independently from each other, then locomotion under the control of the pacemakers shows a perturbed rhythm soon after the animal is held under constant conditions. But, the perturba- tion seldom occurs. That this is attributable to the existence of a bilateral connection between the postulated pacemakers was demonstrated both functionally and morphologically. The functional demonstration was based on two lines of evidence [27]. First, the free-running period in locomotion shown by the cockroach with a unilateral lobe ablated did not change signif- icantly under DD, no matter which of the bilateral lobes was left intact, but was significantly longer than that shown by the intact cockroach. Second, the postulated pacemaker entrained to the LD cycle received by the contralateral photoreceptor. From a morphological aspect [28], abundant axons and axon terminals were observed degener- ated in one optic lobe following extirpation of the other. Roth and Sokolove [28] also utilized a postaxotomy change in RNA distribution and retrograde axonal transport of the enzyme horse- radish peroxidase to visualize the perikarya of neurons which directly interconnect the optic lobes of the cockroach, Leucophaea maderae. The cricket, Teleogryllus commodus, which had the optic nerves cut on one side during the last larval instar, showed two activity (stridulation) components with different periods in LL; one period was longer, the other shorter than 24 hr. It is likely here that the two underlying pacemakers are only weakly coupled [29]. EXTRA- “OPTIC LOBE” PACEMAKER The locomotor rhythm persisted in the silk- moths, Antheraea pernyi and Hyalophora cecro- pia, after ablation of the bilateral optic lobes and, in addition, the unilateral cerebral lobe. Because the rhythm vanished by total brain ablation or severance of the circumesophageal connectives, the pacemaker may be situated not in the optic lobes, but in the cerebral lobes [30]. The postulated pacemaker receives photic in- formation through the extraocular photoreceptor for entrainment and emits temporal information toward the thoracic nervous system through the neuronal pathway to control the flight rhythm. The housefly, Musca domestica, [31] and the mosquito, Culex pipiens pallens, [32] may also have the pacemaker in the cerebral lobes. The mosquito, deprived of the greater part of the optic lobes (Fig. 6), still maintained a circadian activity rhythm free-running both in DD (Fig. 7) and in LL (Fig. 8) but synchronizing with a 24 hr LD cycle. Surgical operations such as brain 950 Y. CHIBA AND K. TOMIOKA BA Fic. 6. The structure of the mosquito brain; a horizontal section at level C of the head indicated at bottom left. At this level, all neuropiles in the optic lobe are seen in one section, but the cerebral lobes do not appear. a opt t: anterior optic tract, ce: compound eye, cib p: cibarial pump, d cib p: dilators of cibarial pump, dd: dorsal dilators, f opt ch: first optic chiasma, la: lamina, Id: lateral dilators, lo: lobula, lop: lobula plate, me: medulla, opt nsc: optic lobe neurosecretory cell, opt n: optic nerve, ph p: pharyngeal pump, p opt t: posterior optic tract, sog: suboesophageal ganglion, s opt ch: second optic chiasma (Kasai and Chiba, unpublished). ablation to eliminate rhythm were not successful because of high mortality. Therefore, the parts of central nervous system crucial for controlling the overt rhythm are unknown, although they might well be in the cerebral lobes, as in the silkmoth and housefly. The adult mosquito has no ocelli [33]; there might be an extraocular photoreceptor enabling the LD cycle to entrain the rhythm. The moquito’s circadian activity is characterized by a diphasic pattern with two peaks per cycle [34— 36]. The peaks are assumed to be of a physiologi- cally different nature [37] or to be controlled by separate oscillators [35,36]. Whatever the mechanism is, they occurred in the altered mos- quitoes, suggesting that the optic lobe is not involved in producing the diphase. However, persistence of the rhythm in the optic-lobe-ablated insects does not in fact mean that the optic lobes contain no oscillator, in view of the idea that multiple oscillators form hierarchy underlying an overt rhythm [10, 38]. If the hier- archy exists, it is not unusual for the overt rhythm to linger at least for a few days, even though in a- typical form, in the absence of the master oscillator (pacemaker). The possibility that the rhythm observed in the optic-lobe-ablated mosquito is residual is highly unlikely, because the rhythm persisted in all altered individuals in a typical form, and for as long as measurement continued (i.e., at least 10 days). Cymborowski [40,41] has insisted that the cricket, Acheta domesticus, has an intra-“cerebral lobe” pacemaker. This is based on the disappear- ance of rhythm and the occurrence of hyperactivity Insect Circadian Pacemaker 951 Intact 12 24 12 Time of day Fic. 7. Activity records of two typical female mos- quitoes Culex pipiens pallens, held under LD 16:8 followed by constant darkness (DD). Lighting conditions indicated by white and black at top of figure. The post-operative mosquitoes (lower panels) showed significant rhythms under DD with taus of 23.7. ‘LB-MD-ablated’ mosquitoes lost the greater part of both optic lobes (including the lobula, lobula plate, and medulla) but retained, bilaterally, the distal portion with lamina [32]. =z (RENN es Days Intact |= ree en “1 = z aan | 5- a a TD | SUT IO | a oH Mn eer oes ie aa == T aa LL 10- Saas T Gl aaa a es Sue ea SE SS SS LD 15- TTL Tar wr — LB-MD-ablated Us L201 ee Lae EAs [wD ee : aaa LL Trae Serr TT TIT TTT 3T Tilam TTT aT CU 10-77—> ITT 11 Ta D Time (hours) Fic. 8. Activity records of two typical female mos- quitoes Culex pipiens pallens, under LD 16:8 fol- lowed by constant light (LL). The LL records for the post-operative mosquito (lower two panels) are replotted on ac. 30 hr time axis in the lower panel to clarify the presence of a rhythm with a tau of c. 15 hr. For further explanation, see Fig. 7. caused by the ablation of neurosecretory cells in the pars intercerebralis. The rhythm resumed, however, with a brain transplanted from a normal animal. In the grasshopper, Ephippiger ephippi- ger, the bilateral removal of the optic lobes did not essentially affect the sound production but caused a change in its waveform [42]—another example alluding to the existence of a cerebral lobe pacemaker. CIRCADIAN TIMING OF BEHAVIORS DURING POST-EMBRYONIC DEVELOPMENT Once-in-a-lifetime developmental behaviors such as molting and eclosion in many insects are under circadian control. Remarkable evidence has been accumulated for endocrinological mecha- nisms of the circadian timing of adult eclosion [43], larval-larval molting [44], larval-prepupal develop- ment [45, 46], and pupation-associated gut-purge [47, 48]. The adult ecdysis (eclosion) clock of the silk- moth is located in the cerebral lobe [43]. This was demontrated by the exchange of brains between two different species with different daily timings of emergence. An extraocular photoreceptor in the cerebral lobes functions for synchronization with the LD cycle. The eclosion involving a stereotyped sequence of behaviors is triggered by a peptide hormone—the eclosion hormone released from the brain under control of the circadian clock. This peptide acts directly on the CNS to release the ecdysial motor programs [49-51]. The eclosion is preceded by movements to detach the old cuticle from the new one. In the moth, Manduca sexta, this preparatory behavior, triggered by the declin- ing titer of ecdysteroids, is timed by the action of a temperature-sensitive clock residing outside the brain, whereas the timing of eclosion is influenced by both the phase of the ecdysteroid decline and by the brain clock. The two distinct clocks thus act in this moth late in development. In the moth, Samia cynthia, a brain clock times the prothoracicotropic hormone release which is necessary for gut purge. However, the final timing of this preparatory behavior for pupation is con- trolled by a clock in the prothoracic gland that 952 Y. CHIBA AND K. TOMIOKA gates the release of ecdysone. The prothoracic gland itself includes the photoreceptor for entrain- ment. These conclusions came from experiments which localized illuminations and transplanted glands from one larva to another [48, 49]. SUMMARY AND PERSPECTIVE Optic lobe and midbrain Insects may be divided into two groups depend- ing on whether or not circadian locomotor rhythm persists after optic lobe ablation. One group includes the cockroach and cricket which un- doubtedly have a pacemaker in the optic lobes [19, 20]. In the other group, however, which includes the silkmoth [43], housefly [31] and mosquito [32], proof of the existence of a pacemaker in the midbrain still eludes us. Therefore, in comparing the two groups, one cannot say more than that the crucial part in controlling the locomotor rhythm is different. An interesting view is that the difference is related to the method of metamorphosis [43]; the locus of the pacemaker is the optic lobes in insects undergoing incomplete metamorphosis and the midbrain in those undergoing complete meta- morphosis. A study with the beetle [23] casts doubts on this view, which still deserves further study. Neural and hormonal controls As to the pathway conveying a time cue (tem- poral information) from the optic lobe pacemaker to the outside of the brain, there have been conflicting views. Nishiitsutsuji-Uwo and Pitten- drigh [9] postulated that the lateral neurosecretory cells (LNSC) in the pars intercerebralis function as a junction station where the pathway is changed from nervous to hormonal. This postulation was soon superseded by the view that the pathway is all neural and is never changed to hormonal in the brain [53, 54]. However, things are not quite settled; recent reports say that surgical operations on the pars intercerebralis frequently lead to abolishment of the rhythm [15, 40, 41,55]. Connection of the optic lobe pacemaker to the pars intercerebralis still leaves much room for investigation, especially from the standpoint, appearing in the following section, that multiple oscillators form a circadian system. In Drosophila melanogaster, an arrhythmic mutant gained rhythmicity with the donor’s period when receiving a brain from a short period mutant by transplantation [39]. Although it was not mentioned whether the transplanted brain in- cluded the optic lobe or not, in any event, this result suggests that a humoral control is involved in the manifestation of locomotor rhythm. Multiple oscillators Kinetic studies using a Drosophila pupae population have resulted in the two-oscillator model to explain the mechanism of daily timing of eclosion [56]. It says that a temperature sensitive oscillation controls the timing of eclosion directly but is entrained to a light sensitive one which is defined as the master oscillation. The definite phases of oscillation, recurring at the interval of about a day, are called “gates” which allow insects to emerge if developmental maturation is attained. The concrete feature corresponding to the assumed oscillators is still unknown, but the concept of “multiple oscillators underlying an overt rhythm shown by a single physiological process” has been accepted as a most attractive paradigm for experimentation. It has contributed toward the analysis of circadian systems related not only to post-embryonic behaviors [44, 47, 48] but also to adult locomotion [19, 29,55]. In the cricket, T. commodus, the neurosecretory cells in the pars intercerebralis may be related in some way to the manifestation of rhythm, since their ablation sometimes abolished stridulation and locomotor rhythm [15, 56]. Singing activity of the cricket lost circadian rhythmicity, which reap- peared in synchrony with a 24hr temperature cycle. The reappearance was accompanied by some characteristics exemplified by the presence of transients, suggesting the existence of an extra- “optic lobe” temperature sensitive oscillation [58]. On the other hand, the existence of “separate oscillators for multiple physiological processes” has been assumed in Drosophila (adult locomotion and eclosion) [59], the moth Pectinophora (ovi- position, eclosion and hatching [60]), the flesh fly Insect Circadian Pacemaker 953 Sarcophaga (the initiation of larval wandering, pupal eclosion and the induction of pupal diapause [61]) and the cockroach (locomotion and growth layer formation in the endocuticle [62]). How these oscillators, if they really exist, are organized to form a circadian system is a problem arising after the localization of oscillators. Cellular mechanisms The localization of the oscillator (pacemaker) is a step further into the study of the cellular mechanisms of oscillation. This analytical step, already taken in rodent suprachiasmatic nuclei [1, 2] and the bird pineal body [3, 4], has not been reported in insects. However, a Drosophila study yielded an in- teresting view concerning the cellular mechanism of behavior rhythm, although it is not directly related to localization of the pacemaker. Fluores- cence intensity of isolated salivary gland cells of wild-type D. melanogaster larvae showed signif- icant rhythmicity in the cytoplasmic and nuclear areas. But in arrhythmic mutant (per) cells, arrhythmic and rhythmic changes were registered, the latter showing lower amplitudes. The de- creased amplitude and the lower number of significant cellular rhythms in per® mutants might be viewed as leading to an apparent arrythmicity at the behavioral level [63]. Gene control To use biotechnological means for revealing the essential mechanism of oscillation would have great impact. In D. melanogaster, three mutants are known with respect to a free-running period in emergence and locomotion; one is arrhythmic (per°), the other two having free-running periods of 19hr (pers) and 28hr (per') [64]. These mutants are all related to a gene per located between zeste and white on the distal in the X chromosome, one of the most extensively investi- gated region of Drosophila genome [65]. The per gene DNA region was almost identified after much controversy [66,67]. Transfer of this gene from the wild type to an arrhythmic mutant restored circadian behavioral rhythm [68, 69]. It is highly possible that this DNA codes for a proteoglycan [70]. ACKNOWLEDGMENTS We thank Dr. Terry Page of Vanderbilt University who kindly permitted us to reproduce Figure 3 and provided us with a glossy print. Thanks are also due to Mr. Yutaka Yamamoto for his help in preparing the manuscript. This work was supported by grants from the Japanese Ministry of Education, Science and Culture (Nos. 60304010, 60304011, 61840024). REFERENCES 1 Inouye, S. T. and Kawamura, H. (1979) Proc. Natl. Acad. Sci. USA, 76: 5962-5966. 2 Sawaki, Y., Nihonmatsu,I. and Kawamura, H. (1984) Neurosci. Res., 1: 67-72. 3 Deguchi, T. (1979) Nature, 282: 94-96. Takahashi, J.S., Hamm, H. and Menaker, M. (1980) Proc. Natl. Acad. Sci. USA, 77: 2319-2322. 5 Block, G.D. and McMahon, D.G. (1984) J. Comp. Physiol., A, 155: 387-395. 6 Jacklet, J. W. (1969) Science, 164: 562-563. 7 Strumwasser, F. (1974) In “The Neurosciences: Third Study Program”. Ed. by F.O. Schmitt and F.G. Worden, MIT Press, Cambridge, Mass., pp. 459-478. 8 Jacklet, J. W. (1984) Int. Rev. Cytol., 89: 251-294. 9 Nishiitsutsuji-Uwo, J. and Pittendrigh, C. S. (1968) Z. vergl. Physiol., 58: 14-46. 10 Brady, J. (1974) Adv. Insect Physiol., 10: 1-115. 11 Saunders, D.S. (1976) Insect Clocks. Pergamon Press, Oxford. 12 Page,T.L. (1981) In “Handbook of Behavioral Neurobiology, Vol. 4 (Biological Rhythms)”. Ed. by J. Aschoff, Plenum Press, New York, pp. 145-172. 13 Sokolove, P. G. (1975) Brain Res., 87: 13-21. 14 Roberts, S. K. (1974) J. Comp. Physiol. , 88 : 21-30. 15 Loher, W..(1972) J. Comp. Physiol. ,79: 173-190. 16 Tomioka, K. and Chiba, Y. (1984) Zool. Sci., 1: 375-382. 17 Balkenol, M. and Weber, F. (1981) Mitt. Dtsch. Ges. allg. angew. Entomol., 3: 223-227. 18 Page, T. L. (1982) Science, 216: 73-75. 19 Page, T. L. (1983) J. Comp. Physiol., 153 : 353-363. 20 Tomioka, K. and Chiba, Y. (1986) J. Insect Physiol. ,32 : 747-755. 21 Page, T. L. (1986) Japan-US Seminar on Biological Rhythm (July 30-Aug. 2, 1986, Hawaii), organized by Y. Chiba and J. Feldman. 22 Koehler, W. K. and Fleissner, G. (1978) Nature, 274: 708-710. 23 Fleissner, G. (1982) J. Comp. Physiol., 149: 311- 316. 24 Tomioka,K. and Chiba, Y. (1982) Naturwissen- 25 26 yi 28 29 30 Sil 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 954 schaften, 69: 395-396. Tomioka, K. and Chiba, Y. (1985) J. Interdiscipl. Cycle Res., 16: 73-76. Wills, S. A., Page, T. L. and Colwell, C. S. (1985) J. Biol. Rhythm, 1: 25-37. Page, T. L., Caldarola, P. C. and Pittendrigh, C. S. (1977) Proc. Natl. Acad. Sci. USA, 74: 1277-1281. Roth, R. L. and Sokolove, P. G. (1975) Brain Res., 87: 23-39. Wiedenmann, G. (1983) J. Comp. Physiol., 150: 51-60. Truman, J. W. (1974) J. Comp. Physiol., A, 95: 281-296. Helfrich, C., Cymborowski, B. and Engelmann, W. (1985) Chronobiol. Int., 2: 19-32. Kasai, M. and Chiba, Y. (1987) Physiol. Entomol., 12: 59-65. Clements, A. N. (1963) The Physiology of Mos- quitoes. Pergamon Press, Oxford. Chiba, Y., Yamakado, C. and Kubota, M. (1981) Int. J. Chronobiol., 7: 153-164. Clopton, J. R. (1985) J. Comp. Physiol., A, 155: 1- 2, Jones, M. D. R. (1982) Physiol. Entomol., 7: 281- 289. Chiba, Y., Kubota, M. and Nakamura, Y. (1982) J. Interdiscipl. Cycle Res., 13: 55-60. Moore-Ede, M. C., Scemelzer, W. S., Kass, D. A. and Herd, J. A. (1976) Fed. Proc., 35: 2333-2338. Handler, A. M. and Konopka, R. J. (1979) Nature, 179: 236-238. Cymborowski, B. (1973) J. Insect Physiol., 19: 1423-1440. Cymborowski, B. (1981) J. Interdiscipl. Cycle Res.., 12: 133-140. Dumortier, B. (1972) J. Comp. Physiol., 77: 80- i? Truman, J. W. (1972) In “Circadian Rhythmicity”. Ed. by J. F. Bierhuizen, Centre for Agricultural Publishing and Documentation, Washington, pp. 111-135. Truman, J. W. (1972) J. Exp. Biol., 57: 805-820. Truman, J. W. and Riddiford, L. M. (1974) J. Exp. Biol., 60: 371-382. Fujishita, M. and Ishizaki,H. (1982) J. Insect Physiol., 28: 77-84. Mizoguchi, A. and Ishizaki, H. (1982) Proc. Natl. Acad. Sci. USA, 79: 2726-2730. 48 49 50 >i SY 53 54 55 56 ST 58 59 60 61 62 63 64 65 66 67 68 69 70 Y. CHIBA AND K. TOMIOKA Mizoguchi, A. and Ishizaki, H. (1984) J. Comp. Physiol., A, 155: 639-647. Truman, J. W. (1978) J. Exp. Biol., 74: 151-174. Truman, J. W. (1971) Am. Zool., 21: 655-661. Truman, J. W. and Weeks, J. C. (1983) Soc. Exp. Biol. Symp., 37: 223-241. Truman, J. W. (1984) J. Comp. Physiol., A, 155: 521-528. Bardy, J. (1971) In “Biochronometry”. Ed. by M. Menaker, National Academy of Sciences, Washing- ton, D.C., pp. 517-524. Robert, S.K., Skopik,S.D. and Driskill, R. J. (1971) In “Biochronometry”. Ed. by M. Menaker, National Academy of Sciences, Washington, D.C., pp. 505-515. Tomioka, K. (1985) J. Insect Physiol., 31: 653-657. Sokolove, P.G. and Loher, W. (1975) J. Insect Physiol., 21: 781-799. Pittendrigh, C. S. (1960) Cold Spring Harbor Symp. Quant. Biol., 25: 159-184. Rence, B. and Loher, W. (1975) Science, 190: 385- 387. Engelmann, W. and Mack, J. (1978) J. Comp. Physiol., 127: 229-237. Pittendrigh,C.S. and Minis,D.H. (1978) In “Biochronometry”. Ed. by M. Menaker, National Academy of Sciences, Washington, D.C., pp. 212- 250. Saunders, D. S. (1986) Chronobiol. Int., 3: 71-83. Lukat, R. (1978) Experientia, 34: 477. Weizel,G. and Rensing,L. (1981) Physiol. 143: 229-235. Konopka, R. J. and Benzer, S. (1971) Proc. Natl. Acad. Sci. USA, 68: 2112-2116. Konopka, R. J. (1979) Fed. Proc., 38: 2602-2605. Bargiello, T. A. and Young, N. W. (1984) Proc. Natl. Acad. Sci. USA, 81: 2142-2146. Reddy, P., Zehring, W. A., Wheeler, D. A., Pirrot- ta, V., Hadfield, C., Hall, J.C. and Rosbash, M. (1984) Cell, 38: 701-710. Bargiello, T. A., Jackson, F. R. and Young, M. W. (1984) Nature, 312: 752-754. Zehring, W. A., Wheeler, D.A., Reddy, P., Konopka, R. J., Kiriacou, C. P., Rosbash, M. and Hall, J. C. (1984) Cell, 39: 369-376. Reddy, P., Jacquier, A. C., Abovich, N., Peterson, G. and Rosbash, M. (1986) Cell, 46: 53-61. J. Comp. Proceedings of the Fifty-Eighth Annual Meeting of the Zoological Society of Japan October 7-9, 1987 Toyama Suffixal letters of abstract number refer to the abbreviated subfields of zoology PH : physiology CB: cell biology GE: genetics IM : immunology BI : biochemistry DB: developmental biology EN : endocrinology MO: morphology BB : behavior biology E@e: ecology TS : taxonomy and systematics ee a Bi 7 ‘ete Naarl ere ry ds ‘: f abi oy y\ = $ | pa it PEE ODES EY I Pie BALE 1G) sab ms ‘esi MSINSIRVe DUR ALO ORRE fie an A TY : | th Oe =F j PL | 5) Bah Z cach ya. 2 ae a We one ate Ki x Ae rea ( ‘ PNG ; We ey rt ah ae ee \ a yt at i 1), Ablasidive WoC wea | PLETE re fe ae eae eee we | PLZ 0 ED Aat a ia ve * Pe if copa: YOU sg Soyer ZOOLOGICAL SCIENCE 4: 957 (1987) © 1987 Zoological Society of Japan PRESIDENTS MESSAGE I would like to take the opportunity to make a few comments on the occasion of the Fifty-eighth Annual Meeting of the Zoological Society of Japan, held in Toyama. First of all, I would like to express my sincere gratitude to the chairman of the Organizing Committee, Professor Manabu K. Kojima of Toyama University, as well as to the members of the local committee who have endeavored to produce such a fruitful meeting. In addition, I would like to thank the nearly 1,000 members in attendance for their contribution to the success of this meeting. As you know, our Society is now enjoying its 108th year since being founded through the efforts of our predecessors, and serves as the focal point of zoolgical research in Japan. It goes without saying, however, that our Society’s importance arises not from its venerable age, but from its many members who are active at the forefront of research, and I want to emphasize that we should remain a youthful, fresh society which promotes the exchange of information and understanding among members. The progress being made recently in Life Sciences is quite rapid, and researchers now have great expectations of the information emanating from our Society. I believe we all should make aggressive efforts toward the progress of the Zoological Society of Japan. In 1984, on the occasion of the 100th anniversary of the founding of the Society and after many years of preparation, we began publishing our new official journal, Zoological Science. At the 1984 annual meeting, our former president Dr. Hideshi Kobayashi discussed six goals of our Society in the president’s message (see Zoological Science, 1 (6): 852-853). Upon my being elected president last year, I vowed to continue to pursue those goals which were established in the pivotal year 1984. I am pleased to note that Dr. Kobayashi will henceforth work towards the progress of Zoological Science as Editor-in-Chief. For the enhanced, international development of our journal, I hope our members will support the publication and continue to submit thier high quality manuscripts for the journal. Our Society is characterized by its dedication to the very wide field of Zoology, and by a membership comprising both elder and young members. It is my desire that our members do not restrict themselves to one narrow field, but instead have an interest in all of biology. With a view towards broadening our scope from the minutely detailed experiments in our laboratories, to the observation of biological phenomena on a global scale, we should strive to improve communication among biologists of different fields. Recently, the Science Council of Japan inquired about the progress being made by our Society. We must not let ourselves become satisfied only with the existence of our Society; instead, it is necessary for us to assert to the world in this day of strong expectations for Life Sciences, our important role and compelling reason for existence. We must preserve the free atmosphere and flavor of our Society in which every member can devote himself to his basic and independent research. At the same time, we would like our Society to be a humane, professional group which can contribute to the many problems human beings confront, from bioethical issues which arise in the genetic engineering pursued in biotechnology, to the problems of population, food, and environment. My sincere wishes for the great success of each and every one of our members. October 8, 1987 NosBuo EGAMI The President of the Zoological Society of Japan ZOOLOGICAL SCIENCE 4: 958 (1987) [THE ZOOLOGICAL SOCIETY PRIZE] © 1987 Zoological Society of Japan Biochemical Studies on Fertilization MorTonor!i Hosni Biological Laboratory, Faculty of Science, Tokyo Institute of Technology, Tokyo 152 Fertilization is one of a few exceptional events in which allogeneic cell interactions are allowed to take place. The process is achieved through successive changes of two highly differentiated cells, eggs and spermatozoa. These changes are both spatially and temporally well regulated and can be synchronously induced in large populations of homogeneous cells at least in some marine invertebrates like sea urchins and starfish. This provides us good experimental systems to under- stand the molecular mechanisms underlying cell- to-cell interactions and other important cellular events. While spermatozoa are naked cells, eggs are generally encased within one or a few egg coats mostly made of glycoconjugates. Egg coats are the place where major events ending up in the gamete fusion are performed. By using suitable marine invertebrates, we have studied the three principal events; sperm binding, acrosome reaction and sperm penetration. Sperm Binding to the Vitelline Coat Sperm bind species-specifically to the vitelline coat as a transitory but obligatory step for fertilization in various animals. In the ascidian Ciona intestinalis, a hermaphrodite, the binding is a step of self/not- self recognition and is possible to be analyzed separately from other events like the acrosome reaction. Ciona sperm are known to recognize the molecular architecture, especially the terminal L-fucose, of a glycoprotein (the sperm receptor) in the vitelline coat. It was attempted to identify a sperm surface protein that binds complementarily to the sperm receptor. On the basis of ex- perimental evidence, we have proposed a theory that a glycosidase on the surface of ascidian sperm, a-L- fucosidase in the specific case of Ciona, serves as the binding protein by forming a quasi-stable complex in sea water with a potential substrate, the sperm receptor. Egg-Jelly Components Responsible for the In- duction of Acrosome Reaction Where fertiliz- ing sperm undergo the acrosome reaction is yet an Open question in almost all animals. In the starfish, however, it is well established that fertiliz- ing sperm undergo the acrosome reaction when they meet the egg-jelly coat. Thus jelly compo- nents responsible for inducing the acrosome reac- tion were searched in Asterias amurensis. Three components are proved responsible for the induc- tion; acrosome _ reaction-inducing substance (ARIS), its co-factor (Co-ARIS) and an oli- gopeptide(s). ARIS and Co-ARIS are indispen- sable for the induction in normal sea water and presumably activate Ca-channels in the sperm plasma membrane. The peptide is not obligatory but it facilitates the reaction by increasing the intracellular pH of sperm. ARIS is a sulfated glycoprotein of a extremely high molecular weight, and sulfate residues and saccharide portions are essential for its activity. Three sulfated steroid saponins are chemically identified as Co-ARIS. The activity of Co-ARIS depends mainly on the side chain of steroid and sulfate residue. Enzymatic Lysins Enzymatic lysins were screened by comparing effects of various enzyme inhibitors on the fertilization of intact and naked eggs. Lysins for the vitelline coat were searched in an ascidian Halocynthia roretzi where the coat is rather easily removed by using needles without injuring eggs. Either or both of two trypsin-like proteases, spermosin and acrosin, and a chymo- trypsin-like protease are shown to be vitelline coat lysins. It is also shown by a similar method that sea urchin sperm use an arylsulfatase and a sialidase as jelly coat lysins, and an arylsulfatase and a chymotrypsin-like protease as vitelline coat lysins. ZOOLOGICAL SCIENCE 4: 959 (1987) [THE ZOOLOGICAL SOCIETY PRIZE] © 1987 Zoological Society of Japan Chronobiology of Behavior YOSHIHIKO CHIBA Environmental Biology Laboratory, Yamaguchi University, Yamaguchi 753 Behaviors are timed with reference to 24-h day as the result of synchronization of circadian rhythm with its environmental counterpart. The timing is affected also by the physiological state related to the purpose of behavior. We have been investigat- ing how these three factors are involved in timing host-seeking or biting behavior of the female mosquito, Culex pipiens pallens. Virgin females show a diphasic activity under LD 16:8h, peaking at lights-off (‘dusk’) and lights-on (‘dawn’). After insemination, however, she becomes active from ‘dusk’ to midnight and lowers the ‘dawn’ peak. This modification is caused by the male accessory gland (MAG) substance in the seminal fluid. The effect of MAG homogenate, assayed by injection into virgin female, shows a dose-dependency; 0.5MAG equivalent is critical, eliciting a half-maximal response. More than 95% of the females captured on baits in the field are inseminated prior to host-seeking and biting. Modification of the circadian pattern by insemination may have some adaptive significance in achieving the purpose of the ensuing behaviors. The virgin female activity is under circadian control, free-running under con- stant conditions, synchronizing with environmen- tal cycles or showing a temperature compensation (Qi;9=0.91-0.96). The internal mechanism of circadian rhythm has been postulated to include, at the very least, a pacemaker (primary generator of temporal information) and photoreceptor which disperses photic information toward the pacemak- er for entrainment. Mosquitoes deprived of the greater part of their optic lobes still maintained the circadian rhythm in DD and LL, and showed the diphasic activity entrained roughly to a 24-h LD cycle. Thus, the pacemaker or a crucial part for controlling the rhythm is presumably within the cerebral lobes, but not within the optic lobes we demonstated almost conclusively to house the pacemaker in the cricket. The entrainment may be attained primarily by the photic information con- veyed through an extraocular receptor. The other photoreceptive pathway, ‘compound eye plus optic lobe’ (CE-—OL), may function either to strengthen the synchronization or to mask the overt rhythm. The surgical experiment provided a clue to a question of subspecific difference in sensitivity to light between pallens distributed over the ground and its subspecies molestus inhabiting under- ground constructions. The spatially scotophilous molestus is temporarily photophilous, being very active in the light fraction of a 24-h LD cycle or under LL, where pallens is hardly active. But the optic lobe ablation makes pallens photophilous, indicating that the subspecific difference is related to ‘CE-OL’ function. Our interest covers the connections between the physiology of individual females and the field observations on population or community. In individual females, mating and biting are not daily events; the former occurs once in a lifetime and the latter recurs at the interval of a few days while eggs are matured and laid. The daily activity peaks observed in the laboratory correspond to the so-called ‘gate’ which allows mosquito to assume one of these behaviors. How does, for example, each inseminated female, select her own biting time in such a wide gate that lasts for several hours from dusk to midnight? On the other hand, biting populations of multiple species and their host form a community revealing a 24-h rhythm in, for example, diversity. Is the community rhythm explained simply as the addition of the rhythms of component species? These ecological questions also deserve future study. 960 Physiology Pal dl COMPARISON OF OSMOREGULATORY CAPACITY BE- TWEEN THE AMPHIDROMOUS AND LANDLOCKED RINO- GOBIUS (GOBIIDAE) AFTER TRANSFER TO 50% SEA WATER. Ko IV/MUN ghoicl Ws SATINOS BuO, IbaloO. 5 RAC. of Edu. , Wakyama Univ., Wakayama. Among the genus of Rinogobius living in Japan), Re culminus sande bmunncus sa nemene amphidromous species, and R. brunneus liv- ing in Lake Biwa and R. flumineus are the landlocked species. When the fishes were directly transferred to 50% SW, a high mor- tality was observed in the landlocked spe- cies, but no fish was died in the amphi- dromous species. In the landlocked species the blood osmolarity increased to 400 mOsm within 24 hrs after transfer. However, the lacustrine species surviving for 7 days in SW slightly surpassed R. flumineus in the hypoosmoregulatory capacity. In the amphi- dromous species, the blood osmolarity de- creased within 48 hrs, although the levels were somewhat higher than the FW levels. Na-K-ATPase activities in the gill of the amphidromous species increased much above the FW levels for 7days in SW, but any change was not observed in the landlocked species. On the other hand, those levels increased greatly when all the species ex- amined were adapted to deionized water. It is suggested that the amphidromous species preserve an ability to survive well in SW during their FW lives, but the land- locked ones lose some function to remove an excess ion from their bodies during SW emieaavas PH 2 WATER INFLUX THROUGH THE INTEGUMENT OF TREEFROG, Hyla arborea japonica. H. Nakashima and Y. Kamishima. Dept. of \aLOUba py ACM Ikon wOdy Seas 7 Oleeypaimne , Ural we okayama 700 Anurans take the water they need through the integument. The water intake in japanese treefrogs was mainly confined to the ventral pelvic skin. Water influx through this portoin was 0.3ul/em?/min. The water influx was markedly increased in animals kept under dehydrated condition, showing 1.12ul/em?/min. Isoproterenol, an adrenergic 8-agonist, enhanced the influx 7 times as much as the controls, showing 2.13u1/em*/min. However, the influx rate One) jamntimnay listens | biele dlianve: | ¥siera) slomaweals 0.08ul/em?/min which was much lower than the animals in off-breeding season. Enhan- cement by the B-agonist was also reduced in these animals. Prolactin administra- tion to the animal in off-breeding season caused the same depriviation of the water influx with that of animals in breeding season. Morphological modifications of the tissue was observed in accordance with the physiological activities. When water influx was high,the skin appeared thinner and flatter, and the outermost layer of the epidermis was strongly stained with sieviemady dyes, Uwhille the! skin wath desis water influx appeared granular in texture, and the epidermal cells were larger and showed no special chromophilic nature. ri AMINO ACIDS METABOLISM STIMULATES WATER AB- SORPTION ACROSS THE SEAWATER EEL INTESTINE. M. Ando. Lab. of Physiol. Fac. of In- tegrated Arts & Sci., Hiroshima Univ., Hiroshima. we ee eee To elucidate how intracellular L-alanine enhances water absorption across the eel intestine, effects of metabolic inhibitors were examined. The enhancement by 5 mM L- alanine was inhibited by aminooxyacetate, an aminotransferase inhibitor. After block- ing pyruvate synthesis by the drug, the water absorption was accelerated by pyruvate, whose effects were inhibited by oxythiamine, a dehydrogenase inhibitor. Furthermore, the effects of L-alanine was inhibited by 2,4-dinitrophenol, and Oo con- sumption of the tissue increased after treatment with alanine. These results in- dicate that L-alanine is metabolized via the TCA cycle to produce ATP. L-glutamine also enhanced the water absorption through a common metabolic pathway with L-alanine. However, the responses to 1-2 mM L- glutamine was faster and greater than those to 5 mM L-alanine. Therefore, it seems likely that L-glutamine is a major fuel for the water absorption across the seawater eel intestine. PH 4 NEURAL CONTROL OF FERTILIZATION IN _ THE GENITAL CHAMBER OF THE FEMALE CRICKET, TELEOGRYLLUS COMMODUS. T.Sugawara. Dept. Biol., Saitama Med. Sch., Saitama. Stimulation of mechanosensory cells in the postero-lateral wall of the genital chamber elicited reflex contraction in the spermathecal duct via the genital chamber nerve. The contraction of circular muscles of the duct occurred in the basal part connecting with the chamber, and was propagated in the peristaltic manner upward to the spermathecal bulb. It was observed that this contraction produced sperm flow downward to the chamber. The spermatozoa were transported with tails first. Sensory cells were found in the copulatory papilla. They are clustered in both sides of the spermathecal duct, and send dendrites along the duct toward its opening to the chamber. They may respond to the sperm descent. The process of fertilization during the rest phase of oviposition in the female cricket was thus revealed as follows: (1) the egg introduced into the genital chamber stimulates the mechanosensory cells in the chamber wall, (2) the reflex contractions occur both in the muscles covering the chamber and in the spermathecal duct, (3) the sperm is squeezed out and the egg is fertilized, (4) the sperm stimulates the sensory cells in the copulatory papilla, (5) the gate-opener muscles (M9s) reflexly Contwact, Vand fLamally (GO) woe ecg is ejected into the ovipositor. PH 5 RESPONSES OF NONSPIKING GIANT [NTERNEURONS TO MAGNETIC FIELD AND VISUAL STIMULATION IN THE CRAYFISH. Y.Okada and T.Yamaguchi. Dep. of Biol., Fac. of Sci., Okayama Univ., Okayama. In the protocerebrum of the crayfish, there are five pairs of nonspiking giant interneurons (NGIS) which are presynaptic to the oculomotor neurons. Three of them form a cluster and respond with graded hyper- or depolarizing potentials to ipsi- or contralateral eye illumination, respec- tively (Okada and Yamaguchi,1986). The present study of the responsiveness of these neurons to the magnetic field stimulation (MFS) and visual stimulation (VS) revealed the following features. (1) The NGIs respond with heyuP Gy Ose depolarizing potentials without action potentials to MFS of each statocyst. (2) Hyperpolarizing current injection through a recording electrode into a NGI increases the amplitude of depolarizing response elicited by MFS or VS and decreases that of hyperpolarizing response. Depolarizing GuUErent injection shows the reversed effect on the responses. (3) When MFS and VS are applied in succession, the polarity of response to preceding stimulation affects on the amplitude of the response of following stimulation. (4) WineSe results suggest that the responses of NGIs to MFS are modulated by those to VS OG Vice Versa. PH 6 MORPHOLOGICAL AND FUNCTIONAL FEATURES OF NEURONS INNERVATING THE REPRODUCTIVE ORGANS IN THE FEMALE CRICKET. becumunra, YY. Kotoh and T. Yamaguchi. DeaMoemOr bVOl., Fae. of Sei. , Okayama Univ., Okayama. a Pnieeenes: female~cri eke tjyoErhy thmne movements of the spermathecal duct are induced “by 107>8M 5-HT and they are inhibited by 107~8M_octopamine. Nit* Pace lint ne mevealis sthat) the female reproductive organs (genital chamber, spermathecal duct, spermatheca) are innervated by several dorsal unpaired median neurons (DUMR4 neurons) emerging through the fourth nerve roots from the terminal abdominal ganglion and two paired neurons extending the axons through one of the nerve roots. One of the paired neurons is a 5HT-like immuno- me Neate a one Winn.) and, stihe sslimm uno = Merne biVilsteyn 1S. 1Se ene ein asks sssoma: aind terminals which are distributed among the muscle layers of genital chamber, copulatory papilla and the spermathecal duct near the junction with the genital chamber. The most probable conclusion from another immunohistochemical evidence is that the DUMR4 neurons are octopamine-like immunoreactive. Physiology 961 PH 7 FUNCTIONAL ROLE OF NONSPIKING NEURONS IN THE CERCUS-TO-GIANT INTERNEURON SYSTEM OF CRICKETS. Y.Baba and T.Yamaguchi. Dept. of Biol., Fac. of Sci., Okayama Univ., Okayama The nonspiking interneurons (NIs) responding only with slow potentials to wind stimulation of the filiform hairs CT] sand harrs») lone thier -cier ¢ iv same present in the terminal abdominal ganglion of crickets, and 15 NIs (NI-1 - NI-15) are distinguishable by the Heat wees ofe thie Gamo mpho lossy rand responsiveness to the stimulation. The directional sensitivity of NIs is rather complex: for instance, NI-2 responds well with depolarizing potential to anterior wind, and NI-7 responds with depolarizing potential to lateral wind in the ipsilateral side to its soma as well as with hyperpolarizing potential to lateral wind in the contralateral Side. The results of selective wind stimulation of T- and L-hairs on the cerci shows that each NI integrates the Sensory inputs from these hairs ina characteristic way. Double staining of a medial giant interneuron and a NI reveals that the dendrite of medial f€iant interneuron is wound with branchlets of NI-1, NI-2 and NI-5. These results imply that the NIs may play an Mmpow tant: erase sr nie tihes es iprantiea, | information processing in the cercus-to -giant interneuron system. PH 8 SEXUAL DIMORPHISM AND PROGRAMMING CELL DEGENERATION IN THE FLIGHT MUSCLES OF ADULT CRICKETS. T.Yamaguchi, S.Kogawauchi, S. Shiga, K. Yamada and T.Kimura. Dept.of Biol., Fac. of Sci., Okayama Univ., Okayama. The depressor muscle, M90 of male adult is larger than that of female adult, and the elevator muscles, Mll2a and M119, and depressor muscle, M129a, of female adult is larger than those of male adult. In both sexes, an increase in weight of the muscles, Ml12a, M119 and M129a, is first seen during four days following the imaginal ecdysis. After the weight reaches the maximum value, a rapid decrease in the weight and the marked change in the color of muscles take place: the young muscles assume pale-red color, and old muscles do white color. In the case of Ml12a, the muscle histolysis with accompanying the disappearance of birefringence occurs about 20 days after the ecdysis. However, in the motoneurons innervating the muscle, Ml1l2a , there is nov -stivein f 1 cra net: ant iriosp hhys, sand) = tihve morphological profiles are well preserved, and they fire normally during flight performance. 962 Physiology PH 9 DESCENDING STATOCYST INPUT TO THE UROPOD MOTOR SYSTEM OF CRAYFISH M.Takahata and M. Hisada. Zool. Inst., Fac. Sci., Hokkaido Univ., Sapporo 060. The responses of uropod motoneurons and related interneurons to magnetic field stimulation were analysed intracellularly in an animal with its statolith replaced with fine ferrite granules. The motoneuron activity was recorded from the dendritic branches in the terminal abdominal gan- glion while the activity of descending statocyst interneurons was monitored at the circumesophageal commissure. All the motoneurons examined showed subthreshold, depolarizing PSPs in response to descending interneuron activity. No hyperpolariz- ing response was observed. The PSPs seemed to be mediated polysynaptically since there was no 1:1 correspondence between the descending spikes and the PSPs. The only candidate at present for the neuron interposed between the descending statocyst interneuron and the uropod motoneuron is the local nonspiking interneuron: no other class of neurons in the terminal abdominal ganglion such as local spiking interneurons and interganglionic interneu- rons responded to the magnetic field stimulation except the descending statocyst interneurons. The response of nonspiking interneurons was either depolarizing or hyperpolarizing, but in neither case the 1:1 correspondence was observed between the descending spikes and the PSPs of a nonspiking interneuron. The connection between the descending Sstatocyst interneurons and the uropod motoneurons thus seems to be mediated by a network of interneurons probably involving both the non- spiking and spiking interneurons. PH 10 CRAYFISH STATOCYST INTERNEURONS HAVE OVERLAPPING DENDRITIC FIELDS IN SEVERAL SENSORY NEUROPILES IN THE BRAIN. H. Nakagawa and M. Hisada, Zool. Inst., Fac. Sci., Hokkaido Univ., Sapporo Seven descending interneurons (Sl to S2) which respond tonically to statocyst stimulation were identified in the crayfish brain. Statocyst was stimulated by artificially bending the statocyst hairs in situ while recording intracellularly from candidate neurons at the circumesophageal connective. After physiological identification, the neurons were stained with intracellular injection of NiCly and subsequent silver intensification. Morphological characteristics of these seven interneurons are (1) the cell bodies in the anterior cluster, (2) dendrites projecting to the optic, parolfactory and antennal lobes and to the dorsal parts of both the deuto- and tritocerebrum, (3) lack of dendrites projecting to the olfactory and accessory lobes except S3. The projections of these interneurons in the optic lobe and the deutocerebral area agree well with the fact that the equilibrium responses are controlled by both statocyst and visual inputs. The function of the additional tritocerebral projections remain to be analyzed. [he morphology of these neurons clearly indicates that they are multimodal, highly complex interneurons rather than simple relay interneurons. by comparing the input statocyst and the axonal location in the circumesophageal connective, Sl, 52 and S7 are thought to be respectively identical with Cy, Cy and I5 among four statocyst interneurons described by Takahata and Hisada (1982). PH 11 A WHOLE-MOUNT SILVER INTENSIFICATION METHOD FOR ELECTRON MICROSCOPY M.Sato and M.Hisada. Zool. Inst., Fac. of Sci., Hokkaido Univ. Sapporo. Nickel chloride filled neurons of the crayfish can be examined using both light (LM) and electron microscopy (EM). The method of whole mount silver intensifica- tion for LM was improved by changing the schedule and the ratio of the contents, pH and osmotic mol, of the developer base solution. The injected nickel cations were precipitated with rubeanic acid. The tissue, which included the neuron, was pre-fixed and treated with that solution. After treatment, it was post-fixed and embedded in epoxy resin, and then sec- tioned (50pm thick). The sections were analyzed under LM, and resectioned for EM. LM observation showed that the neurites were clearly blackened by fine silver grains, and under EM, they contained electron dense deposits of 10-100nm in diameter. This method makes it possible to observe both the gross morphology and the ultra structure of one neuron, which until now has required the observation of 2 or more neurons. Moreover, to investigate connections between two distinct neurons by using together this method and the HRP- method, Gt) is possibile ) fo dalsitaimomersia neurites of the different cells on the basis of differences in staining charac- teristics. PH 12 LASER DESTRUCTION OF DENDRITE TO FACILITATE THE ANALYSIS OF NEURAL CIRCUIT. M. Hisada, M. Takahata and T. Toga. Zool. Inst., Hokkaido Univ., Sapporo. We have devised a laser irradiation apparatus in combination with a dissection microscope. By manipulating a single lens in the optical pathway the device works either as a epi-illumination fluorescence microscope or a microbeam irradiation source of about 30 yum in diameter. By staining a single neuron in situ intracellularly with Lucifer Yellow dye, we are able to observe the morphology of the whole neuron for identification during the intracellular recording of the neural activity when the device is used as a fluores- cence microscope. With the microbeam, a desired portion of the stained neuron can be destructed and eliminated out from the functional entity in a few seconds by the photochemical reaction. The severance does not affect the function of the rest of the cell appreciably except a temporal slight depolarization. We have tested this method in the crayfish ganglion to assess the role of some neurites in the local nonspiking, ascending and motor neurons. In the nonspiking and the ascending neurons, the differential distribution of sensory input terminals among the neurites was revealed by the selective elimination of the neurites. In the flexor inhibitor motoneuron, excitatory interaction between the sibling neurons remained intact after the severance of axon indicating the interaction is in a local, nonspiking mode. The device has hence proved to be quite useful to assess the functional differentiation of individual dendrites in a single neuron. Potential use of this device includes the elimination of an entire cell in a nerve ganglion or in a developing embryo to assess the role of the cell as a whole. Physiology 963 PH. 13 THE EFFECTS OF CALCIUM AND CALMODULIN OF THE INITIATION OF SPERM MOTILITY IN RAINBOW TROUT. W. Yamao and M. Morisawa. Ocean Research K efflux and subsequent Ca influx trigger the first step of DC On | On trout sperm motility. In order to know the second step, which may be regulated by the intruded Ca : we studied the effects of calmodulin antagonists on the motility of rainbow trout sperm. There was no effect of Woon but W-7 and trifluoperagine GURE) ait the concentration around 10 M inhibited the motility of live sperm. On the other hand, W-7 didn't inhibited the motlity of demembranated sperm, suggesting that calmo- dulin regulates the sperm motility at the plasma membrane. In contrast, TFP inhibited the motility of demembranated sperm, impli- cating the presence of other regulatory mechanism in the axoneme which is sensiti € tor PEP With regard to the effects of Ca concentrations’ on the live and demembra- nated sperm, the former were motile in the medium containing Ca at the concentration mors than 10 M and immotile in less’ than HOE Mi, but the latte were immotile in the medium contaiping Ca at the concentration more than 10 -M and motile in less than, 10 Moca). these results suggest that (a at the concentration between 10 M-10 M_ af- fects the plasma membrane or axoneme, and regulated sperm motility of trout sperm. The yegulatory mechanism may be controled by Ca regulatory protein, calmodulin. PH 14 EFFECTS OF PROTEIN KINASE INHIBITORS ON THE PHOSPHORYLATION OF PROTEINS AND MOTILITY IN RAINBOW TROUT SPERMATOZOA. 9 M.Morisawa » 71T-.Hirabayashi and H.Hayashi Ocean Res. Institute, Univ. Tokyo, Tokyo and Institute of Mol. Bios, hace KON, c Nagoya Univ., Nagoya. We showed that cyclic AMP-dependent phos- phorylation of tyrosine residues in 15K protein triggers the final step of the initiation of trout sperm motility. We investigated here the effects of protein Kinase inhibitors on the cAMP dependent phosphorylation of sperm proteins with molecular weights of 15K, 25K, 32K, 68K and motility of demembranated sperm. n= 7 ¢ a Known inhibitor of protein kinase C did not inhibit both phosphorylation of 15K protein and sperm motility. Inhibitors of cAMP dependent protein kinase such as H-8, Hor, protein kinase corepeptide inhibited phos- phorylation of all proteins including 15K protein and sperm motility. Poly GGitieh yar) 4.3 he, specrive iImhbhibneor Of tyro— sine kinase inhibited only the phospho- rylation of 15K protein and sperm motility. However, it did not inhibit the phospho- rylation of other proteins. Antiserum of phosphotyrosin inhibited sperm motility but not phosphorylation of proteins. These results suggest that tyrosine kinase is Phosphorylated and activated by A-kinase, and phosphorylation of 15K protein through this process causes the initiation of sperm motility in rainbow trout. PH 15 THE PROCESS OF ACQUISITION OF SPERM MOTILI- TY IN SALMONID FISH. Sj .Morisawase K. .wlshida; & M. Morisawa?, ‘Biol. Labie:s St. Marianna Univ., Kawasaki, Teikyo Univ., 7?Ocean Res. Inst., Tokyo Univ. ; ULoKyo. Testis spermatozoa of salmonid fish are morphologically Mature in the breeding season, however, they do not have the po- tential for motility. Spermatozoa acquire the potential during passage through the sperm duct after leaving the testis. During the course the HCO concentration and pH in the seminal plasma increased. When tes- tis sperm of rainbow trout lacking motility were incubated in artificial seminal plasma containing HCO ac pi 8.2, mot Pury poten— tial developed. The maturation of motility potential was less at lower or higher pHs. During the incubation period, the intracel- lular cAMP level increased to a level simi- lar to that of sperm duct spermatozoa. The motility of demembranated testis sperm of chum salmon caught in the bay was much less than for river fish, and demem- branated sperm of river fish were similarly motile in testis and sperm duct spermatozoa These suggest that in the process of acquisition of motility potential 1) axo- nemal maturation 2) membrane maturation and 3) accumulation of cAMP, are each indispen- sable, as the HCO concentration and pH value of seminal plasma increase from. the testis to sperm duct. PH 16 SPERM-ACTIYATING SUBSTANCE FROM HERRING EGG i. Ohtake , S. Tanimoto and M. Morisawa Dept. of Physiol., Dokkyo Univ. Sch. Med., Tochigi, Dept. of Biol., Hac nOtacs Clt.ns Toho Univ., Chiba, abr | Ota, Phys 0) cy, Ocean Res. Inst., Univ. of Tokyo, Tokyo. It is well established that spermatozoa of marine teleosts initiate motility when they are discharged to the fluid of high osmolarity such as sea water. The motility of herring spermatozoa was also initiated in hypertonic sea water. However motility was low. When herring egg or supernatant from egg and sea water suspension (egg water) was added to spermatozoa which exhibited low motility in sea water, spermatozoa become active. Thus it is likely that the motility of spermatozoa was initiated by osmolarity and was then activated with the substance released from the egg. The most effective egg water was obtained when herring eggs were immersed in sea water, While no or less sperm - activating factor was found in distilled water or diluted sea water isotonic to the seminal plasma. This suggest that the substance is released from the eggs when they exposed to sea water at spawning. The activating effect vanished when egg water was incubated with pronase. The factor is heat stable. From the procedure that 6 ml of egg water (10 g eggs / 100 ml sea water) was loaded on Sephadex G-75(1.89 * 40 cm) and eluted with artificial sea water (2 ml / tube), single peak with activity was obtained in the fractions between 40 - 50. 964 Physiology PH 17 INTRACELLULAR CA** INDUCES INITIATION OF LUM. T.Inoda and M.Morisawa. Ocean Research Institute, University of Tokyo, Tokyo. We recently reported that a decrease in osmolality surrounding spermatozoa at spawning causes the initiation of sperm motility in Xenopus. (Inoda and Morisawa, Comp. Biochem. Physiol. in press). We re- ported here that EGTA, chelating agent for divalent cations, had no effect on _ the motility when EGTA was present in external medium, whereas spermatozoa were quiescent in the medium containing calcium jgnophore, A23187. Subsequent addition of Ca induced the initiation of sperm mofjlity, sug- gesting that intracellular Ca plays an important: role in the initiation of sperm motility in frogs. (Dev.Growth.Differ., 1987, 29(4),abstract). We further showed that when spermatozoa of Xenopus were treated with 0.4 % Triton X-100 and sus- pended in the reactivating medium con- taining MgA ; axonemal movement did not occur if Ca was depleted from the medium. However, when Triton-extracted spermatozoa were suspended in_the reactivating medium containing MgATP and Ca , axonemal move- ment occurred. The motility became active concomitant with increase in Ca concen- trations and maximum motility was obtained at the concentration between 0.1-1 mM Ca These results suggest that intracellular Ca regulates on-off switch of the initia- tion mechanism of Xenopus sperm motility. PH 18 ACTIVITY OF THE ACCESSORY OLFACTORY BULB TO CHEMICAL STIMULI DELIVERED TO THE VOMERONASAL MUCOSA IN TURTLES T. Hatanaka! and T. Hanada ; IDept. Biol. G5 Cie Wels, Winti\~o Ore Cision, Clasiiog p IMSio Oit BWalOl, Seeks, Wins, Cie Wesywswoyer ; Ibaraki. To investigate the receptivity of the vomeronasal receptors, induced wave responses and unitary discharge of the accessory olfactory bulb neurons were recorded in three species of aquatic turtles, Geoclemys reevesii, Pseudemys scripta, and Clemmys japonica. Response amplitude and pattern to various stimuli were compared in the three species. And relation of the induced wave pattern and the unitary discharge was discussed. Induced wave responses were yielded by not only vapor stimuli like as iso-amyl acetate and some fatty acids but also liquid stimuli like as some salts solution and acids solution. But amino acids were not so effective with few exceptions. Rank orders of effectiveness of chemicals were almost coincident in the same species, but differed among species. Unitary responses of the bulb neurons were composed with facilitation and suppression of firing. Some discharge patterns resembled to the rise and fall patterns of the induced wave. But unit had a tendency to respond with same pattern to various stimuli. So, particular relation of stimulant and response pattern was not detected. Pal US) CYCLIC NUCLEOTIDE-INDUCED CONDUCTANCE INCREASE IN SOLITARY OLFACTORY CELLS. No SWAWki, AGOl, WmnStt., PACs OF SCi., Hokkaido Univ., Sapporo. To test the possible involvement of cy- clic nucleotides as intracellular messen- gers in the olfactory transduction pro- cess, I have studied the effects of var- 1ous nucleotides, their analogs and phos- phodiesterase inhibitors on the membrane conductance in enzymatically isolated bullfrog olfactory receptor cells by means of a whole-cell clamp perfusion technique. Of six different cyclic nucleotides, cGMP and cAMP were most effective and induced a phasic increase in membrane conductance. However, structurally different cyclic nucleotides, 2',3'-forms, were ineffec- tive. The conductance increases induced by CGMP and CAMP were not influenced by the presence of di- or triphosphate nucleo- tides, indicating a direct action of these cyclic nucleotides on the membrane. The conductance increase by cGMP was identi- fied as being due to an increase in cat- ionic permeability of the receptor mem- brane, and its adaptation process was found to bs controlled by divalent cations such as Ca“* and Mg**t. Dibutyryl cAMP and 8-bromo cAMP induced delayed and prolonged conductance increases. Furthermore, IBMX and theophylline prolonged the decay of CAMP-induced conductance increase. Re- sults suggest that a phosphodiesterase may be distributed in the olfactory receptor membrane. PH 20 ADAPTATION OF ODOR RESPONSES IN THE OLFAC- TORY RECEPTOR CELLS MAY BE CONTROLLED BY INFLUX OF EXTRACELLULAR DIVALENT CATIONS. T. Kurahashi and T. Shibuya. Inst. of Biol. Sci, Univ. of Tsukuba), dibanalkiss In this study, the adaptation phenomena of odor responses were investigated from several point of view, by using solitary cell preparation and patch clamp method. In the standard Ringer's solution, many of the depolarizing responses in the solitary olfactory receptor cells evoked by 10 mM n-amyl acetate showed phasic time course. And the odorant-induced current ( OIC ), which observed under the voltage- clamp condition, showed similar time course. When the double pulse stimulus was applied to the cell, amplitude of OIC which was induced by second stimulus was always smaller than first. The second cur- rent amplitude became larger as the inter- val of each stimulus became longer. The amplitude was almost recovered with a rest of more than 10 seconds. When the extracellular divalent cations ( Ca, Mg ) were removed and double pulse stimulus was applied, in contrast with above observation, the amplitude of second OIC was almost same with first. Moreover, in such an environment, there was little reduction in amplitude of OIC even for the continuous stimulus. These results may indicate that the ex- tracellular divalent cations correlate with the adaptation of the odor response. Physiology 965 PH 21 THE STRUTURE AND ODOR RESPONSE OF OLFATORY BULB IN THE PIGEON Columba livia T.Hanada and T.Shibuya, Inst. of Biol. Sern, wunw. of Tukuba,, Ibaraki . Historogical structure of the pigeon olfactory bulb(OB) was observed by staining with Bodian's silver method. The structure was fundementary similar to that in other vertebrates. However, the mitral and granule cells were smaller than those of rat. From this structure, the pigeon OB seemed to have sufficient functions as the primary olfactory center. OB induced waves(16-17Hz) were remarkably obserbed to two kinds of odors ( n-Amyl acetate and d- Limonene) known as the common chemical substances for olfactory experiment. Unitary spike activities to the odors were mainly recorded at the external plexiform layer and mitral cell layer. Several units had different response patterns, excitation , inhibition and complex types to odors. The same odor evoked with different response patterns among units. Moreover, many units showed variable response patterns with increase of concentration. These response patterns were similar to that of a reptilian OB (Gecko). IN OB units of the pigeon, a response range with_excitation pattern in concentration (10°° - 10°) of n-Amyl acetate narrower than in other animal OB units. PH 22 RESPONSE OF TARSAL CONTACT CHEMORECEPTOR OF THE Citrus-FEEDING SWALLOWTAIL BUTTERFLY Papilionidae RELEASING OVIPOSITION BEHAVIOR Kekhusume: dana [.oShibuya. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. Citrus unshiu is one of the most favorite host plants of the Citrus-feeding swallowtail butterflies, Papilionidae. The stimulation of the extract of C. unshiu leaves to the tarsi of the female Papilio xunthus evoked oviposition behavior clearly. Many chemosensory sensilla (designated as "type-A") were observed with many other cuticular structures ("type-B and -C") by SEM. The "type-A" sensilla were more abundant in the female than in the male, especially on the tarsi of forelegs which were necessary for “drumming", pre- oviposition behavior. The tip recording method was applied to these sensilla of a female P. protenor, it become obvious that the electrical resistance of them was much smatlter than that of others ("“type=Cc"). This fact suggested the presense of pores on the sensilla, which was one of the most remarkable features Of al CoOmeEeacit chemoreceptor. The sensilla initiated larger spikes to the leaf extract.The num- ber of the spikes increased with increase of the extract concentration. It seems that the "type-A" sensilla have a function as contact chemoreceptor to the ovipositional substance. PH 23 RESPONSES OF CONTACT-CHEMORECEPTOR CELLS TO THE WING-RAISING FACTOR ON ANTENNAE IN THE MALE LOBSTER COCKROACH, Nauphoeta cinerea. A.Watanabe and T.Shibuya. Inst. of Biol. Sci, Univ. Of. Esukuba, Ebaraki - It is well known that the contact-sexual pheromone (wing-raising factor) contained in cuticular wax of the lobster cockroach releases a wing-raising display as an initial action pattern in mating behavior of the male cockroach. This investigation was undertaken to study receptive mechanisms in the antennal contact-chemoreceptor cells related to initiation of wing-raising display. The tip recording method was applied to lead out activities of receptor cells ina Single sensillum on the male antenna. A glass capillary was filled with stimulating suspension, which was composed of 0.1 M NaCl and extracted wing-raising factor and treated with ultrasonic waves. Spike- responses (6-20Hz, 10-20mV) to the wing- raising factor were recorded in many sensilla. One type of sensilla contained receptor cells which responded to wing- raising factor. Another type of sensilla contained, at least, two kinds of receptor cell responsible to 0.1M NaCl or wing- raising factor with defferrent spikes in amplitude. Morphological type of sensilla responsible to wing-raising factor may be the sensillum chaeticum. PH 24 a@-GLUCOSIDASE ON RHE, SASTESREGEPEOR MEMBRANE OF THE FLY, Phormia regina. M.Ozaki and T.Amakawa. Dept. of Biol., College of Gen. Edu., Kobe Univ., Kobe. In the labellar chemosensillum of the blowfly, Phormia regina, a&-glucosidase was thought to distribute at the tip of the sensory process with the active site ex- posing outside of the cell. We investi- gated the activity of theaglucosidase in the intact sensilla by floating method. The binding abilities of the a@glucosidase to polysaccharides, sugars and inhibitor were compared with those of sugar receptor (investigated electrophysiologically) or those of a candidate protein for the P site (a sugar receptor site to sucrose, L-fucose etc.)(investigated by affinity electrophoresis). The results on. starch, L-fucose, fructose and nojirimycin suggested that @-glucosidase is different from sugar receptor molecule. After the sensillum tip was dipped in the DOC solution and the tip of the sen- sory process was torn to small vesicles, the sensory process was reconstructed and the responsiveness recovered. When the sensillum tip was dipped ir 10 mM nojiri- mycin bisulfate (oa- glucosidase inhibitor) atter “the s*DO0G “treatment, however, the recovery of the responsiveness was 2 fold later. Thus, @&-glucosidase may have a role concerning with membrane fusion by recognizing sugar chain on the surface of the reseptor membrane. 966 Physiology PH 25 EFFECTS OF PROTEIN KINASE INHIBITORS AND PHORBOL ESTERS ON THE SUGAR- RECEPTOR CELL OF THE FLY. T.Amakawa and M.Ozaki. Div. of Biol., Cols, lof Gens Hhduic, SkobiesUniwi vey Kobe. We gave protein kinase inhibitors and protein kinase Cc activators (phorbol esters) inside the chemo- sensory process of the taste receptor cell of the fly, Phormia regina. Their effects on the electrophysiological response from sugar receptor cell were as follows. Protein kinase C inhibitor (H-7) reduced but phorbol esters (TPA and DPBA) enhanced the decrease of the impulse frequency, during stimulation (adaptation). Cyclic nucleotide dependent protein kinase inhibitors (H-8 and HA1004) decreased the impulse frequency immediately after the beginning of stimulation and depressed overall magnitude of response (excitability). These results suggested that at least two different types of protein kinase, kinase A or G and kinase C, are deeply concerned with the sugar taste response by regulating excitation and adaptation, respectively. Thus, we believe protein phosphor- ylation plays important roles in taste receptor cell as widely recognized in visual and secretory cells. PH 26 ISOLATION OF MUTANTS IN HYGRORECEPTION OF DROSOPHILA MELANOGASTER, F. Yokohari, T. Ito and T. Tanimura Biol. Lab., Fac. Sci., Fukuoka Univ., Fukuoka. We attempted to isolate mutants in hygroreception of Drosophila melanogaster, in order to introduce genetic approaches in study of insect hygroreceptor. For the isolation of X-linked mutants, wild-type males were mutagenized with ethelmethane sulphonate. The treated males were crossed with virgin females of the attached-X chromosome. F, males were individually crossed with attached-X females and the isogenic lines were established. Prior to the experiment, ca. 50 male flies were transferred to a empty vial and kept in a desiccator with dry silica gel for 4 - 5 hrs. They were transferred to transparent acrylic box (200mm long x 15mm wide x 5 or 10 mm high), in which humidity gradient was set up, and kept for 20 min in the dark. Then the distribution of the flies were photographed. The number of flies in each side was counted on the photograph. Among 868 lines tested, flies of 18 strains could not detect the humidity difference. Electroantenograms (EAGs) to humidity stimulus were recorded and compared between the wild-type and the mutant flies. EAGs of 8 strains showed remarkably smaller value than the wild-type flies. Survey of antennal sensilla by SEM found possible hygroreceptive sensilla in the sacculus of antennal flagellum, but no difference was found in the external structure of the sensilla among EAG-small strains tested and wild-type. al Ad THE STRUCTURE OF THE MUSHROOM BODY AND RESPONSES OF THE PROTOCEREBRAL NEURONS IN THE HONEYBEE BRAIN H.Yamamoto and T.Shibuya. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. ‘ The mushroom body in the honeybee brain was morphologically studied. The mushroom body was reconstructed 3-dimensionally from serial sections of the brain stained with Bodian's method. The mushroom bodies were observed as paired structures. Each of them consisted of two cup-like calyces anda stalk with &- and @-lobes. To know the physiological aspect of the mushroom bodies, responses of the protocerebral neurons to odor (geraniol and n-propanol) and light were recorded. Some of them were stained by fluorescent dye in- jection of lucifer yellow CH. 4 classes of neurons were recorded as follows; 1)bimodal neurons responded to both odor and light (OL neurons), 2)unimodal neurons responded to odor (O neurons), 3)unimodal neurons responded to light (L neurons), and 4)neurons responded neither to odor nor light. Among neurons identified morphologically, there were one L neuron and one OL neuron. The L neuron run from right to left bilaterally in the protocerebrum and branched in the lobula and the medulla. The cell body of the OL neuron was observed at the ventral side of calyces and many branches between a&-lobes. The OL neuron, compared with O neurons, ex- hibited weaker responses to odor and showed gentle dose-response curve. PH 28 THE STIMULATING EFFECT OF 1,6-ANHYDRO-86-D- HEXOPYRANOSES ON THE LABELLAR TASTE RE- CEPTORS OF UGE! TEINS) BION. I Shimadaie., Hesilon 1 iyi Ol, ual? fancigae Meguro?. 'Dept. Ose WIOWS Seis, VOlMoOku Wmniw . Binal Cieyes Oe ROOd Clans 4.) Paes of Moric. , Tohoku Univ. > asiendaii. Most- Sugars posess reducing properties duc) tomthcin tReemanomenien cemtenismandms aie generally unstable model substances for the Study of! Structune—activilty rediatwonsihapaam taste. Eight 1,6-anhydro-8-D-hexopyranoses which have stable conformations without free anomeric centers were examined their CULSCCLVOMOSS OM cle Tabelilar caste re- ceptors of the fleshfly. 1,6-Anhydro-8-D- idopyranose with three successive equatorial hydroxyl groups was found the most effective on the sugar receptor, which again supports the validity of the proposed model of pyra- nose(P) site (Shimada et al., 1974 and Shimada, 1987). 1,6-Anhydro-§-D-galacto- pyranose, on the other hand, was revealed COs Situmul ate: sehe ssialkeyireice pitonnaaa Minxeumes of both anhydrohexoses clearly stimulated themsugar vandy sale s;eceptonmsm acs ehes Same time. Furthermore, 1,60-anhydro[8-D-alero- pyranose alone could stimulate both the Sug@iwand: Ss alGierecep Gomsi, FOOn aH hes ama, Suggest the different sites for sweetness and bitterness in human taste. Physiology 967 PH 29 DAILY RHYTHM OF SUCROSE INTAKE IN THE BLOWFLY, Phormia regina M. A.Shiraishi, T.Frusho, S.Okamura and H.Tsutsumil, Dep.Biol.,Fac.Sci.,Kyushu Univ, Fukuoka. lamakusa.Marine Laborato- ry-Kyushu Univ,Kumamoto. aily sucrose intake in the blowfly, Phormia regina M was measured with the apparatus prepared in our laboratory. Three kinds of electrical signals, muscle potential from alimentary canal during sucrose intake, artifact accompanying movement of labellar lobes during intake or during licking of the surface of solution and touch artifact of legs to solution, respectively were measured. These three electrical signales were counted at an interval of every 1 h during 3 or 4 days. Sucrose was dissolved into 0.001 M NaCl to keep electric conductivity of solution. Daily sucrose intake in the blowfly showed the circadian rhythm. Usually, blowflies took sucrose under light condition but did not take the solution under dark condition, even though blow- flies were starved more than 24 h. Blowflies were kept previously under 12 h light cycle (6:00 - 18:00, light) or under constant light for 4 days. These indivi- dual blowflies were put into the appara- tus and were measured the activity under constant dark. These flowflies were started to take sucrose after 12 h and continuously took sucrose during 12 h and stopped. The rhythm of sucrose intake was observed more than 48 h. PH 30 BeBe hy Of PHOTOPERITODTE CYCLES ON THE PHOTOTAXIS OF A WATER STRIDER, GERRIS PALUDUM. T. Harada and K. Taneda. Dept. Of “Biol. BacewOL Sere, Kochi Univ.,Kochi. We have investigated the effect of the breeding photoperiodic cycle on the posi- tive phototaxis of a water strider, Gerris paludum. First instar nymphs just after hatching were bred under long-day (LD=10:14) or short-day (LD=14:10) photo- periodic cycles until they became adults. Then the adult specimens were bred under long-day or short-day photoperiodic cycles. All the breedings and determinations were made at constant temperature(20-22°C). The direction of movement of each adult speci- men with reference to that of the light source was periodically determined during all the breeding periods. The degree of positive phototaxis was expressed as the percent number of specimens whose motile tracks were confined within 30 degrees toward the light source. When the adult specimens were bred under the short-day (or long-day)cycles, they showed a low (or high) degree of positive phototaxis regard- less of the nymphal breeding photoperiodic cycles. Thus, the phototactic behavior of the adult specimen was considerably affect- ed by the photoperiodic cycle. However, the photoperiodic cycle experienced during the nymph stage seemed to have little in- fluence on the phototactic behavior of the adult specimen. PH 31 SEASONAL CHANGE IN THE PHOTOTAXIS OF A WATER STRIDER,GERRIS LACUSTORIS. T. Harada and K. Taneda. Dept. Of Brod: Facwon Sci., Koch Unt v..,Kochi. Seasonal change in the degree of positive phototaxis of the water strider, Gerris lacustoris which had been bred in an out- door aquarium, was investigated using arti- ficial beam light as a stimulus during the full period of occurrence (from April through September). The degree of positive phototaxis was expressed as the percent number of specimens whose motile tracks were confined within 30 degrees toward the light source. In April, the degree was considerably high. The high value per- Sisted until June. Then the degree gradual- ly decreased until the onset of diapause (September). The decreasing curve in the degree of phototaxis was similar to de- creases in the day length. There was a Silght difference in the seasonal change between male and female specimens. Diurnal variation in the degree of phototaxis was not observed on any day. When the speci- mens were bred for more than 15 days under a long-day photoperiodic cycle, they showed a higher degree of phototaxis than the con- trols. The ecological significance of the change in positive phototaxis of this spe- cies was also discussed on the basis of the present findings. PH 32 THE ROLE OF THE PINEAL AND THE EYES IN THE CIRCADIAN ORGANIZATION IN THE NEWT. A.Chiba, M.Kikuchi and K.Aoki. Life Sci. ' Inst., Sophia Univ., Tokyo The roles of the pineal and the eyes in the circadian organization in the newt were studied by examining the effects of pineal lesions and bilateral optic enucleations on locomotor activity? rhythms both under light-dark cycles and in constant darkness. Under light-dark cycles, both pineal le- sioned newts and blinded newts showed. the locomotor activity rhythms which were en- trained to the light-dark cycles. After transfer to constant darkness, pineal-le- sioned newts failed to maintain circadian rhythmicity. The blinded newts showed free- running rhythms in constant darkness. The free-running periods of the blinded newts, however, were different from the periods observed before blindings. The effects of pineal lesions on loco- motor activity rhythms were also examined in the blinded newts. Pineal lesions after blindings often cause arrhythmicity not only in constant darkness but also under light-dark “cycles. These data suggest that both the pineal and the eyes are involved in circadian or- ganization in the newts. 968 Physiology Pa) 3 KEY STIMULI RELEASING COURTSHIP AND SPAWNING BEHAVIOR IN HIME SALMON (LANDLOCKED RED SALMON, ONCORHYNCHUS NERKA) M.Satoul, H.Takeuchil K.Takeil, T.Hasegawal, T.Matsushimal N.Okumoto2 and K.Uedal. 1zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo, 2Nat. Inst. Aquaculture, Tochigi. Signals releasing courtship behavior in male himé salmon were examined in model presentation experiments. To test the courtship behavior, two- dimensional square or rectangular models of various sizes and patterns were presented to the male and the frequency of "quivering" elicited by model was measured. It was found that key stimuli for eliciting courtship behavior consisted of three aspects of visual cues, i.e., shape (horizontally elongated rectangle with a size, 6 cm high x 24 cm long), pattern (black above, white below), and movement which simulated some features of the female sexual behavior. To test the spawning behavior, three-dimensional models (size: 6 cm high x 24 cm long x 4 cm wide) were vibrated vertically (frequency: 21 Hz) by a motor-controlled vibrator and the frequency of "Spawning act" elicited by the model was measured. Two types of three-dimensional model were used: a simplified "female" model with a pattern of black above and white below and a transparent model with exactly the same dimensions. The results showed that both the vibrational and visual cues were important in eliciting spawning. The importance of the position of visual cues relative to that of the vibrational cues was also shown. The results suggest that in addition to the presence of both vibrational and visual targets, the matching of both targets' positions is important for eliciting spawning behavior. PH 34 THANATOSIS OF THE CRICKET T. Katayama and M. Sakai Dept. of Biol., Fac. of Sci., Okayama Univ., Tsushima-Naka-3-1-1, Okayama Thanatosis is a death feigning posture that can be found widely in the animal kingdom. It can be induced artificially when animals are forcibly brought into an abnormal posture. So far, however, Physiological study on thanatosis has been only made in stick insects. (Godden 1972). We found that thanatosis could be induced in crickets (Gryllus bimaculatus) that continued for several minutes. Animals could respond to a weak auditory stimulus by ventilatory movements during thanatosis and woke up quickly any time when a strong stimulus was given. This was not the case in animals under anesthesia with CO2, ethyl ether or low temperature (5°C). The duration of thanatosis was shortened when animals were in a noisy condition or tested after they were disturbed. The thanatosis could be induced as far as the suboesophageal ganglion was not inactivated. Spontaneous discharges of the hindleg motoneurons and descending neurons tended to decrease during thanatosis while those of the cercal giant interneurons were not. These results suggest that inhibitory interneurons impinging upon the motor system underlie the generation of thanatosis. PH 35 SOCIAL BEHAVIOUR OF AMOEBA PROTEUS (V). H. Horikami and K. Ishii. Lab. Biol., Hosei Univ., Tokyo We found that Amoeba proteus (AP), strain G, could form compact groups in higher cell density in fresh saline. Then, the processes of the group formation and the dispersion were investigated to get a clue to the grouping mechanism by photomacrography with interval timer. (1) In higher cell density (more than ca. 2 cells/mm2), a frequency of simultaneous collision of more than 3 cells became high to be a small group in a short time. Such groups grew up to a large compact group by collisions each other within 1 Hr. (2) An isolated group dispersed gradually. In a case of the group of 10 cells it took ca. 30 min before the dispersion, and it was more than 1 Hr in the group of 50 cells. (3) The dispersion time of the all groups was about 3 Hrs in a control experiment. And periodic formation of the group was not observed during 12 Hrs. (4) However, APs after the dispersion of the group could form groups again in the fresh saline. From these results, the group formation in APs is probably due to trapping effect based on reciprocal chemical information. Pal 20) CHICKS BLINDED BY FORMOGUANAMINE DQ NOT DEVELOPE, LID SUTURE MYOBIA. 8 Oishi , J. K. Lauber’ and Y. Obara . Dept. Oe JdtOl 6. Fac Ole WSCA oy Nara Women's Univ., Nara, IDNs O12 OOlls , Univ. of Alberta, Edmonton, Canada, Dept. of Agr. Chem., Fac. of Agr., Meijo Univ., Nagoya. The involvement of the retina in avian lid suture myopia was investigated by use of formoguanamine (FG), which induces blindness in chicks. In control chicks reared under a diurnal lighting schedule of LD 12:12, unilateral eyelid suture at 3 days of age induced a typical lid suture myopia response on the operated side by 4 weeks of age, Gofkos enlarged eye: 1.542+0.056¢ (suture eye) vs. 1.244 +0.017g (control eye); increased anterior chamber depth: 3.48+0.21mm (sutured eye) vs. 2.14+0.03mm (control). On the other hand, in FG-treated blind chicks also reared under LD 12:12, there was no difference in eye parameters between sutured and non-sutured eyes. All globe parameters measured were very similar in FG-treated and control non-sutured chicks. Corneal diameter and anterior chamber depth in the eyes of FG-treated chicks (6.11+0.06mm and 1.46+0.08mm, respecti- vely) were significantly smaller than in control non-sutured eye (6.76+0.03mm and 2.14+0.03mm, respectively). Thus, the retina is necessary for the induction of lid suture myopia, and also for normal corneal growth. Physiology 969 PH 37 CHANGE IN DENDRITIC MORPHOLOGY OF FLIGHT MOTOR NEURONS DURING METAMORPHOSIS IN THE SILK MOTH, BOMBYX MORI. H.TSUJIMURA. Lab. of Boil., Tokyo Univ. of Agr. and Tech., Fuchu, Tokyo. During metamorphosis, some larval motor neurons (DLM motor neurons) which innervate mesothoracic dorsal longitudinal muscles degenerate but others survive and become adult flight motor neurons. In this study we show changes in dendritic morphology of these DLM motor neurons by axonal cobalt back-fill and silver intensification meth- ods. In early stages of metamorphosis some larval dendritic branches degenerated and dendritic field of DLM motor neurons de- creased. Moreover, relative position of the dendrite in the ganglion shifted somewhat. In middle pupal stage new branches started to grow from remaining dendrite and many filopodial extensions occurred on the surface of the dendritic branches. In late pupal stage large field of complex dendrit- ic branches were completed and this became adult one. This result shows that neural circuits for controlling the motor neuron activity are reorganized during metamorphosis. This reorganization may plays a role in altera- tion of behavior from larval pattern to adult one and development of flight behav- ior during metamorphosis. PH 38 STIMULUS SELECTION IN A FOOD REINFORCED COLOR- PATTERN DISCRIMINATION IN THE GOLDFISH, CARASSTUS AURATUS . K. Ohnishi. Dept. of Physiol., Nara Medi. Uni., Kashihara To examine whether the goldfish select an attribute independently from visual stimuli in the learming process, the fish were trained to discrim inate between a pair of the stimuli having two different attributes (color and pattem). Normal and telencephalonless goldfish were given the food only when the fish selected a runway of two rmways training apparatus. The inner walls of the rmways were covered with the stimulating color papers which were positioned according to Gellerman alter- nation sequence. Brightness of the color papers whose spectral reflectance was measured was physi- cally equal. When the fish were trained with green- vertical (green paper with vertical black pattems) and blue-horizontal, the animals showed remarkable learmed behaviors (over 75% correct responses from about the 10th day after the start of training trials): acquisition by the telencephalonless fish was slightly slower than by the normal fish. ‘Two days later from the training trials on the 20th or 40th day, memory tests were performed in these animals. They could discriminate between white— vertical and white-horizontal, but not blve and green, While the fish trained with blue and green or green-vertical and blue-vertical could discrim- nate between blue and green (over 75% correct responses from about the 20th day). These results suggest that the fish select more impressible attribute (patter) from the visual stimuli with multi-attributes (color and pattem) and memorize it alone in the extratelencephalic nervous systems. PH 39 LIGHT REQUIREMENT FOR GREEN PIGMENTATION OF THE WILD SILKWORM ANTHERAEA YAMAMAI COCOONS: LOCAL IRRADIATION AND PARABIOSIS EXPERIMENTS. Y. Kato. Dept. of Biol., International Christian Univ., Mitaka, Tokyo. Green pigmentation of silk glands in A. yamamai, which is followed by green cocoon formation, requires high intensity of light. I tried to determine where photo- receptive site will be localized. When neck-ligated animals or isolated abdomens were partially irradiated after gut purge stage, silk glands exhibited green pig- mentation in any cases. Next, two decapi- tated animals were connected with a vinyl tube, one of the partner exposed to light and the other covered with a card-board box. The results showed that silk glands of covered animals became green as well as those of exposed ones. Surprisingly, light irradiation to the part of connect- ing tube in which haemolymph were flowing was effective. Thus, these suggest that haemolymph may function as a possible photoreceptive site. PH 40 DEVELOPMENT OF THE OPTOKINETIC RESPONSE AND THE ACTIVITY OF THE DIRECTIONALLY SELECTIVE UNITS IN THE MIDBRAIN OF THE MEDAKA (ORYZIAS LATIPES) Y.Kasuya and K.Aoki. Sophia Univ., Tokyo. Bike Sensiinste.,, The development of the behavioral acuity in optokinetic response in the larva and adult Medaka (0-180 days after hatching) was investigated. The be- havioral acuity of optokinetic response improved drastically during 15 days from 55 to 70 days after hatching. In spite of 1-1.5 hr after hatching, 70-80 % of larval fish could swim as the same direction of the rotating stimulus, 1-3 days after hatching there was significant decrease in the direction detecting ability and also significant difference was observed in the appearance of the opposite following fish. Directionally selective visual units of 21 in motion detecting 48 units were recorded in the midbrain in Medaka during stimula- tion by a rotating striped drum. Direc- tionally selective units were also ob- tained in the Medaka in the optic tectum and in and around the anterior dorsal teg- mentum which was already found in the gold fish and in the Japanese dace, respec-— tively. Further, these units were ob- tained in and around the optic tract and posterior part of the midbrain tegmentum, and nucleus rotundus. 970 Physiology PH 4] THE MECHANISMS OF NYSTAGMOGENIC AREA IN THE MESO-DIENCEPHALON OF JAPANESE DACE, TRIBOLODON HAKONENSIS. K. Aoki and M. Takahashi, Life Sci. Inst., Sophia Univ., Tokyo. Optokinetic nystagmus (OKN) of Japanese dace (Tribolodon hakonensis) was induced by the electrical stimulation of the thalamic-pretectal region in the brain. Nystagmus always occured in both eyes and lasted during the stimulation of 10 sec. The amplitude of nystagmus ranged from 3° EO) MAD (7 5 G2 on the average). The stimulation of the right region induced nystagmus with the slow phase to the right, and vice versa. The strengths of the stimulus to induce nystagmus were 5-10 pA, and variation of the pulse repetition rate (10-200/sec) for nystagmus affected on the velocity of the slow phase. Op- timal pulse repetition rates which could induce maximum slow phase velocity were 50-100/sec, and induced slow phase velocities were 4-22°/sec. Although OKN could be induced by electrical stimulation under the light condition, in some cases, OKN was inhibited by ambient room light. In those cases, the nystagmus with spon- taneous eye movement was observed within the evoked eye movements. Under the dark condition and with high stimulus pulse repetition rate (200/sec), the long last- ing after-nystagmus was observed from nys- tagmus continued after the stimulation quite longer than that induced by visual stimulaion. PH 42 EFFECTS OF MOVING STRUCTURED BACKGROUND ON THE NEURONAL ACTIVITIES OF OPTIC TECTUM IN THE JAPANESE TOAD H.-j-Tsail; M.Satou2, A.Shiraishi3 and K.Ueda‘4. lpept. of Biol. Sci. & Biotechn., Tsinghua Univ., Beijing, China,2~4Z00]. Tnsitee,) Hace OF Sci., Univ. of Tokyo, Japan Toad's visual system can extract informations about the object's motion from the background. It can also discriminate the 'real' object's mot ion from the 'self-induced' visual motion. Firstly, to study the underlying neural mechanism of this characteristic, the effects of moving structured background (visual noise) on the responses of single neuron in the optic tectum to a moving object (black square ) were examined. The background was moved either '‘'in- phase' (at the same velocity and in the same direction) or 'anti-phase' (at the same velocity, but in opposite direction) with the object. Class T5 neurons responded to an object moving against the stationary background. When the object and the background were moved 'in- phase,' the responses were strongly inhibited in most cases. On the other hand, in the case of "anti-phase' movements, the responses were inhibited only miidly, not inhibited at all or facilitated. These in-phase and anti-phase effects were considerably modulated when the neuron's excitatory receptive field (ERF) or its surrounding area was masked. Secondly, the neuron's responsiveness was tested under a condition that the object was maintained stationary state in the center of ERF, while the background was moved and then stopped. Many neurons started to respond with a burst of spikes within a few sec after the moving background was stopped. This 'motion after effect' became stronger, when the object large enough to mask the whole ERF was used. PH 43 DIAGNOSIS OF THE LEVEL OF VIGILANCE +N IN A FREE MOVING MOUSE. A.Higashi, Natl.Inst.for Physiol. Sci Okazaki,444, Japan. Ot Sleep-wake phenomina might be expressed as the momentarily changing reverse vectors, and each vector corresponding to the acti- vity to reach at the absolute sleep and wake equilibrium potentials spontaneously by way of the body surface. Based on such an assumption, it will be presumed that the momentarily changing vigilance level might also be generated from the momentar- ily changing sleep-wake vectors. In order to analyze the relation between the con- scious activity and sleep-wake phenomina, the method of dynamic behavioral stage analysis (DBSAM) and the method of micro- sleep and micro-wakefulness processing (MMP) in a free moving mouse have _ been developed. In this report, the relation between the practical measurements’7 and logics of data processing for DBSAM and MMP, and a working hypothesis of conscious activity is outlined. The first step to consider this relation could be shown as ay alin Wal ee's Ib, Cleck time of Fig.1; Relation coneciousness-flew between sleep- prea "| wake phenomina x aie and conscious wtage(or) © tj activity. C;Vig- 25 ilance O. X and Sy a Y; boundaries. 1,2,3 and 4; entering sleep, awaking, quiet and light sleep stage, respectively. PH 44 PROSTAGLANDINS FOR MOVEMENT ON SPERMATOZOA AND OVIPOSITION BEHAVIOR IN CRICKET,GRYLLUS BIMACULATUS. M.Ishii, N.Yamaguchi and N.Ai. Dept. of Bi- ol.,Tokyo Gakugei Univ., Koganei, Tokyo. We clearly observed that full-matured sperm taken out from spermatophore just after copulation in G.bimaculatus adult fe- male had not any mobility of spermatozoa at all. But after transportation of sperm from spermatophore to female spermatheca,sperm- atozoa stored up inside of spermatheca rose gradually flagellar movement with high fre- quent stroke. In this experiment, we had attention to show effective concentration to rise spermatozoa movements. In this case flagellum movement and relative velocity of spermatozoon were measured as the indicator of mobility. Otherwise,PGE2, PGD2 and PGF2- alfa were prepared as activators for sperm- atozoal movement. When fresh spermatozoa were soaked in several kind of PG solution with different concentration, reletive vel- ocity of spermatozoon was measured by using of blood count set. As the experimental re- sult, PGD2 and PGF2-alifa were not effective to the movement with .low concentration. But PGE2,on the contrary, was more effective to it with lower concentration as @z2 Oi IESS. In this experiment, we could get graph of dose-response. So, in circadian rhythm of oviposition, inthe period of egg deposition had not any PGE2 at all(.,,-7 or less),but oviposition period had ntBner conc. PGE2 (397? or more). Physiology 971 PH 45 EVIDENCE FOR A ROLE OF THE CENTRAL PAIR COMPLEX IN FORMING PLANAR WAVEFORMS IN FLAGELLA, S. Ishijima!, K. Sekiguchi’ and Y. Hiramoto~. ‘Biological Laboratory, Tokyo Institute of eisiclsike oleh O-okayama, Meguro-ku, Tokyo, “Jobu University, Shin- machi and ~The University of the Air, Wakaba, Chiba-shi. Electron micrography has revealed that American horseshoe crab sperm flagella have the typical 9+2 structure, whereas Asian horseshoe crab sperm flagella have a 9+0 axoneme which lacks central pair and central sheaths but possesses other structures; inner and outer arms, radial spokes and nexin links. Beat patterns of the American and the Asian horseshoe crab sperm were recorded by means of a Nac microscopic high speed video at the rate of 200 fields per second and were analyzed in order to study the role of the central pair of the axoneme in flagellar movement. The American horseshoe crab sperm beat with planar waves, similar to those of sea urchin and starfish sperm flagella, at the BaeerOor 2929 Hz. On the other hand, the Asian horseshoe crab sperm beat with right-handed helical waves at the rate of 17.6 Hz. These results suggest that the central complex (central pair and central sheaths) plays an important role in forming planar waves, whereas it is not essential for the conversion of microtubule sliding into axonemal bends. PH 46 EFFECTS OF SEMINAL PLASMA FACTOR ON THE DEMEMBRANATED SEA URCHIN SPERM FLAGELLA. MeOkimo. Dept: Biol., .Coll. Arts & Sei. Univ. Tokyo, Tokyo, Japan. The mammalian seminal plasma contains a factor (SPF) which inhibits the movement of the reactivated mammalian (Lamirande et al, PuoloeeeReproduet., 23, 68a, 1983), salmonid fish and sea urchin (Okuno et al, ZnisGTa 3. 916, 1986) sperm flagella. The factor inhibited the reactivated flagellar Movement at approximately 30 pg/ml. However, the preexisting bends on the flagellar axoneme propagated very slowly (less than 1 pm/sec) near. the critical concentration. The bend angle and the curvature of both the principal and the reverse bend maintained constant during the propagation. The length of the bent region Maintained also constant while the straight inter-bend region elongated. These results seems to suggest the active area of the axoneme propagates the axoneme maintaining its length and the distortion regardless of the propagation velocity. When the axoneme was digested with trypsin followed by the incubation with SPF the sliding disintegration of the outer doublet was inhibited. However, considerable number of the axoneme peeled off at the distal region. In addition the preliminary experiment revealed that the stiffness of the axoneme was increased by the incubation with SPF. These results suggested that the SPF increased the viscoelastic resistance of the axoneme. PH 47 ROLES OF CALCIUM AND CYCLIC AMP ON MOTILITY INITIATION OF HAMSTER SPERMATOZOA. K. Ishida’, M. Waku' and M. MoriSawa.'Dept. of Urol. Teikyo Univ. Sch. of Med. and*Lab. of Physiol. Ocean Res. Inst. Univ. of Tokyo, Tokyo. When calcium was removed from sperm cells of hamster by diluting them into the medium containing both EGTA and calcium ionophore A23187, they are completely ques- cent. Spermatozoa initiated motility by an addition of excess amount of calcium, suggesting that calcium plays an important role in the initiation of sperm motility. The level of cyclic AMP content and acti- vity of adenylate cyclase and phosphodi- esterase were reduced by removing calcium from the cell by the above method. Addition of calcium enhanced both activity of the adenylate cyclase and phosphodiesterase, and the intra-spermatozoal cyclic AMP level. These results suggest that activation of the adenylate cyclase resulting an increase in cyclic AMP by the presence of calcium in the cell is a prerequisite to the initia- tion of sperm motility. Since both cyclic AMP generating system and sperm motility were stimulated by the presence of external calcium, it is likely that accumulation of intraspermatozoal calcium by transport from the outside into the cell causes the synthesis of cyclic AMP through the acti- vation of the adenylate cyclase and then, cyclic AMP induces the initiation of sperm motility in hamster. PH 48 EFFECTS OF ORGANIC SOLVENTS ON THE SLIDIN DISINTEGRATION OF CHLAMYDOMONAS FLAGELLA. M. Noguchi, M. Takahashi and H. Segawa. DEepE. OF Biola, hace (Of SC.) Loyama Univ., Toyama. Effects of organic solvents on the sliding disintegration of Chlamydomonas flagella and on hydrolysis of ATP during the disintegration were examined. The sliding disintegration was monitored by changes in turbidity. Isolated flagella were demembranated and used after a brief treatment with trypsin. The maximal turbidity change was observed at 30 uM ATP in the standard solution. In the presence of methanol or glycerol, turbidity change decreased with increasing concentration of the solvents. On the other hand, the amount of hydrolyzed ATP increased with increasing methanol concentration, whereas it decreased with increasing glycerol concentration. Thus the inhibitory effect of glycerol on sliding disintegration was accompanied with the inhibition of the ATPase activity. -In contrast the inhibitory effect of methanol on sliding disintegration was accompanied with the activation of the ATPase. These results suggest that methanol inhibit the sliding by uncoupling the mechanochemical coupling on the interaction between dynein and adjacent microtubule, and that glycerol inhibit the sliding by reducing the rate of ATP hydrolysis. 972 Physiology PH 49 FLAGELLAR BENDING PATTERNS DURING RE- SPONSES TO ELECTRICAL STIMULATION OF THE OUTER DYNEIN ARM LESS CHLAMYDOMONAS MUTANT, ODA38. Kenjiro Yoshimura and Keiichi Takahashi. Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo. Flagellar responses to electrical stimu- lation were analysed in the oda38 mutant of Chlamydomonas reinhardtii which lacks the outer dynein arms. The flagella of the mutant normally beat with the ciliary pattern. When the cell was held on a suction electrode and stimulated by an electric pulse (ims, 0.04-0.07u)A), the flagella beat with the flagellar pattern for about 0.6sec, after which the ciliary type beating was resumed. The changes in bending pattern at the onset of the re- sponse were analysed quantitatively. We identified the following two kinds of changes. First, the sliding velocity of the microtubules in the propagating reverse bend decreased uniformly along the length of the bend. This change was different from that in the wild type cell, in which the sliding velocity decreased more in the more distal region of the reverse bend. This suggests that the inner and outer dynein arms play different roles at the onset of the response. Second, there were the interruptions of the effective stroke and prolongation of the recovery stroke similar to those ob- served in the wild type cells. PH 50 RELATIONSHIP BETWEEN INTRACELLULAR cAMP LEVEL AND SWIMMING DIRECTION IN PARAMECIUM CELL MODEL. A.Izumi! and Y.Nakaoka?. MiGs HOI (C@ili Bitolla,) Unive sO thie nAdTs sa Chebamandrs—Dermite of Biophys. Engineer., Fac. of Engineer. Sci., Oosaka Univ., Toyonaka. We have reported that cAMP regulates the swimming direction in Paramecium cell Models as welk ag Gasy . In one type of triton-extracted model(model II) calmodulin antagonists inhibit Ca*t-induced backward swimming and cAMP enhances the inhibitory effect. The other type of triton-extracted model(model I) is insensitive to calmodulin antagonists and the sensitivity is obtained by addition of cAMP. Sucrose-treated model cannot swim forward without cAMP, even in the absence of Ca?*+. These cAMP effects are inhibited by an inhibitor of cAMP-dependent protein phosphorylation. An intracellular protein(forward swimming-inducing substance : FSIS)had equivalent effects to cAMP. These results suggest that FSIS increases intracellular cAMP level. To clear this suggestion, intracellular cAMP levels of three types of cell model in various condition, were measured by a radio- immunoassay. cAMP levels were higher, when the models swam forward. It was suggested that cAMP formed by intracellular FSIS (and perhaps cAMP-dependent protein phosphory- lation) is required for Paramecium cells to swim forward. Pal Sl EFFECTS OF THE LENGTH OF A FLAGELLUM ON ITS MOVEMENT. S.ABaba!, Y.Mogami’, A.Yogo? and Y.Imamura?, Dep. of Biol. Fac. of Sci. Ochanomizu Univ., Tokyo, *Dep. of Regul. Biol. Fac. of § Sci., Saitama Univ., Urawa and #Terumo Co., Tokyo. The length of a flagellum is a crucial phys- ical quantity that may restrict mechanical per- formance of the contractile machinery of the flagellum. To study the length effect on fla- gellar movement, we analyzed bending waves in echinoderm sperm flagella of various lengths obtained by severing their distal part. The bending waves in flagella of all lengths ana- lyzed, from some 2 wm through the full length of intact flagella, could be fairly accurately described by equations of the same form as used to describe those in intact flagella, in which the angular direction g is a sine function of the distance s measured along the flagellum and time t as follows, G(s, t). = 6.(s)) costa ft + Sos) eogmishe where f is the beat frequency, and @a, Sos, Dp are the amplitude, phase and bias of @. The beat frequency of severed flagella longer than half the full length remained essentially at the normal level of intact flagella, whereas that of shorter flagella increased linearly with their decreasing length. The curves of maximum rate of anglular change, 2zfg.(s), and of phase, Se(s), were shifted upward and downward, respec- tively, in the range of length shorter than half the full length. However, the curve of bias %»(s) remained unchanged within the whole range of length. PH 52 COMPUTER SIMULATION OF TACTIC BEHAVIOR IN PARAMECIUM. TRTronimaga, and Yep Nasszoh, instep saonee Sci., Univ. Tsukuba, Ibaraki 305 To understand mechanisms governing tactic behaviors of free-swimming unicellular or- ganisms, a computer model was constructed to simulate distribution of the cells in two different environmental conditions ad- joining with each other. Basic assump- tions for constructing the model were, (1) the cells first distribute at random ona finite line, (2) then environmental condi- tion in one half side of the line is sud- denly changed, (3) each cell passes over a border point between two halves of the line with a certain probability depending on its pass direction, (4) moving rate of the cell changes with time after it passes over the border and (5) the cells on the line do not disturbe their movements with each other. Thus simulated distribution was compared with the actual distribution of the specimens of Paramecium caudatum in a thin square chamber. Temperature of half of the chamber was suddenly lowered to a certain degree. Time change in the distribution of the specimens was ex- amined. We found some discrepancies be- tween simulated and actual distributions. The discrepancies were mostly dependent on the presence of the cells swimming along the border between two Gl al sé 12 @ie IM te temperature-regions. PH? 55 CORRELATION BETWEEN TENTACLE MOVEMENT AND MEMBRANE POTENTIAL RESPONSES IN NOCTILUCA MILIARIS. MewOantecand Y. Nartoh, Inst. o£ Biol: Scueeunivye OF TSukuba, Ibaraki 305. To understand mechanisms controlling ten- tacle movement of the marine dinoflagel- late Noctiluca miliaris, membrane poten- tial responses and tentacle movement were recorded simultaneously, and examined their time relationship. A slow flexion took place during a slow depolarization of the tentacle regulating potentials (TRPs). Rate of the flexion markedly increased after the specimen showed a negative spike. The flexion reached its maximum several hundreds milliseconds after the negative spike. The tentacle extended during a hyperpolarization after the nega- tive spike. The flexion was markedly en- hanced by raising external Ca concentra- tion, while the TRPs were little affected. On the contrary, the tentacle stopped its movement at its most extended state when the external Ca ions were eliminated, while the TRPs could be seen. A quick flexion was associated with the flash triggering potential (FTP). In contrast to the TRPs-associated flexion, the FTP- associated flexion did not need external Ca ions. PH 54 CILIARY RESPONSES IN THE GILL OF THE ABALONE. Akira Murakami and Keiichi Takahashi. ZOOleeinsica,.. Hace Of, SCi., Univ. Of Tokyo), Tokyo. Responses of cilia on the gill of the abalone, Nordotis gigantea, to electrical stimulation and to 5-hydroxytryptamine (SHT) were investigated. A single gill plate was isolated and folded around one of a pair of platinum electrodes in the perfusion chamber. The lateral cilia on the isolated gill plate were motionless or beat slowly with a beat frequency <10 Hz. When stimulated with an electric pulse (duration 0.5 ms), the cilia were ar- rested. The duration of the arrest changed from < a second to > 2 minutes as the stimulus intensity increased from 10 to 20 V.. The response was Ca-dependent. 5HT (10 ° M) stimulated the lateral cilia to beat faster; the frequency of the stimulated beat was usually > 15 Hz. The duration of the arrest response was re- duced in the medium containing 5HT. The ciliary beat activation by 5HT also ocur- red in Ca-free sea water. The results indicate that the respiratory current of the abalone is regulated by mechanisms similar to those reported for the feeding current in Mytilus (Takahashi and Mura- Kam, eo 68 etc.) Physiology 973 PHeS> CILIARY RESPONSES IN SEA URCHIN LARVAE OF LATE DEVELOPMENTAL STAGES. Yoshihiro Mogami’, Kikuyo Fujima? and Shoji A. Baba’. ‘Dept. Biol. Ochanomizu Univ. Tokyo, *7NEC Micom. Tech. Tokyo. Spontaneous’ ciliary responses of cilia of major ciliary bands (epaulets) in free swimming eight-armed pluteus of sea urchin (Hemicentrotus pulcherrimus ) were analized with the aid of high speed video-microscopy. Larvae swimming forwards’ frequently showed backward swimming with reversal beating of epaulet cilia. Revarsal beating usually returned to normal beating via intermediate beating with less polarized bend formation. It was, in some cases, followed by the cessation of beating in a position near the end of recovery stroke of reversal beating. After a period of cessation arrested cilia slowly inclined posteriorly to the position of the end of effective stroke of normal beating, where they beat with small amplitude (inactive beating) and then quickly moved to the arrest position. After several times of flip-flop between arrest and inactive beating the cilia resumed normal beating, never associated with reversal beating. The difference between the changeover to normal beating from reversal beat and from ciliary arrest suggests the functionally irreversible changes of _ the ciliary motile machinery during the’ transition from reversal beating to arrest response. Unusual metachronal coordination of co- exsitense of dexioplectic and laeoplectic waves in individual larvae was confirmed. The direction of bidirectional wave propagation was maintained also in reversal beating. PH 56 MULTIMODAL INHIBITORY INNERVATION OF THE GILL OF APLYSIA JULIANA. M. Kurokawa* and K. Kuwasawa. Deptew Brok®, Tokyo Metropolitan Univ., Tokyo. *Present address: Meiji Inst.Health Science. , Odawara. We have reported that the gill musculature of Aplysia receives excitatory innervation by motor neurons located in three neural classes, the abdominal ganglion, the branchial ganglion and the gill neural plexus. In this study, four types of SUEY innervation were found in the jabillic (1) IJPs were induced in gill muscle cells by the branchial nerve stimulation. (2) Motor neurons of the neural plexus in the gill received inhibitory innervation from the abdominal ganglion. The inhibition persisted for a long period after cessation of the branchial nerve stimulation. (3) A certain neuron in the branchial ganglion received IPSP input from the branchial nerve. (4) The motor neuron in the abdominal ganglion (AGN) received presynaptic “inhibition at °its periphery to gill muscle cells. As AGN also received periodic bursts of IPSPs inthe abdominal ganglion, this central inhibition of AGN may result in depression of both longitudinal gill shortening, which is directly evoked by AGN, and pinnule contraction evoked by the motor neurons in the branchial ganglion, which are activated by EPSPs from AGN. The presynaptic inhibition may allow AGN to cause the pinnule contraction without the longitudinal shortening. 974 Physiology PH 5/7 SYNAPTIC TRANSMISSION BETWEEN SECOND- AND THIRD-ORDER NEURONS OF COCKROACH OCELLI. - M. Mizunami and H. Tateda. Dept. of Biol. Fac. of Sci. Kyushu Univ. Fukuoka. We examined how the steady (tonic) and dynamic (transient) potential changes in the second-order ocellar neurons are transmitted to the third-order neurons of the cockroach, Periplaneta americana. The transfer function to steady inputs, measured by simultaneous recordings from second- and third-order neurons, was. Sigmoidal. The threshold of transmission was a little negative than the membrane potential of dark- or light-adapted state. Dynamics of transmission has been studied by comparing the responses of second and third-order neurons using Sinusoidally-modulated light. We found that 1) the waveforms of responses of third-order neurons were more transient than those of second-order neurons, 2) the amplitudes of responses of third-order neurons increased exponentially with the increase in the amplitudes of potential changes in second-order neurons and 3) potential changes of higher frequencies in second-order neurons produced larger res-— ponses in third-order neurons. The former two retained after an application of TTX, and were due to sigmoidal nature of input- output characteristics of the synapse. The enhancement of responses to high-frequency inputs, the latter one, was mainly due to active membrane properties of third-order neurons, because it was reduced by an SjojollaSerestOjoy Oe Wd, PH 58 DESCENDING OCELLAR INTERNEURONS OF THE AMERICAN COCKROACH. T. OHYAMA and Y. TOH. Dept. of Biol., Fac. of Sci., Kyushu Univ., Fukuoka. In the cockroach, ocellar interneurons descending directly from the ocellar nerve to the thorax have been reported (Toh & Sagara, 1984; Mizunami & Tateda, 1986). However, their morphology in the thorax and detailed physiology were not known. In the present study, two descending ocellar interneurons were morphologically and physiologically identified. They are identical with D-I and D-II neurons of Mizunami and Tateda (1986). The D-I and II neurons have a cell body in the brain and extend distally into the ocellar nerve. The D-I neuron sends an axon ipsilaterally through the suboesophageal ganglion. The D-I axon was confirmed to reach the PrOEnOLaAciICG ganglion.) fhe) D=l rE neuron sends an axon contralaterally through the suboesophageal ganglion. The D-II axon was confirmed to reach the second abdominal ganglion. Both axons extend side-branches and fine processes in the hemisphere of each ganglion ipsilateral to the axon. These neurons responded with only a few off spikes to ocellar illumination, but responded with a train of spikes to cercal stimulation. The train of spikes was MALOs eC Joy, CeSGllilare swiliwinmamMercwOims We Ines been proposed in many insects that the ocellus controls activities of the CNS. The present data appears to comprise physiological evidence of such ocellar KUMIS Ee WOM. Fimo RESPONSE OF THE ’3HMPORAL ORGAN TO THE AIR-BORNE CH&M=SCALS IN JAPANESE HOUSE CENTIPEDS. No WWXeal EiaGl YW -4tOIN, MESNes O12 BilOls, PAC. of Sci., Kyushu Univ., Fukuoka. a eee The temporal organ of the Japanese house centipede, Thereuonema hilgendorfi, has been identified to be CO, receptors: the receptor cell decreases impulse frequencies by CO, stimulation. The temporal organ also Fesponds to many air-borne chemicals. Acidic chemicals(eg., fatty acid) decrease impulse frequencies whereas basic chemicals (eg., amine) increase them. Yamana et al. (1986) supposed that the effect of air-_ borne chemicals may be secondary effects: air-borne chemicals change pH at receptor site, which results in changes of molar wIec(Siealeja) Cie CO 5 wie 1S Slaeyie alin lac present study, however, that presence or absence of CO, do not affect responses of the receptor Cells to air-borne chemicals. This result suggests changes of molar fraction of CO, may not be always involved in chemical reSponses of the temporal organ. Yamana et al.(1986) also supposed that the possibility of pH effect is intrinsic to the receptor cell. However, in the present study, increasing of pH of air-born chemicals did not always result in increasing impulse frequencies. These results suggest that the possibility of the air-borne chemicals may be directly accepted by the receptor cell. PH 60 VISUAL SYSTEM OF THE TIGER BEETLE (CICINDELA CHINENSIS) LARVA I.STRUCTURE Y.Toh and A.Mizutani, Dept. of Biol., Fac. OLRSCHe) ehyuShUm Una vier UkuUokaye Larvae of the tiger beetles live in the burrows. They amdush and catch preys. The neural mechanism underlying such unique visual behavior appears an interesting subject to examine. In the present study structure of the ocellar system of the Cicindela larva has been examined. The larva possesses six ocelli on either side of the head. The six ocelli are classed into two large ocelli (ca. 450 um), two medium ones (ca. 150 um) and two small ones (ca. 75 um). Each ocellus possesses a corneal lens, beneath which a rhabdom layer occurs. The number of retinular cells included in the six ocelli is estimated about 20,000. Retinular axons enter the optic neuropil located beneath the ocelli. The optic neuropil is stratified into two layers (lamina and medulla). Retinular axons synapse with monopolar neurons in the lamina. Lateral connections among lamina monopolar neurons also occur. Lamina monopolar neurons extend into medulla, and synapse with branches of medulla intrinsic neurons. The medulla intrinsic neurons swell up to 10 um in the posterior region of the neuropil, and extend to the brain as an optic nerve. The optic nerve contains about 150 axons. These results suggest occurrence of the neural circuit for movement detection in the optic neuropil. Physiology 975 PH 61 VISUAL SYSTEM OF THE TIGER BEETLE (CICINDELA CHINENSIS) LARVA II.ELECTRO- PHYSIOLOGY, A.Mizutani and Y.Toh, Dept. Gees volen hac. Of Sci, Kyushu Unity), Fukuoka. The ocellar system of the Cicindela larva was studied electrophysiologically. Of the six ocelli two large ocelli and one medium ocellus have been examined. The two large ocelli are directed dorso-anteriorly and dorso-posteriorly, respectively, and the medium ocellus is directed anteriorly. Visual fields (measured by ERG amplitudes) of adjacent ocelli partially overlap each other. The flicker fusion frequency of large ocelli measured by ERG is about 50 Hz. Spike responses of the higher order neurons to illumination were intracellular- ly recorded in the optic neuropil. Response patterns are as follows: 1)sustained inhibition of spontaneous discharges during illumination 2)phasic-tonic discharges during illumination 3)phasic-tonic inhibition during illumination 4)off- discharges 5)on- and off- discharges. Receptive fields of some higher order neurons were coincident to or included in that of some ocellus, whereas receptive fields of other neurons were wide enough to cover those of more than two ocelli. It is inferred from observed variation in response pattern and receptive field of the higher order neurons that extensive visual information processing would take place in the optic neuropil. PH 62 MICROVILLI-BEARING GANGLION CELLS IN ONCHIDIUM STALK EYE. Y.KATAGIRI’, N.KATAGIRI°and Y.SHIMATANI Dept. of Physiol’. and Anatomy”, Tokyo Women's Medical College, Tokyo. The ganglion cells (GCs) in the Onchidium stalk eye are found separately or in a cluster in the neuropil along the periphery of the retina which consists of visual cell type 1, type 2 and supporting cells. GC is approximatly oval in shape and has a large oval nucleus and an axon. It is distinguished from other retinal cents, “by large Sate (ip S=240) yam in diameter), a position in the eye, electron lucent cytoplasm, and absence of photic vesicles and pigment granules. In serial sections, a mass OIE. iskereey7alIL eat (MV ) surrounded by its own cytoplasm are found in the GC. MV are short and less compare to those of visual cells. MV in GC has not been known in other molluscan eyes. GC contains many dense lamellar bodies in the cytoplasm which are also rich in other organelles; Golgi apparatus, mitochondria, rough endoplasmic reticulum, ribosomes, glycogen granules, dense vesicles, filaments and microtubules. Fine structure of GCs is similar to those of GCs in eyes of Bulla and Aplysia except for MV. Although it is unknown whether MV of GC in the Onchidium stalk eye are a photo- sensitive organelle or not, large action potentials respond to light are recorded from the GC. PH 63 NEUROANATOMY OF THE TERMINAI, NERVE SYSTEM IN THE DWARF GOURAMI . Y.Oka!, M.Ichikawa2?,M.Satou! and K. Ueda!. PA OO en DSi IReCrarrO | 1S,C leer Und v rs Ort Tokyo,?Dept. of Anat. & Embryol., Tokyo Metro. Inst. of Neurosci., Tokyo. Tt has recently been suggested that the terminal nerve (NT) system is involved in the control of sexual behavior in tele- osts. We have examined neuroanatomically the NT system in a tropical fish, dwarf gourami, as a basis to study its involve- ment in the sexual behavior. The ganglion cells of the NT (NT cells)in the transi- tional area between the olfactory bulb (OB) and the telencephalon strongly reacted to a monoclonal anti-LHRH (LRH13, a gift from Dr. Wakabayashi of Gunma Univ.) as well as to a polyclonal anti- LHRH. The LHRH-ir fibers were abundant in the OB, ventral telencephalon (V) and several other areas. A distinct bundle of axons emanating from the NT cells ran through the V and the preoptic area (POA), ., and some of them entered the optic nerve ~ (ON). The NT cells could also be labelted by the cobaltic lysine applied to the cut end of the ON. The dendrites ramified in the OB, while the axons ran through the V and the lateral POA, and entered the ON. Thus, the LHRH-ir NT system might affect the olfactory and visual functions as a neuromodulator and/or neurotransmitter. It might also affect the V and POA, which have been suggested to be involved in the control of sexual behavior in teleosts. PH 64 EFFECT OF DENERVATION ON THE EXCITATORY AND INHIBITORY INNERVATION IN THE INSECT MUSCLE FIBERS. H. Washio, Lab. Neurophysiol. Mitsubishi-Kasei Inst. of Life Sci. Machida, Tokyo Insect skeletal muscle fibers are known to be innervated multiterminally and sometimes polynerrally. The coxal depressor muscle (177d and e) of the cockroach, Periplaneta americana receives innervation from a slow excitatory (Ds) and three common inhibitory motoneurons, possiblly from one of the dorsal unpaired median neurons. In the previous work on denervated coxal muscles of the cockroach it was shown that the excitatory neuromuscular transmission disappeared in about 2 days and resumed in about 3 weeks after nerve SeeCeEvonT dite Zoe intr ein sms bludiy?, ele havc demonstrated that there is the different time course of these failure and resumption of excitatory and inhibitory inputs in the cockroach leg muscle fibers. Although almost simultaneous failure of those innervations took place at 26°C, the disappearance of the excitatory input has been found to preceede the inhibitory's a few days at 15°C. Also the resumption of the excitatory transmission took place more than two weeks before the one of the inhibitory after motor nerve crush at 26°C, The result suggests that the neurotrophic influence of nerve on the muscle may be specific for the type of innervation in insect muscles. 976 Physiology PH 65 STRUCTURAL DIVERSITY OF THE HEMOGLOBINS FROM THE TWO CLOSELY RELATED BIVALVES - BAR- BATIA VIRESCENS AND BARBATIA LIMA T.Suzukix, M.Shiba*, T.Furukohri* and M. Kobayashi** *Dep.Biol., Fac.Sci., Kochi Univ., Kochi, *xkDep.Biol.,Fac.Sci.,Niigata Univ., Niigata The subunit structures of intracellular hemoglobins from Barbatia lima and B.vire- scens were investigated. B.virescens hemo- globin consisted of only 30,000 daltons component, whose structure was determined to be heterodimer. The two constituent chains were isolated by HPLC. The Mr of the chains was estimated to be 17,000 by SDS- PAGE. On the other hand, B.lima hemoglobin consisted of two components. One was a subunit with Mr of 60,000, whose structure was determined to be tetramer (usual A585 structure). The two chains were isolated by HPLC. The other was a polymeric subunit. SDS-PAGE showed that the Mr of this subunit is 32,000, therefore the didomain structure was suggested. No N-terminal residue was found by Edman method. We made CNBr clea- vage and enzymatic digestion of the di- domain subunit, and isolated the peptides containing N-terminal region. The sequence was determined as follows; N-terminal 36 residues of the first domain are Ac- (BVSZ)KIEEVTQPANKNLIRSTWNVMAGDRGNGVELM, and the first 37 residues of the second domain are MGVTERIEEVTQPANKGLIRETWNIVAGDRKNGVELM. The homology between domains was very high. PH 66 HATCHABILITY AND HEMOGLOBIN OF DAPHNIA MAGNA EMBRYO M. Ninomiya and M. Kobayashi. Dept. of Biol, hac Of sSGiae fn Naalgatal UnulvsasNlecalta The developmental rate of embryos in the brood pouch of Daphnia magna was markedly affected by temperature. The hatchability of embryos stayed constantly high at temperature below 27 C but decreased acutely with a temperature increase above this point. Fifty percent hatchability point was obtained at 29 C for embryos in the brood pouch of pale animals and at 30 C for embryos in the brood pouch of red animals. The hatch- ability of embryos in the brood pouch increased with an increase in oxygen tension and reached a steady high level. Fifty percent hatchability was obtained at oxygen tension of 12.6 torr for embryos in the brood pouch of pale animals and 8.6 torr for embryos in the brood pouch of red animals. The oxygen tension for 50 &% hatchability of embryos isolated from the brood pouch was 32.7 torr, and no sig- nificant difference was found between embryos isolated from pale and red animals D. magna hemoglobin was separated electrophoretically into at least 6 dis- tinct components. There was a difference between the relative amount of hemoglobin components in pale animals and that in red ones. Parthenogenetic eggs also had dif- ferent amount of hemoglobin components, compared to that in adult animals. Fal (ey) APLYSIA MYOGLOBIN WITH AN UNUSUAL HEME ENVIRONMENT A. Matsuoka and kK. Shikama. Biol. Inst., Tohoku Univ., Sendai. The radular muscle of Aplysia, the gastropod sea mollusc, is one of the most remarkable red muscles found in inver- tebrates. We have succeeded in isolating native oxymyoglobin directly from the radular muscle of Aplysia kurodai, a common species around the Japanese coast, and have examined its structural, spectral and stability properties. Unlike mammalian myoglobins, Aplysia kurodai myoglobin contains only a single histidine residue at the proximal position, lacking the usual distal one. Moreover, the hydropathy profile along the amino acid sequence of Aplysia Mb is completely different from that of sperm whale Mb, especially on the distal side of the heme. Apdlysital MbOR "ais ivenay Sal maelcurammeiar meters spectrum to Baose of mammalian oxy- myoglobins; the 4-peak being higher than the §-peak and the absorbance ratio (CT Iain ISOs, Lies Sicalon lity, however, is quite different from those of mammalian oxymyoglobins, and Aplysia MbO> is found to be extremely susceptible to autoxidation. Its rate is one-hundred SAMS) Ihalelieie ie jos YO, Amel LES pal dependence is unusual and much less steep, when compared with sperm whale MbOy as a reference. PH 68 SEASONAL VARIATION OF SPECTRAL SENSITIVITY EN GRAVE US REGUNULA CELLS AND ITS MONOCOLNAL ANTIBODIES. T.Hariyama, Y.Tsukahara! H.Yamamoto, T.Takeuchi2 and F.Tokunaga3 Research Center for Applied Information Sciences, Dept of Biol.,Fac.of Sci., Sept P F it Sea Tohoku Univ., Sendai Spectral sensitivity curves (SSC) were obtained from crayfish retinula cells by intracellular recordings with on axis stimulation throughout the year. During summer, only one type of SSC(}, max 600 nm) was obtained and the eye has retinal. In other seasons, four types of SSC were obtained. These eyes contain both retinal and 3-dehydroretinal. The SSC of the two of the four and of the summer's were well fit to the absorption spectra computed from Ebrey's (1977) nomogram for retinal (A max 560, 600 nm). The rest of the four were also fit for 3-dehydroretinal ( )max 600, 640 nm). The selective light adapta- tion for the isolated rhabdom showed that the visual pigment based on retinal ab- sorbed shorter wavelenth than the visual pigments of the rhabdom with 3-dehydro- retinal and retinal. These results suggest that the synthesis of the winter type opsin was induced by 3-dehydroretinal, Sumer type opsin was induced by retinal. In order to confirm these hypothesis, we prepared monoclonal antibody against rho- JOPS IMS. IWhere 15° MO STGHiPIicamt diffrence of molecular weight of rho- dopsins in summer and in winter. Physiology 977 PH 69 RETINYL AND 3-DEHYDRORETINYL ESTERS IN THE CRAYFISH RETINA Tesuzuki: - Y.Maeda2, Y.Toh° and E.Eguchi * ‘Dept. of Pharmacol.,Hyogo Coll. of Med., Nishinomiya,*Dept. of Chem.,Yokohama Natl. Univ.,Yokohama,*?Dept. of Biol.,Kyushu Univ. Fukuoka, *Dept. of Biol.,Yokohama City Univ. Yokohama. The crayfish Procambarus clarkii has rhodopsin and porphyropsin in the retina, and the ratio of the two pigments varies with seasons. In order to clarify the Origin of visual pigment chromophore we studied retinyl esters in the crayfish retina. 3-Dehydroretinyl and retinyl esters were found in the retina and more than 95% of them were in 11-cis configuration in dark-adapted animals. When light-adapted animals were kept in dark, visual pigments gradualiy regenerated and all-trans retinoids accumulated in the stored ester. A major fatty acid of the stored ester was docosahexaenoic acid (C22:6). The results of fractionation experiment and microscopic observation indicated that the retinyl esters were stored in photoreceptor cells as oil droplet. The ratio of dehydroretinal /retinal in the visual pigment was always higher than that of dehydroretinol/retinol in the stored ester. The present results show that the retinyl and dehydroretinyl esters are the precursor of visual pigment chromophore and suggest a mechanism of selective utilization of 3-dehydroretinyl ester for pigment synthesis. PH 70 CLONING OF C-DNA’S FOR OCTOPUS RHODOPSIN AND RETINOCHROME Uematsu: , 1. Hara’ and F. Tokunaga“ THepte Oi. buol.,0kac..of Sci:, Osaka imuve,lovoenaka, — Dept... of Phys;, Fac: Omescue. shohoku Unive, Sendai. In order to elucidate the amino acid sequences of octopus rhodopsin and retino- chrome, we have tried to prepare their c-DNAs. The c-DNA library was constructed by Okayama-Berg method. About 300 positive transformants for bovine rhodopsin c-DNA were isolated. By further screening 15 transformants were expected to encode rhodopsin. Restriction enzyme digestions showed their lengths were 300 - 600 bp. Ye synthesised the oligonucleotides induced from partial amino acid sequences of the squid retinochrome. These oligo- nucleotides were used as probes for screening colonies containing retinochrome c-DNA. The oligonucleotide encoding the amino-terminal region of retinochrome did not give positive signals for any colo- nies. The oligonucleotide encoding one of BrCN fragments of retinochrome provided 14 positive colonies expected to contain retinochrome c-DNA. Restriction enzyme digestions showed the lengths of their c-JNA were 500 = 750° bp. We are now trying subcloning of their c-DNA fragments to the plasmid vector, pTZ, to determine their base sequences. PH 71 THE ROLE OF RETINAL-BINDING PROTEIN IN THE CHROMOPHORE EXCAHNGE BETWEEN RHODOPSIN AND RTETINOCHROME SYSTEMS OF THE SQUID RETINA. Aw Lerakita, Rw Had ang: Le Hata Dept. OL Blow pace Of scaly. OSaka Univ, Toyonaka, Osaka 560, Japan Our previous works on retinal-binding protein (RALBP) with aporetinochrome and Oopsin suggested that RALBP plays important roles on retinoid turnover in the squid retina. The role of RALBP on the exchange of retinal between metaretinochrome (MRet) and metarhodopsin (MRh) was then examined by the use of S-dehydroretinal 9 (netinall>) as a tracer of retinal chromophore. In the fresh dark-adapted retina, RALBP dominantly combines with 11-cis-retinal. When mixed with excess all-trans— retinal and retinol, RALBP takes up each of them, releasing endogenous retinoid ligands. When an all-trans-retinal-rich RALBP was incubated in the dark with MRet2-carrying membranes (retinalz2 as chromophore), the RALBP took up 11-cis-retinal2 from MRet?2 and gave the all-trans-retinal to produce retinochrome in the membranes. On further incubataion of y thas) WiiSeits= retinal2-rich RALBP with MRh-carrying mem- branes, the RALBP took up all-trans-reti- nal from MRh and gave the 11-cis-retinal) to form Rh2 in the membranes. These findings showed that RALBP may act as a shuttle of retinal between MRh and MRet to regenerate each of the photopig- ments, Rh and Ret, in the visual cells. PH 72 RHODOPSIN AND RETINOCHROME IN THE NAUTILUS RETINA A. Kishigami, R. Hara and T. Hara IDISJONE 6 Oak. WWalEWlo 5 Wes Ot. SEs peOsaleal Winsi75 , Toyonaka, Osaka 560, Japan Just like the dibranchiate cephalopods, the nautilus (Nautilus pomptlius) has two kinds of photopigments, rhodopsin and ret- inochrome, in the retina. Retinochrome roughly amounts to about 4% of rhodopsin. These pigments were examined in digitonin extracts as follows. The Amax Of rhodopsin lies at very short wavelengths (465nm). On irradiation with blue light, rhodopsin yields a photosteady state mixture with metarhodopsin (v510nm), which is photoregenerated to rhodopsin by orange light. Rhodopsin is stable in the presence of 100mM NH20OH, whereas metarho- dopsin is slowly decomposed into retinal- oxime, showing a time-course consisting of two phases, fast and slow. On irradiation with orange light, reti- nochrome (Amax:v510nm) readily bleaches to metaretinochrome, which again yields reti- nochrome on addition of all-trans-retinal. As the result, nautilus retinochrome can catalyse the conversion of free all-trans- reeinal sco ii ers in the sight: Retino- chrome is fairly stable in 20mM NH20H, but metaretinochrome is destroyed quickly. According to the analysis by SDS-PAGE, the molecular weight of retinochrome was 26K, while that of rhodopsin was 84K, far larger than 45K in the squid, Yodarodes. 978 Physiology Pi J DEPENDENCY OF RHODOPSIN PHOTOLYSIS ON PHO- TON DENSITY OF PICOSECOND LASER PULSE T.Yoshizawa, Y.Shichida, S.Matuoka and H.Kandori. Dept. of Biophysics., Fac. of Science, Kyoto University, Kyoto. A photon density of picosecond laser pulse is essentially so high that it often causes unphysiolosical events due to multi- photon effect in investigating the primary processes of rhodopsin. Three typical ex- amples of the multi-photon effect were shown in the following. Excitation of cattle rhodopsin witha weak pulse induced the generation of photo- rhodopsin which decayed to bathorhodopsin with time constant of 45 psec, while exci- tation with an intense pulse induced the generation of another bathochromic product due to multi-photon effect. This product was stable in the picosecond time scale. Relative quantum yield of isorhodopsin to rhodopsin was 0.39 which was obtained by irradiation with a weak laser pulse. The value was close to that obtained by steady illumination. Irradiation with intense pulse gave bigger ratios due to multi- photon effect. This result was close toa previous result by other groups. 11-cis-locked 7-membered rhodopsin was excited with a weak pulse, and one interme- diate was observed in the early picosecond time scale. Excitation with an intense pulse induced the generation of another product due to multi-photon effect. PH 74 CHICKEN RHODOPSIN GENE. Mig WES! 5 NG WASUILO ancl) iy Uolwinagel™ 4 “DAs OG Pisa, BACs OF Si, aC “Pee. Mase for Tuberculosis and Cancer, Tohoku Univ., Sendai. Chicken genomic library was screened with a bovine opsin cDNA probe. Aclone isolated under high stringency hybridization condi- tions contained DNA sequence highly homolo- gous to all the five exons of bovine and human opsin genes. Sequence comparison of the putative open reading frame in the chicken DNA fragment of 4.3 kb with bovine opsin cDNA revealed 82 % identity for the nucleotide and 84 % for the deduced amino acid sequence, indicating that this DNA fragment contains the whole chicken opsin gene. In the 5'-noncoding region upstream of TATA box, several separated segments of 8 to 13 nucleotides long arecommom in chicken and mammalian opsin genes, suggest- ing that these sequences might be related to the cell type specific expression of the opsin genes in these organisms. Pil 7 OPSIN LOCALIZATION DURING RETINAL DEVELOPMENT IN THE GALE“C AND ADS MUTANT MOUSE. J. Usukura | and D. Bok 2. | Dept. of Anatomy, Univ, of Tokyo, Tokyo. 2 JSEI UCLA, USA. Electron microscope immunocytochemistry anc Western blot were used to detect the presence anc localization of opsin in the developing photoreceptor of ras mutant and their BALB/c normal control. Western blot analysis of isolated retinal membranes detected first at 10 postnatal days in both strains. Opsin levels rose progressively with development. in normal retinas. In contrast, levels in the ras retina became undetectable by 30 days after peaking at15 days. Specific binding of anti-opsin antibodies was first observed by immunocytochemistry at postnatal 5 days in the distal plasma membrane of the connecting cilium in both BALG/%c and ras retinas. Thereafter, labeling intensity increased progressively with development in the G4ZG/c retina. Anti-opsin binding to photoreceptors in the vas mouse retina predominated in the plasma membrane of the connecting cilium at 5 postnatal days, but opsin was present at higher density in the inner segment plasma membrane at 5 - 20 postnatal days when compared to BALG/c photo- receptors. Opsin rich vesicles were observed in the subretinal space of the vas retina during this period. By 30 days, labeling of the ciliary and inner segment plasma membrane decreased to near background levels. PH 76 EFFECT OF VITAMIN-A DEFICIENCY ON ERG RESPONSE AND 3-OH-RETINAL CONTENT IN THE SILKWORM COMPOUND EYES. S.Hori!, I.Shimizu!, S.Yoshida2, T.Yoshizawa*. ‘Lab. for Plant Ecological SiGuichy pwAC> C12 SCiloy IMvOcroO Wmiwao, 2Dept. of Buophys-.,) Hace ‘Of Sci) Kyoto Unanve Identification and analysis of 3-OH- retinal with HPLC was carried out on various stages of Bombyx mori pupa and adults reared on mulberry leaf. 3-OH-retinal first appeared on 7 day after pupation in the eye region of pupa, and its amount rapidlly increased during 2 days before ecolosion. ERG response of developing compound eye was also detected from 7 day pupa. When reared on vitamin-A-deprived artifi- cial diet, the silkworm adults lost the ERG response of their compound eye. 3-OH- retinal was not detected in the vitamin-A- deficientcompound eyes. They recovered ERG response by infection of lutein at middle pupal stage. However, 3-OH-retinal and retinal was not detected from the adult compound eye of injected animals. Animals injected with beta-caroten, retinoié acid and bicyclic retinal did not show the recovery of ERG response. Physiology 979 PH 77 Effects of Ca2* and Zn2+ on the ERP of octopus retina. K. Ohtsu. Ushimado Marine Laboratory, Fac. of Sci., Okayama Univ., Okayama. In a Ca“*-free condition, the early receptor potential (ERP) of the retina of Octopus ocellatus reacts normally to the first test flash but is depressed upon following test flashes (flash-induced depression, FID). Recovery from FID usually takes an hour but strikingly accelerates to within several minutes upon addition of 1 mM CaCl9. If the retina is transferred to not only Ca2+—- but Nat-free artificial sea water (ASW) following test flashes in ae -free ASW, however, FID is greatly reduced. This suggests in view of the Na-Ca exchanger that Ca“" which flowed into the photoreceptor cell through membrane channels opened by the test flashes in Na -free al could not be pumped out in the following Na*/Ca2+-free ASW because of the lack of the counter ion, Na’. On the other hand, immediate immersion of the retina into Na‘/Ca2+-free ASW without the two test flashes in Nat-free ASW resulted in remarkable FID but recovered in a few minutes. This suggest a Ca2+ release from the internal calcium store, which causes quick recovery from FID, as well as the inability for Na-Ca exchange. FID was also abolished by the addition of 1 mM es Zn2+ might substitute partially for Ca2t+ APE CTDAUeA Ye Zn¢++ might inhibit the pumping out of Ca2* by affecting the Na-Ca exchanger because it has been known that the ion inhibits the function of Ca-ATPase. PH 78 BIPOLAR-BIPOLAR COUPLING AND RECEPTIVE FIELD STZES OF CARP BIPOLAR, CELLS £osartoy and T.Kujiraoka,. Inst. jof IBiod)- Sci., The Univ. of Tsukuba, Niihari-Gun, Ibaraki, Dept. of Physiol. St. Marianna Univ. Sch. of Med.,Miyamae-ku, Kawasaki ON and OFF bipolar cells were identi- Racd iim che (carp. retina .-by means -of intracellular recording and dye injection. The receptive field centers, determined by measuring the response amplitudes obtained by centered spots of different diameters were 0.3-1.0 mm in diameter for ON bipolars and 0.3-0.4 mm for OFF bipolars These central receptive field values were much larger than dendritic field diameters measured from histological methods. Simultaneous intracellular recordings were made from pairs of neighboring bipolar cells. Current of either polarity injected into one member of bipolar cell pair elicited a sign-conserving, sustained potential change Bn pe thiessco therm | bapolLar eel. The coupling efficiency was nearly identical for both depolarizing and hyperpolarizing currents. Phis electrical coupling was reciprocal, summative, and it was observed between cell types of similar function and morphology. Dye coupling was observed in 4 cases out of 34 stained eel ks, These results suggest that there is a spatial summation of signals at the level of bipolar cells, which makes central receptive fields much larger than thew dendritic fielidsr. PH 79 TRANSMITTER RELEASED FROM PHOTORECEPTORS IN THE CARP RETINA. K.-I. Takahashi and M. stele Baa Dept. Physiol., Keio Univ. Sch. Med., Tokyo. We have developed a new technique which enabled us to measure a reversal potential in cone horizontal cells despite of the presence of electrical coupling between Cellswi (ie) ehyStoOlne sss. cGrn Loosing eo eia i. This technique was applied to rod hori- zontal cells. The carp retina was super- fused with a solution containing high- calcium, barium, and potassium channel blockers. A calcium action potential was triggered in the cells in situ when they were depolarized electrically or by application of the excitatory amino acid (EAA). At its overshoot, light responses and electrically evoked synaptic poten- tials appeared with polarities reversed to those elicited at the resting state. In addition, their reversal potentials coincided with the maximal depolarization of the EAA-induced response. Since our previous report revealed that the maximal depolarization was also the reversal potential of the EAA response, the ionic mechanisms of three kinds of responses in the rod horizontal cells were identical. From these results, it is suggested that the EAA is a leading candidate for the transmitter released not only from cones but also from rods, and that the synaptic mechanism from rods to horizontal cells is the same as that of the cone system. PH 80 POSSIBLE in situ INACTIVATION MECHANISM OF CGMP PHOSPHODIESTERASE IN FROG ROD OUTER SEGMENTS. S. Kawamura and M. Murakami Dept. Physiol., Keio Univ. Sch. Med., Tokyo. Activation of cGMP phosphodiesterase (PDE) in rod disk membrane is an inter- mediary process of phototransduction. Though the turn-on (activation) mechanism of this enzyme is now well understood, the turn-off process has not been well elucidated: previous results so far done under in vitro condition showed that the inactivation of this enzyme is a much slower process than the recovery of the photoreceptor potential. We suggest a possible rapid turn-off mechanism of this enzyme that is attained under in situ condition. aS The Michaelis constant (Km) of PDE is about 0.1 mM in the dark. When PDE is activated and the maximum PDE activity (Vmax) increases in the light, the Km also increases to about 1 mM. This Km increase is observed only in a crude preparation that probably preserves cell intactness. Using the crude preparation, we found that the Km increase does not seem to require components other than the proteins that are known to be involved in PDE activation. The Km increases within 200 msec after a light flash. When one takes the in situ cGMP concentration (<10 pM) into account, the rapid Km increase may function as a rapid turn-off mechanism of PDE in situ. 980 Physiology PH 81 THE EFFECT OF LIGHT DEPRIVATION ON THE FLY VISUAL SYSTEM DEMONSTRABLE WITH CYTOCHROME OXIDASE HISTOCHEMISTRY K. Mimura, Fac. of Liberal Arts, Nagasaki University, Nagasaki. Cytochrome oxidase histochemistry was used to examine the effect of the visual deprivation on the development of the neuron network in the optic lobe (Mimura, 1986). Flies, in which unilateral compound eye were covered immediately after emer-— gence, were raised under normal light and dark conditions (LD). The results indi- cated that asymmetry in staining between the covered and uncovered sides waS pro- duced and, then, deprivation produced a decrease in the cytochrome oxidase stain- ing | of «the retinavand) opticyy lobes. sthus difference in staining between both sides was not seen in 1-5 days post-emergence, but became evident after the sixth day post-emergence. No difference was seen in the central part of the brain. When flies were raised in LD after being raised in DD until the 5th day post-emergence, cyto- chrome oxidase staining was delayed. These results confirm the previous behavioral experiments of visual deprivation (Mimura, 1987), and suggest that visual depriva- tion during the early period of post- emergence leads to a long-lasting decrease in neuronal activity. PH 82 SPECTRAL SENSITIVITIES OF THE COMPOUND EYE OF SOME BEETLES. Wo Yo Iain, Mi, MHILPAUINVAMIOE, 375 UMONE ~iocCl Ist, WVAUUEDVN, IDEONES Oi IWWOIL6>, Wele>s CO: Seis; Kyushu Univ., Fukuoka. Some coleoptera show bright colorful appearance, whereas some show somber monochromatic appearance. It appears to be an interesting subject to see relation between body colors and spectral types of the compound eye in such coleoptera. However, there have been few electrophysiological accounts about spectral sensitivities of coleopteran photoreceptors. In the present study, spectral sensitivities of the compound eye in three species of beetles,Cicindela chinensis D., Liocola brevitarsis L. and Coccinella septempunctata bruckii M., have been studied by intracellular recordings. Three types of color receptors,A.max at Gels SOO iin (4 cells), 420 im (6 Cells) ain 520 nm (9 cells), were recorded in C. septempunctata. Two types of color | receptors were found in the other two; X.max cue Cele S/O ion (45 CSS) aac! SSO im (Ail cells) in C. chinensis and A max at ca. 375 nm (5 cells) and 515 nm (12 cells) in L. brevitarsis. Blue cells were not recorded in the latter two insects, but they would be included. The failure of recordings in blue cells may be due to their small sine. Tt 1S conyzectuned from present data that color receptor types may be rather independent of color appearance of beetles. PH 83 SPECTRAL SENSITIVITIES OF THE ANTERIOR LATERAL EYES OF ORB WEAVING SPIDERS. S. Yamashita. Biol. Lab., Kyushu Inst. of Design, Fukuoka. Spectral sensitivities of the anterior lateral eyes of the orb weaving spiders, Argiope bruennichii and A. amoena were examined by recording extracellular ERGs and intracellular receptor potentials. Both ERGs and intracellular receptor potentials consisted of two rapid initial peaks followed by a slow phase. In the red-adapted eye, the amplitude of the first peak decreased markedly and the maximum amplitude for the second peak was observed at about 480-520 nm. In contrast, the second peak was markedly depressed in the blue-adapted eye and the maximum amplitude for the first peak was observed at about 540-580 nm. These observations suggest that the anterior lateral eye contains two types of receptors and that they are coupled to each other. Receptor cells were Simultaneously impaled in a pair and electrical coupling was demonstrated between the receptors by passing a current into one cell and recording a resulting potential in the other cell. PH 84 ELECTRON MICROSCOPIC IMMUNOCYTOCHEMISTRY OF THE DROSOPHILA VISUAL PIGMENT. Y. TOMINAGA and T. TANIMURA. Dept. Biol., Fac. Sei., Fukuoka Univ. Fukuoka, The peripheral retinula cells (R1-6) of Drosophila compound eyes contain different types of rhodopsin from those of the central retinula cells (R7 & 8). Using a monoclonal antibody specific for R1-6 rhodopsin, we have studied the subcellular localization of R1-6 rhodopsin and the turnover mechanism of photoreceptor membranes by electron microscopic immunocytochemistry. Heavy labelling was seen on the rhabdomeric microvilli of R1-6. Multivesicular bodies in R1-6 were also heavily labelled by the anti-opsin antibody, and coated vesicles near the bases of microvilli were sometimes labelled. Secondary lysosomes presumably originated from multivesicular bodies were also labelled. These cytoplasmic organelles must be involved in the breakdown processes of rhabdomeric microvilli membranes. i Flies grown on the carotenoid-deficient medium lacks rhodopsin. R1-6 cells of these flies were scarcely stained by the antibody. By feeding retinoids, opsin synthesis can be triggerd in these flies. We examined the time course of opsin synthesis. The number of gold particles on the rhabdomeric microvilli membranes gradually increased. The labelling was found on the same cytoplasmic membrane components, such as rough ER, Golgi bodies, smooth ER, suggesting that these organelles may be related to the renewal of photoreceptor membranes. Physiology 981 PH 85 THE EFFECTS OF VITAMIN A DEFICIENCY ON THE COMPOUND EYES OF SILKWORM MOTH. E.Eguchi, S.Hori* and I.Shimizu*. Dept. of Biol., Yokohama City Univ., Yokohama. *Lab. for Plant Ecological Studies, Fac. of Science, Kyoto Univ., Kyoto. The structure and function of the compound eyes of silkworm moths (Bombyx mori) raised with vitamin A free diet (A free moth) were investigated and compar- ed with those raised with normal diet (normal moth). From the observations of thin-sections by electron microscope, it was found that the structure of rhab- doms of A free moth were maintained almost the same as that of normal moth, but the disorganization and vacuolation were occationally seen in the rhabdom. The observations of freeze-fractured specimens revealed the significant difference on the densities of intramembra- ne particles on P-face of rhabdom microvilli membrane between them, namely the density of A free moth is ca.300/u? which is less than 10% of normal moth. Electrophysiological measurements of the response amplitudes to graded stimulus light intensities by ERG method showed that the eyes of A free moth had sig- nificantly higher threshold (approximately 2-3 in log unit of light intensity) than the normal moths, and some showed no response even to the brightest light stimulus used in the experiment. PH 386 SUBCLASSIFICATION OF a- AND B-ADRENOCEPTORS IN MELANOPHORES OF GOBY, TRIDENTIGER OBSCURUS. 2 3 H. Katayama , Y. Isogai’, F. Morishita’ and K. Yamada3, 1Mukaishima Mar. Biol. Stat., Hiroshima Univ., Mitsugi-gun,Hiroshima-ken, 2Toyo-Jozo Co. LTD., Tagata-gun, Shizuoka- hKemeandeo4oo0L. Inst., Fac. Sci., Hiroshima Univ., Hiroshima. Melanophores of the goby have both a- and B-adrenoceptors. Adrenergic a-agonosts in- duce pigment aggregation of melanophores, while both a-antagonists and B-agonists tend to inhibit pigment aggregation. To study effects of drugs on melanophores, re- sponses of denervated melanophores in an isolated, split caudal fin were recorded photoelectrically with a microscope. The order of pigment aggregating potency of the a-agonists used was norepinephrine (NE)>clo- nidine (CL)>naphazoline (NP)>tetrahydrozoline (TH)>methoxamine (MX)>phenylephrine(PH). CL and NP are selective az-agonists. TH, MX and PH are aj-agonists. Yohimbine, a speci- fic ajg-antagonist more potently inhibited pigment aggregation caused by NE than pra- zosin, an aj-antagonist. These results in- dicate that a-receptor of the melanophores is a2 type. Specific B2-agonists, salbuta- mol(SAL) and terbutaline(TER), and a speci- fic Bj-agonist dobtamine(DOB) inhibited pigment aggregation induced by NE. The or- der of the lower limit of effective concen- tration that inhibit pigment aggregation was SAL 2-chloroadenosine = aden- osine = ATP> N -L-phenylisopropyladenosine (PIA) > cyclohexyladenosine (CHA). The po- tency of NECA (an A, agonists) was about 100-fold greater than that of A, agonists (PIA and CHA). Dipyridamole,~ a purine transport inhibitor, had no effect on the purine actions, suggesting that P-site, a intracellular purine-sensitive site, is not involved in the cell responses. Theophyl- line competitively inhibited the melano- some-dispersing action of adenosine, while forskolin, an activator of adeneylate cy- clase, augmented the adenosine action. These findings suggest that adenosine re- ceptors in the medaka melanophores are A in nature and further that the stimulation of the receptor mediates activation of ade- nylate cyclase, which induces the disper- sion of melanosomes through increase in the intracellular level of cAMP. PH 90 ADENOSINE INHIBITS THE RELEASE OF NEURO- TRANSMITTER FROM ADRENERGIC MELANOSOME- AGGREGATING NERVES IN TILAPIA. N. Oshima, H. Kasukawa and R. Fujii. Dept. O« BHiOloe, Paco Ot SEi,, Tone Uniy,, Chiba, In order to study the mechanism of pre- synaptic inhibition in adrenergic chromatic nervous system, the action of adenosine on the release of neurotransmitter from ner- vous elements was examined. Sp jae wil PieSQpeisahiewowis Oi tele tesla iter, Sarotherodon niloticus, were loaded with SH-norepinephrine (NE), and rinsed with fresh saline. After they were transferred into a small plastic chamber, sampling of the bathing medium was carried out every 2 min. The number of disintegrations of 3H in the sampling medium was counted in a LPigquid sicints lataon icounters. When electrical stimulation was applied, the release of “°H=NE was Significantly increased. The increase was suppressed by adenosine dose-dependently, and the minimal effective concentration was about 10 © M, Such inhibitory effect of adenosine was blocked by theophylline, amplying that adenosine interacts with presynaptic adeno- sine receptors. ATP also showed the same effect. Since ATP (adenosine) has been shown to be liberated as a co-transmitter from nerve” terminalis jallong wath) NE (ek. Fujii and Oshima, 1986), the mechanism of auto-regulation is suggested in chromatic nervous system in fish. On the other hand, NE itself did not repress the °H-NE release from adrenergic nerves of the tilapia. Pa Sul CHANGES IN BODY COLOR AND MECHANISMS CON- TROLLING MELANOPHORE MOVEMENT IN UPSIDE- DOWN CATFISH. H.Nagaishi, H.Nishi, R.Fujii and N.Oshima. Des Oz WilOls, PAC, OF SeI5, Woo Windy, Chiba. of swimming and resting bottom up. Their ventral side is dark alike the dorsal part. When the fish were adapted to the white or black background, the brightness of both dorsal and ventral parts changed. Melanophores present in the dorsal and ventral skins were shown to be controlled adrenergically, since they responded to norepinephrine by a remarkable pigment aggregation, which was readily antagonized by the specific blocker, phentolamine. in, che naghit, thie idorsalpanstommsteine body surface seemed to be paler than the ventral part. This is probably due to the G@ifference in the size and number of melanophores existing in each side: In ventral part, smaller melanophores are present in larger numbers. When melatonin was added into the aquarium water, the fish were blanched in spite that they were on the black background, and swam actively. In addition, upside-down catfish was found to prefer the black background to the white one. Thus, the intimate relationship between the behavior and the change in the body brightness was suggested. ial IZ SKIN COLORATION AND ITS CONTROLLING MECHA- NISMS IN THE BLUE-GREEN DAMSELFISH. H.Kasukawa, K.Miyaji, N.Oshima and R.Fujii. DENE, Cie Walol., WAC, Oi SCig, WoOla® Warwo, Chiba. In the blue-green damselfish (Chromis caerules) skin, which normally displays yellowish green hue, three sorts of chromatophores, i.e. xanthophores, irido- phores and a melanophore are present, and constitute a dermal chromatophore unit resembling that of anurans or reptiles. The iridophores showed analoguos fine structures as those in the blue damselfish, Chrysiptera cyanea. Those cells are categorized into the motile and non-motile ones, and each cell reflected rays of varying, but a single spectral peak by a non-ideal multilayer thin-film inter- ference. The motile cell possessed only alpha- and beta-adrenoceptors, mediating changes of spectral shifts towards longer or shorter wavelength, respectively. The changes may be due to the simultaneous increase or decrease in the distance between adjoining reflecting platelets. On the other hand, the non-motile iridophore reflecting rays of single spectral peak may possess neither intracellular motile machinery nor receptors involved. Although the integument of this fish reflects several colors microscopically, it looks greenish as a whole, being due to an integrative effect of different purer colors reflected from iridophore sorts. The darkening is induced by a reverse process. Physiology 983 PH 93 THE ROLE OF Ca IONS IN PIGMENT-AGGREGATION RESPONSES OF MEDAKA XANTHOPHORES. M. Iwakiri, Dept. of Biol., Fukuoka Univ. of Educ., Munakata. Using denervated xanthophores in isolat- ed scales of he medaka, Oryzias latipes, effects of Ca antagonists and EGTA were examined on the cell responses to various pigment aggregating reagents. KCl (more than 30 mM) and noradrenaline (more than 1 uM) induced a full aggregation of pigment within the cells. Caffeine at concentra- tions less than 1 uM induced a weak pigment aggregation, while at higher doses it caus- ed a marked pigment dispersion. Pigment-ag- gregation response of xanthophgres EouXed was inhibited by verapamil, La and EGTA, respectively, but not by procaine. The cell response to noradrenaline was also inhibit- ed by verapamil, La and EGTA, respective- ly. However in this case, the inhibitory effect of EGTA was considerably low and besides verapamil was observed to inhibit the cell response through its competitive action to noradrenaline at the receptor site. Caffeine-induced aggregation respon g of the cell was inhibited by procaine, La and EGTA, respectively, but not by verapa- mil. These findings suggest that each cell response to the respective reagent requires Ca from different sourses, i.e. the cell response to KCl requires the extgacellular Ca , while the intracellular Ca is in- volved in both noradrenaline- and caffeine- induced cell responses. PH 94 TRANSMISSION OF LIGHT-SENSITIVE RESPONSE OF XANTHOPHORES IN SCALES OF THE MEDAKA. meta te Archa Weret., Col. of Nursing,, Nagoya. Usually, xanthophores in isolated scales of the medaka, Oryzias latipes, maintained a fully pigment-dispersed state in physio- logical saline under darkness. The cells responded to light with a rapid and full aggregation of pigment granules and then with a gradual redispersion, finally at- taining the half pigment-aggregated state. When a part of the scale was illuminated locally, in some xanthophores located in the outside of the illuminated area, aggre- gation of pigment also occurred to a cer- tain extent. The subsequent illumination of the whole scale induced not only the aggre- gation response of non-stimulated xantho- phores but also aggregation of the prestim- ulated cells. These light-sensitive re- sponses of xanthophores were inhibited by the presence of Ca” antagonists, such as papaverine and verapamil. The findings sug- gest that the light-sensitive response is transmitted from the stimulated xantho- phores to the other cells. The data also suggest that the chromatic nerves are not involved in the transmission of cell re- sponse, because such phenomena were con- sistently observed in both innervated and denervated cells. Ca~ might take part in the transmission of the light-sensitive re- sponse as well as in the aggregation re- sponse itself. PH 95 THE METABOLIC PATH TO FOR CHROMOPHORES OF INSECT VISUAL PIGMENTS. T.Seki, S.Fujishita. Dept. of Health Sci., Osaka Kyoiku Univ., Hirano Campus, Osaka. Retinoids and carotenoids in a compound eye of insects in several orders were ana- lysed by using HPLC-system detecting absor- bances at two wavelengths, UV and visible. Xanthophyll was found in all the insects whether they use retinal or 3-OH retinal as the chromophore of visual pigments. This result indicate that the xanthophyll pres- ent in the compound eye does not necessary- ly become the precursor of the chromophore of visual pigments. In Mantodea, Orthoptera and Phasmida, far more retinyl ester than retinal was found in the compound eye, and in the case of Odonata which uses both retinal and 3-OH retinal as the visual pigment chromophore, far more retinyl ester and 3-OH retinyl ester than chromophore retinals were found in the compound eye. The predominant geo- metric isomer of the esters was ll-cis. These esters must be the precursor and the stock form of the chromophore retinals. In Diptera and Lepidoptera which are the higher orders in Insecta and use 3-OH reti- nal as the chromophore retinal, little 3-OH retinyl ester was found in some species. In these species xanthophyll must be the precursor of the chromophore retinal. They may have obtained the ability to isomerize all-trans 3-OH retinal, which is the produ- ct by the imaginal xanthophyll dioxygenase ACEIVAL ty; eNO it — CPSs —OH ere amales PH 96 IOINT Cae MCE CHAIN eS) M Sie O.H sale Hee Sul OW) SeEONSnle SAYONPAVH al. Onuuky Nite OAS Gel N aon hn EB VAVE lun BX CITATORY NEURONE OF THE AFRICAN GIANT SNAIL, Achatina fulica Férussac. Y.Furukawa and M.Kobayashi. Physiol. Io g IieWes sGi? me Siiecncecl! Nets: fr Seue 5 Hiroshima University, Hiroshima. Ilonic mechanisms of the slow ex- citatory post-synaptic potential (SEPSP} of the heart excitatory neurone (PON) in- duced by the activity of two cerebral neurones were investigated under the voltage-clamped condition. By experi- ments of the ionic substitution and of the Cs*-injection, SEPSP was found to be diuke! sembau ni liye at iome itahves » dre ecmrlenarsien se 1Ont *>background’ K*+-conductance. In some preparations, the transient increase of conductance was also seen. The increased conductance was assumed to be Ca?#?*- conductance as synaptic response was in- rem sied: jbsyasstihiewel nej.e GatelaO mss, O) teu Ph GeleA. Serotonin (5-HT) produced similar slow depolarization in PON, and both slow depolarizations induced by 5-HT and the cerebral cells were depressed by the 5-HT antagonist, methysergide. The slow depolarizaion by 5-HT was also mainly due to the decrease of ’°background’ K*t-— conductance and two different 5-HT- sensitive K+-channels were identified by the patch-clamp experiments. Serotonin also increased the voltage-dependent Ca2+-—conductance in PON. These results suggest that the neurotransmitter of the two cerebral cells may be 5-HT. 984 Physiology Pll Si REGULATION OF HEART ACTIVITY IN THE GIANT AFRICAN SNAIL, ACHATINA FULICA. 6 fats IWejies Cie iiO@lGs PEG OL Seao7 Wolo Univ., Chiba. Molluscan heart is known to be myogenic, being regulated by the central nervous system. Working on the African giant snail, Achatina fulica, the controlling mechanism for the heart activities was studied. First, the presence of serotonin and FMRFamide in both the central nervous system and the heart was immunohisto- chemically examined and confirmed. The FMRFamide-positive nerve fibers in the heart were larger in number than the serotonin-positive nerve fibers, especially in the auricle. Using isolated heart preparations, the effects of the above- mentioned and the related substances were then studied. Serotonin, FMRFamide, dopamine and norepinephrine were found to augment the activities, while ACh acted inhibitorily. Serotonin acted excitatorily on the heart beat and amplitude. FMRFamide also acted excitatorily on the heart beat, but showed no effect on the amplitude. Effect of serotonin was more potent than FMRFamide. Dopamine showed a similar effect to that of serotonin. Norepinephrine capsed a decrease of AUN plas len ten ee alate eu in Ol miae Mirs ATP at higher concentrations lowered the amplitude. Adenosine had the same effect but to a lesser degree. The receptor analysis of these drugs is under investigation. PH 98 EXCITATORY AND INHIBITORY PATHWAYS OF CARDIO- REGULATORY NERVES IN THE CENTRAL NERVOUS SYSTEM IN AN ISOPOD, BATHYNOMUS DOEDERLEINI. K.Tanaka and K.Kuwasawa. Dept. of Biol., Fac. of Sci., Tokyo Metropolitan Univ., Tokyo. Each of two anterior cardiac nerves contains three cardio-regulatory axons, a cardio-inhibitor ( CI ) and two cardio- accelerators( CAl and CA2 ). We found that CI, CAl1 and CA2 arise from, respectively, the 3rd LOO oLuther sts 2ndsand Mrd\ithomacweigangla ( T1G, T2G and T3G). In fact, we identified a candidate cell for CA2 neurone in T3G. In isolated single-ganglion( T2G or T3G ) preparation, impulse discharge of CAl or CA2 was activated by stimuli applied to anterior and posterior connectives of T2G or T3G, and ON? Sheds Cre wis Usic,, Zovel ebocl Sigel coors. Oe T2G or T3G. CA1l and CA2 activities were inhibited by stimuli toconnectives. T1G was necessary for the inhibition of CAl and CA2 produced by stimuli of peripheral nerves. CI was activated by stimuli of connectives anterior and posteriortoT1G. In preparat- ions containing T1G to T3G, CAl and CA2 were inhibited by stimuli of peripheral nerves, while CI was activated by the stimulation. These results show that there are excitat- ory inputs were transmitted to CAl and CA2 in their own ganglia. Reciprocal relation between CI and CAs activities in response to stimuli applied to the periphery was eliminated in the preparation whose cerebral ganglion was removed. The reciprocal coupling mechanism between excitation of Cl. and inhibition of CAs may exsist in T1G. Pil NEUROPHYSIOLOGICAL AND PHARMACOLOGICAL STUDIES ON THE CARDIO-ARTERIAL VALVE OF BATHYNOMUS DOEDERLE INI Y.Fujiwara and K.Kuwasawa. Dept. of Biol., Fac. of Sei., Tokyo Metropolitan Univ. , Tokyo. The haemolymph flow to the arteries is modulated with dilation and constriction of their own valves. The lateral cardiac nerves(LCNs) running along lateral arteries(LAs) were found to consist of one or two axons innervating the valve. The repetitive stimulation applied to LCNs always increased the haemolymph Flow. All LCNs may be valve’ inhibitory (dilator) nerves. When the heart was perfused with ACh, pressure pulses of the outflow into LAs were increased. This shows that ACh dilated the valve. Such inhibitory effects were also somewhat observed with muscarine, arecoline and atropine. o-HT and octopamine decreased amp! itude of pressure _ pulses. This shows that these drugs caused costriction of the valves. Nor- adrenalin, adrenalin, dopamine and glutam-— ate produced no effect on pressure pulses. We supposed that ACh may be a candidate for, at least, a transmitter of the inhibitory (dilatory) valve nerve. Since constriction of, the valves should not be produced by neuronal mechanisms, it was suggested that 5-HT and octopamine may act the part of the excitatory humora | modulator on the cardio-arterial valves of the lateral arteries. PH 100 NEURAL CONTROL OF THE HEART AND THE PERICARDIUM IN AN AMPHINEURAN MOLLUSC ( LIOLOPHURA JAPONICA ). S.Matsumura, K.Kuwasawa. Dept. Bilol ales O£ Sei. Tokyo Metropolitan Univ. lokyor Simultaneous extracellular recording from the heart and the pericardium showed that the heart and the pericardium beat at different rates. It is likely that the heart and the per- icardium possess myogenic nature. ACh(107 Om) produced inhibitory effects on the heart, 5-HT(1078m) produced excitatory effects on the heart. Inhibitory and excitatory nervous control of the heart may involve cholinergic and serotonergic mechanisms, respectively. Cardioexcitatory responses were elicited by repetitive electrical stimuli applied to the ventral and the lateral nervecords ( VNC and LNC ). The responses elicited by stimuli to VNC were observed even after the dener- vation of LNC. Cardioinhibitory responses were elicited only by stimulitothe LNC. [n Cryptochiton, it has beendetermined whether the cardioexcitation caused by nerve stimu- lation is a direct effect of innervation to the heart or not. We confirmed that the exci- tatory effects were caused by cardioregurato- ry nerves, running from LNC and VNC. The in- hibitory nerve possibly arises from LNC alone. The pericardium was activated by stimuli to LNC and VNC and inhibited by stimuli to the LNC. The effects of nerve stimulation on the heart and the pericardium did not always coincide. Excitatory and inhibitory nerves to the heart are not common with those to the pericardium. Physiology 985 PH 101 INNERVATION OF IDENTIFIED NEURONS IN THE VISCERAL-GANGLIONIC NERVOUS SYSTEM OF PLEUROBRANCHAEA NOVAEZEALANDIAE K. Kuwasawa and M. Kurokawa. Dept. of Biol., Fac. of Sci., Tokyo Metropolitan Univ. , Tokyo. The visceral ganglion has a pair of the connectives connected with the cerebro- pleural ganglion and the branchial nerve running to the gill and the visceral nerve to visceral organs including the circulatory system. Microscopic sections cut from the ganglion showed that’ the ganglion consists of about twelve ganglio- nic neurons. It was observed anatomically and electrophysiologically that the visceral nerve sends branches to the penis ganglion, arteries, the hermaphroditic duct, the pericardium, the renal pore, the ventricle and auricle of the heart. There is a single-cell ganglion on the visceral nerve usually at the site from where the nerve to the hermaphroditic duct arises. The cells in this nervous system were filled with Co‘t* and/or Ni** by simul- taneous pressure and iontophoretic inject-— ion. Some processes of the cells were stained through a distance of 20mm which reach their effectors. Two of the largest neurons sends axons to the aorta and the hermaphroditic duct. There is an electrical coupling between them. One medium sized cell sends an axon to a region of the renal pore. The cell in the single-cell ganglion sends axons to the visceral ganglion, the pericardium and the glandular sac located at the branchial end of the auricle. PH 102 DISTRIBUTION OF BIOACTIVE AMINES IN THE CENTRAL NERVOUS SYSTEM OF A MOLLUSC, PLEUROBRANCHAEA NOVAEZEALANDIAE M. Otokawa! and K. Kuwasawa ? ‘Fac. of Social Sci., Hosei Univ.,Tokyo. *Dept. of Biol., Fac. of Sei., Tokyo Metropolitan Univ., Tokyo. Tissue extracts from the central nervous system were analysed for bioactive amines by HPLC on a column (Chemcosorb 5-ODS-H) with an electrochemical detector (BAS, HEG—AB)= Eleectrochemically active subs tances were identified by the retention time. In the cerebro-pleural ganglion average amounts of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) were, respectively, 33.0,265.9 and 170.1 pmoles. In the buceal ganglion averages of NE, DA and 5-HT were 3.5, 39.6 and 30.0 pmoles, respectively. In the left pedal ganglion averages of NE, DA and 5-HT were 4.8, 15.6 and 228.5, respectively. In the right pedal ganglion they were 15.7, 166.2 and 144.5 pmoles, respectively. In the visceral ganglion trace amounts of NE and DA were detected, but 5-HT was not observed. Immunocytochemichal study also suggested the absence of 5-HT containing neuron in the ganglion. The glandular sac and pinnules of the gill innervated by nerves of the visceral ganglion were shown to have considerable amounts of DA and 5-HT. PH 103 ANALYSIS OF RHYTHM COORDINATION BETWEEN THE AURICLE AND VENTRICLE IN THE OYSTER CRASSOSTREA GIGAS H. Uesaka~, H. Yamagishi~ and A. Ebara>. RacomeHa taeslvtdc sl biainalkee. IEE Ge lehat co) ly. Sich.) Umer Vie One liste (i bic remel aD labial Kenia enisitere HEM IMYV SEE Sty Wins wae Wolreo, Aolerypexr The mechanism of coordination of the auricular and ventricular rhythms mediated by mutual stretching was analyzed in the oyster heart from phase-response curves G2RG) Stonmabirwet stretches shies PRCsi were biphasic showing phase delay and advance as a function of the phase where the stretch was applied. The auricle and ventricle were considered as cardiac ODSGCULINLAEOHRS Awely (Suma ibe Glavin erinsine properties for brief stretches, due to their PRCs being remarkably alike. Pinttas asannile nye Ole ems Dledits easlinyst im pabay; repeated stretches was predicted from the PRC and confirmed. When the interval of the stretches was close to the beat TimEoe val», “ice OSenililaeor @Seablasnecd ei Fixed Phage rElAeLTOMSNID Wied Ene stretches at a phase predicted from the PRC and the beat rhythm was stably entrained. Alterations of the auricular anid a venmtrne telat seme Wales meme con ded ann ssilutat ine Mew Ui mMcolo is den ait eda ss tale came Omleldia Die interpreted as repeated phase responses. The regular heart rhythm of the oyster must be achieved by mutual stable entrainment of the auricular and ventricu- lar rhythms mediated by mutual stretching. PH 104 HIBERNATION-LIKE STATE IN CARDIAC FUNCTION OF NONHIBERNATING CHIPMUNKS. N.Kondo. Dept. of Muscle Physiol. Mitsubishi-kasei Inst. of Life Sci., Machida, Tokyo In an attempt to elucidate whether changes in cardiac properties observed during hibernation occur before entering into hibernation, seasonal variations in electromechanical responses were studied on myocardium of nonhibernating chipmunks. Characteristic properties of hibernating preparations were as follows: 1)frequency- dependent decrease in tension, 2)reduced amplitude of action potential plateau(APp) which is augmented by 4-aminopyridine(4- AP), 3)inhibition of APp and tension by substitution of Ca by Sr. In nonhibernating preparations, a frequency-dependent increase in tension was observed between June and September, while between October and next May, tension showed a frequency- dependent decrease. APp showed a high amplitude between May and September, but a reduced amplitude between October and next April which was increased by 4-AP. Sr markedly increased tension with a prolongation of APp in preparations obtained during summer season. In those obtained during winter season, Sr caused a marked inhibition of tension and APp. These indicate that changes in cardiac function are already triggered before hibernation begins, suggesting possible involvement of as yet unknown substance(s). 986 Physiology PH 105 EFFECT OF HYPOXIC CONDITION ON THE ISOLATED HEART BEAT OF THERMALLY ACCLIMATED GOLDFISH io Weulkwcla>g Wee, Oi WiOGl op FASS OF SGio, Osaka City Univ. Osaka. The heart excised from 10°C- or 25°C- acclimated goldfish was immersed in physiological saline at 10°C with or without glucose and the frequency and the amplitude of ventricular contraction was recorded under hypoxic condition (about 25% oxygen) for three hours. The ventricular activity was evaluated by the "power" which is the product of frequency and amplitude. The relative power (percentage to the initial value) was significantly higher and the decreasing rate was smaller in 25°C-acclimated fish than in 10°C- acclimated fish. After 30, 60, 120 and 180 min, the power decreased irrespective of exogenous glucose supply to 36, 19, 8 and 3% in average in 10°C-acclimated fish and 63, 55, 47 and 39% in 25°C-acclimated fish. These results indicate that the ventricular activity of cold-acclimated fish is more sensitive to hypoxic condition than that of warm-acclimated fish and that exogenous glucose is scarcely available to ventricular contraction under hypoxic condition, especially in warm-acclimated fish, in contrast with the case under normoxic condition. PH 106 ACTIVATION OF CONTRACTURES IN INSECT SKELETAL MUSCLES. T.Yamaguchi!,H.Washio*. 'Dept.Bjol. Intern. Christ. Univ., Mitaka, Tokyo, Lab. Neuro- physiology, Mitsubishi-Kasei Inst. Life Science, Machida, Tokyo. To clarify the activating mechanism of contractures of insect skeletal muscle, 4X 10-4M L-glutamate- and 100mM potassium- induced contractures were examined in the retractor unguis muscle of cockroach (Peri- planeta americana). Both contractures were completely eliminated when the muscles were immersed in calcium free saline containing 2 mM EGTA. If then, calcium ions were added to these muscles, a delayed glutamate and a tonic potassium contractures were initi- ated, respectively. If the muscles were previously immersed for 10 min in a saline containing 10mM Mntt+, glutamate contracture was reduced by ca. 70%, while the phasic contracture of potassium was reduced by about half of the control. 1pM Nifedipine reduced both L-glutamate and potassium con- tractures at different rates, whereas pro- caine at the same rate. The latter drug suppressed the phasic component of potas- Sium contracture. These results support the hypothesis that in insect muscle, contrac- tures are initiated by external calcium ions which enter the membrane and then, act by two ways, one to SR and the other di- Hecivily, sto) Contractile japparavuls)., Ghas) was also supported by the fact that a phasic contracture was induced by caffeine. PH 107 ADRENERGIC PATHWAY IN NEURO-MUSCULAR TRANS- MISSION IN THE RED MUSCLE OF PUFFER FISH. S.Yukiyama and T.Hidaka* Dept. of Biol.Fac. of Sei., and *Fac. of Gen. Edu., Kumamoto Univ., Kumamoto. In the pectoral fin red muscle of puffer fish, Takifugu poecilonotus and T. rubripes, excitatory junction potential(ejp), inhibi- tory junction potential(ijp) and the dipha- Sic junction potential composed of ejp and ijp were elicited by the single nerve stimu- lation. Miniature ejp (mejp) and miniature ijp (mijp) were observed in the resting muscle. The previous study showed that this transmission was mediated by the cholinergic pathway. Besides this pathway, the presence of another pathway was suggested from effects of adrenergic agonists and antagonists. While a-agonist, noradrenaline and phenyle- phrine, increased the amplitude of ejp and ijp, a-antagonist, phentolamine and prazosin increased both ejp and ijp. While B-agonist, isoproterenol and terbutaline, enhanced ijp, B-antagonist, propranolol, reduced it. These results suggested that a1i-receptor related to the generation of ejp and ai- and 82- receptors related to that of ijp. On the other hands , a-agonist, noradre- naline, decreased the frequency of mejp and B-agonist , terbutaline , accelerated the generation of mijp. These results suggest the possibility that a- and 8-receptors are present not only on the postjunctional membrane but also on the presynaptic memb- rane. PH 108 EFFECTS OF PICROTOXIN ON CA CURRENT AND TENSION IN ISOLATED FROG MUSCLE FIBRES. V. Jacquemond, K. Takeda! and 0. Rougier. Lab. Physiol. Elements Excitables, Univ. Claude Bernard, Villeurbanne, France. ‘Visitor from labs Physiol.) ihaceecluerre Tottori Univ., Tottori. Single twitch fibres were isolated from semitendinosus muscle of Rana esculenta and prepared for double-mannitol-gap voltage- clamp method (Ildefonse et al., 1985, Biochem. Biophys. Res. Commun. 129, 904). Test solution used contained 1.8 mM CaCl>2, 122 mM TEA-CH3S03H, 3 mM 4-aminopyridine, 10 mM HEPES-KOH and 5 mM glucose. It had normal Ca but most Cl was substituted, and most K channels were blocked in it. Holding potential (estimated to be ca. -90 mV) adjusted so as to have a contractile threshold at -50 mV was applied to maintain normal resting potential. 5 mM picrotoxin inhibited slow inward Ca current greatly (to below ca. 30% of control values in a fibre), but simultaneously recorded peak tension was increased (ca. 2.5 times in the fibre), provided the interval between applied depolarizing voltage steps (50 and 70 mV in amplitude) was kept long enough (2 minutes). These effects of picrotoxin on Ca current and tension were reversible and could be repeated in the same fibre. The results have shown that, in the experimental conditions used, picrotoxin potentiates contraction when it is inhibiting Ca current. Physiology 987 PH 109 MODULATORY ACTIONS OF THE CATCH-RELAXING PEPTIDE ON MOLLUSCAN MUSCLES. T.Hirata and Y.Muneoka. Physiol. Lab., Fac. Integrated Arts and Sci., Hiroshima Univ., Hiroshima. The catch-relaxing peptide (CARP: H-Ala- Met-—Pro-Met-Leu-Arg-Leu-NH.,) iS a novel neuropeptide isolated from™the pedal gang- lia of the mussel Mytilus edulis (Hirata et al., 1987). This peptide showed not only relaxing action on catch tension in the anterior byssus retractor muscle (ABRM) of the mussel at low concentrations (thre- shold, 0.3-1.0 nM) but also some other actions on various molluscan muscles. In the ABRM, CARP potentiated or inhibited phasic contraction in response to repeti- tive electrical pulses of stimulation. In general, the peptide showed potentiating action at lower concentrations (threshold, OeS-OeoenM)y) vand) inhibitory action lat higher concentrations (threshold, 1-50 nM). CARP also inhibited contractions of the ABRM in response to ACh and FMRFamide. In the radula protractor and retractor muscles of the prosobranch mollusc Rapana, cont- ractions in response to electrical pulses, ACh and glutamate were inhibited by CARP. Cardiac activities of Meretrix, Tapes and Mytilus were also inhibited by CARP. Thus, CARP seems to have modulatory actions on many molluscan muscles, and the actions seem to be brought about by direct effect of the peptide on muscle fibres. PH 110 PHARMACOLOGICAL PROPERTIES OF RELAXING RESPONSE OF MYTILUS SMOOTH MUSCLE TO THE CATCH-RELAXING PEPTIDE. T.Hirata,N.Fujimoto and Y.Muneoka. Physiol. habe bac integrated Arts “and: Scil., Hiroshima Univ., Hiroshima. Pharmacological properties of relaxation of catch in the ABRM of Mytilus in response to the catch-relaxing peptide (CARP) were examined and compared with those of relaxa- tion in response to 5-HT, dopamine and repetitive electrical pulses of stimulation. Butaclamol (107° M), a dopamine blocker,did not affect relaxing responses to CARP, 5-HT and Bee eLve electrical pulses. Mersalyl (5x107~* M), a 5-HT blocker, slightly depre- ssed but did not blocked relaxing response to CARP. Propranolol (107-3 M) depressed or blocked relaxing responses to 5-HT,dopamine and repetitive electrical pulses, whereas it enhanced that to CARP. Phenoxybenzamine (10-4 M) enhanced relaxation by CARP, 5-HT and repetitive electrical stimulation, but it did not affect relaxation by dopamine. Simple exposure of the ABRM to La3+ (5 mM) did not affect relaxing response to CARP, but, after the muscle had been exposed to Bo Fe La in the presence of a depolarizing agent, relaxing response to CARP was aboli- shed, as in the cases of 5-HT and dopamine. Cyclic AMP levels in the ABRMwere increased by 5-HT, but were not significantly affected by CARP. CARP seems to relax catch tension by acting directly on muscle fibres, though its presynaptic action could not be ruled out. Bee ete EFFECTS OF NEUROPEPTIDES ON THE HEART BEAT OF MOLLUSCS M.Kobayashi, K.Hori, T.Hirata and Y.Muneoka Phystol slab. aac. rom Integrated Arts and Sciences, Hiroshima Univ., Hiroshima Effects of several neuropeptides on the heart beat of a prosobranch Rapana thomasi- ana and a pulmonate Achatina fulica were studied. From the structure-activity rela- tions of Phe-Met-Arg-Phe-NH» (FMRFamide) related PeDenCes on the heart it was found that Arg3, Phe4 and the C-terminal amide were essential to FMRFamide-like effects both in Rapana and Achatina. In Rapana, FMRFamide enhanced the amplitude of beat in atrium and ventricle, while in Achatina it enhanced the beat of ventricle but reduced that of atrium. A novel neuro- peptide (Catch-relaxing peptide, CARP) extracted from Mytilus had potent inhibi- tory effects on the Rapana heart, with the 10 threshold of 3 xX 1019-107? mM. tt appeared to be acting postsynaptically. Benzoquino- nium blocked the inhibition of heart beat by acetylcholine and nerve stimulation but not blocked the inhibitory effects of CARP. CARP may be acting as a neurohormone to the Rapana heart. This peptide had no significant effects on the Achatina heart. PH 112 IN THE LOBSTER, PANULIRUS JAPONICUS; EFFECTS OF PERICARDIAL NEUROHORMONES. Ho IMUPKAMOCOo LMSW WilOll, SCioyg WmiVo Ot Tsukuba, Ibaraki 305. Hlectrical activity of the heart and blood pressure in the lateral pericardial Cavity were recorded during various behavioral states of the lobster. Each beat of the heart was followed by a fall in the pericardial pressure. Them pizcyslel: was regarded as one pumping action of the heart in vivo. Maomnguuidiel tos ast ne sical: altered significantly from state to state Ale RAMeeC EMOm Os2 wo To e/ cut (S Insole change in maximum). The amount of fall gradually increased after an increase in heart rate,’ and decreased with decrease of the heart rate. The fall was decreased by a transient inhibition of the heart beat roOlNWOniMe a AOOC! idol weenatO@ia 5 vyldst le feeding behavior caused it to increase up oO fei Giny oe Sisal dlaedyy ale VielS Ohio enhanced after vigorous body movements. Injection of pericardial hormones, octopamine or proctolin (10 pM), into the joSieaLCGkeCllA I CEI iny “CAUISSCl Wag wellbibs we) increase to over 1 g/cm. The increase had a slow time course similar to that observed in the feeding behavior. Injec- tion of serotonin (10 pM) or physiological solution had no significant effect on the ifg) IE IL. Thus, proctolin-and octopamine might increase the pumping efficiency of the lobster heart in vivo. 988 Pin) JUILS) CA-BINDING PROTEINS IN GUINEA PIG SMOOTH MUSCLES. N.Oishi! and H.Sugi?. 1RI Center and 2Dept. Physiol., Seh. Med., Teikyo Univ., Tokyo. In order to understand the regulation of Ca2t ion in excitation-contraction coupling, investigations of the activator Ca2+ that triggers cotraction are es- sential. By histochemical studies, the sub-sarcolemmal store of the activator Ca2+* that translocates to contractile machinery during contraction is demon- strated in vertebrate and invertebrate smooth muscles (Atsumi and Sugi, 1976; Sugi and Daimon, USN )) To search for the candidates for the Ca?+t-accumulator, we surveyed Ca2+-binding proteins (CaBPs) from plasma membranes of guinea pig smooth muscles, taenia coli and aorta. An plasma membrane-enriched fraction was processed for sodium dodecylsulfate-poly- acrylamide gel electrophoresis, and CaBPs were detected on the electroblot by 45Ca2+-autoradiography. CaBPs with Mr of about 58,000 and 100,000 were routine- ly detected. And that with about 37,000, 55,000 and 140,000 were occasionally observed. The 37,000 protein was the most abundant CaBP in homogenate, while the large part of it was recovered in the soluble fraction. Further identification is in progress. PH 114 PHYSIOLOGICAL AND MORPHOLOGICAL STUDY OF FISH SWIMBLADDER MUSCLE. Nor hanigueht eS. ssuzukawandehs SucieasDept Physiol., Sch. Med., Teikyo Univ., Tokyo. ~The swimbladder muscle of a crying fish, Sebastiscus marmoratus, was _ studied physiologically and morphologically. A single electric stimulus gave rise to an isometric twitch lasting 15 msec at 20° C. With repetitive stimuli, the tension showed a_ rapid spontaneous decrease to zero. High external potassium caused only a small contracture tension at 4° C but notvat. 202 Gs These facts suggest that the Tate sof JCa~ reuptake bythe | sarcoplasmic reticulum (SR) is very large. On the other hand, the muscle did not respond to a single electric stimulus at low temperature, suggesting that part of excitation-contraction coupling processes is inhibited at low temperature because the action potential and the Ca2+-activated tension in the skinned fibers are mot affected by temperature. Electron microscope observations revealed that, while the muscle was producing the maximal tension by caffeine, the SR was dilated and formed many small vesicles, and the electron-opaque pyroantimonate-Ca2+ complex was scattered on myofibrils, suggesting possible structural changes of the SR during the Ca2t+ release. Physiology Ppl LS) TWO MECHANICALLY DIS TIN Cit Siluyaee, Sa @)cam MUSCLE FIBERS IN THE TIBIALIS ANTERIOR OF THE FROG. H. Iwamoto and H. Sugi. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo In the tibialis anterior muscle of Rana japonica were found two types of muscle fibers which had distinct mechanical properties. The fibers of one type produced net work within a certain range of frequencies when they were oscillated sinusoidally and showed a large loop in the Nyquist plot, while the fibers of the other type did not produce work at any frequency and showed only a small loop in the plot. The effect of external pH on their mechanical performance was Studied because it has been reported to affect the intracellular “concentrationsaen inorganic phosphate, which is known to promote work production. Their mechanical properties were invariable in the pH range between 6.6 and 7.8, suggesting that they are not readily altered by external conditions. The two types of fibers were not found in a muscle at the same time. Since the work-producing type _ has hitherto been obtained in spring and summer while the other in the “rest “of Va yearn sthose differences may reflect seasonal modifications of the kinetics of the crossbridge action. PH 116 COMPARISON OF THE EFFECT OF OUABAGENIN AND BAY K 8644 ON SCATTERED LIGHT INTENSITY FLUCTUATION IN VENTRICULAR MUSCLE. T.Kobayashi! and D.Bose@. \Dept. of Phy- siol., Sch. of Med., Teikyo Univ., Tokyo and Ipept. of Pharmacol. and Ther., Fac. of Med., Univ. of Manitoba, Winnipeg. Light intensity fluctuation of coherent light scattered by a strip of ventricular muscle during diastole (SLIF) is believed to be due to asynchronous cellular motion within the myocyte as result of sponteneous release of calcium from sarcoplasmic reti- culum (SR). Ouabagenin produces’ inotropy by increasing intracellular free Ca, and in toxic concentration it produces abnormal aftercontractions (AC) by sponteneous Ca release from the SR. On the other hand the Ca channel agonist BAY k 8644 also produces inotropy but this is associated with a decrease in Ca release from SR, indicated by inhibition of ‘post-rest potentiation’. This study was performed to see if the effect of these agents on the power spectra of SLIF were different. Both frequency and amplitude of SLIF were increased after 1 uM ouabagenin but these changes were most Marked after onset of toxicity rather than during the positive inotropic response and were reversed by 2mM EGTA. In contrast, 1uM Bay k 8644 decreased SLIF at all level of inotropic response. These results suggest that SLIF is a better indicator of intra- cellular Ca overload and toxic oscillatory contraction rather than an increase in contractile state. Physiology 989 PH 117 EFFECTS OF THIOCYANATE ION ON THE CONTRACTILE AND MEMBRANE PROPERTIES OF RAT CARDIAC MUSCLE R. Sugaya, H. Iwamoto and H. Sugi. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo The effects of SCN- on the contractile and membrane properties of the rat papillary muscle were studied. Equimolar substitution of seeretor Clin’ Tyrode solution ‘caused a monotonic decrease in the resting potential. Meanwhile the amplitude of the = action potentials decreased with the resting potential but the force showed a transient enhancement followed by a gradual decrease. The deporalization caused by varying external Kt concentration could basically reproduce these changes in the action potential and the force. However, the amplitude of the action potential and the force were smaller in SCN--Tyrode. During the recovery period after the removal of SCN-, further suppression of the force was observed. These results suggest at least three actions of SCN-- on the cardiac muscle: 1) reduction of resting potential, by which force is enhanced, 2) suppression of the generation of action potentials, and 3) suppression of the contractile force, presumably due to. the incorporation of SCN- into the muscle cell. PH 118 LOCAL MOVEMENT OF INTACT SINGLE MUSCLE FIBERS OF THE FROG DURING INCREASE IN LOAD. T. Takei, T. Tsuchiya and H. Sugi, Dept. of Physiole, Sch. of liede, Teikyo Unive, Tokyo. Local movement of a muscle fiber is known to take place during the creep in isometric contraction and during ramp re- lease and its role in muscle contraction has long been discussede To know it fur- ther, local movement during step or ramp inrease in load in intact single muscle fibers of the frog was measured at low temperature (3-4 C). Carbon powders were attached at the several points on the surface of a fiber so that the total muscle length might be divided eaqually and the local movement was recorded using high speed video system (200 frames/ sec). Following step increase in load from isometric tension (Po) to 1.2-1.4Po, length of all segments increased equally. When the step increase in load, however, was large (1.6-1.8Po), a fiber tended to show "give" and only one of several segments was remarkably extended. In ramp increase in load to 1.3-1.4Po, the velocity of lenth- ening continued to increse and all segments were lengthened nearly equally. These results provide the evidence that large load induces localized elongation of a fiber. PH 119 COOPERATIVE INTERACTIONS OF MYOSIN HEADS WITH ACTIN FILAMENT STUDIED USING SLIDING MOVEMENT OF MYOSIN-COATED BEADS ALONG ACTIN CABLES. ait He .f BSUS se IS). @haede anid: 4h. eh ines “hone. Phivsmowls,) Sieh Medic. Meakyvom Una si soko and 2Dept. Biota (mac ssichtes seUmeivis wlolksy ov. Tokyo. To study the role of double-headed myo- sin molecule structure in muscle contrac- Guon ibhesmotitonvwon Latexsbeads coated aw bh partially p-phenylenedimaleimide (p-PDM)- inactivated rabbit skeletal muscle myosin along sthicevactin jeables vor vowan teaCGhara seesull was examined. One kind of myosin samples consisted of myosin molecules with two native or two inactivated heads, while the other kind of samples consisted of myosin molecules with zero, one or two inactivat- ed heads. For a given level of ATPase ac- tivity, the motion of beads was faster when they were coated with the former sample than when they were coated with the latter sample. If the velocity of bead motion was plotted against the fraction of myosin molecules with two native heads, all the data points fell around the same curve, suggesting that the bead motion is caused only by myosin molecules with two native heads. This implies that the two heads of each myosin molecule in muscle interact with actin filament in a cooperative man- ner to produce force and motion. PH 120 PROPERTIES OF A HIGH-MOLECULAR WEIGHT CAL- CIUM BINDING PROTEIN ISOLATED FROM PLASMA MEMBRANE-ENRICHED FRACTION OF A MOLLUSCAN SMOOTH MUSCLE T.Yamanobe’, T.Mimura’ and H.Sugi’. *Central Lab. Analyt. Biochem. and *Dept. Bhyswol; Sch. Med., Teakyo UWniv.; Tokyo. In the anterior byssal retractor muscle (ABRM) of Mytilus edulis, Ca ions are accu- mulated at the inner surface of the plasma membrane, and are released into the myoplasm to cause contraction (Atsumi and Sugi, 1976) To study the mechanism of Ca accumulation and release at the plasma membrane, we have isolated a 450K Ca-binding protein charac- teristic of the plasma membrane-enriched fraction of ABRM (Yamanobe, Mimura and Sugi, 1985). SDS polyacrylamide gel electrophore- sis indicates a decrease in the mobility of the 450K protein when Ca is removed, as with other Ca-binding proteins reported. The pro- tein exhibits distinct changes in fluores- cence and UV absorption by the change in pCa from 7 to 5. Ca binding assay shows that the protein has 7~9 Ca-binding sites with half- maximal binding at pCa 5.5. These results are consistent with the view that the 450K protein may be involved in the regulation of mechanical activity of the ABR M in physiological range of pCa. Experiments are progress to elucidate the physiological role of the 450K protein in the contraction- relaxation cycle of the ABRM. 990 Physiology, Cell Biology eg} WAL GENETIC ANALYSIS OF SYNAPTIC TRANSMISSION R. HOSONO Dept. Biochem. Sch. Med. Kanazawa Univ. Kanazawa. The cha-l-unc-l7 complex gene of C. elegans consists of at least two comple- mentation groups, cha-1l and unc-17. The cha-1 region encodes ChAT, but the function of the unc-17 region is unclear. We measured ChAT activity, and choline and ACh levels of the gene mutant alleles. We found that in the unc-17 mutants the ChAT activity and choline levels are normal but ACh levels are abnormally high. Experi- mental results suggested that the unc-1l7 region encode functions necessary for storage and/or release of ACh. To find related genes with unc-17, we further isolated resistants to trichlorfon. Six strains were allocated to unc-13(cn490), unc-17(cn355), tcf-1(cen252), unc-18(cn347), unc-10(cn257), and unc-3(cn4146). In addi- tion to the unc-17 region, mutations in unc-13, tcf-1, and unc-18 caused abnormal accumulation of ACh. These genes seem to be functionally related because they share common phenotypes, that is, uncoordinated movement, resistance to inhibitors to acetylcholine esterase, growth retardation and small body size in adulthood. These mutations were induced by insertion of transposable element Tcl, making it possi- ble to clone the gene with the element as probe. We found an unique Tcl-containing 6.8 kb Bgl fragment in TN347 strain (unc-18(cn347)) and are now trying to clone the DNA fragment. (C)s} al INTRA- AND INTERSPECIES COMPARISON OF HEAT-EXTRACTED PROTEINS IN Paramecium. Wo Tsukii, Lab. ALOIL G 7 Hosei Univ., Chiyoda-ku, Tokyo 102 cient = a el eee See Morphological species of Ciliates consists of number of sibling species called syngens. Though syngens show little morphological diversity, advances in com- parative studies, especially of isozyme patterns, gave a taxonomical basis for the identification of syngens of P. aurelia and Tetrahymena pyriformis. On the other hand, isozyme studies have so far failed to discriminate syngens of P. caudatum. So, I attempted to compare heat-extracted proteins among syngens of P. caudatum as well as those among other ciliate species. Moderately-starved cells were boiled for a few minutes and then centrifuged. The supernatants were compared by SDS-PAGE. Twenty to 30 protein bands were identified in each sample of 16 strains from four different syngens of P. caudatum. A few syngen specific bands were observed. Nearly all bands were, however, common among them, and, thus, it was unsuccessful to identify each syngen. Morphological species (P. caudatum, P. aurelia, P. jenningsi, P. multimicronucleatum, P. trichium, Tetrahynema thermophila, Bleharisma japonicum) were discriminated well by this method, and their evolutionary relationships based on the percentages of common bands between different species coincide with those already known. CB 2 DEGENERATION OF EPIDERMAL MELANOPHORES DURING METAMORPHOSIS IN THE FROG, HYLA ARBOREA M. Yasutomi. Lab. of Biol., Aichi Medical College, Nagakute, Aichi. Epidermal melanophores of the skin decrease in number or disappear during metamorphosis in many species of frog. This phenomenon is thought to be an adapta- tion for rapid color change (physiological color change), a property which the animals acquire after metamorphosis. We have reported that the decrease was due to the migration of them to the dermis in Rana japonica. In this frog, at metamorphic stage epidermal melanophores rather loosely associated with epidermal cells. The base- ment membrane became thinner and sometimes small breaks were seen in it. In Hyla arborea, epidermal melanophores disappear after metamorphosis. Through metamorphic climax the basement membrane was compact and epidermal melanophores were surrounded tightly by epidermal cells. Some of the epidermal melanophores included dense and round bodies which were probably lysosomes. Occasionally, the lysosome-like bodies contained melanosomes. Such cells were electron dense and the cytoplasm showed features of autolysis. From these facts, we concluded that the disappearance of epidermal melanophores during metamorphosis in H. arborea was not due to the migration of them to the dermis but to the degenera- tion in the epidermis. CBS Mvosin in normal and neoplastic fish pigment cells: Its occurrence and a mode of intracellular localization. Akivama , S. Hirai”~ and,J. Matsumoto Dept. Biol, Keio Univ., ~I{nst. Parsteur, France. The occurrence of myosin in swordtail erythrophores, goldfish melanophores and erythrophoroma (GEM 81) cells was demonstrated by means of polyacrylamide gel electrophoresis (PAGE) using sodium dodecylsulfate (SDS) and sodium pyro- phosphate (Nappi ) and immunoblot using the antibody against chick gizzard myosin Cheavy chain). The band appearing at the position of approximately 200 kd in SDS-PAGE was equivocally decorated with the antibody and Nappi electro- phoresis had separated out native myosin free from any degradation. Myosin was also detectable in the two melanogenic variants induced from GEM 81, which were capable or not of pigment motile response. Immunofluorescence disclosed that myosin occurs at least in two forms in fish pigment cells; 1) a loose meshwork connecting dot-shaped aggregates, 2) stress fiber type-filaments. The former was prominent in normal Pigment cells and heavily melanized GEM cells’ with motile response whereas the latter was so in non- or less-7 melanized, poorly responsive GEM cells. This would suggest involvement of myosin in pigment translocation. Cell Biology 991 CB 4 IMMUNOCYTOCHEMICAL LOCALIZATION OF LAMININ IN THE ANTERIOR PITUITARY OF MALE RATS AT PUBERTY. S.Kusaka,S.Yamashita,S.Yamaguchi ,S.Kusunoki Life Science,Lab.,Advance CO.,LTD. ,Tokyo Laminin, one of the major components of basement membrane(BM), was detected immuno- cytochemically in various tissues or organs. Recently, Tougard et al reported that laminin was localized in the BMs and inside grandular cells, especially gonadotrophs, of the rat anterior pituitary. We have immunochemically demonstrated the localiza- tion of laminin in the BMs and LH cells of 10,20,30,40,50,60 and 90 day old rat pitu- itaries. After decapitation, pituitary glands were removed and fixed in a formol- sublimate(3:7) solution at 4C and embedded in Paraplast. 3um thick sections were then prepared and immersed in 0.4% pepsin in 0.01N HCl at 37C for 60 minutes. Sections were processed by the immunoperoxidase method. Laminin was localized in the BM of the capillaries in all days’ pituitary. In the 30 day old pituitary, there was a slight detection of laminin in some LH cells, the number of which increased with Hesse oF the pituitaries, but decreased in the 90 day old. In addetion, in the over 50 day old pituitary, laminin was detected in the BM of the lobule-like structure. these LTesultis suggested : 1) LH cells produced the laminin, possibly reflecting sexual maturation in rat, and 2) the BM may have formed at a particular stage in the growth of functional development of the pituitary gland. CB 5 USE OF NUCLEOTIDES IN ENZYME-IMMUNO-HISTO IN SITU HYBRIDIZATION. T.Koji, M.Tanno, T.Moriuchi and P.K.Nakane Dept. of Cell Biol., Tokai Univ. Sch. Med. Sete DieeIpeICAM AD AW Ais To understand better the physiology of cells and tissues, one requires studies on the states of nucleic acids and protein metabolism at the cell level. To localize specific mRNA, we developed a new method using T-T dimerized haptenic cDNA probe. Subsequent to hybridization with cell mRNAS, the T-T cDNA was localized immuno- histochemically. In this study while usin the T-T cDNA, our efforts were focused on to eliminate the deteriorative effects of formamide (e.g. instability, nonspecific reaction, morphological damage), the reagent usually used to decrease the melting points of double stranded nucleic acids. As an alternative to the formamide we examined the use of a mixture (Nm) of nucleotides (AMP, GMP, UMP, CMP) to reduce the melting point. The effects of Nm on re-annealing of single-stranded PBR 322 were investigated by agarose gel-electro- Phoresis. It was found that Nm (20-200 mg/ml) reduced the rate of the reannealinc On dot blot hybridization using Nm, we obtained Similar sensitive and specific results to that with formamide. On frozer sections of rat pituitary glands fixed by perfusion of 4% paraformaldehyde/PBS, Prolactin mRNA may be localized using Nm instead of formamide and the tissues were better preserved when Nm was used than whe formamide was used. CB 6 TRANSFORMATION OF MOUSE C3H 10T1/2 CELLS BY LOW-DOSE-RATE 7 - AND @ -IRRADIATION T. Yawagucha: BrGlainists ss Fac. Gene Hane. Ehime Univ., Matsuyama. Cultured C3H 10T1/2 cells in confluency were irradiated with either B-rays from tritiated water ((HT0) or Sco 7 rays. The duration of exposure was 20 h at’ either ASC Or 3.7 “Ca tand =the: total ‘dosie was’ varied by changing the dose-rate. The absorbed dose from HTO was calculated using a water content value of 0. 84. The value of 0. 84 water was determined in a mass culture of the cells by a method in which [carboxyl- 14C}-inulin was used to measure the amount of extracellular water. The dose-survival and dose-transformation curves for 7 -ir- radiaion at 4°C were nearly the same as thosie®atter a single acute Xray dose: When y -irradiation was administered at SyiaaGe both the lethality and transforma- tion-induction were lower than those after the corresponding doses at 4°C, indicating the existence of repair from damage during iMnadinat von tate 3.74 C: The same effect of temperature was observed in the case of HTO-exposure. The effect per Gy, however was hieherm tor GB Srays than fore jan ayse in both the responses. Resulting relative biological effectctiveness (RBE) values of Gein ASB pay s-= Were ser 4dsrate Dina hore ceel kusllaiinigs and 6y lve for eel ly cE ansmor ma. tion within the dose range examined (1 to 6 Gy of B-rays), irrespective of the tem- perature during irradiation. CBs 7 A CYTOPLASMIC FACTOR WHICH CONTROLS THE SEROTYPE EXPRESSION IN SEROTYPE MUTANT STRAIN OF PARAMECIUM TETRAURELIA. S.Koizumi and S.Kobayashi. Dept. of Biol., Miyagi Coll. of Educ., Sendai. Mutant strain d48 in Paramecium tetr- aurelia cannot produce immobilization anti- gen A. Gene A encoding i-antigen A is pre- sent in micronucleus, but not in macro- nucleus because of lacking during macro- nuclear reorganization. To clarify the cell cycle stage at which gives rise to the lack of gene A, wild 51 macronucleoplasm from cells at about 10th cell cycle was trans- planted into various stages of d48 cells: Recipient cells of d48 received 51 nucleo- plasm by 2-3hr after the sensitive period were possible to express A in high frequen- cies (50-70%). Exconjugant clones of d48 derived from crosses with 51 frequently ex- pressed serotype A when pair separation was delayed. The results exhibit that some pro- tective substance for lacking gene A is also present in cytoplasm of wild type cells. To obtain the direct evidence for such a substance in cytoplasm, transfer of cytoplasm at various stages of 51 cells into d48 cells 1-2hr after the sensitive period were carried out: Transfer of cyto- plasm during macronuclear reorganization caused the frequent expression of serotype A in recipient cell lines, but other stages of cytoplasm did not. Transplantation of -nucleoplasm throughout the vegetative stage was effective to express serotype A in the recipient cells of d48. 992 Cell Biology CB 8 PREVENTION OF RADIATION-INDUCED INTERPHASE DEATH BY RNA- AND PROTEIN-SYNTHESIS INHIBITORS T.Yamada!, H.Ohyama* and K.Inui*. Divs. 1Biol. & *Radiat.Health, National Institute of Radiological Sciences, Chiba. Mammalian thymocytes are highly radiosensitive and show "interphase death" within a few hours after low doses of irradiation. However, the mechanisms of this type of death remain ill-defined. We have demonstrated that rat thymocyte interphase death involves a discrete, abrupt transition from the normal _ state, such as cell volume reduction to a definite small size, and is not merely the consequence of progressive and degenerative changes, and is an example of "apoptosis", the mode of death frequently observed when cell deletion is programmed. In the present study, the effect of inhibitors of RNA- and protein-biosynthesis on interphase death as an expression of the death program was examined using thymocytes prepared from Wistar strain rats. Thymocyte suspension was incubated for 4 hours immediately after X irradiation, and dead cells were counted at the end of the incubation. Inhibitors such as cycloheximide, puromycin, chromomycin and actinomycin D given to the suspension suppressed completely appearance of the stained (dead) cells, and transition to small-sized cells. These results provide further support 1E@36 the view that interphase death is apoptosis. Chess A STUDY ON TRIPLOID HYBRID CELLS IN RHODEINE CYPRINID FISHES. K.Yamamoto and Y.Ojima. Dept. of Biol., Fac. of Sci., Kwansei Gakuin Univ., Nishinomiya. Two kinds of triploid hybrids by reciprocal cross between Rhodeus ocellatus ocellatus and Acheilognathus limbata (RA3n:RZ@ X Aw; AR3n: Ro X AP ) were ob- tained by cold shock treatment of feritl- ized eggs. A whole of the triploid hybrid fingerling was treated by trypsin solution and cells obtained were cultured. The cells after 5 times of subcultivation were used. Both of the triploid hybrid cells had 72 chromosomes consisting of diploid sets of the female and a haploid set of the male. Ag-NORs were located on the 3 acrocentric chromosomes. The multiplication rate of RA3n was relatively high in comparison with that of cultured cells of R.o.0o. carrying two NORs. The directly prepared or the primary cul- tured cells had two active NORs, despite of triploidy. However, the cultured cells in this study had three active NORs. There Might be a certain correlation between the high multiplication rate of RA3n and reac- tivation of a NORs. Furthermore decrease of the chromosome number and increase of Ag-NORS were ob- served in RA3n after further subculture. CB 10 THE ORIGIN OF THE NATIONAL MONUMENT, "SCARLET CRUCIAN CARP" (HIBUNA) IN THE HARUTORI LAKE, KUSHIRO, HOKKAIDO, JAPAN. Y.Ojma. Dept. of Biology, Faculty of Sci. Kwansei Gakuin Univ. Nishinomiya. " Scarlet Crucian Carp " (Hibuna) in the Harutori Lake of Kushiro, Hokkaido was designated a Natural Monument of Japan in 1937. Since then, however, no one had the opportunity to closely ex- amine the origin of this special Hibuna because of its protected status. The present study was undertaken in an aim to deal cytogenetically with the origin of the Hibuna, in comparison with the Japanese genus Carassius and the Gold-fish. The results clearly showed that there are two kinds of Hibuna, diploidy and triploidy, in the Harutori Lake. The diploid Hibuna, 2n=100, showed the same chromosome constitution and C-band pat- tern as the Gold-fish. The individuals with triploid chromosomes, 2n=154, were the same as that of the C. a. langsdor- fii in chromosome constitutions and C- band patterns. 3) Ju MICROINJECTION METHOD AND ITS APPLICATION IN TETRAHYMENA , H. Ohba and Y. Watanabe, Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki Microinjection of cytoplasm, antibody, protein or DNA into cells has been proved to be very useful method for assaying the in vivo activity of introduced substances. But, this method was not easily practiced in Tetrahymena mnainly because of stiff layers of the cell membranes. Here we have succeeded in injection using a glass micropipette of keen tip ( less than 0.5 um in diameter ) at the angle of 65 degree and using the individual cells immobilized in proteose-peptone medium containing 2.5% methyl cellulose. Maximal injection volume was about 20 % of cell volume ( 3 nl ). We used this method to inject a guinea pig antiserum against p85 which was the CjSine joycoclwieie Oi CCleyrl ( CSilil CGivisiom arrest mutant ) into wild-type cells. p85 has been proved to be localized in basal bodies at cell equator just before cell Ca WSO in incl alin Oral Ajjjaratws (( Ta Biochem. 100, 797, '86 ), suggesting the involvement of p85 in determination of cell division plane and in food uptake. By the antiserum injection, inhibition of cell division occurred probably due to ceasing of food vacuole formation, whereas no cdaA1 phenocopy was exhibited. We are now testing the effects of other antisera against p85 to demonstrate the involvement of p85 in division plane determination. Cell Biology 993 CB a2 EFFECTS OF MACRONUCLEAR ELIMINATION ON GERMINAL MICRONUCLEUS DURING CONJUGATION OF PARAMECIUM. K. Mikami, Res. Inst.for Sci. Educ., Miyagi UniweOmwHGuUC.), cendal. During conjugation, the micronucleus (MIC) produces gametic nuclei after meiosis, Then the nuclei exchange reciprocally and fertilize. To probe function of the macro- nucleus (MAC) on the micronuclear behavior, MAC was pipetted out by glass needle at various stages of conjugation in Paramecium caudatum. MIC shows crescent shape at meiotic prophase. When MAC was removed at 2-2.5 hours of conjugation, however, MIC skipped over typical crescent stage and then divided once or twice. Results showed that MAC gave information for"crescent"just prior to the crescent stage and information for nuclear division just prior to dividing stage of meiosis. It is likely that macro- nuclear gene products required for micro- nuclear division transfer easily through boundary of mating cells. After meiosis, one nucleus remains and other 3 degenerate at about 12 hours of conjugation. The results suggest that the information for it is given 4 to 6 hours (crescent stage) of conjugation. When MAC was removed before 2nd meiotic division, MIC did not enter into the partner. When both of MIC and MAC were removed from a cell at 6 hours, gametic MIC of the partner migrated to the enucleated cells. The results suggests that MAC about at the 2nd meiotic stage is indispensable for micronuclear migration. CB} ells MORPHOGENESIS OF MIRROR-IMAGE SYMMETRY DOUBLET CELLS IN THE CILIATE STYLONYCHIA PUSTULATA. Io MAMO> ZOOdILs MWASiEop WAG. Ot SGilo, Hiroshima Univ., Hiroshima. Mirror-image symmetry doublets were obtained when dividing cells were exposed to heat shocks. Two ventral surfaces of doublets were located on the same side. The adoral zone of membranelles (AZM) and the cirri in the left component were arranged in the same pattern as the normal singlets, except for the lack of the right marginal cirri. In the right component, the ciliary organelles were mirror-symmetrically disposed as compared to those in the left component. However, the right component had no cytostome, and the membranelles of its AZM were antero- POSterntorly = sotated 1804. In cell divi- sion of doublets, the oral primordium appeared just posterior to the AZM in each components. The sites of primordia of the other ciliary organelles were arranged in mirror-image symmetry. In almost all the right components, the membranelles of the new AZM were inverted. The normal and inverted mem- branelles rarely developed from the oral Pprimordium of the right component. Therefore, the pattern formation of ciliary organelles in the right component seems to be undergone by the reversal of the right-left axis. CB 14 SOME CHARACTERS OF ENCYSTMENT-INDUCING SUB- STANCES OF THE CILIATE EUPLOTES ENCYSTICUS. Ee VOMEeIZiaWidia OO. lnc Er acl mtorr Sisal, 6 p Hiroshima Univ., Hiroshima. It has been reported that cell-free fluid (CFF) contains some substances which are responsible for encystment of E. encys- ticus (Yonezawa, 1986). The purpose of this work is to partially characterize the substances. 20 liters of CFF was concen- trated at a reduced pressure to about 50 ml DYNdmrOtary evaporatorlat #400 nc. After centrifuging concentrated CFF (conc-CFF) at 3000rpm for 20 minutes, the supernatant was dialyzed against redistilled water. Encystment-inducing activity was detected in dialyzed conc-CFF but not in external fluid. Heating to 100 © did not apprecia- bly affect inducing activity within the first one hour of exposition, whereas after three hours the activity dropped to about a half of the original. Incubation of dia- lyzed conc-CFF with lipase (200 wg/ml) or o-glucosidase (25 wg/ml) for 24 hours at 30 C did not lead to a complete inactiva- tion of the substances. On the contrary, activity was completely destroyed after 24- hour-treatment with trypsin (200 ug/ml) at 30 TC. After centrifuging dialyzed conc- CFF at 10000g for 30 minutes, the superna- tant was chromatographed with Sephadex G-75 column. The peak of elution pattern (OD at 28:0 nm) appeared in the fraction 62, whereas the peak of activity was detected iin INS iiceCieseia. 7/2. CB 15 CYTOSKELETON OF HELIOZOAN AXOPODIA IN ECHINOSPHAERIUM AKAMAE. S.Ikegawal, H.Ishida!, Y.Shigenaka! and E.Masuyama’. !Lab. Cell Biol.. Fac. In- tegr. Arts & Sci., Hiroshima Univ. and 2Dept. Life Sci., Hiroshima Women's Univ.. Hiroshima. Heliozoan axopodia of EcChinosphaerium akamae radiate from the spherical cell body. The core of each axopodia is formed of the regular arrangement of many microtubules as main cytoskeletal ele- ments. By treatment with 65% Do0, we found that the axopodia could be isolated com- Pletely from cell body. Electron micros- copy revealed that the isolated axopodium maintained the regular arrangement of microtubules. We subjected the isolated axopodia and the cell body extracts to SDS-PAGE. The result showed that two remarkable bands appeared in isolated axopodia extract, corresponding to tubulin on account of the electron microscopical results. To identify the proteins of these two bands further, we subjected the heliozoan extract to Western blot, and analyzed by using antibody against tubulin of rat brain (ADVANCE Co., Ltd.). This an- tibody reacted with only one band (Mr=46000). Althogh another band (Mr=50000) was not recognized, it behaved together with the low band during the process of isolation axopodia. These results strongly suggest that the proteins of two bands must be tubulin of axopodia of Echinosphaerium akamae. 994 Cell Biology CB 16 MECHANISM OF AXOPODIAL RETRACTION AND CON- TRACTION IN HELIOZOA. Y. Shigenaka, S. Ikegawa, H. Ishida and H. Terada. Lab. Cell Biol., Fac. Integr. Arts & Sci., Hiroshima Univ., Hiroshima Against various types of stimuli, helio- zOoan axopodia react by demonstrating slow retraction and/or instantaneous contraction. In general, the former retraction occurs when certain chemical drugs or big prey or- ganisms are given to the cell. On the other hand, the latter contraction might be indu- ced as small-sized protozoans are attached to the distal portions of axopodia. In this case, the contractile tubules are consider- ed to induce the contraction by self-twist- ing and supercoiling of them. At this time, the axonemal microtubules, which are conta- ined as cytoskeletal elements in the axopo- dium, depolymerize simultaneously at the final step. We also examined the localizat- ion of calcium during axopodial contraction using a potassium pyroantimonate assay. Ca- Sb deposits were detected on the contract- ile tubules only during twisting and coil- ing of this organelle, and on the axonemal microtubules during their disassembly. Our results indicate that axopodial contraction is enforced by twisting and coiling of con- tractile tubules and by folding of axonemal microtubules, which might be mediated by influx of Cat through axopodial membrane and efflux of Ca2+ stored in vesicles. (3 ULTRASTRUCTURAL CHANGES IN THE PARORAL REGION DURING CONJUGATION BETWEEN DOUBLET CELLS AND SINGLET CELLS IN PARAMECIUM TETRAURELIA Y.Yashima. Dept. of Biol., Sch. of Lib. Arts and Sci., Iwate Med. Univ., Morioka. During nuclear migration, one of the meiotic nuclei lies in the paroral region and survives, but the remaining seven nuclei lie outside the paroral region and degenerate. To better understand the morphological changes associated with conjugation, conjugation was induced between singlet cell and doublet cell, and was observed by TEM. According to TEM observation, 2-3 hours after the beginning of conjugation, the paroral region was unchanged with the exception of the contact region. In particular, there were no microtubules in the paroral region at this time. When a micronucleus moved into the paroral region, microtubules were observed around the nucleus, and as the micronuclei in the paroral regions moved in the direction of the contact membrane, the microtubules tended to increase around these nuclei Just before the beginning of the nuclear exchange, a microtubule meshwork had formed around the micronuclei in the paroral regions, but there were almost no microtubules around the micronuclei outside these regions. There were some electron dense granules (0. 08- 0.1242m in diameter) around these extraparoral nuclei, but no electron dense granules were present around the intraparoral micronuclei. These results suggest that 1) the microtubules are related to the nuclear movement in the paroral region and 2) electron dense granules may be related to nuclear degeneration CB 18 ISOLATION AND CHARACTERIZATION OF REPRO- DUCTIVE FORM OF HOLOSPOLA OBTUSA, A MACRO- NUCLEUS-SPECIFIC BACTERIUM OF PARAMECIUM CAUDATUM. Y.KOJIMA and M.FUJISHIMA. Dept. of Biology, Fac. Of Sci, Univ. of Yamaguchi, Yamagueha The gram-negative bacterium Holospola obtusa is a macronucleus-specific symbiont of Paramecium caudatum. This bacterium shows molphologically distinct five types in its life cycle: an infectious long form of buoyant density 1.16 g/ml, three inter- mediate forms of buoyant densities 1.13 g/ml, 1.11 g/ml and 1.09 g/ml, and a repro- ductive short form. The former four types have been isolated by Percoll density gra- dient centrifugations. But the reproductive form is not yet isolated by the same meth- od, because the bacterium is easily di- gested by host lysosomal enzymes in the homogenate. We intend to extract and char- acterize holospolas DNA, but the long forms were hardly lysed by ordinary methods for DNA extraction. Therefore, an isolation method of the reproductive form is needed to establish. In this study, we succeeded to isolate the reproductive short forms. Cells borne the reproductive forms were mildly lysed in NP-40 containing medium and their macronuclei were isolated. Then, the nuclei were homogenized, centrifuged in Percoll, and reproductive forms in a band of the tube were harvested. The isolated bacterium was found to be easily lysed in a lysozyme, SDS-containing medium. CBaus CHANGES OF PROTEIN COMPOSITION IN THE LIFE CYCLE OF HOLOSPORA OBTUSA, A MACRONUCLEAR SPECIFIC SYMBIONT OF PARAMECIUM CAUDATUM. M. Fujishima and Y. Kojima. Biol. Inst. Fac. of Sci., Yamaguchi Univ., Yamaguchi. The gram-negative bacterium H. obtusa invades into the host cytoplasm via the food vacuole, infects its macronucleus ex- clusively and grows in the nucleus. The bacterium can be isolated from host homoge- nates by Percoll density gradient centrifu- gations. We have ever clarified that the bacterium changes its length, morphology of nucleoid and buoyant density in the life cycle: four long forms of which longitudi- nal lengths are 15 ~m and buoyant densities are 1.16, 1.13, 1.11 and 1.09 g/ml anda short form of which length and buoyant density are 1.5 ~m and 1.09 g/ml. During early stage of the infection process, the long form of 1.16 g/ml decreases its buoy- ant density to 1.09 g/ml. Then, each long form divides to become about 10 short forms at the third day after the infection. When the host grows, this short form also grows by binary fissions. But, when the host starves the short form stops to grow and becomes long forms. In the present study, we compared protein compositions among the bacteria of buoyant densities of 1.16, 1.11 and 1.09 g/ml by 2D-PAGE. It has been found that the composition is similar be- tween those of 1.16 and 1.11 g/ml, but that of 1.09 g/ml is very different from others. Cell Biology 995 CB 20 EFFECT OF GLUCOSE ON FOOD VACUOLE FORMA- TION IN PARAMECIUM MULTIMICRONUCLEATUM. S.Mishima!, Y.Harada2. IBiol. Lab., Coll. Gen. Educ., Ibaraki Univ., Mito and 2Dept. GESETOph YS. HNGnia, Hace OL Engh.) SCis), Osaka Unv.,Toyonaka. Paramecium multimicronucleatum usually ingests bacteria, which are tightly packed into food vacuoles. However, we found that when glucose was added to the culture medium in concentrations of 20 mM or more, paramecia did not form tightly packed vacuoles but formed loosely packed ones. In the glucose solution, paramecia made food vacuoles. The volume of each vacuole in the acid stage was measured in three media: (1) bacteria (2) glucose (3) bac- teria and glucose. When compared, the volume in medium (3) was the sum of (1) and (2). Furthermore, though it is known that starved paramecia first make a huge vacuole after the addition of large amounts of bacteria, in medium (3) starved paramecia still formed large initial vacuoles even at lower bacteria concentra- tions. These results suggest that glucose as well as bacteria may be recognized as food or nutrient by paramecia. Other sugars, such as ribose and sucrose, had similar effects, but the other important soluble nutrients, such as amino acids, had no effect on food vacuole formation. CBe21 EXPRESSION OF JUMYO GENE OF PARAMECIUM TETRAURELIA IN DIFFERENT CULTURE CONDI- TIONS. Y. TAKAGI. Dept. of Biol., Nara Women's Univ., Nara. Jumyo (jum) gene of Paramecium tetrau- relia is thought to be a pleiotropic gene ConeEollminge = Shore “clonal latespan (Sh) and slow division (S) (Y.Takagi & K.Izumi, 1987, Biomed. Gerontol.11:115-116.). Under condition of the daily isolation culture, the jum/jum clones usually show lifespans shorter than 20 fissions and have fission rates slower than 1.5 fissions a day. Al- though an intraclonal variation of life- spans was commonly observed as shown by some lines with lifespans longer than 50 fissions while some other lines with very short lifespans, the longer lifespans were not due to the phenotypic reversion of Sh but instead due to the two or more life cycles linked by missing autogamy. Thus, the jum gene shows a complete penetrance with a stable expressivity well distin- guishable from long lifespan (L) and fast division (F) of the wild type. Under con- dition of the mass culture, however, the phenotypic reversion from S to F was real- ly the case. Mutants (d4-SL4), wild types and their F, segregants of S, all showed the F phenotype of 3-4 fissions a day dur- ing the logarithmic phase of growth in 200 ml flask cultures. The reversing factor(s) appears to be associated with cell density yet remains completely unknown. CBaz22Z PHAGOCYTIC RESPONSE TOWARD FATTY ACIDS IN AMOEBA PROTEUS A.Kihara,K.Ishii Lab.Biol.,Hosei Univ., Tokyo Amoeba could be extended its pseudopodium around a oil drop of some water-soluble fatty acid (e.g. nonanoic acid), and food- cup did not contact directly with it. This response was elicited from any surface of advancing amoeba. Amoeba which immersed in nonanoic acid solution altered prominently its shape depending on the concentration. When the concentration was lower than 100nM, amoeba moved normally. At the concentration of 100nM or 150nM, amoeba first extended many pseudopodia vigorously all around then became the flatly spread shape, however the pseudopodia were getting scanty and were devoid of amoeboid movement in the solution beyond 150nM. According to these results, it seems that the pseudopodium formation is accelerated by the fatty acid at the moderate concentration, though it is inhibited at the higher concentration. It was established that the larger food- cup was produced toward the bigger oil drop. Some stable concentration gradation -caused by diffusion may be produced in the medium around the fatty acid drop, and these gradation, the food-cup formation seems to depend on, should be expanded as the in- crease of diameter of the oil drops. This feeding mechanism may make it possible that amoeba adjust the food-cup width to the various size of edible materials lacking of direct contact. OBZ REGULATION OF THE INTRACELLULAR CALCIUM STORAGE IN AN AMOEBA, COCHLIOPODIUM SP. I. Yamaoka, S. Yamashita and R. Murakami. BILOl, WMSto, Paes Silo, VAWAGguela Winily. , Yamaguchi. Amoeba Cochliopodium sp. has an ability to store of calcium in the body. Calcium are stored in spherical granules (about 0.3 Am in diameter) which are enveloped by a limiting membrane and situated at the peri-nuclear region. Phosphorus also are detected in the granules by the X-ray microanalysis. A factor to regulate the calcium storage in the granule was examined. Alkaline phosphatase activities were detected histochemically in the inside of the membrane of the granules. When the amoeba was cultured on the agar containing 2.0 mM EGTA the granules disappeared within 6 hours of culture. When the amoeba treated with EGTA was transferred to normal medium, these granules reappeared, restoration of the granules was inhibited by 2.5mM levamisol, a specific inhibitor of alkaline phosphatase. These results suggest that alkaline phosphatase is involved in calcium storage in the granules and that by using of inhibitor of the enzyme activity the calcium storage can be inhibited. 996 Cell Biology CB 24 COMPARISON OF ULTRASTRUCTURES AMONG RESTING CYSTS OF SEVERAL HYPOTRICH CILIATES. T. Matsusaka. Dept. of Biol., Fac. of Sci., Kumamoto Univ., Kumamoto. Among hypotrich ciliates, the resting cysts of the family Oxytrichidae, so far reported, have common ultrastructures; e.g. 3-layered cyst wall, electron dense cyto- plasm, and total absence of ciliary organ- elles. These features seem to be a useful marker to consider hypotrich phylogeny. The cysts of 4 Oxytrichid ciliates exam- ined demonstrated typical ultrastructures described above and their endocysts reacted positively to anti-CW190 (an endocyst pro- tein isolated from Histriculus cavicola) as detected by immunoelectron microscopy, al- though the intensity of reaction was varia- ble among species. In Holosticha sp., its cyst showed similar ultrastructures and reaction to anti-CW190 to those of Oxy- trichidae. The cysts of Holosticha adami and Pseudourostyla levis had 2-layered cyst wall, cytoplasm of moderate electron dens- ity, and basal bodies but not ciliary shafts, and was negative to anti-CW190. In Euplotes encysticus, the cyst had 4-layered cyst wall, cytoplasm of moderate electron density, and intact ciliary organelles, and was negative to anti-CW190. These findings may suggest that Oxytrichidae may be a fairly homogeneous group, that Urostylidae may be a relative to Oxytrichidae, and that Euplotidae may be a quite different group from the other 2 families. (3 25 CORTICAL MORPHOGENESIS DURING BINARY FISSION IN SEVERAL HYPOTRICH CILIATES: A COMPALATIVE STUDY. T. Matsusaka, O. Noguchi, N. Shimizu. DES5 WaOil~, WAG Silo, KKMMENOIES Wins , Kumamoto. In many hypotrich ciliates, cortical morphogenesis during binary fission have been reported, but only a few papers have compared it among different families. In the present study, cortical morphogenesis were compared on protargol impregnated specimens of 5 different hypotrich families including 11 species (Oxytrichidae, 4 spe- cies; Urostylidae, 4 species; Pseudourosty- lidae, 1 species; Strongylidiidae, 1 spe- cies; Euplotidae, 1 species). The present observations and earlier pa- pers clearly distinguished Euplotidae from the other 4 families by its subsurface sto- matogenesis and different cirral migratory pattern. The other 4 families might have common ancestor, Since their stomatogenesis occur at cell surface and cirral migratory pattern is fundamentally the same. Among these 4 families, Oxytrichidae may be quite homogeneous group, because they showed es- sentially the same patterns of stomatogene- sis, cirral migration and dorsal bristle formation. On the contrary, Urostylidae may be divided into several groups, since they showed variations in stomatogenetic and dorsal bristle formation. CB 26 AGE-RELATED CHANGES IN THE RELEASE OF SEROTONIN FROM RAT PLATELETS Y. Yonezawa, H. Kondo and T. A. Nomaguchi Dept. of Biol., Tokyo Metropolitan Inst. of Gerontol., Itabashiku, Tokyo. Platelets contain various coagulation factors, cell growth regulators, serotonin (a vasoconstriction factor), and others. In order to determine whether platelet functions change with aging, we studied serotonin release from Wistar rat platelets with thrombin. Serotonin contents in young male(7M) and female(6-7M) eal platelets were) 17/40 ng) and s660\ ng /3x 10> pita telkerasr respectively. In males, those increased at middle age(12-14M) and significantly decreased at old age(24-25M). The same changes were obtained when those in serum were measured. In females, serotonin contents in both platelets and serum did not change during aging. No serotonin was detected in plasma of all rats measured. Serotonin release was induced from rat platelets with 1.0-2.0 U/ml thrombin and reached a maximum with 4.0 U/ml. In males, the amount of this release was lower at miciditesvage tit hiantmarte myoumnamiccier but increased at old age to much higher level than at younger ages. In females, the release from platelts of young and middle rats was greater about 1.5-2 times than that from male rats, but decreased markedly at old age(25-26M). These results conclude that serotonin contents in platelets and its release from platelets of Wistar rats with thrombin greatly changed during aging. CBez/ ANALYSES OF (CELL SUREACE ANG ERNES IN INTERDIGITATING CELLS OF MICE USING MONOCLONAL ANTIBODY. S.Tanaka , H.Uda!, T.Maruyama-. lDep. of Pathology, Kagawa Med. School, Kagawa, Shionogi Lab., Toyonaka, Osaka. Recently it has become known that the skin is an immunological organ: Langerhans' cells (Lc) play a major role of immunological response in the skin. After sensitization by antigens, Le are assumed to migrate to draining lymph nodes Encl abimieSiejyicoGie elle Ainiewdcgein icoQ lymphocytes. But there is no obvious evidence. We prepared an antibody for interdigitating cells (IDC) of mice. The obtained antibody recognized a cell surface antigen which is a glycoprotein of about 15K daltons. According to study by Laigistie Amc GQleccwoOm mMmilCGicosCoOpie immunohistochemistry, this antibody reacts to Lec, IDC, marginal zone histiocytes (MZH) and some undetermined histiocytes. However at reacts with almost no LEI liewibase Clemcietietie Ceils (O?DC)) , lymphocytes and fibroblasts. Because our antibody attached to IDC as readily as to Lc and MZH, then we can assume that there is the same antigen on IDC, Le and MZH, and that this antigen is different from those on FDC, lymphocytes and fibroblasts to which the antibody would not attach. The fact that this antibody reacted some undetermined histiocytes will require further study and clarification. Cell Biology 997 CB 28 FUNCTIONAL MORPHOLOGY OF THE AGRANULAR CELL OF THE SALIVARY GLAND IN THE CATTLE TICK HAEMAPHYSALIS LONGICORNIS, WITH SPECIAL EM- PHASIS ON THE DIFFERENCE BETWEEN BOTH SEXES, Hae venacawa, S. Shiraishi -and fT. A. Uchida. Agee. 4 bac. of Agr., KyushusAlnive; Fukuoka. Elimination of the excess water and chloride during condensation of blood con- ponents is a major physioldgical problem for blood-sucking ticks. In the cattle tick Hee loupreornis, the. excess water and ‘ellec= trolytes are returned to the host by means of salivary secretion. In female ticks, the agranular cells of Type III acini change conspicuously their volume and structure during feeding; especially, the e-cell and the abluminal interstitial cell develop greatly at the late stage of feeding, end- ing up with an elaborate system of membra- neous infoldings containing numerous free ribosomes and elongated mitochondria, which is a common feature of fluid-transporting epithelia. Thus, these cells may contribute to maintaining osmotic and ionic balance. On the other hand, in male ticks which need not to suck a large quantity of blood, the e-cell and the abluminal interstitial cell are poorly developed in volume, although the fundamental structure of both cells is similar to those in females. This corre- sponds well with the much lower abilities in fluid secretion of male salivary glands. CB 29 ULTRASTRUCTURES OF ADDUCTOR MUSCLES OF BI- VALVE,Lima vulgaris. A.Matsuno and H.Kuga. Dept. of Biol., Fac. of Sci., Shimane Univ., Matsue. The adductor of Lima vulgaris (bivalve) shows long-distanced contraction and relax- ations to open the valves wide. It seems that the adductor works well in this way compared with ones of other bivalves. The adductor is composed of translucent and Opaque portions. The translucent portion is composed of cross-striated muscle cells that are similar to those of vertebrates: They have regular array of sarcomeres, and have thick (about 14 nm in diameter) and thin ( about 7 nm) myofilaments. The opaque portion is composed of smooth muscle cells that show hexagonal profiles in cross sec- tion and elongated shapes. These cells con- tain also thick and thin myofilaments homo- geneously distributed. Thin ones measure about 7 nm in diameter. Thick ones show various sizes (10-80 nm) as they distribute disordered way in longitudinal direction of the cell. We tried to estimate the size of thick myofilaments by the statistical anal- ysis: Diameters measured from micrographs were classified into groups at 10 nm inter- vals, and the frequency in each class was shown along the vertical axis. The distri- bution of diameters semmed to fit the normal distribution curve that has a peak at 45-55 nm in diameter. Thick myofilaments of these sizes are commonly observed in molluskan retractor, but they are rare in adductors of bivalves. CB 30 RUNNING PATTERN OF IO-NM FILAMENTS IN ASCIDIAN SMOOTH MUSCLE. K. Terakado. Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa. The organization of intermediate (10-nm) filaments in smooth muscle cells was ex- amined under an electron microscope. The material used was a body-wall muscle of the ascidian Halocynthia roretzi. The in- termediate filaments ran in groups through Spaces in a continuous network of myo- fibrils, connecting them at the regions of dense bodies longitudinally, obliquely and transversely to form an intimately associ- ated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones (analogous regions to the I-bands of striated muscle) exclusively, interconnecting successive dense bodies. The pattern of attachment of intermediate filaments to dense bodies was predominantly "one-sided". The filament themselves were not incorporated into the contractile apparatus, and they remained folded or unfolded between myofibrils in Synchrony with the contraction-relaxation cycies. The filaments terminated, usually in small groups, in the cell surface. These results suggest that the inter- mediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myo- fibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function. CB 31 CHANGES OF VOLUME AND BIREFRINGENCE OF SPERM NUCLEI DURING SPERMIOGENESIS IN TESTES OF CEPHALOPODS . 2 3 1 Hewoatom, he Katom sand Yweasatore Sugashima M: Be E-, Nagoya Univ., lobar, SBiol. tab. , Nagoya City Univ., Nagoya and ~Biol. Lab., Shoin Womens Univ., Kobe. Process of sperm maturation was followed during spermiogenesis of Octopus vulgaris by Nomarski and polarization optics and tr- ansmitting EM. During the spermiogenesis, sperm nuclei elongated from 9.0 pm to 21 pn, width reduced from 8.9 pm to 1.2 pM. , Thus, nuclear ,volume decreased from 364 pm to 21.5 pm’, indicating 1/16 condensation. Sperm heads gradually gained birefringence (BR). Same sequencial trends were confirm- ed in Octopus ochellatus and Ommatostrephes sloani pacificus. Sign of BR of these sperm are negative. From the series of imbibition experiments with various refractive indices, nature of the sperm BR was determined as in- trinsic with slight contribution of positive BR due to the oriented manchette microtubu- les. Based on results obtained from pronase and DNase treatments, we confirmed that the BR of sperm nucleiwas yielded by highly ori- ented DNA molecules and the co-efficient of BR, (n,-n,), was measured as 0.8 to 2 x 10 , almost idéntical with the pure DNA gel. However, we presume the molecular alignment of DNA in mature sperm head could not be a simple straight bundles but should be a form of tightly packed coiled-coil chromonema with DNA molecules as their backbone. 998 Cell Biology C3} Bz USE OF LASER RAYS FOR PRESERVING DROSO- PHILA EMBRYOS BY FREEZING. Y. Kuroda! Y. Takada'and T. Kasuya’ ! Lab. of Phenogenet., Dept. of Ontogenet., Nat. Inst. Genet., Mishima. ’Inst. of Phys. and Chem. Res., Wako, Saitama. An attempt was mede to use the laser rays for preserving early embryos of Drosophila melanogaster at -196 C. The point to be solved for freezing Drosophila embryos was how to introduce protective agents into the inside of eggs through the vitelline membrane. In the present study, UV laser microbeam was applyed to cut the micropile to allow glycerol to enter into the inside of eggs. Dechorionated eggs were exposed to laser microbeam at the micropile under a microscope, incubated in culture medium involving 15% glycerol at 25°C for 16 hours, and frozen in liquid Nienogen atl —196. Co Attemesevenmal days, eggs were thawed and incubated at 25 C. Larvae hatched at a low frequency and grew to adult flies. The entrance of glycerol into the inside of eggs after exposure to laser rays was confirmed by the stain- ability of eggs with neutral red. The survival of eggs after freezing was better when irradiated eggs were incubated with glycerol for more than 16 hours. The appropriate stage of embryos, the incu- bation medium and the speed of freezing and thawing are now under stady to obtain higher viability of eggs after freezing. CBs ANALYSIS OF CELL PROLIFERATION DURING THE HATCHING PROCESS OF THE FRESHWATER SPONGE Ephydatia fluviatilis. T.Matsuzaki and A.Shima. Zool.Inst., Fac. Oi SCilo, Wmtyo Oi WOO 5 MOK WO > The gemmules of the freshwater sponge were exposed to gamma-rays of Cs at various time points during hatching incuba- tion at 27 C and delay in hatching was examined. There were two radiation sensi- tive periods, one around the beginning of incubation and the other at about 27 hrs prior to hatching. The former period cor- responds to the beginning of the transition of cells from resting stage to prolifer— ating phase, the latter to the onset of DNA replication in S phase as revealed by DNA content measurement using DAPI-DNA micro- fluorometry. With regard to hatchability, irradiation at the latter period only was effective for reduction. Further, changes in number of nuclei per cell were counted on photomicrographs of gemmular cells at different time points throughout hatching incubation. The fraction of binucleated diploid cells decreased and that of mono- nucleated diploid cells increased with the advancement of incubation time. From these results, we presumed that upon the incuba- tion of gemmules diploid twin nuclei in one thesocyte replicated synchronously their DNA, and then formed 4 diploid nuclei via nuclear division. Thereafter a tetra- nucleated cell divided into 4 mononucleated diploid cells just before hatching. CB 34 CHARACTARIZATION OF THE PERMANENT PORCINE DIPLOID ENDOTHELIAL CELL LINE IN CULTURE. K.Yamamoto! , M.Yamamoto! and N.Hasegawa2. lpDept.Biol.,Tokyo Metropol.Inst.Gerontol., Tokyo; 2DivsGell. (Biolsyvakult. InsteMicto= biol.Res., Kunitachi. The characterization of a porcine aortic endothelial cell line PAE-20 is described. This cell line underwent spontaneous trans- formation at about 80 PDL. The transformed cells are presently growing indefinitely ( 510 PDL) and exhibit normal mode of chromo- some number. An abnormal chromosome, 3p+, were observed in all metaphase chromosomes in the transformed cells (120-492 PDL). The plating efficiency in the transformed cells was higher than that in the normal cells. The transformed cells at 474 PDL exhibited low efficiency of colony formation in soft agar. The transformed cells, however, possessed antibodies to Factor VIII-related antigen and high potential to produce prostacyclin up to 495 PDL. The angiotensin converting enzyme activity of the cells decreased linealy up to 81 PDL, and main- tained barely detectable levels in the transformed cells. Fibronectin of the cell surface decreased significantly after the cells spontaneously transformed, but the transformed cells possessed the same antibody to the fibronectin antigen as the normal cells when cells were cultured in dishes coated with human fibronectin. CB 35 ALTERATIONS IN ISOZYMES OF A SPONTANEOUSLY TRANSFORMED PORCINE ENDOTHELIAL CELL LINE DURING LONG-TERM SERIAL CULTURE. M. Yamamoto and K.Yamamoto. Dept.Biol., Tokyo Metropol.Inst.Gerontol., Tokyo. Tweleve isozymes were determined in the porcine diploid endothelial cell line PAE- 20 that spontaneously transformed at about 80 PDL and continuously subcultivated over a 3-year period. lULactate dehydrogenase B( LDHB) gradually decreased up to 69 PDL and lost at 84 PDL. Lactate dehydrogenase A ( LDHA) increased in the transformed cells (84-474 PDL). The transformed cells demon- strated a distinctly decreased ratio of LDHB to LDHA compared to the normal cells. A lower migrating isozyme of malic enzyme gradually decreased in the transformed cells. Isozyme expressions of malate dehy- drogenase, isocitrate dehydrogenase and mannose phosphate isomerase varied in the transtormeds cells.) Expressions, jot) iseven isozymes (phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, phosphogluco- mutase, purine nucleoside phosphorylase, glutamate oxaloacetate transaminase and peptidase A and B) were stable in all PDL examined here. Total choresterol contents lineally increased at 21-69 PDL but gradu- ally decreased up to 510 PDL. Changes in the choresterol contents may be related to isozyme phenotypes of malic enzyme and malate dehydrogenase (lipid synthetic enzymes). Cell Biology 999 CB 56 EFFECTS OF CHEMICAL CARCINOGENS ON THE CULTURED CELLS DERIVED FROM THE FINS OF THE CENTRAL MUDMINNOW (UMBRA LIMI; PISCES). I. Suyama and HH. Etoh. Natl. Inst. Radzol: Sci.;, Chiba. The ULF-23, a cell line from the fins of U. limi, was established and cultivated at 25°C. From the growth curve the doubl- ing time was estimated to be 72 h. Fluores- cense profiles of the cells stained with PI were obtained by a cytofluorometer. From these data the cell cycle times were estimated as follows; GotGi= 59 h, S=94h, and G2+M = 4h. The ULF-23 and ULO, another cell line from the ovary of Umbra, were treated with chemical carcinogens for sis- ter chromatid exchange (SCE) analyses. Chemicals used were MNNG (107° to 2x107°M), MAM acetate (2x10~’ to 107°M) = and ENU (2x10-® to 2x10-°M). After treatments with the chemicals SCE in second mitosis was observed. SCE values of control cells were 9-12/cell. SCEs in ULF cells treated with MNNG were increased with increasing concen- trations and reached to 27 at 107°M. No increases in SCE were observed in ULF cells treated with MAM acetate below 10-°M and in ENU-treated ULO cells within the range tested. The yield of SCE with chemi- cals was lower than in cultured mammalian cells, but the usefulness in cytogenetic studies of cultured Umbra cells was con- firmed. CB 37 Effects of human serum from various ages on proliferation and migration of human lung and skin fibroblasts. H. Kondo, Y. Yonezawa and T. A. Nomaguchi. Drejoisa— sirie yd See Lokyo..Meterioped. Ahnst. Gerontol., Itabashiku, Tokyo. Tt has been reported that both serum and blood plasma from old animals inhibit cell growth in plasma clot culture. Jlowever, we have demonstrated that sera from rabbits and most rats in old age did not inhibit cell proliferation. In order to determine whether human scrum from the old stimulates or inhibits cell proliferation, we measured cell proliferation activity of human serum from various ages (16-89 years) on the 3rd and 7th days. The results showed that sera from the old of either sex stimulated proliferation of human fetal lung fibroblasts (TIG-1) as well as FBS. This is also the case when uSing human old skin fibroblasts. Next, we tried to determine cell migration activity of human serum (16-64 years) since cell growth with plasma clot culture method includes cell proliferation, cell migration and others. Human sera from their 60s caused lower migration of TIG-1 cells, human adult and old skin fibroblasts. There was statistical significance between both age groups in female only when TIG-1 cells were tested. These results implied that serum from the old did not greatly inhibit proliferation and migration of human fetal lung fibroblasts, adult and old skin fibroblasts. CB 38 CHROMOSOME REPLICATION OF SYNCHRONIZED INDIAN MUNTJAC CELLS M.Ikeda! & T.Ueda?.1Dept. of Biol.,Keio Univ., Yokohama.Dept.ofDevelop. Biol., Mitsubishi Kasei Inst. of Life Sciences, Tokyo. Fusion between mitotic and S-phase cells induces the formation of prematurely con- denced chromosomes (PCC) in the inter- phase partner. Mitotic HeLa cells were fused with S-phase muntjac cells which were syncronised with excess thymidine and N20 high-pressure. Viewed through a light microscope, S-phase PCC appears to be fragmented and heterogenous; early S- phase is characterized by extremely at- tenuated filaments. Mid-S-phase is heterogenous, with attenuated regions of little staining containing some large and small blocks of stained material anda late S-phase PCC has elongated duplicated chromosomes with gaps. Autoradiographs of S-phase PCC by pulse and pulse-chase ex- periments showed 3H thymidine incor- porated first into unstained region, then into lightly stained fragments, and finally into dark stained chromosomes. Stained with Chromomycin A3(a G-C specific dye), the fiberous material can be clearly observed in Giemsa unstained regions. On the other hand, no staining can be observed when the same PCC is treated. wath» Hoeehs t) 3/3258. (a -A—T specific dye). CB 39 CHNGES IN CHARACTER OCCURRING DURING SUG- CESSITVE SUBCULTURES IN SMF CELL LINE. T.KUBO!, N.EBITANI? and S.NADAMITSU?, Biol. abe. sophia Univ., Tokyo, “Biol. Lab., Shohoku Coll., Atsugi and *Biol. Lab. Hiroshima Women's Univ., Hiroshima. The SMF cell line has been established in the fins of the marine teleost fish, Sebastiscus marmoratus. Two clonal cell lines were obtained from the above cells at different PDNs viz (a) TN-1 from the cells at small PDN and (b) TN-2 from the cells at large PDN. Some characteristics were con- pared among primary cells (P) and the SMF cells at small and large PDNs (S-SMF and L- SMF) and also the TN-1 and TN-2 cells. MORPHOLOGY - P and TN-1 cells were fibro- blast-like and showed regular arrangement. S-SMF cells were fibroblast-like but didn't show regular arrangement. L-SMF cells were fibroblast-like but were short and wide to some extent, and their arrangement was ir- regular. TN-2 cells were not fihbroblast- like, but rather epithelial-like. MODAL NUMBER OF CHROMOSOMES - P: 2n=48, S-SMF: 2n =96, L-SMF: 2n=50, TN-1: 2n=98, TN-2: 2n=50 NUMBER OF METACENTRIC CHROMOSOMES AND AgNORs = Ps 2and 2. S-SMF: "2 and "8, U=sMrs 27% and LNs seamen or N= as 2 aire: Are Chromo- some loss was observed in SMF cells subcul- tured successively for a long tern. In view of the number of metacentric chromo- somes and AgNORs, the chromosome loss was not random, but meiotic chromosomal segre- gation might have taken place. 1000 Cell Biology CB 40 CB 42 CONDITIONED MEDIUM OF BOVINE AORTIC _ENDO- ESTABLISHING A D-GALACTOSAMINE INDUCED THELIAL CELLS CONTAINS A GROWTH FACTOR(S) HEPATIC INJURY MODEL USING PRIMARY CULTURED THAT STIMULATES THE PROLIFERATION OF HUMAN RAT HEPATOCYTES ENDOTHELIAL CELLS, J.Kijima’, J.Arai, S.Kusunoki!) Toruruyas K, Kaji and M, Matsuo, Isotopes Lab,, ‘Life Science Lab., Advance CO, pL. 5 MOlAvO, Tokyo Metro, Inst, Gerontol,, Tokyo, “Dept. of Pharmaco., Kitasato Univ., Tokyo. ae eee Ee The In vitro hepatic injury model using Bovine aortic endothelial cells (ECs) primary cultured rat hepatocytes was shown grew in MEM supplemented with 19 % fetal to ~~ be’ “quite ‘Suitablley for the Se aimse bovine serum (FBS), but human ECs derived screening of some natural extracts because from the umbilical cord could not grow in many samples of these extracts could be the same culture medium, We tested the screened in a short time and the amounts of possibility that bovine ECs produce a endo- samples required were very small. thelial cell growth factor(s) (ECGF), and After 24 h preincubation, the hepatocytes the factor acts in an autocrine fashion to were exposed to a medium containing either stimulate ECs proliferation, Quiescent D-galactosamine (GalN) or GalN and a bovine aortic ECs were exposed to MEM for natural extract. After a lapse of 72 h the 4 days and the conditioned medium was har- GPT and LDH activity and the albumin vested, The conditioned medium added 10- concentration in the medium were measured. 40 % to MEM plus 10 % FBS stronly stimulated We had thus established a GalN induced the proliferation of human ECs, and the cell hepatic injury model using primary cultured number increased by 2-4 times in 3 days, rat hepatocytes. We compared the effect of The ECGF activity was nondialyzable, trypsin Ganoderma lucidum mycelium extract (GLM1), sensitive, acid unstable and heat stable, Ganoderma lucidum fruits body extract The ECGF activity eluted from heparin- (GLF1, GLF2) and Alismatis rhizoma extract Sepharose at 0,6 M and 1,5 M NaCl, and the (AR1) with Glycyrrhetinic acid using this activity of the latter fraction was inhibit- model. ytbicse ed by 10-100 ig/ml] of heparin, The cel] (This model showed a significant increase homogenate of hovine ECs also contained in GPT and LDH activity and a decrease in albumin synthesis in the GalN challenge medium. Four samples where 300 pg/ml were added to hepatocytes inhibited the increase ECGF activity which eluted from heparin- Sepharose at_1,5 M NaCl, — On the other hand, human ECs homogenate contained very litwle ECGE de uIViItV,. In any, of GPT activity. Only GLF2 significantly These data showed existing evidence prevented the increase of LDH activity by for autocrine growth control by bovine ECs, GalN. It seems that this assay was therefore effective. but not by human ECs, CB 41 CB 43 WOUND HEALING OF RAT FETUS SKIN. PROTECTIVE EFFECT OF NATURAL EXTRACTS ON S.Ihara, E.Nagao, Y.Motobayashi, A.Kist— TWO TYPES OF HEPATIC INJURY MODELS ler and N.Shioya. Dept. of Plast. Surg. T.Nakamura’, H.Miyazaki!, S.Kusunoki}, Te Kitasato Univ. Sch. of Med., Sagamihara. _ Furuya’, 'Life Science Lab.,Advance Co.LTD., As the’ "first step to! make clear the Tokyo, Dept. of Pharmaco. Kitasato Univ., feature of fetus wound healing process, Tokyo we have made a model in which wounded rat fetus skin was cultured in vitro. When a small open wound (2mm ¢) was made in the 18a fetus skin and the skin piece was cultured, kept floating, in DMEM plus 1% Ultroser G, a thin network (NW) was formed horizontally across the wound edge within 24h after the beginning of cul- IGUNIAS 6 Two to 4 days after, fibroblast- like cells appeared and increased in NW. NW skonmation, did note sdepend. Jonmsache pRBesence Of —Ultrosery | Ga buts the sicels: growth depended. SEM study showed that NW consisted of thin filaments (40-60nm é@) which were branched in places. Its appearance resembled collagen fibrils or fibrin fibers. However, no fibrous collagen-specific striation could be observed by TEM. SDS-PAGE analysis showed that the major component of NW was a 55kd polypeptide (P55) which was insensitive to bacterial collagenase, and no a-chain of collagen was detected in NW. Meanwhile, the amino acid sequence (9 amino acids) of a tryptic digest from P55 was moe iiacemcleal wo wae waole sequences of human fibrinogens a, 8B and Go Therefore, P55 may be a new kind of ECM component which is produced by the healing fetus skin. We observed that some natural extracts have a protective effect on primary cul- tured rat hepatocytes against hepatic injury induced by D-galactosamine (GalN). The effects of GLM1, GLF1,GLF2 and AR1 were studied through an in vivo hepatic injury model in rats induced by carbon tetrachlo- ride (CCl4) and GalN. Eight week old, 180-220g male Wistar rats were used and permitted free access to food and water. Samples of the natural extracts (dosage 1, 10, 100 mg/kg bw ip) were admin- istered 30 minutes before treatment with CCl4 (dosage 1.0 ml/kg bw po) and GalN ( dosage 500 mg/kg bw ip). An increase in serum transaminase (GOT, GPT) and LDH activities was observed after CCl4 and GalN treatment. All samples, dosage of 100 mg/kg, inhibited the increase of GOT, GPT and LDH activities in the CCl4 induction hepatic injury model. On the other hand, an increase of GOT,GPT and LDH activities induced by GalN was in- hibited by the administration of GLM1 and GLF2 (dosage 100 mg/kg). Histopathological findings resembled biochemical findings. These results demonstrated a correspond- ence between in vivo and in vitro findings. GE 1 RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA IN GENUS APODEMUS H.Suzuki K.Tsuchiya,, M.Sakaizumi;, SO UU aes it, \Walilsueeiaileun 6 K.Moriwaki Cenmtnamw Res. labis, he Jakes!) Univ. School of ,Med., Tokyo, Miyazaki Med. College, ~The Tokyo Metropol. Inst. Med. Seuas cNath. Inst. Genet. , Mishima’. Southern blot analysis for ribosomal DNA (rDNA) in six Apodemus species ( A. Siva wensie tA © hlaviicolilsiis,.» Ans Semoitus, A. agrarius, A. argenteus, A. speciosus) using 14 restriction enzymes revealed that a major rDNA repeating unit type (repetype) of each species was specified by characteristic topological arrangement of restriction sites. In the non-transcribed spacer region near the 28S gene, most of A. speciosus collected from 20 different locations in Japan gave two EcoRI bands (1.3 kb and about Hikab)s) aibenabuted 0) eoRT= isite variation within individual. No correlation was noticed between two karyotype groups (2n=46 and 48) and _ the EcoRI site variation. Small island populations, however, had either one of two EcoRI bands in their genome predominantly: Tokara, Yaku and Miyake had@weo, band Lsushima: 1:3: kb. These results suggest that fixation of variant repetypes occurs rapidly in a small population, irrespective of repetypes. Genetics 1001 GE ENZYMATIC ANALYSIS OF THE INSEMINATION REACTION fN DROSOPHILA I. Now MAsadaiy.pavandy TER ak « Watanabe. ldept. of Biology, Faculty of Scjence, Okayama Univ. of Science, Okayama, Genet. Stock Center, Nat. Inst. of Genetics, Mishima. Phenoloxidase and protease are concerned in the insemination reaction which is’ the phenomenon occurs in copulated female accompanied by reaction mass formation and plays important roles on isolation in Drosophila. The production of reaction mass takes place within a few minutes after copulation both in intra- and interspecific crosses. In the latter crosses, reaction mass was maintained in uterus, then melanized in vivo and in vitro following the detection of phenoloxidase activity resulted in restraining the fecundity. In the former crosses, however, reaction mass remained the original color, then reduced in size gradually and disappeared by proteolysis prior to oviposition. Because formation and disappearance of reaction mass show temperature dependency, specific enzyme-blockers injected in vivo significantly inhibited above phenomena, and enzyme activities were detected, each enzyme(s) are thought to be key one(s) for formation and transition of reaction mass in intraspecific crosses. Above phenomena seem to be the consequence of the female's discrimination system of male's secretion by humoral immune involved in phenoloxidase- protease system in Drosophila. GE 2 GE 4 EXPRESSION OF AY GENE IN THE SKIN OF THE HETEROMORPHIC CHROMOSOMES IN SEA URCHIN, MOUSE. TYPE B OF ECHINOMETRA MATHAEI, Gesaro, lskakewehies Biols;Enst.,> fohoku FROM OKINAWA Univ., Sendai. T.Uehara! and K. Taira’. ‘Dept. of Biol., Univ. of Ryukyus, Okinawa. “Shuri Junior High Sch., Okinawa eee Mammalian melanocytes are capable of producing two types of melanins, black pigment (eumelanin) and yellow pigment (pheomelanin). In the house mouse, the Since sea urchins have many chromosomes of small size, it is difficult to distinguish the homologus ones. Therefore, existence of the heteromorphic : : : chromosomes in sea urchins has been left unsolved type oe meet: SSemown Peas $e SUS until now. Type B urchin is one of the four types Deon Ours (a) LEUSS ee ae EbeuenE of E. mathaei. Diploid chromosome number in all ERs BP TESS sit aoeme Dene Le bomeGL Cc of the four types is 42 in which one strikingly large eee wen genotype (a/a) exibits eae chromosome pair exists. The largest chromosomes mee whereas genotype iGo ha) ext bats consist of two metacentric ones in three urchins LS ae ea ; of A,C and D type. In Type B, karyotypes of one pees eos cep er ments SLO hae is largrst chromosome pair of 52 fertilized eggs from ee eeuce dapheome lanogencsis ay ae three pairs were analysed. Twenty five cells with cells ae CEU eG CORLSRSS of puis Poot homomorphic chromosome pair and 27 cells with aden PROB eNeS Ls Mone) Cocamecd heteromorphic chromosome one were counted. Fur- pees ofp yellow mouse and Brack , theremore, in Type D§ x Boe two kinds of the morse, Us ees oe aaa Es (ails heteromorphic chromosome pairs were observed. oe eee ban. Ehatiqin ala pigia- One consists of a metacentric chromosome from Fnapenake suid gue S8aE oe 272 Seyi Type D and a telocentric one from Type B, and the Page oda cues, used: fora mnund Zing athe other composed of a metacentric chromosome from MOSS OF (a/a) genotype. Phen pthe »spleen Type D and a submetacentric one from Type 3. cells from (A fay MOUSE weperaSe! ati EG These results show that there are dimorphic chromo- myeloma cells. PSS hes NORES GERAE) ORE somes in the sperm from Type B. hybridoma cell line secreted antibody which react solely to (A’/a) mouse skin. By immunofluorescence technique, this antibody was found to react to epidermis, hair follicles, dermal cells in the skin of 2.5 day newborn mouse. 1002 Genetics GENS ESTABLISHMENT OF THE MULTIPLE RECESSIVE TESTER MEDAKA HOMOZYGOUS FOR THE 3 LOCI. A.Shimada and A.Shima. Zool.Inst., Fac. of Sci., Univ. of Tokyo, Tokyo. The establishment of a multiple reces- sive tester stock is the prerequisite for using specific-locus method in the Medaka Oryzias latipes. We have previously produced a multiple recessive tester stock INOMOryZOUS IOI 5. LOC, aoSo, Wy WE, . fall. joll and r. For practical use, however, we found some problems like low viability and low fecundity in this stock. Then attempts were made to change some loci in order to obtain more suitable tester stock. We deleted the Da, pl and r loci and introduced the 1f locus as a new marker. The homozygotes for the locus lf are known to have no leucophores throughout the life, while the leucophores in the wild type can be recognized around brain of embryo about 3 days after fertilization. The new tester stock Medaka thus obtained are homozygous for the b, gu and If loci, have high viability and fecundity and are judged to be suitable for use in the specific-—locus test. The data on numbers of F, offspring obtained during the production of this tester were used for segregation analyses of the 3 locus-pairs, b-gu, b-lf and gu-lf, respectively. Since P values were clearly greater than 0.05, the observed results were in good agreement with those to be expected for the independent assortment of each two pairs of alleles. GE 6 RADIATION INDUCTION OF SPECIFIC-LOCUS MUTATIONS IN THE MULTIPLE RECESSIVE TESTER MEDAKA. Ay Sihtitmay anid Ava Sihaimadar, = Zool inisiter.peliae eauon: Sci., Univ. of Tokyo, Tokyo. We succeeded in establishing the multiple recessive tester Medaka Oryzias latipes homozygous for 3 or 5 loci. In order to determine if our tester Medaka are valid for detection of induced recessive muta- tions by the specific-locus method, we set out to examine gamma-ray-induced mutation BeOGTUEncikses Bie iene js Ike, ew, ginal foil Loew o The total numbers of loci examined were 187,073 and 147,713 for the postspermato- gonial and spermatogonial cells, respec- tively. No spontaneous mutant that survived more than 4 days after hatching has yet been obtained. Because we chose as markers genes whose phenotypes are expressed rather early during development, we very often observed deaths of mutant embryos prior to hatching. The approximate ratio of numbers of such mutants as died before hatching to those of viable (survived more than 4 days after hatching) mutants was 10:1, indi- cating that the vast majority of the mutation are not viable probably due to deletions rather than point mutations. We presented data which showed that gamma- irradiation induced specific locus muta- tions in the Medaka were fairly well in accordance with the comparable results on mice obtained above all by W.L. Russell. Ge NEW MUTANT IN THE MEDAKA. H.Tomita. Lab. of Freshwater Fish Stocks, Fac.gf Sci., Nagoya Univ., Nagoya. The mutant(ha), deformity of inner ears was already established. The new mutant which is similar pheotype to the mutant(ha)was found. The offspring of crosses between the new mutant and the ha mutant were all normal. These mutants _ belong to different alleles each others. The new mutant is recessive autosomal and polymeric(ha-~2,ha-3). At the embryonic and larval stages, the auditory vesicle swelled and protruded. This protrusion turned to normal at about 10 mm body length. At about 20mm body length, the fishes swimmed round. In the inner ears, the ultriculus fused with the scculuse and became swelled vesicle and the semicircular cannals developed incompletely and sometimes were absent. The rs-2 -and rs-3 mutants were estab- lished at last year. The former had small scales and the latter was partial lack. of scales(partial naked). They are autosomal and recessive. The rs-2 is independent of the b and dm alleles. The rs-3 is independent of the’ b, ci, dm,rs—2 and ve alleles. eines)... wi GE 8 KARYOTYPE EVOLUTION AND GEOGRAPHICAL DISTRIBUTION OF BIARMED CHROMOSOME GROUP OF RICEFISH, GENUS ORYZIAS, IN YUNNAN, CHINA AND EASTERN ASIA. Ho Uwal, R.-F. Wang and Y.-R. Chen: Deptenon Biol., Shinshu Univ., Matsumoto and *Kunming Inst. of Zool., Acad. Sinica, Kunming, China. Karyotypic studies were made in two species of ricefishes collected from Yunnan, south-western China. Oryzias latipes collected from the eastern Yunnan Plateau had 2n, 46 chromosomes consisting of 3 metacentric, 8 submetacentric, 2 subtelocentric, and 10 acrocentric pairs , the arm number (NF) being 68 (2n=46, NF=68, 3M+8SM+2ST+10A). The karyotype was characteristic by having a "large" metacentric pair and nucleolus organizer regions (NORs) on the short arms of a submetacentric pair. A diminutive species 0. minutillus collected from Xishiangbanna, the southern low mountain areas of Yunnan, showed 2n, 42 chromosomes consisting of 21 acrocentric pairs, NF being 42 (2n=42, NF=42, 21A). NORs were located at the telomeric regions of an acrocentric pair. Geographical distribution of these ricefishes were seemed to be correlated with river systems and climatic zones. Karyotype of 0. latipes from Yunnan was closely related to that from eastern China and western Korea (2n=46, NF=68-70), but distinct from that from eastern Korea and Japan (2n=48, NF=68-70). Relationship between karyotype evolution and geographical distribution of ricefishes was discussed among four species of the biarmed chromosome group collected from eastern Asia; 0. mekongensis (2n=48, NF=58), 0. latipes (2n=46-48, NF=68-70), 0. curvinotus (2n=48, NF=82), and 0. luzonensis (2n=48, NF=96). Ged GENETIC DIVERSITY OF THE EAST ASIAN POPULA- TIONS FOR THE FRESHWATER FISH, ORYZIAS aimal Soy Re pThe Tokyo Metropol. Inst. Med. Sci., Tokyo Hace sere. Shinshu Univ., Matsumoto, and College of Nat. Sci., Sangmyung Women's Univer. TOeowl ¢ M. Sakaisunin Jal Uwa-, eons Allozyme studies have revealed that the wild populations of medaka are composed of four major groups, the Northern Population and the Southern Population from Japan, the East Korean Population, and the China-West Korean Population. In Korean Peninsula two groups were separated by the backbone moun- tain, which is a major boundary of Korean freshwater fish fauna. Introgression of "western'’ alleles into the East Korean Population was observed in the southern area of the peninsula. But little traits of Japanese two major populations were found in Korea, suggesting strong isolation between the populations of the Japan Islands and Korean Peninsula. The popula- tions of Yunnan District in China showed a Similar genetic profile to that of the China-West Korean Population. On the other hand, the medaka collected in Hong Kong was different from any of the four major groups. These results suggest that the China-West Korean Population has a great distribution range and that the population of Hong Kong belongs either to the fifth group of OQ. tatapes or to a different species closely relABeGy EO abies GE 10 KARYOTYPE POLYMORPHISM OF A SMALL RICEFISH, ORYZIAS MINUTILLUS. T. Ashida and H. Uwa’*. Nagano-ken Fujimi High School, Suwa-gun and “Dept. of Biol., Fac. of Sci., Shinshu Univ., Matsumoto. /Mémbers of the genus Oryzias so far studied could be karyotypically divided into three groups; monoarmed chromosome type, biarmed chromosome type, and fused chromosome type. Karyotypes of 0. minutillus from Bangkok (2n=34, NF=44; 4M+1SM+12A) and Chiang Mai (2n=30, NF=44; 6M+1SM+8A), Thailand, had 4 and 6 pairs of "large" metacentric chromosomes, respectively, and it had been considered to be a member of the fused chromosome group (Magtoon and Uwa, 1985). Nucleolus organizer regions (NORs) were located on the short arms of a submetacentric pair. Recently, we found specimens of O. minutillus collected from Phuket in southern Thailand had 2n, 42 chromosomes consisting of 21 acrocentric pairs (2n=42, NF=42; 21A). NORs were located at the telomeric regions of an acrocentric pair. The hybridization test among specimens from three localities revealed that they could breed and F1 offspring were fertile. 0. minutillus collected from Xishiangbanna in Yunnan, China, also had a karyotype (2n=42, NF=42; 21A) similar to that from Phuket. From these results, karyotype of specimens from Phuket (and Xishiangbanna) might be basic for QO. minutillus, and those from Bangkok and Chiang Mai might be caused by Robertsonian centric fusion and pericenrtic inversion. Genetics . 1003 GE 11 CHROMOSOME ANALYSIS OF THE MOUSE SPERMATO- ZOA BY THE INTERSPECIFIC IN-VITRO FERTIL- IZATION METHOD M. Shimada, Y. Kamiguchi, H. Tateno and K. Mikamo, Dept. of Biol. Sci., Asahikawa Med. Col., Asahikawa. We modified our interspecific in-vitro fertilization method with zona-free hamster ova for analyzing human sperm chromosomes in order to study mouse sperm chromosomes. The main points of the modification are as follows: (1) Use of TYH medium for sperm capacitation instead of mBWW medium used previously. (2) Use of MEM alpha medium containing 30 % fetal bovine serum for postinsemination culture instead of TC medium 199 containing 10 % fetal bovine serum. (3) Determination of the optimum moment of insemination by observing the changing aspects of movement patterns of spermatozoa. (4) Insemination of ova with sperm suspension of a low concentration, 1 XS Oe sperms /imise for a short. tume,, = 56 minutes. Most of mono-, di- and trispermic eggs were successfully activated by this method, showing expulsion of the 2nd polar body and active growth of male and female pronuclei. Among them, 71 % of both mono- and dispermic eggs developed up to the stage of the 1st cleavage metaphase. We described the procedures of this method and some results thereby obtained. GE 12 ACUTE AND LATE EFFECTS ON OOCYTES IN CHINESE HAMSTERS X-IRRADIATED DURING NEONATAL STAGES H. Tateno_, K. Mikamol, Wo Kamiguchi! and K. Tanaka“. IDept. Gz Isalolls S@alo- and Central Lab. for Res. & Ed., Asahikawa Med. Col., Asahikawa. In our previous studies, it was shown that diplotene and early dictyate oocytes of neonatal Chinese hamsters were highly radiosensitive and they were completely destroyed by 100 rad X-rays. In the present study, we exposed females at 6 days of age to three doses of X-rays (10, 25 and 50 rad) in order to examine acute and late effects of these lower dosages on their oocytes. Nearly 95% of oocytes survived 10 and 25 rad X-rays, but only 30% of oocytes could survive 50 rad. Fertility span of females exposed to 10 and 25 rad X-rays was not significantly different from that of non- irradiated females. On the other hand, the fertility span was significantly shortened in the females exposed to 50 rad. They clearly showed delay in sexual maturation and precocious cessation of estrous cycles owing to the shortage of oocytes. There was no significant increase in incidences of chromosome aberrations and dominant lethal mutations in the oocytes ovulated from all irradiated females. These results showed that the X-rays irradiated on diplotene and early dictyate oocytes induced only acute oocyte-killing, but did not induce late effects due to genic damages. 1004 Genetics, Immunology GES ASSESSMENT OF GENETIC EFFECTS OF ENVIRON- MENTAL MUTAGENS USING HUMAN SPERM CHROMO- SOME ANALYSIS Y. Kamiguchi, H. Tateno, M. Shimada and kK. Mikamo, Dept. of Biol. Sci., Asahikawa Med. Col., Asahikawa. Using our interspecific in-vitro fertil- ization system between human spermatozoa and zona-free hamster ova, we studied cyto- genetic effects of X-rays and an alkylat- ing agent, methylmethansulfonate (MMS), on human sperm chromosomes. Total number of 4959 spermatozoa were karyotyped in the former experiment,and 3095 in the latter. (1) Incidences of spermatozoa with structural chromosome aberrations were 14.1 & (0 seach), W8sO B= (25 sacl), 2oo5 S (SO rad), 42.6 % (100 rad) and 68.0 % (200 rad) in the X-ray experiment, and were 14.6 % (GOMES \ A VAAde SS mem); 295 BS Clo IS /MIL) A Bos (2S sisi) eine Oil oT 3 (SO ug/ml) in the MMS experiment. Dose-depen- dent increase of affected spermatozoa was linear in both experiments. (2) In the X- ray experiment, the incidence of breakage- type aberrations was far higher than that of exchange-type aberrations, both of them showing linear increase with increase of dosage. (3) In the MMS experiment, break- age-type aberrations increased linearly with increasing dosage, whereas exchange- type aberrations increased quadratically, reaching 32 % of total aberrations in the highest dose group. Importance of this method in assessing genetic effects of mutagens on human was stressed. GE 14 COMPARATIVE CHROMOSOMAL STUDIES OF ANEMONEFISHES (PERCIFORMES ) A.Takai, S.Kosuga, and Y.Ojima, Dept. of Balonl. | Hac) sof. Seis, Kwanisiea Gakwwien Univ., Nishinomiya. Karyotypes and distribution of nucleolus organizer regions (NORs) and C-banded heterochromatin of seven anemonefishes were comparatively studied. Chromosome number was 2n=48 in all species and karyotypes were similar as follows : Am- phyprion clarkii, A.ocellaris, and A. polymunus (12M+24SM+12ST,A, NF=84); A. frenatus and A.perideraion (12M+22SM+ 14ST,A, NF= 82); A. ephippium (10M+28SM+ 10ST,A, NF=86); Premnas biaculeatus (12M +30SM+6ST,A, NF= 90). NORsS were located at terminal regions of the short arms of 2nd or 3rd largest subtelocentric pair in all species except A.ocellaris in which NORs were located at pericentric regions of the long arms of the 2nd largest submetacentrics. C-bands were located in centromeric regions of several chromosomes, but the distribu- tional patterns were different among the species. Only A.polymnus had many telomeric C-bands. The present results suggest that the karyotypes of A. ephip-—- pium and P. biaculeatus are somewhat differentiated from the other four species and a close relation between changes of C-banded heterochromatin dis- tribution and species differentiation. NORs seem to be located in the identical chromosomes among the species. Uh at LATE LARVAL HEMOPOIESIS OF BULLFROG RANA CATESBEIANA, USING MONOCLONAL ANTIBODIES. K.Hatakeyama and K.Sugiyama. Dept.Biol., Fae Sei. ,Hiro-aki Univ. -Hirosaki. Four mouse monocional antibodies ,named mAb Rc-T-l(reported as T1-3A5 previously), mAb Rc-Gl(as 2A-4) ,mAb Rc-G2(as 2B-9) and mAb Re-P(as 1B-10),were prepared against lymphocytes of bullfrog Rana catesbeiana previously and have been characterized of their reactivities among various lymphoid tissues of frog by indirect imnunofluores- cence antibody staining. An application of these mAbs to the late larval hemopoietic cells during the metamorphosis(Cooper Sta- ges 25-34)demonstrated that both neutroph- ils(recognizabie by mAb Rc-Gl and mAb Rc- G2) and small monocyte-like celis(recog.by mAb Rc-G2)were mainly originated from the larval mesonephros and from the liver in part. Whereas,the platelet antigen Rc-P positive cells were originated from the larval liver and showed a considerable hi- gher cell population in PBL during larval stages,but a noticeable decreasing of the- se cells was shown at the postmetamorphic froglet,in contrast to the Rc-Gi and Rc-G2 positive cells. Much of thymocyte antigen (Pc-T-1) was detected at the begining sta- ge(st.25/26)of hind limb formation aiready Rce-T-1 positive cells were also detected significantly on the lymphoid cells from mesonephros(st.25/26)and PBL with a faint reactivity. IM 2 C-REACTIVE PROTEIN (CRP) IN RAT. -THE SITE FOR ITS SYNTHESIS AND INTER- ACTION WITH MACROPHAGE (M@) - W. NUNOMURA!?, H. WATANABE! and H. HIRAI?. 1) Tumour Laboratory and 2) Nippon Bio- Test Laboratories Inc., Tokyo. Although CRP is one of typical acute phase proteins, its physiological role(s) and mechanism for the synthesis have not been fully elucidated. In this study, we investigated the site(s) for the synthesis and interaction with M@ in rat CRP. By immunohistochemical method, CRP was strongly stained in liver by chemical inflammation provoked injection of turpen- tine. CRP was localized in cytoplasm of hepatocytes and also detected on the sur- face of polynuclear leucocytes infiltrated in the injured tissues. In primary culture of rat hepatocytes, CRP was synthesized and released into the medium. Cytokines from rat M@ faciliated the production of CRP, while those from thymocytes and splenocytes had no effect. On the other hand, the incubation of rat CRP with M@ resulted in the generation of O2. These results suggest that the restorat- ion of injured tissues are facilitated by cooperation of CRP and M@. Immunology 1005 IM 3 ENHANCED RNA SYNTHESIS IN ACTIVATED RAT PERITONEAL MAST CELLS (PMC) H.Fujimaki t and A.D.Befus DavyeOrebasie Medi Sci. , Natl. Tnsit . fox Environ. Studies, Ibaraki “Dept. of Micro.g Infect Dis., Univ. of Calgary, Canada. The well recognized heterogeneity in rat mast cell populations may be due to dif- ferences in the phases of differentiation in a single cell lineage, or to microenvi- ronmental influences. Therefore, to deter- mine the molecular basis of mast cell heterogeneity we have initiated investi- gation of mast cell RNA. Preliminary experiments showed that RNA levels are low in normal adult PMC.Thus we have attempted to increase RNA synthesis of PMC activated with 48/80 or worm antigen. MILIESeELW Sprague-Dawley rats were injected IP with 48/80. One,5,10 and 25 days later rats were killed and PMC were isolated and purified using a discontinuous Percoll Geaduent One uday betone the isaits were killed ~H-uridine was also given IP. In vivo labeling with uridine showed no aay enhanced RNA synthesis in purified PMC. Secondly, purified PMC from normal rats were treated with 48/80 or sensitizing antigen in vitro and were cultured for 1, 3 and 24 hours in the presence of 3y- Uist LNe Uptake Of UBITdine | in acti— WcieoGme CmEuncTEeased) compared | sto) non activated PMC. These observations provide important tool to study underlying mechanisms of mast cell heterogeneity. IM 4 ANTI-MOPC-315(H-2¢) PLASMACYTOMA RESPONSES IN MAJOR HISTOCOMPATIBILITY COMPLEX -IDENTICAL STRAINS OF MICE: Natural Tolerance to the Tumor-Specific Antigenic Determinants in a Susceptible Strain of Mice M.Hosono, K.Yano, Y.Katsura and K.Takahashi* Dept. Bacteriol.& Serol.,Chest Dis. Res. Inst,, Kyoto University, Kyoto and *Aburabi Lab., Shionogi-Seiyaku Co., Ltd., Koga-gun, Shiga. BALB/c-originated MOPC-315 plasmacytoma bears tumor specific transplantation antigens(TSTA) recognizable by and rejected in the syngeneic BALB/c mice as well as inthe other H-2-iden- tical allogeneic strains except for DBA/2 mice. The trait of unresponsiveness to the TSTA in the latter mice is genetically in- herited codominantly. Among BALB/c-DBA/2 hybrids, white mice, homozygous at the BALB/ c-albino locus, were entirely resistant to the tumor. Preimmunization of adult BALB/c mice with nucleated and anucleated cells of DBA/2 but not of any other mice used, pro- moted tumor regression, indicating an in- duction of positive immunological memory. In contrast, administration of those antigens, into neonatal BALB/c mice rendered them un- responsiveness to the TSTA at adulthood, indi- cating that immunological tolerance or nega- tive immunological memory was induced. These results provide evidence that in susceptible mice, natural tolerance has already occurred with regard to their own bodies' antigenic determinants which cross-react with the TSTA, supporting the "clonal deletion model" for an unresponsiveness to "foreign" antigens. Ties STUDIES ON THE PLATELET-LIKE STRUCT URES(PL S) IN HEMOLYMPH FROM LAND SLUG, INCILARIA SPP. USING BIOTINYL LECTINS AND AVIDIN- | HORSERADISH PEROXIDASES(TI) E.Furuta', K.Yamaguchi’? and A.Shimozawa' Dept. of Anat. and “Lab. of Med. Sex. Dokkyo Univ. Sch. of Med. ,Tochigi Three morphologically and functionally distinct hemolymph cells, Type I,II and III, were present in collected hemolymph from land slug, Incilaria spp,,In additon, vast numbers of PLS can be seen in most of the light and electron micrographs. In onder to know the origin of Pls, four dit— ferent biotinyl lectins and avidin-horse- radish peroxidases were used. Type I cell (phagocyte) and PLS were preferentially stained with wheat germ agglutinin, Ricimus lutinin in vitro. However, they were nega- tive for Concanavalin A staining and Type II cells were not positively stained with each lectin. Moreover, when hemolymph cells were cultured with SH7 medium, Type I cells gradually split into several PLS during 3rd or 4th day incubation. And in vivo, PLS are not normally present in hemolymph but they may be formed rapidly in response to trauma, Because, after injection of a high concent- ration of carbon or latex beads into the hemocoel of land slug, great numbers of PLS are present. These results suggest that PLS are derived from Type I cell and retain. ed same recognizing ability of Type I cell, so they can clamp foreign materials. IM 6 STUDIES ON THE PLATELET-LIKE STRUCTURES IN HEMOLYMPH FROM LAND SLUG, INCILARIA SP. USING BIOTINYL LECTINS AND AVIDIN-HRPO (II) . K. Yamaguchi', E. Furuta? and A. Shimozawa% TBab. of Med. Sci. and?Dept. of Anat., Dokkyo Univ. Sch. of Med., Tochigi. We reported previously that the cells of 3 morphologically distinct types were pre- sent commonly in the hemolymph of land slug, Incilaria fruhstorferi and bilineata; Type I cell was macrophage-like, Type II cell was lymphocyte-like and Type III was fibro- blast-like. Besides these 3 type-cells, nu- merous platelet-like structures(PLS) were usually observed in collected hemolymph. In order to define the origin of PLS, biotinyl lectin-avidin horseradish peroxi- dase method was used. In light microscopi- cal examination, Type I cell and also PLS were stained positively with WGA, UEA and RCA. However, in electron microscopic ob- servation, only PLS was weakly stained with WGA, but Type I cell was not stained with any lectin. These results suggest that the condition of fixation may not be suitable for the hemolymph cells in the present ex- periment. Since the hemolymph was prefixed with very low concentration of glutaralde- hyde (0.08%) for long time(20hrs) at room temperature as the same method in our pre- vious experiments, the hemolymph cells might not be fixed enough before DAB treat— ment was done. Another reason may be con- Siderable that the concentration of bioti- nyl lectins(20yg/ml) is lower than the suf- ficient one. 1006 Immunology, Biochemistry Wil 7 MELANO-MACROPHAGE CENTER LIKE STRUCTURES IN THE HEART OF THE MEDAKA,ORYZIAS LATIPES. H.Nakamura! , A.Shimozawa' and S.Kikuchi? . Dept. Of vAnaite DokkyonUnunye SChe sor eMedia Shimotsuga-gun and “Kominato Marine Biol. agp HEC ol Ore SSiLo pn Caadloe UimIWon Caioes It is known that the endocardial lining cells (ECCs) in the heart of some teleosts take up foreign particles such as carbon and yeasts. In the medaka, Oryzias latipes, not only ECCs but also the cells in the subendocardial spaces showed phagocytosis and the ingested carbon particles persisted in these cells for over 2 years. Most of the subendocardial cells (SECs) contained yellowish-brown pigments and often accumu- lated to form nodules resembling in appear- ance that of melano-macrophage centers (MMC) observed within the spleen and kidney. By light microscopy, SECS stained positive by PAS and Schmorl reactions suggesting that they contained lipofuscin and ceroid series, Perl's reaction was slightly positive (but not constantly). By electron microscopy, macrophages containing large heterogeneous inclusions together with some lymphocytes were observed. No sheath-like structures were found around them. Such MMC-like structures were also found in the atrium of the black molly, Poecilia sphenops, but not in the flounder, Para- lichthys olivaceus. The possible homology between these structures and the MMCs in the hemopoietic organs has still to be resolved. IM 8 INTERNAL DEFENSE FACTORS IN THE APPLE SNAIL, POMACEA CANALICULATA. A.Shozawa, C.Suto & N.Kumada. Dep. of Med. Zool. ,Nagoya Univ. Sch. of Med., Nagoya. Several workers have recently reported the production of active oxygen species by molluscan hemocytes. They suggested possibilities of cell-mediated cytotoxici- ty by the hemocytes as observed in verte- brate leucocytes. Hemocytes of the fresh water snail, P. canaliculata, show active phagocytosis, a major role of the host defense. We tried to detect active oxygen species produced by the snail hemocytes. Hemagglutination activity of the hemolymph was also examined. Active oxygen species were detected by cytochemical methods using nitroblue tetra- zolium (NBT) or diaminobenzidine (DAB). The hemocytes showed NBT-reduction and DAB-reaction. Those responses were inhib- ited by superoxide dismutase or sodium avide, respecuively. The cells stimulated with phorbol myristate acetate or zymosan showed significantly higher responses than resting cells, This suggests that the Snail hemocytes produce active oxygen spe- eies during phagocytosis or upon stimula- tion by membrane-activators. Hemolymph samples were tested for hem- agglutination against rabbit, chicken and SMSC Sir welmeOewwoss Oily talolosis COdis were agglutinated and the titers were 8- 32.) this ceacvion wes) anhiibaved™ by EDINA. Bie AMINO ACID SEQUENCE OF STARFISH OOCYTE DEPACTIN. T. Takagi ,. K. onishi!, I. Mabuchi2+3 and H. Hosoya?. BiOd 6. WASetop MACs SCio; Tohoku Univ., Sendai, “Dept. Biol. College Cys Mees EliNcl SOEio5p Winawo Wolryvo, Wolks7©- Natl. Inst. for Basic Biol., Okazaki. a a LG A Amino acid sequence of starfish (Asterias amurensis) oocyte depactin was determined. It is composed of 150 amino acid residues and the N-terminus is free proline. The molecular weight is calcu- lated to be 17,590, in good agreement with the value estimated by SDS-PAGE. Predic- tion of the secondary structure shows that depactin contains 64 % alpha helix. Com- parison of depactin sequence with those of other proteins shows no significant homo- logy. The amino acid sequence of starfish oocyte depactin is shown in Fig. 1. 1 10 20 30 40 PQSGTALDENVKEEITRAFKMDQOSKVKVPWMLLEI VOQNDDR 50 60 70 80 IDVVKVTKKAGPSDNLETLREELKQREVVYFVLDYEPSEE 90 100 110 120 KRAKHNIPKGKTYPLTCFWSMETANIKLKMKYSSTVGTLK 130 140 150 SATSTLKTYLEAHDFDDLSEEAIGDKIKNF Fig. 1. Amino acid sequence of starfish oocyte depactin. Bile IIH GWS OW LRAOS 2 AAI DL, LNOSUWOL, “A, 5= DIPHOSPHATE (PIP,) ON F-ACTIN-SEVERING ACTIVITY OF 45K PROTEIN FROM SEA URCHIN EGGS M.Ohnuma and I.Mabuchi. Dept. of Biol., Coll. of VAmtis 1& Sicily, LU nal v2 ofS okiyio) mu Wolaor se Shialsbicensire poi ed ithe yey Wal ecieias actin-modulating activities of two actin- modulating POC HNS Wwis@ ex Lan and gelsolin. 45K protein is a Ca*t-dependent F-actin- severing protein obtained from sea urchin egies. Wey invelsiet gated) ef fects pola ral E mom the actin-severing activity of 45K protein by measuring high-shear viscosity and the rate of actin depolymerization induced by dilution. F-actin solution was diluted with a solution that contained 45K protein in the presence of Ca F-actin depolymerized rapidly. This was considered to be due to the severing activity of 45K protein. However when 45K protein was preincubated with PIP, before dilution, it did not seem to, sever F-actin even in the presence of Cac Thus, it was considered that PIP blocked the severing activity of 45k protein. On the other hand, other related MAEGRLAILSG, IPI, IPI, IP3 AlmGl IPS, Gaal more affect the severing activity of 45K protein. 45K protein forms 1 to. 1 complex with actin in the presence of Ca + and this complex (45K-A) caps the barbed end of F- actin. 45K-A reduced the depolymerization rate induced by dilution. Preincubation of 45K-A with PIP, before dilution did not affect the Lassa dees the rate. Therefore, PIP5 may not influence the capping activity of 45K-A. Biochemistry 1007 BI 3 2D-PAGE ANALYSES OF C-PROTEIN ISOFORMS EXPRESSED IN DEVELOPING, DENERVATED AND DYSTROPHIC CHICKEN PECTORALIS MUSCLES T. Kojima, K. Sano, and T. Obinata. Dept. Of Bial., Fac. of Sci.) Chiba Univ., Chiba C-Proteins in chicken slow, fast, and cardiac muscles have been distinguished by immunochemical methods, and developmental changes in the expression of C-protein isoforms have been pointed out (Reinach et Hee oGesObrnata ect als; 1984). 9 in’ this investigation, we established a method to characterize C-protein variants on 2D-gel by a combination of the electrophoretic system which were deviced by Hirabayshi (1981) and immunoblotting. We were able to distinguish four C-protein isoforms in chicken striated muscles, based on the difference in size and pI, namely, a fast-type (FC), a cardiac-type (CC), and two slow-type (SC-1 & SC-2) C-proteins. At neonatal ages, both pectoralis (PM) and posterior latissimus dorsi (PLD) muscles expressed FC and SC-1. During postnatal development, expression of SC-1 was down-regulated in PM, while in PLD, SC-1 was replaced by SC-2. Therefore, adult PM contained solely FC, and adult PLD did FC as well as SC-2. Interestingly, however, when the muscles were suffered with muscular dystrophy or denervated, expression of both SC-1 and SC-2 was induced in PM, whereas SC-1 was re-expressed in PLD. As a result, PM and PLD became almost the same with regard to the expression of C-protein isoform. BI 4 DIFFERENCE BETWEEN MAMMALIAN AND CHICKEN MUSCULAR DYSTROPHY AS REVEALED BY TROPONIN T ISOFORM EXPRESSION. K. Takeuchi*, S. Ohshima*, T. Totsuka+, T. Shimizu# and T. Obinata*. *Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba, +Inst. of Develop. Res., Aichi Pref. Colony, Aichi, and #Dept. of Neurol., Fac. of Med., Univ. of Tokyo, Tokyo. We have shown that progression of chicken muscular dystrophy is accompanied with change in troponin T (TNT) isoform expression (Zool Sci, 3, 1066, 1986). The present work was carried out to clarify whether the TNT isoform-change occurs also in mammalian muscular dystrophy. Variations of TNT was detected by 2D-PAGE in combination with immunoblotting. We observed that TNT changes during postnatal development of normal mouse skeletal muscle; at neonatal ages, several spots of TNT were detected, which focused ina acidic pH region, while mature muscle gave more spots which differed in both MW and pl. Progression of muscular dystrophy in two strains of mice, namely dy and mdx, did not cause change in TNT isoform, even 3 months after birth when the symptom of dystrophy was obvious. Several types of human muscular dystrophy were also examined, but the difference in TNT isoform was scarcely detected between normal and dystrophic muscles. These results indicate that the change in TNT isoform can be a marker of the muscular dystrophy of chicken but not of mammals. BI 5 ANALYSIS OF PHOSPHORYLATED GIZZARD MYOSIN WITH PTOTEIN KINASE C BY NATIVE PPi PAGE. H.Takano-Ohmuro, T.Kaminuma and K.Kohama* Tokyo Met. Inst. Med. Sci., Bunkyo-ki, Tokyo and *Dept. of Pharmacol., Fac. of Med., Univ. of Tokyo, Tokyo We previously reported that of the gizzard smooth muscle myosin (GM, un- phosphorylated form) phosphorylated at its regulatory myosin light chian (20,009 Mr, L 0) with myosin light chain kinase (MLCK§ Produces faster moving bands (GMP l:heterodimer myosin with 1 unphosphory- lated Iy5g and 1 mono-phosphorylated Log, GMP2: homodimer myosin with 2 mono- phosphorylated Log S) On navive pyrophos- Phate polyacrylamide gel electrophoresis (PPi PAGE) [J.Biochem. 140, 259-268 & 1681-1684]. However, when the GM was phosphorylated with protein kinase C (PKC) at its Log » Only one band which comigrated with GM was observed on PPi PAGE. In addition, GM sequentially phosphorylated with PKC and MLCK or with MLCK and PKC gave only one band which comigrated with GM on PPi PAGE, We conclude that the mobility increase shown by GM phosphorylated with MLCK on PPi PAGE is not attributable to an increase in the negative charge of Lag due to phosphorylation. This conclusion is also supported by the result that phosphorylated skeletal or cardiac myosin with MLCK did not show mobility increase on PPi PAGE (J.Biochem. 180, 259-268). BI 6 CHANGES OF TROPOMYOSIN ISOFORMS DURING EMBRYONIC DEVELOPMENT OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS. T.Ishimoda-Takagi, S.Koui, T.Maruyama and Y.Fukushima. Dept. of Biol., Tokyo Gakugei Univ., Tokyo. Multiple isoforms of tropomyosin (TM) are present in sea urchin eggs. We examined changes of TM isoforms during embryonic de- velopment of the sea urchin, Hemtcentrotus pulcherrtmus. TM included in the crude egg and embryo extracts were co-precipitated with exogenous F-actin by ultracentrifuga- tion. Egg and embryonic TM isoforms were analyzed by two-dimensional urea-shift gel electrophoresis. Three isoforms of TM were detected in the egg extract. These three TM isoforms were also detected until mesenchyme blastula stage. However, in gastrula extract TM isoforms decreased drastically, and two other components bound to actin instead. Although a third of specimens from prism to early pluteus larvae revealed the gastrula- type pattern, three TM isoforms were restor- ed in the rest of specimens from these stage larvae. The extracts from late pluteus lar- vae retained three TM isoforms. The two com- ponents observed in the gastrula extract were not likely to be degraded products of TM isoforms or stage-specific TM isoforms. We could obtain three TM isoforms from gas- trula embryos as well as unfertilized eggs. These results might suggest that the stage- specific actin-binding proteins which com- peted with the binding of TM isoforms with actin appeared in gastrula stage embryos. 1008 Biochemistry lV LOCALIZATION OF 400 kDa FRAGMENT OF CONNECTIN FILAMENT IN MYOFIBRILS OF CHICKEN BREAST MUSCLE. Y. Itoh, T. Suzuki,K. Ohashi and K. Maru- Veliko Dijendo Enl@ils, was Seis, Clasvoey winslye , Chiba. Connectin is an elastic filamentous protein of MW of three million. It links myosin filaments to Z lines in myofibrils. By proteolytic action, 400 kDa fragments are easily formed and can be isolated by gel filtration. We have prepared polyclon- al antibodies against the 400 kDa peptides. There are two kinds of monoclonal anti- bodies reactive with this fragment (SM1 and 3B9). With an indirect immunofluorescence technique, fluorescent bands in A-I junct- ion region moved toward Z lines, when myo- fibrils were stretched from 2.5 to 3.5 pm sarcomere length. SM1l label also moved, but 3B9 one did not. Immunoelectron micro- scopic investigations are under way. The present work clearly shows that the 400 kDa fragment exists in the connectin filament between the end of myosin filam- ent and A-I junction region. The latter portion is elastic. BI 8 PURIFICATION AND CHARACTERIZATION OF 45kDa ACTIN BINDING PROTEIN FROM BOVINE AORTA SMOOTH MUSCLE. M. Oosawa, S. Shimaoka and K. Maruyama. Dejoes BIOs 7» WAGs SG. , Caailog Unis, Clalloei. During the purification of 84kDa gelso- lin from bovine aorta smooth muscle by the procedure of Ebisawa et al. (Biomed. Res., 6:161-173,1985), a 45 kDa protein was iso- lated by hydroxyapatite, DEAE cellulose, and gelfiltration chromatography. The 45 kDa protein markedly slowed down the polymerization process of actin and elongation step of actin nuclei only in the presence of Ca ions. Peptide mapping clearly showed that it is different from actin. However, the amino acid composition was very similar to that of gelsolin. Gelsolin was easily split into 45 and 47kDa fragments (CT 45; CT 47) by the action of chymotrypsin. The present 45kDa protein had two isoforms one of which was very similar to CT 47, though the chain weights were slightly different. The IP of the 45 kDa protein differed from that of CT 45 fragment of gelsolin. A further work is in progress to clari- fy the relationship of the 45kDa protein with the gelsolin fragments. Bes HEEECLS OE ACHlGKEN I-PROTEIN ON THE RECONSTITUTION OF MYOSIN FILAMENTS. K.Ohashi, K.Ishikawa and K.Maruyama. Dept. Ow WioOl>, WAGs Of Selo, Choa Unaiw.,, Clailoa. I-protein 1S an myofibrillar protein that, as -decalizing ~at) the A-Il junetaonal region of myofibrils and has inhibitory action on the actomyosin ATPase activity under low ionic conditions. Chicken myosin was assembled into filaments under various PH and concentration of KCl. When myosin assembly was carried out by dilution in the I-protein containing solutions, myosin filaments were constituted under higher pH and higher KCl concentration than when assembled without I-protein. For example, myosin filaments were constituted in the solution of 70 mM KCl and 10 mM potassium phosphate buffer, pH 6.5 when equimolar I-protein was added, while not without I-protein. In the solution of 20 mM KCl and 10 mM potassium phosphate buffer, pH 6.5, almost all I-protein could bind to myosin filaments when the molar ratio of added I-protein to myosin was lower than 5% and unbound I-protein coexisted when I-protein was added more than 5%. Approximately equimolar I-protein could bind to myosin filaments when excess of I-protein was added. In this case, myosin filaments were aggregated into higher density clusters than the control experiment without I-protein. Electron microscopic observation revealed that I-protein assembled myosin filaments side by side and end “to end) antopgm@ilarge bundles and sheets. BI 10 PHOSPHORYLATION OF DROSOPHILA MYOSIN LIGHT CHAINS AND ITS EFFECTS ON ATPase PN CUUIEW/ EARNS o S. Takahashil, H. Takano-Ohmuro2 and K. Maruyamal. lpept. Biol., Fac. Sci., Chiba Univ., Chiba, and 2Tokyo Metropol. Inst. Med. Sci.,Bunkyo, Tokyo. Takano-Ohmuro et al. (J. Biochem. ,94: 967-974,1983) reported that Drosophila myosins have different light chain iso- forms in fibrillar(f) and tubular(t) muscl- es; L£1(34kDa), L£2, L£2"(30), L£3(20) and Ie, (91), Wie, Wie2Q" ,_ Wied (2) « The present P32-ATP incorporation experi- ments showed that Lfl, Ltl, Lf2'and Lt2' were phosphorylated. Dephosphorylations of these light chains by the treatment of myosin with two kinds of phophatases resu- IeSrel abso Mela! ilMalaeteys) Mod wae We, AGKe IL IeE Y incl IjeDY GOES iO) IEA, Ibe Siaxores< We haye been able to isolate myosins light chains of which were either complet- ely phosphorylated or dephosphorylated from adult Drosophila flies by modifying preparation procedures. The Ca-activated and Mg-activated ATPase activities of phosphorylated myosin was markedly higher than those of dephosphorylated ones. The present results suggest that Drosophila myosin ATPase activity is regulated by phosphorylation of light chains. Biochemistry 1009 BI 11 CONNECTIN LOCALIZATION AND PASSIVE TENSION GENERATION IN MYOSIN-EXTRACTED MYOFIBRILS OF SKINNED FIBERS OF FROG SKELETAL MUSCLE. T. Suzukil, H. Higuchi*, S. Kimural and kK. Maruyamal. stpept. Biol., Fac. Sci., Chiba Univ. and 2Dept. Physiol., Jikei Univ. Med. EO SOLO De ss Nas iin Sy) ale 51 aay When myosin was partially extracted with 0.35 M KCl leaving the center region of A band in frog skinned fibers, connectin fil- aments remained between unextracted myosin filaments and Z lines. Immuno electron microscopic studies revealed that three stripes in the regions from 0.65 to 0.8 um from the center of A band remained as in intact myofibrils. Resting tension devel- opment decreased only a little. On the other hand, when skinned fibers were treated with 0.6 M KCl, all the myosin molecules were dissolved away leaving the I band intact. Connectin stripes disappeared completely. Instead, there were deposits of antibodies around the region 0.3 um apart from the Z lines. It appears that connectin filaments freed from the M band region were pulled back toward Z lines. The resting tension generation dropped completely by this 0.6 M KCl treatment. Thus the present observations strongly support the view that connectin filaments linking myosin filaments to Z lines are responsible for passive tension generation. Bl 12 MAGNESIUM POLYMERS OF ACTIN FORMED UNDER THE INFLUENCE OF #-ACTININ AND GELSOLIN. S. Shimaoka, M. Oosawa and K. Maruyama, DeSic, BIOL, , MACS Seis, Chaos lpi, Claalloere It is known that Mg polymer is formed when actin is polymerized by Mg ions in the Peesence Of B-actinin, or fragmin. . The Mg polymer shows low viscosity and amorphous aggregates under an electron microscope. This is due to formation of fragile actin filaments of short length. In the present study, the effect of gel- solin was tested if Mg polymer is formed. Viscosity and flow birefringence measure- ments suggested its formation, but these were due to short actin filament formation under electron microscope. -actinin was effective to produce Mg polymers under the same conditions. It was observed that, when phalloidin had been added to actin monomers, Mg poly- mer was not formed in the presence of £- actinin. Short actin filaments were formed. When phalloidin was added to preformed Mg polymer, partial changes into short actin filaments took place. In the presence of #-actinin short and fragile actin filaments are formed and they were deteriorated by uranyl acetate used for negative staining. Phalloidin may stabilize the fragile actin filaments. It is of interest that gelsolin did not result in such weak structure of actin filaments. BI 15 THE ISOMERASE ACTIVITY OF SEPIAPTERIN REDUC- TASE S. Katoh and T. Sueoka. Department of Bio- chemistry, Josai Dental University, Sakado, Saitama 350-02 Sepiapterin reductase is known as. an NADPH-oxidoreductase required for catalyzing the last two step of the biosynthetic pathway of tetrahydrobiopterin (BH,), an essential cofactor for the formation of biological monoamines, from GTP. In the presence of NADPH, sepiapterin reductase can reduce two monoketo tetrahydropterin (PH,) intermediates of BH, such as C1'-keto PH, (6- llactoy VP). and! G2" —ketosrH, (6-16 “hydroxy — 2 -OxXoOpropy PH.) "to “form BH; On! cher other hand, we have found now that the large amount of this enzyme convert Cl1'-keto PH, glee)! Gl -joron~ell Ish, Cideeie osteo Jesh, alin elo absence of NADPH by HPLC-analysis of PH, using amperometric detector. The retention time of the novel PH, on HPLC column was Similar to that of C2'-keto PH,. The novel PH, converted into 6(R)-L-erythro-BH, by the enzyme with NADPH and the oxide of this PEOGUGE ysby., sl, ins yacGad.) \wasy sel erychnro biopterin in HPLC analysis using authentic isomers of four 6(R)-BH, and four biopterins as control. The novel PH, has the keto group since it formed hydrazone. By these results and the recent analysis on '!H-NMR, the structure of the novel PH, was identified as 6 (R) -L-erythro-1'-hydroxy-2'-oxopropyl tetrahydropterin (C2'-keto Daly} c Thus sepiapterin reductase has 6-lactoyl tetra- hydropterin isomerase activity besides the activity of an NADPH-oxidoreductase. BI 14 REACTION PROCEDURE IN THE FORMATION OF TETRA- HYDROBIOPTERIN FROM DIKETO TETRAHYDROPTERIN INTERMEDIATE T.Sueoka', S.Katoh', M.Masada’* and M.Akino®. 'Dept.of Biochem.,Josai Dental Univ.,Sakado, 2Depesok iBiole yy Tokyo) (MetropolactangsUnawe,, Tokyo, *Dept.of Industrial Chem., Kokugakuin Univ. , Tokyo. In the biosynthetic pathway of tetrahydro- biopterin(BH,) from GTP, two monoketo tetra- hydropterin (PH,), C€1'’-keto PH, and C2 -keto PH, were found as the intermediate between pyruvoyl PH, (dyspropterin) and BH,. We have analyzed the reaction procedure in _ the reduction of pyruvoyl PH, to BH, by the function of sepiapterin reductase in vitro. Pyruvoyl PH, was synthesized from dihydro- neopterin P, by the incubation with MgCl,, and pyruvoyl PH, synthase purified from fat bodies of the silkworm. Pyruvoyl PH, sample, which was prepared from its synthesizing reaction mixture after deproteinization through Millipore filter, was added with homogeneous sepiapterin reductase prepared from rat erythrocytes in the presence of NADPH at 37 °C in Tris-HCl buffer (pH 7.4 and 8.6). Time-dependent variation of the PH, components in -the reaction system was measured by HPLC-electrochemical analysis. And C2'-keto PH, was certainely recognized as the intermediate between pyruvoyl PH, and BH, from the observation of the pattern of a typical successive reaction. However, C2'- keto PH, also appeared in addition of the final product BH, when C1'-keto PH, was incubated with this enzyme and NADPH . It might be undeniable that, besides C2'-keto PH,, Cl1'-keto PH, is also the intermediate of this reaction. 1010 Biochemistry Blgels THE RELATIONSHIP BETWEEN PAPILIOCHROME AND THE TYPE A RED) PIGMENT: T. Okabe, K. Morimoto and Y, Umebachi. Dept. of Biol., Fac. of Sci., Kanazawa Univ., Kanazawa. Papiliochromes are the wing-pigments of Papi lo IMiieEciricilies, Wiere are tlmeee kia nds of Papiliochrome, II (pale yellow), M (deep yellow), and R (reddish brown). These pigments are all related to kynuren- ine, dopamine, and @-alanine. The II and M are formed from kynurenine and N-f-ala- nyldopamine. On the other hand, the type A red pigment of Pachliopta aristolochiae Contains A-alanine but is not related) ito kynurenine. The pigment shows a remarkab- le color change with pH, that is, red in alkaline and yellow in acid. The color change occurs at about pH 3. This sugges- ts that the pigment is a kind of quinone. Onvacaduhydroliysas Gl Ne HCI lOOl Ch ae= (5 hr) the pigment releases @-alanine and in- dole compounds. But the possibility also exists that the indole compounds may be secondary products during acid hydrolysis. But there is almost no doubt that the pig- ment is a @-alanine-containing quinone pigment. ‘The possibility has been sugges- ted that the type A red pigment is formed from N-S-alanyldopamine or N-f#-alanylnor- epinephrine. When N-@-alanyldopamine is incubated with tyrosinase, an orange pig- ment is formed. The pigment also shows the color change with pH and releases f- alanine on acid hydrolysis. BI 16 SYNTHESIS OF PAPILIOCHROME II BY PHENOLO- XIDASE FROM A PRAYING MANTID. M.Yago, H.Sato and H.Kawasaki. Dept. of Biochem., School of Dentistry, Iwate Medi- cal Coll., Morioka. Papiliochrome II(P-II), a yellow pig- ment of the wings of Papilio butterflies, was found and has been studied in detail by Y.Umebachi and his coworkers. According to their results, the pigment is composed of kynurenine(Kyn) and N-B-alanyldopamine (NBAD), and decomposes into Kyn and N-8- alanylarterenol(NBANA). It seems that in Papilio xuthus, free Kyn and free NBAD enter the wings from haemolymph and com- bine there. It is unclear which enzymes are involved in the combining process. The results of the present experiment indicated that phenoloxidase from a praying mantid catalyzed the synthesis of P-II from L-Kyn and NBAD. The enzyme from the left colle- terial gland of Tenodera aridifolia sinen- Sis Saussure is involved in oothecal scle- rotization and hydroxylates the sidechain of N-acyldopamines. When L-Kyn and NBAD were incubated with the enzyme in 0.2M 6 phosphate buffer(pH 5.0) for 24hrs at 28°C, yellow pigment was formed. After being purified, it was subjected to various ana- lyses. Its uv absorption spectra and its chromotographical results were the same as that Ort Weil, Oya laverwatiayer alia Ihaldl ielGIL exe 100~C for 40min, Kyn and NBANA were identi- fied as hydrolysis products. Thus the yellow pigment was found to be P-II1. Blealy ACTIVATION OF LACCASE-TYPE PROPHENOLOXIDASE IN THE CUTICLE OF INSECT. IV. PROPERTIES OF THE ACTIVATOR H.1.Yamazaki. Biol.,Lab., Atomi Gakuen Women's Univ., Saitama The phenoloxidase activating system in insect cuticle is supposed to play acentral role of the hardening and darkening process in the course of larval-pupal ecdysis. In this system, the laccase-type of phenol- Oxidase seems to be a key enzyme. The previous observations suggested that laccase in the cuticle might be formed from the pro-form by an activator with proteolytic activity. The changes of the laccase inthe cuticle were analysed by SDS-PAGE during larval- pupal ecdysis. After electrophoresis, laccase including the pro-form was located by immunoblotting method using anti-laccase antiserum. Three immuno-reactive bands were released from the cuticle by SDS treatment at 2 hours after ecdysis, but no bands were detectable after 24 hours. As a preliminary attempt to elucidate the activation mechanism, the activator for pro-laccase was partially purified by ammonium sulfate precipitation and gel filtration from early pupal cuticles. The Suitable temperature for activation was 0°C and the pH was 6-7. Under the conditions, the partial purified activator, which contained proteinase activity, could activate pro-laccase to the active form. BI 18 A FEW ADDITIONAL PIECES OF INFORMATION ON THE B-ALANINE IN INSECT CUTICLE. Y. Umebachi, Y. Tagi and M. Sakai. Dept. of Biol., Fac. of Sci., Kanazawa Univ., Kanazawa. Umebachi (1979, 1984) reported in lepi- dopteran scales and dipteran exuviae that the #-alanine content and the amount of the ketocatechol released on acid hydroly- sis are inversely related to each other. After that, an attempt to investigate the subject using the protein fraction solubi- lyzed from exuviae has been made. As the material, the mutant strains yellow, black and ebony of Drosophila melanogaster were used. The 4 4 HCl-methanol extract of the exuviae were hydrolyzed in acid, and the @-alanine and ketocatechol were determin- ed, The above-mentioned relationship bet- ween #-alanine and ketocatechol has proved to hold true here also. This shows that the HCl-methanol extract can be useful for the investigation of the cross-linking in cuticle. After the extraction with HCl- methanol, the residue was further extract- ed with 1 N NaOH at 25°C. The extract was suitable for the investigation of #-alani- ne but not for the study of ketocatechol. After the extraction with NaOH, the final residue also contained B-alanine, which was small in quantity but high in molar ratio. The final residue may be useful in studying the nature of the bonding bet- ween protein and chitin. Biochemistry 1011 BI 19 STRUCTURES AND ORGANIZATION OF GENES FOR THE MAJOR PLASMA PROTEINS OF Bombyx mori S.Mori, S.Izumi and S.Tomino Dept. Biol. Tokyo Metropol. Univ., Tokyo Genomic sequences for the major plasma proteins (30K proteins) of B. mori were cloned and their structures were analyzed. An EMBL 3 library was constructed from the Mbo I partial digests of larval fat body DNA and was screened for the 30K protein- mRNA sequence with 30K-C6 cDNA probe. Among seven clones isolated, three clones carried tandem array of 30K protein genes 7 Kb apart on a genomic DNA segment, implying that these genes constitute a family. Sequence analysis provided evidence that one of these clones, termed 30K-El bears two 30K protein genes, 6Gl and 21G1 corre- sponding to 30K-C6 and 30K-C21 cDNAS, respectively, in an opposite orientation. mRNA sequencing demonstrated occurence in 6Gl gene of an intron with approximately 800 bases in length in the upstream region adjacent to the protein-coding sequence. Initiation site of the 6Gl gene transcrip- tion was determined at nucleotide level. The 5' flanking segment of the transcrip- tion initiation site contained sequences highly homologous to the regulattory ele- ments predicted for the eukaryotic class II genes, including TATA-box, CAT-box, en- hancer elements and ecdysteroid-receptor binding site. BI 20 HYDROCARBONS OF PHILOSAMTA SILKWORM LIPO- PHORIN C. Katagiri and H. Kihara. Biochem. Lab., Inst. of Low Temp. Sci., Hokkaido Univ., Sapporo. Hydrocarbons are important consti- tuents of insect cuticle and are known to provide a water-proofing layer to protect the insect from desication. Considerable amounts of hydrocarbons have been reported to be present in a circulating lipopro- tein, lipophorin, of cockroach and locust in addition to diacylglycerol and chole- sterol. Lipophorin is known to transport hydrocarbons from the site of synthesis to PiCmESHste On —deposmivon, esg., Cubicle. Lipophorin of Philosamia silkworm diapau- sing pupae, on the other hand, contains only a trace of hydrocarbons. The pre- sent study demonstrates that silkworm lipophorin also transport hydrocarbons and that, after pupal ecdysis, it contains appreciable amounts of hydrocarbons (15% iE E@IEZL IL. Waljojatol jis The pupal lipophorin which contains hydrocarbons was subjected to physicochemical measurements. The data suggest that the silkworm lipohorin after pupal ecdysis is composed of centrosymmet- rical three layers which has been proposed as the possible structure of the cockroach and locust lipophorins; an outer shell with apolipophorin I and phospholipid, a middle layer with diacylglycerol and apo- lipophorin II, and a core with hydrocar- bons. BI 21 SOLUBLE TYPE OF VITELLIN BINDING PROTEIN IN OOCYTE FROM LOCUSTA MIGRATORIA. K.Yamasaki. Dept.Biol.Tokyo Metropol.Univ. Tokyo. Vitellin binding protein(VBP) is involved in the uptake mechanism of vitellogenin into oocyte. VBP locallizes mainly on the surface area of oocyte during vitellogene- Sis and is found in membrane fraction. The VBP is insoluble in aqueous solution. On the other hand,soluble type of VBP exists in the soluble fraction from oocyte homo- genate. One of the functions of the soluble VBP is supposed as a responsible element in condensation apparatus of vitellin. Soluble VBP was precipitated by (NHq4) 2S0q4 from soluble fraction of oocyte homogenate. The VBP was soluble in aqueous solution and the binding activity was immobilized by cellulose acetate. The VB activity was estimated by electro-washing method on cellulose acetate membrane. The VBP frac- tion was treated with 1% sodium dodecyl sulfate(SDS) and VBP was precipitated by the addition of cold acetone. After removal of residual SDS, VBP was found in the pre- cipitate. The precipitate was reextracted with octyl-8-glucoside. VB activity was found in the precipitate and was specific for vitellin. In the extract,microvitellin binding activity was detected and could not find binding activity for vitellin. Ble22 DEVELOPMENTAL CHANGES IN PROPERTIES OF LIPOPHORIN OF THE SILKWORM, BOMBYX MORI. K. Miura and I. Shimizu. Research Section of Environmental Biology, Laboratory for Plant Ecological Studies, Faculty of Science, Kyoto University, Kyoto. Lipophorins were isolated from the hemo- lymph of the males of the silkworm by KBr density gradient ultracentrifugation. The larve and pupae of an early stage had a Single lipophorin of a density 1.12g/ml, which contained two apoproteins. On the other hand adult moths had a low density lipophorin (d=1.06) in addition to the high density type (d=1.12). Total fatty acid content of adult low density lipophorin was more than twice those of larval, pupal and adult high density lipophorin. Adult low density lipophorin contained a large amount of the third apoprotein, apolipophorin-III (apo-III) in addition to the two apoprotein -s. Apo-III was also detected in larval and pupal hemolymph and lipophorin-free Subnatant fraction obtained by the centri- fugation using immunoblotting. It was demonstrated that apo-III content in hemo- lymph increased suddenly on the day before eclosion and that an apo-III-rich low density lipophorin was already constructed in the pharate moths. 1012 Biochemistry Bl 25 LIPID TRANSFER PROTEIN FROM LOCUST, LOCUSTA MIGRATORIA Y.Hirayama, H.Chino. Biochem.Lab.,Inst. of Low Temper.Science, Hokkaido Uiv.,Sapporo. A new protein was found in the hemolymph of the locust, migratoria, which serves to transfer lipid between resting lipophorin and adipokinetic hormone (AKH) activated lipophorin; therefore named lipid transfer protein (IEE) G W@ LEONLACS wis wmwP, a Low Tomiie precipitation , DEAE-sepharose CL-6B column chromatography and density gradient ultracentrifugation were employed. After these steps, an electrophoretically pure protein was obtained. The purified LTP displayed a density of about 1.21 c/ml; thus, LTP may be a lipoprotein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that this protein is comprised of three apoproteins, apo-LTP- I ( MW 310,000), apo-UTP-Ir (MW 89,000), and apo-LTP-III (MW 78,000). Lipid transfer protein may play a role in equilibrating the density of lipophorin by transferring lipids, mostly diacylglycerol, between lipophorin molecules. The ability of LTP to transfer diacylglycerol between AKH-activated lipophorin and resting lipophorin suggests that LTP may also facilitate diacylglycerol mobilization from fat body to flight muscle during flight. BI 24 IN VITRO STUDY OF THE ADIPOKINETIC HORMONE ACTION ON LIPOPHORIN IN LOCUSTS. H. Chino, Y. Kiyomoto and T. Hiraoka. Inst. of Low Temperature Sci., Hokkaido Uni Sapporor Experiments were performed to demon- strate in vitro the action of adipokinetic hormone (AKH) on lipophorin in locusts. The hemolymph was first collected from male a- dult locusts by injecting EDTA-Ringer (flush method) and dialysed against insect Ringer to remove EDTA. The dialysed hemolymph was then incubated with fat body in the pre- sence of AKH. After incubation, the medium was subjected to density gradient centri- fugation for futher analyses. We made the following observations after incubation: 1) The density of lipophorin was shifted from 1.12g/ml to 1.06g/ml. 2) the content of diacylglycerol in lipophorin was ele- vated by several times. 3) Apolipophorin- III (apoIII) became bound with lipophorin. 4) The size of lipophorin became larger and heterogeneous. All these changes are very Similar to those observed in vivo for lipo- phorin after injection of AKH. It was also demonstrated that the above changes require Ca+t essentially. We further tested a re- construction system in which purified lipo- phorin and apolII were incubated with fat body in the presence of AKH. Similar but incomplete changes were observed; we failed to demonstrate clearly the dramatical shift of the density of lipophorin after incuba- tion. Locusta Billy 25 PHOSPHORYLATION OF VITELLOGENIN (Vg) AND VITELLIN (Vn) FROM THE SILKWORM, BOMBYX MORI. S.Y.Takahashi, Dept.of Biol. ,Fac.of Liberal Arts, Yamaguchi Univ. ,753 Yamaguchi. In our previous paper, two types of cyclic nucleo- tide-dependent protein kinases, namely G- and A- Kinases, in silkworm eggs have been reported. The relative activities of them varied significantly during the course of development. In developing egg G-kinase activity was relatively high in the middle of embryonic development, and diminished just be- fore hatching, indicating that the kinase played an important role during embryonic development. In order to know the biological significance of this kinase we have studied about endogenous substrate proteins of the kinase. The silkworm Vn and Egg specific protein ( ESP ) were effectively phospho- rylated both by the highly purified G-kinase. Two moles of phosphate were incorporated in- to vitellin. When the ovarian extract was incubated in the acetate buffer,pH5.2,one of the major ovari- an protein,ESP, was rapidly degraded, whereas Vn was not, indicating that Vn was resistant to prote- olysis by theprotease in the ovary, which had been also characterized in our previous paper as a thiol proteinase. By contrast, if ovarian extract was in- cubated in the phosphorylation reaction mixture with either A-or G-kinase before incubation in the digestion mixture, Vn becameto be degraded. The degradation of vitellin appeared to be closely re- lated to the phosphorylation. From the results we suggested that’ the phos- phorylation of Vn probably lead to the structural changes of the protein molecule and enhanced its susceptibility to digestion by endogenous protease. BI 26 HEMOLYMPH AND EGG PROTEINS OF BEAN BUG. II. PROPERTIES OF INSECTICYANIN FROM EGG AND HEMOLYMPH. Wo Obabaaen! 5S lerewnaa ) 5 ul. Numata? and S. Nakayama? , ' Dept. of Med. Zool., Mie Unive, 2 Dept. of Baolly,/ Osaka CaltiyasUneinvaer. 3Tezukayama Coll., Nara. We purified a blue-protein, insecticyanin (INS egg) from eggs of the bean bug, Riptortus clavatus. Eggs were from 1 to 24 hours old and INS egg was purified by gel filtration and ion exchange chromatography. Molecular weight (MW) of INS egg was esti- mated 320,000 daltons by polyacrylamide gel electrophoresis (PAGE) and 335,000 by gel filtration chromatography. By SDS-PAGE analysis, INS oge showed only one band, MW of which was 76,000. This indicates INS egg is composed of four subunits. INS has absorbance peaks at 280, 370 and 620 nm of wave length which suggestes that INS egg is associated with biliverdin. INS egg does not contain lipid but does sugars; Gas- liquid chromatography analysis shows 13 % neutral sugar, mainly mannose. INS egg has high mole % (17 %) of aromatic amino acids, which suggests close similarity to aryl- phorin type storage proteins. Isoelectric pornbiee Cpl) MoLiNS tere wis 7ach a) (Onelot multe four INS in hemolymph (INS hem) has the same pl as INS egg, but the others are 7.60,'7.40 and 7.25. Almost alll properties of INS hem are the same as INS egg except Jol. Biochemistry 1013 BL. 27 HEMOLYMPH AND EGG PROTEINS OF BEAN BUG. III. IMMUNOLOGICAL PROPERTIES AND QUANTITATIVE CHANGES oF INSECTICYANIN. > H. Numata , Y. Chinzei~ and T. Haruna adept. of Biol., Osaka City Univ., Osaka, Dept. of Med. Zool., Mie Univ., Tsu, We found four insecticyanins (INS 1-4) in the hemolymph of the bean bug, Riptortus clavatus by Western immunoblotting analysis with the antiserum against purified egg insecticyanin (INS egg). Hemolymph INS 1-4 were grouped into two types of rocket (A and B) by rocket immunoelectrophoresis using the same anti-INS egg serum. INS 1 -3 and INS egg was shown to be an A type rocket, but INS 4 was a B type. Quantita- tive changes of hemolymph INS were analysed by rocket immunoelectrophoresis during the diapause (D) and nondiapause (ND) develop- ment of females. In the nymphal stage, both INS A and B were observed at high levels but decreased toward adult emergence to undetected levels. In ND female adults, INS A increased again but INS B did not; in D female adults, INS B did and INS A did not. We also analysed quantitative changes of INS A and B of ND and D female and male whole body. INS A and B at high levels in nymphal stage decreased at adult emergence in all kind of insects (ND and D, or female and male ). In ND female adults, INS A increased again, but not in males. ita 1D females and males both INS A and B increased significantly and accumulated gradually during diapause development. BI 28 CHARACTERIZATION OF DYSPROPTERIN SYNTHETASE FROM FAT BODIES OF SILKWORM LARVAE M.Masada and M.Akino* * Dept.Biol., Tokyo Metrop.Univ., Tokyo Dept. Ind.Chem., Kougakuin Univ., Tokyo The properties of the purified dyspropterin synthetase that catalyzes the conversion of dihydroneopterin triphosphate to dyspropterin in the presence of Mg2+ were further studied. Effect of Mg++ concentration on the activity showed a biphasic pattern. The Km values of Mg2+ for the enzyme were calculated to be 2.0 x 10-3 and 2.1 x 1074M, respectively. The Km patos ©: dihydroncopterin triphosphate at 1 and 5 mM of Mgt were almost the same. Some divalent cations such as Ca2t, Mn2+ and Ba++ also stimulated the enzyme activity. However, when Ca2+ or Ba2+ was added to the reaction mixture containing a low concentra- tion of Mg2+(1 mM), the activity was greatly accelerated. A rabbit was immunized with the homogeneous preparation of dyspropterin synthetase.When examined by Ouchterlony double immunodiffu- sion method the anti-serum preparation formed a Single precipitation line with both the purified dyspropterin synthetase and crude preparation from fat bodies of silkworm larvae. The specificity of anti-serum for dyspropterin synthetase was confirmed by its ability to precipitate the dyspropterin synthetase activity. BI 29 MIP, MICROTUBULE-ASSEMBLY INHIBITOR PROTEIN. IMMUNOLOGICAL AND PHYSICOCHEMICAL STUDIES. S.Kotani, A.Ikai, S.Maekawa and H.Sakai. Dept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo. Se ee ee ee Microtubule-assembly inhibitor protein (MIP) is an acidic protein with an apparent molecular weight of 33,000 which inhibits microtubule assembly in vitro (Kotani, S Murofushi, H., Nishida, E., and Sakai, slo's J. Biochem. 96, 959-969). Anti-MIP anti- body was affinity purified from rabbit anti- MIP antisera raized against chemically mod- ified MIP. MIP localizes in cell nuclei in interphase cultured cells as revealed by immunofluorescent light microscopy. Immu- noblotting shows that MIP exists in a vari- ety of mammalian cells and tissues. Kidney appears to be a better source of TIP than brain, an original source of MIP. Kidney MIP, which is indistinguishable from brain MIP in several respects, is isolated by the same procedure as for brain TIP and physi- cochemically characterized. MIP contains 20% aspartic acid and 25% glutamic acid in consistent with its acidic nature. Hydro- dynamically, MIP is a monomer with Sooow = 1.9 and My = 30,000. The frictional ratio, ftp eindicatrese that. tlPetc mob aa globular molecule but has either an elon- gated or an expanded structure. Spectro- chemical analysis reveals that MIP has a largely unordered structure. *?) BI 30 ANALYSIS OF TUBULIN ISOTYPES IN CILIA AND FLAGELLA. K.Nakamura, Y.Kubo, E.Masuyama and WIKWSAMO, DEE O22 Ibuwvwiing; Seileneces, Hiroshima Women's Univ. Uzina, Hiroshima. Tubulin from diverse sources including cilia and flagella exists in its native form as a heterodimer of one A-tubulin and one @B-tubulin. Moreover there has been much evidence from isoelectric focusing (IEF), immunological methods, and genetic analysis that each tubulin has several isotypes. Recent reports have suggested that the heterogeneity is correlated to the assembly and the various functions of microtubules. Thus, it is important to analyze the isotypes of tubulins of axonemes for understanding the roles of EWMlowIbiing alin CEilmileie inom tsies7, Wim tele present experiments, we have developed an method to analyze the isotypes of tubulin using IEF and SDS-PAGE. This technique has been applied to compare the isotypes of axonemal tubulin between gill cilia and Spermesal age lekalcor josie m, sand aaehic following results have been obtained. (1) Both axonemes consist of identical seven isotypes, but the densitometric profiles of isotypes revealed slight difference between them. (2) Immunoblotting after IEF have revealed the presence of at least three isotypes which react with an antibody against chicken brain tubulin. 1014 Biochemistry Bil St NUCLEOSIDE TRIPHOSPHATE SPECIFICITY OF CILIARY MOTILITY OF TETRAHYMENA ToesHiemiaZule K. FURUSAWAT, Mio BRO U.S? TANAKA! AND M. OKUNO®. ‘Res. Inst. Polym. Text., Tsukuba, Ibaraki and 2Dept. Biol., Coll. Arts & Sci., Univ. Tokyo, Tokyo. The nucleoside triphosphate specificity of ciliary motility was investigated by using Tetrahymena models prepared by NP-40 (detergent) treatment after U. Goodenough (do CSl WiloOls YO C19SS) 1OIOIc Whe Cazyc matic specificity of 22S dynein was also studied with respect to its apparent Km and Vmax for each nucleotide. Some of the nuc- leotides were synthesized chemically and/or enzymatically and their purity was checked by HPLC. Among 11 ATP analogues tested at 1 mM, only 2'-dATP, 3'-dATP and N-methy1l-ATP induced ciliary reactivation but others, 8- Br-ATP, 8-azido-ATP, N,N-dimethyl-ATP, @ATP, formycin-triphosphate (FTP), purine riboside triphosphate (PRTP), periodate- treated ATP (oATP), periodate- and NaBHy- treated ATP (orATP), were ineffective. Those which induced the reactivation were good substrates for dynein with apparent Km's of 10 uM or less and with Vmax compa- rable to that of ATP itself. The apparent Km's for those unable to induce the reacti- vation were higher except FTP. The Vmax value for each nucleotide was lower that that for ATP except for FTP, oATP or 8-Br- ATP. The study indicates high specificity of dynein and ciliary motility toward intact adenine ring. BileoZ ANT-ATP AS A SUBSTRATE OF DYNEIN ATPase AND FLAGELLAR MOTILITY K.Inaba, M.Okuno, and H.Mohri. Department Oe BUIOLOMy,GOMMEeS Ore Wires eine SeuSmees , University of Tokyo,Komaba,Meguro-ku, Tokyo. We synthesized Ant-ATP,which has fluorescent anthraniloyl moiety at OH group of ribose,to elucidate the mechanism of flagellar bend formation and its propagation in relation to mechanochemical Cvele Ox chyneiin AUPASS, Wats elwOrescemec analogue of ATP was efficiently hydrolized by 21S dynein from sea urchin sperm flagella,with Km=50yM which was half of that when ATP was used as a substrate. Vmax was the same with both ATP and Ant-ATP. Inhibition of the hydrolysis of Ant-ATP was a) Itiwelle weelkere Unein thee si wae Case @se ATP,using vanadate.UV cleavage of 215 dynein heavy chains in the presence of Ant- ATP and vanadate was less efficient and the rate of the decrease of dynein heavy chains was 60% of that in the presence of ATP and vanadate.Ant-ATP also induced the motility of demembranated sperm.When ATP was used the apparent Michaelis constant for beat frequency(Kmf) was 0.22mM and the maximum frequency(fmax) was 36Hz,whereas Kmf was 0.14mM and fmax was 20Hz for Ant-ATP.ATP- induced sperm motility was inhibited in the presence of Ant-ATP uncompetitively.These TESUILES SilgeOSt whee ele liseS ieime Ose dynein-ADP-Pi complex is essential for flagellar motility. Bil SD THE PROPERTIES OF INNER ARM ATPASE FROM SEA URCHIN SPERM FLAGELLA S.WADA, M.OKUNO and H.MOHRI Dept., Biol., Coll. Arts & Sci., Univ. Tokyo, Tokyo. pee a Ee ne a, es a In order to clarify the role of inner arms on flagellar movement of sea urchin spermatozoa, an ATPase fraction probably originating from the inner arms was’. par- tially purified. We named it 12S, since the sedimentation coefficient of this fraction was 12S. The properties and functions of 12S were compared with those of 21S dynein (the outer arms). The Michaelis Constants (Km) of 12S was about 4 times of that of 21S ATPase. This indicated that the kinetic characteristic of 12S on ATPase activity was different from that of 21S. And the A-band polypep- tide of 12S was different from that of 21S based on the peptide mappings by V-8 protease. As reported last year, 12S fraction has the ability of rebinding to the outer doub- let microtubules of the axonemes, when it was observed the electron microscopy. That was confirmed not only by electron micrographs but also by SDS-PAGE and the measurement of ATPase activity. The low- salt-extracted axoneme which was incubated with 12S fraction and digested with trypsin, the axoneme regain the ability to slide out the microtubules. This indicate that 12S may be one of the element in the sliding systems of flagellar movement. BI 34 IMMUNOLOGICAL ANALYSIS OF DYNEIN HEAVY CHAINS BETWEEN CILIARY AND FLAGELLAR OUTER-ROW ARMS. E.Masuyama , H.Ishida’, K.Nakamura!, and Y.Shigenaka“. DEI5" O22 Ibo walime Sie@., Hiroshima Women's Univ., Fac. Integr. Arts & Sic., Hiroshima Univ., Hiroshima. “When Tetrahymena ciliary 22S dynein was digested with thermolysin, two protease- resistant fragments which were designated TH-1 and TH-2 were obtained. The fragments were characterized by high ATPase activities and consisted of two principal polypeptides (TH-1: 173K and 80K and TH-2: 173K and 120K). Polyclonal antibodies against these dynein fragments were prepared and used to investigate the Origin of these fragments and the structure relationships between the polypeptides of the fragments. By immunoblotting analysis, anti-TH-1 and anti-TH-2 cross-reacted strongly with y- and @-heavy chain of 22S dynein, respectively. The two antibodies recognized only the 173K polypeptide of each fragment. We also used these antibodies to further investigate the immunological similarities of dynein polypeptides between cilia and flagella. Anti-TH-2(@) cross-reacted with heavy chain of flagellar dynein from oyster sperm, but anti-TH-1(§) showed no cross-reactivity. Thermolytic active fragment originating from @-heavy chain of flagellar dynein was prepared and compared with TH-2 about these polypeptide components. Biochemistry 1015 Bl 35 BeEBets, OF AN ACYLPEPTIDE(K26-S5) ON THE TRITON-EXTRACTED SPERM MODEL. E. YOKOTA'?, IT. MABUCHI', A. KOBAYASHI?, and H. SATO?. Diepaeentol., Coll. Ants and Sci, Univ. Tokyo, 2Sugashima MBL., Nagoya Univ., Toba, and 3Dept. Agric. Chem., Okayama Univ., Okayama. An acylpeptide called K26-5, which was isolated from the culture medium of a soil bacterium Bacillus sp. 503, has been shown to inhibit motility of the Triton-extracted sea urchin sperm models at concentrations below 10 pM, although it does not affect the sliding between outer doublet microtubules in the trypsin-treated axoneme. We investigated the mechanism of K26-5 inhibition of the Triton models of sperm Anthocidaris crassispina. The inhibition of motility was canceled by the addition of both cAMP at concentrations above 1 pM and Pierrot ssOlubhe traction of the wlagel ium: We tried to purify active factors in the fraction with hydroxylapatite and DEAE- cellulose (DE 52) column chromatographies. Consequently, fractions that canceled the Most bteron colncrded wath peaks of activity of cAMP-dependent protein kinase. BoEneImcorporLation Of Pi into the Triton ModcmeanGdetne activity of the partially purified cAMP-dependent protein kinase were tiie DyeKA0-5- From these results ,i1t imeopeemhaced that KZ6-5 affects the cAMP- dependent phosphorylation in the Triton model, and thereby inhibits the mechanism Eitemeconvencs Sliding into bending. BI 36 CONDITIONS FOR MINIMIZING THE DEGRADATION OF PROTEINS IN TETRAHYMENA CELL HOMOGENATE M. Nakamura, M. Hirono and Y. Watanabe leralsite = IBILOAL 6 S@GiLop Univ. of Tsukuba, Ibaraki. As a first step for isolating various actin-binding proteins from Tetrahymena cells, we investigated the conditions for Minimizing the degradation of proteins in the cell homogenate because of awn POwenit endogenous protease activity in Tetrahymena cells. We first adopted the homogenizing solution in which Dictyos- telium actin was successfully purified Giyenusasct als (1978) 0. Biol» Chem. 253, 9088-9096 ), and degradation of Tetrahymena proteins in various conditions, such as presence of triethanolamine, concentra- tions of sucrose and tetra-sodium pyrophosphate, pHs, and temperatures were examined by densitometric analysis of SDS-PAGE. As a result, we finally found that most Tetrahymena proteins detectable on SDS-PAGE remained unchanged aie 4) tore 5 lous alin, ei Solljeiestoyal containing 0.5 % Nonidet P-40, 0.4 mM dithiothreitol, 10 po/ml leupeptin, and 80 mM tetra- sodium pyrophosphate, pH 8.0 Bile 57 ISOLATION AND PARTIAL CHARACTERIZATION OF TETRAHYMENA ACTIN. M. Hirono, Y. Kumagai and Y. Watanabe. Institute of Biological Sciences, Univ. of Tsukuba, Ibaraki. ee Bs De Ded ee a CA BT We have previously reported the cloning and sequencing of the Tetrahymena actin gene and the localization of the gene product. In this report, we succeeded in isolating Tetrahymena actin using an antiserum against the synthetic N-terminal peptide deduced from the DNA sequence as an indicator. The method is as follows. The acetone powder of Tetrahymena cells was extracted "with “a buster ofl dllow aontlc strength. Then, the extract was frac- tionated by the Q-Sepharose chromatography. By further gel filtration (Superose12 prep grade or Sephacryl S-200 HR) and ion exchange (Q-Sepharose) chromatographies and ammonium sulfate precipitation, the actin Was) pumifived “nearly! homegene: ty... The isolated actin could be polymerized into filaments in the presence of 100 mM IXCIL , which showed typical features of ubiquitous actin filaments. We also found that Tetrahymena actin was less effective than muscle actin as an activator of the Mg“t- ATPase of muscle myosin subfragment 1. The results consolidate our notion that Tetrahymena actin we found from gene Eley Sobey Shi eel Use bEY ENCE alin Inavaling, considerable divergency in its amino acid sequence. BI 38 CALMODULIN-BINDING PROTEINS IN TETRAHYMENA CILIA WHICH CAN INTERACT WITH MICROTUBULES J. Ohnishi and Y. Watanabe, Inst. of Biol. pci., Univ. of Tsukuba, Ibaraki. Previously we detected at least 36 kinds of calmodulin-binding proteins (CaMBPs) in Tetrahymena cilia by ‘'5I-CaM overlay method. Therefore, we tried to purify native CaMBPs by CaM affinity column chromatography. However, the yield of CaMBPs was unexpectedly low. When a sample for CaM column was applied on ethyl N-phenylcarbamate column which was known to be used for purification of tubulin, most CaMBPs behaved along with tubulin. So we speculated that the association of CaMBPs with microtubules or tubulin occurred and this was the cause of low yield. To validate the speculation, interaction between CaMBPs and Tetrahymena axonemal microtubules polymerized in vitro was examined by cosedimentation experiment. As a result, at least 6 CaMBPs were cosedimented with microtubules and the ways of interactions were classified into two groups: i) CaMBPs which interact with microtubules only during polymerization (30kD, 26kD, 22kD), ii) CaMBPs which interact with microtubules during polymerization and with polymerized microtubules (69kD, 45kD, 37kD). Such CaMBPs were integrated in and exposed on outerdoublet microtubules. Moaws, wie aS laalelnilyy Ibsakeilsy ieleie ela eee involved ina Ca2+dependent regulation of axonemal motility of Tetrahymena cilia. 1016 Biochemistry Biles DYNEIN-MICROTUBULE COMPLEX. S.OHBA,K.OHHATA AND T.MIKI-NOUMURA. Dept of Biology,Ochanomizu Universit ,TOkKyo. We studied dynein-microtubule inter-— action,using binding of dyuge ATPase iso— lated from Tetrahymena cilia to singlet MTs reassembled from porcin brain tubulin. The interaction was analysed by measuring turbidity with spectrophotometer and den— sity of gel bands in erectrophoresis with densitometer. Sometimes dynein-microtubule complex was confirmed and visualized by electron and dark-field microscopes. At first,we examined whether dynein ATPase promots or increases polymerization of tubulin into MTs. Various conc.of dynein were incubated with 0.8mg/ml tubu— lin. The pellet of formed complex was applied to gel electorophoresis to ana— lyse the amounts of polymerized MTs after centrifugation. We could not find any dif- ference among the amounts of polymerized MTs. When prewarmed solution containing 0.25 mg/ml dynein was added to polymerized MTs of 0O.3mg/ml,turbidity incresed, indicatimg binding of dynein to MTs to form complex. We found that MTs in the complex did not depolymerize after dilution of specimen with buffer solution to below critical concentration of tubulin,while MTs in con- trol experiment depolymerized into tubulin completly. ATP addition to the diluted specimen dissociated dynein from the complex,and brought about complete depoly-— merization of the MTs. On the other hand, addition of Ca ions or incubation at OC decomposed complex into each molecule, dynein and tubulin. These behabior indi- cated that bound dynein molecules kept MTs from depolymerization by dilution,but did not protect them from depolymerizing effect by Ca ion or OC. We are now studying on mechanism of these different response of dynein-microtubule complex to the depolymerizing effects. BI 40 CLONING AND SEQUENCING OF THE GENE FOR TETRAHYMENA CALCIUM BINDING PROTEIN (TCBP). T.Takem sal, Ko Olnartsins , T.Kobayashi~, T.Takagi~, K.Konishi~, Y.Watanabe . Inst. LOW, Salo Winttw7o.) O12 WSwWoVoEey - ibgraki. Natl. Inst. of Health, Japan. Biol. WAS 5, WAGs Ox Selo, Wihivo OL WOMOKY p Sendai. We previously reported the purification and characterization of calcium-binding protein (TCBP) from Tetrahymena (J.B.C.,258,13978-13985,1983). Here, we attempted cloning and sequencing of the gene for TCBP. To design several oligonucleotide probes for the screening of a Tetrahymena genomic library, regions of minimal codon degeneracy were selected from the amino acid sequence of TCBP. Using four different probes, we detected only one common hybridizing band by Southern blot analysis of Tetrahymena genomic DNA fragments digested with restriction enzymes. Under these conditions, the genomic library constructed with pBR322 was screened and several hybridizing clones were isolated. Partial DNA sequence of a clone has revealed that, i) Molecular weight of TCBP is larger than 10 KD, ii) TCBP has at least 3 calcium binding sites, iii) TCBP gene is interrupted by at least one intron (87 nucleotide long) having consensus sequence at both ends (GT-AG), iv) TAA codes for glutamine. BI 41 A RELATION BETWEEN CHANGES IN THE LOCALIZA- TION OF HEAT SHOCK PROTEINS (HSPS) AND AQUISITION OF THERMOTOLERANCE OsNuMatalln) Ge voshuldas |. He Ohballlancd YoWatanabe. ) inst. of Biol. (Seis. Une enor TSM, Aoeicelie , Eiaxcl “Dee, OF BLO. , Joetsu Univ. of Education. Using immunofluorescence microscopy, we have characterized changes in the localiza- tion of hsp71 of T. pyriformis after sub- lethal heat treatment (HT)(34°C). After 1h, hsp71 was localized around the nucleoli. About 2h after the HT, hsp71 migrated from nuclei to cytoplasms, and 3h after the HT, it existed only in a cytoplasmic aggregate. To examine the relationship between these changes and aquisition of thermotolerance, we investigated the effect of a prior sub- lethal HT(34°C) on the viability of cells at 37°C. When Tetrahymena cells in log phase at 26°C were shifted directly to 37C, they did not substantially survive (<13% viability). However, when treated at 34°C for 1h and then shifted to 37°C they sur- vived normally for at least 3h. The length of the 34°C treatment of the cells (1h to 3h) had no differential effect on their subsequent survival properties at 37°C. This indicates no relationship between changes in the localization of hsp71 and aquisition of thermotolerance. On the other hand, in thermotolerant cells, we observed that all of hsp26, 33 and 71 were localized in the large cytoplasmic aggregates and that intensity of the immunofluorescence for each hsp pronouncedly increased. BI 42 THE PROPERTIES OF PROTEASE Tl 3 ¥.Motobayashi ; S.Ihgra , and K.Yoshizato “Dept. of Biochem., Develop. Biol. lLab., Dept. of Plastic Surg.,Kitasato Univ. Sch. of Med., Kanagawa and Dept. of Biol., Tokyo Metro. Univ., Tokyo The tail of a metamorphosing tadpole of Bullfrog, Rana catesbeiana, contains Pro- tease Tl which is a cystein endoproteinase and preferentially degrades actin. This en- zyme hydrolyzes Boc-Val-Leu-Lys-MCA and Bz- Phe-Arg-MCA but neither Arg-MCA (hydrolyzed by cathepsin B) nor Z-Arg-Arg-MCA (hydro- lyzed by cathepsin H). Therefore it differs from these cathepsins. The amino acid composition of this enzyme was compared with those of cathepsinB, H, L and papain according to the composition di- vergence test. Protease Tl was not similar to those cystein proteinase. The purified Protease Tl from the regressing tail muscle was digested by trypsin and three fragments (F3, F12, F23) were obtained. Automatic amino acid sequence determination with these peptides were carried out using an Applied Biosystems sequencer model 470. The sequences of F3, F1l2 and F23 were Asp-Gly- Val-Tyr-Gly-Ala-Glu-Leu-Arg, Asn-Leu-Val- Tle-x-Phe-Asn-Pro and Phe-Pro-Ile-Glu-Glu- Ile-x-Tyr-Leu-Asn-Ile-Val-Asn-Leu-Gln-Pro- Ile, respectively. Homology was searched be- tween these sequences and all entries of National Biomedical Research Founcation data base library. No significant homology was detected, and thereby Protease Tl was con- ENiMaC! EOQ 19S A MEW jDEOeS@aLIo < Biochemistry 1017 BI 43 THE THIOL PROTEINASE FROM METAMORPHOS ING TADPOLE TAIL OF RANA CATESBEIANA. S.Fujita, K.Kobayashi and S.Horiuchi. Epeeeoersinst., Sophia Univ., Tokyo. The proteinase from the tadpole tail of Rana catesbeiana appears to act on the proteolysis in metamorphic tail resorption, and its activity is inhibited by leupeptin, as reported in the 57th annual meeting of the Zoological Society. This enzyme is more purified (650-fold) by S-Sepharose rechromatography and the yield is 35%. The molecular weight of this enzyme is determined by FPLC on Superose as 20,000. This enzyme is separated to double protein bands by 7.5% acrylamide gel electrophoresis. The enzyme activity is inhibited by leupeptin, monoiodoacetic acid, E-64 and p-chloromercuribenzoic acid, which are known as inhibitors for thiol proteinases. Pepstatin, an inhibitor for cathepsin D, and diisopropyl phosphofluori- date (DFP), an inhibitor for serine pro- teinases have almost no effect. L-cysteine activates the enzyme activity on degrada- tion of hemoglobin, and other proteins especially myoglobin degradation. BI 44 MOLECULAR PROPERTIES OF THE EXTRACELLULAR HEMOGLOBIN FROM THE PLANORBID SNAIL, INDOPLANORBIS EXUSTUS. T. Ochiail, I. Usuki2 and Y. Enoki%.!Dept. GbeBlotle, Face of Scil., 2Dept. Gir JBWOl. , Coll. of Gen. Educ., Niigata Univ.,Niigata 950-21. 32nd Dept. of Physiol., Nara Med. Univ., Kashihara, Nara 634 Sedimentation pattern of the native hemoglobin of the planorbid snail, Indoplanorbis exustus, gave a symmetric single boundary of soos (sigh aoe: The molecular weight was determined to be DrOay et 1.0 by sedimentation equilibrium. Tron and heme contents were 0.300 and 2.89%, corresponding to minimum molecular WemeiassOr, a 6), 600 and 21,300). respec— tively. The millimolar coefficient of cyanomet derivative at 537nm was 10.3, and the absorbancy at 280nm of 1% solution of oxyhemoglobin was 21.1. SDS-PAGE in the absence of 2-mercaptoethanol gave one major band, corresponding to a molecular weight of 2.8 x 10°, and many minor bands. On the other hand, in the presence of 2- mercaptoethanol, one major band, cor- responding to a molecular weight of 1.7 x 10°, and several minor bands appeared. thes formes value is) too small for the consideration that the former is the dimer of the latter. From these results, we propose a tentative model that the native molecule is composed of 12 polypeptide chains and each chain contains 8 hemes. BI 45 CHARACTERIZATION OF SARCOPLASMIC RETICULUM FROM SCALLOP Sab see ADDUCTOR MUSCLE M. Abe, J. Nakamura , K. Konishi-an 1G WWensehaiecy a isin(oyls atia\sieg Sleaa? Dept. of Biol., Tohoku Univ., Sendai. A highly active preparation of sarco- plasmic reticulum (SR) from scallop cross- striated adductor muscle was obtained according to the method reported at the 57th Annual Meeting and characterized. Although, fragmented mitochondria were seen in the electron micrograph of the preperation and succinate-cyt.c reductase activity was detected, both Ca uptake and Ca-ATPase activities were not affected by NaN3. Na,K-ATPase and 5'-Nucleotidase activities were undetectable. The optipmum pH for Ca uptake was 7.9 and the uptake was rapidly decreased by increasing pH. At pH 7.5 which was optimum pH for Ca-ATPase, only very low activity of Ca uptake was obtained. Half maximum activation of Ca-ATPase was obtained at ISS x lOG aM free /Ca.PThas value awasvalmos te Simiv longco that for Ca) uptake active tye The activation energy of Ca-ATPase was 20 kcal/mol estimated by Arrhenius analysis over Ghee mange of 0 to) 20° Cs Laneweaverr— Burk plot of Ca-ATPase activity against ATP concentration showed biphasic dependency, two values of Km were obtained 10 pM and 0.1-0.3 mM. The obtained SR accumulated 20 nmol Ca / mg protein in the absence of Ca precipitating reagents. BI 46 COMPARATIVE BIOCHEMISTRY OF THE EJACULATED SUBSTANCES BETWEEN SILKWORM AND FLUIT FLY. M.OSANAI and P.S.CHEN, Dept. Biol., Tokyo Metropol.Inst. Gerontol., 173-Tokyo, Japan; Zool.inst., Univ.Zurich, 8057Zurich, Swiss. Unlike Bombyx mori, Drosophila melanogaster do not form spermatophore for the sperm ma- turation. The sperm acquire motility in the vesicula seminalis. However, in Bombyx, like Drosophila, high GPT activity was found in the male reproductive system and spermatophore, especially in the v. semi- nalis (44 % of total activity). In the male reproductive system of Drosophila, concen- tration of Arg was low, but those of urea and ammonia were high. Secretion of acces- Sary gland contained much phospho-serine. These substances were transferred to female uterus with sperm during mating. Most amino acids increased distinctly at 30 min after the end of mating and declined, suggesting protein degradation in uterus. Like in Bombyx, urea increased at the post-mating period, while Orn showed a rather low con- centration. Orn must be metabolized in part to Glu. In this connection, it is notable that Ala rose markedly at 30 min following mating. As in the silkmoth, the energy metabolism of fruit fly sperm involves also Arg, Orn, urea, Ala and OXO. This is obvi- ous due to the facts that Drosophila sperm are motile at the time of transfer, whereas Bombyx sperm become functionally mature in the spermatophore after their transfer to the female genital tract, bursa copulatrix. 1018 Biochemistry BI 47 BIOSYNTHESIS OF PUPAL CUTICULAR PROTEINS OF Bombyx mori: cDNA CLONING AND ASSAY OF mRNA LEVEL DURING LARVAL-PUPAL TRANSFORMATION. H.Nakato, M.Toriyama, S.Izumi and S.Tomino Dept. Biol. Tokyo Metropol. Univ., Tokyo We have cloned mRNA sequence coding for the pupal cuticular proteins (PCP) of B. mori and studied the change in the PCP- mRNAS during larval-pupal transformation. Three components of chitin-binding PCPs, termed PCP1, PCP2 and PCP3, were resolved by gel electrophoresis of the extract from the pupal integument. Immunoblot analysis of the cuticular extracts indicated that PCPs accumulate in a stage-specific manner in the integument. A Agtll cDNA library was constructed from poly(A) RNA of the epidermal cells immediately after the larval-pupal ecdysis, and two clones were isolated by screening the recombinant plaques with antibody probe. Restriction mapping analysis indicated that cDNA inserts in these clones are structurally different. RNA blot analysis demonstrated that mRNAs for PCP accumulate in the epidermal cells immediately before larval- pupal ecdysis then disappear within two days. The PCP-mRNAs complementary to two CDNA clones were different not only in their molecular size but also in the developmental profiles. These results suggest the possibility that genes for PCPs constitute multi-gene family and each gene in the family responds independently, yet in a concerted manner to the change in endocrine environment. BI 48 CHARACTERIZATION OF THE CONSTITUENT FATTY ACIDS IN PHOSPHATIDYLINOSITOL DURING EARLY DEVELOPMENT IN RANA NIGROMACULATA. M. Ryuzaki. Dept. of Biol., Kitasato Univ. School of Medicine, Sagamihara. Qualitative and quantitative changes in phosphatidylinositol (PI) were analyzed in the eggs, embryos and tadpoles of R. nigro- maculata, at various stages of development. Total lipids were extracted by the method of Folch ('54). Individual phospholipids were separated by thin-layer chromatography on layer of silica gel HR impregnated with sodium carbonate. The weight percentage of PI to total phospholipid and to total lipid was about 8-15% and 1-3%, respectively, during embryonic life. At the early stages of unfertilized egg and two-cell embryo, the predominant fatty acids were palmitic, stearic, oleic and linoleic acid. From the early gastrula stage and beyond, the per- centage of linoleic acid declines and there is an increase in palmitic acid at the 1- position of PI. A large amount of arachi- donic acid was observed at the 2-position of PI in the unfertilized egg, hatching em- bryo and post-hatching tadpole stages, rel- ative to palmitic and stearic acid. Pal- mitic and stearic acid were increased at the 2-position of PI in the other embryo and feeding tadpole stages, relative to arachidonic acid, indicating a shift in these molecular species. Thus, there were marked changes in the positional distribu- tion of the constituent fatty acids in PI during early development of R.nigromaculata. BI 49 EFFECTS OF CYTOSINE ARABINOSIDE ON 2',3!-C- YCLIC NUCLEOTIDE 3'-PHOSPHODIESTERASE (CNP) AND GLUTAMATE DECARBOXYLASE (GAD) IN THE O- RGANOTYPIC CULTURE. OF NEWBORN MOUSE CEREBE- LLUM. D.SATOMI. Dept. of Biol., Coll. of Arts and Sci., Univ. of Tokyo, Tokyo. The quantitative changes of CNP, a mark- er enzyme of myelin, and GAD, the rate lim- iting enzyme for the biosynthesis of y-ami- nobutyric acid (GABA), during early develo- pmental stages in the organotypic culture of newborn mouse cerebellum were examined by HPLC. Explants were incubated in the pr- esence and absence of cytosine arabinoside, an inhibitor of DNA synthesis. Under stand- ard conditions, the activity of CNP increa- sed rapidly from 8 day in vitro (8 DIV) to 22 DIV. Myelin was observed after 15 DIV u- nder light microscope. GAD activity increa- sed linearly from 2 DIV to 22 DIV. In the same period, the amount of GABA also incre- ased and synapse formation occurred. In the presence of cytosine arabinoside(10 ug/ml), the level of CNP activity was low during i- ncubation. Whereas GAD activity increased nearly same manner as control. Morphologic-— ally, myelination did not occur and ependy- mal cells could not observed. From these results, it was supposed that the low CNP activity and the lack of myelin were due to the failure of oligodendroglial differentiation, and that the GABAergic ne- urons in explants could differentiate and synthesize GABA without axonal myelination. BI 50 LIPIDS IN TWO FRACTIONS OF HAMSTER CAUDA EPIDIDYMAL SPERMATOZOA M. Awanol, S. Oshio2, A. Kawaguchi! and H. Mohril. Haves Role, Coll, Airks ame Sci., Univ. Tokyo and 2Dept. Urol., Teikyo Uniiivemnscchian Medi malolksyOn. Hamster cauda epididymal spermatozoa were separated into two fractions in a con- tinuous Percoll-gradient centrifugation. The density of the upper and the lower fractions were 1.04 and 1.10 g/ml, respec- tively. We analyzed the lipids in the two fractions. In both fractions, palmitic (16:0), stearic (18:0), docosapentaenoic (22815) 5 docosahexaenoic(22:6) were found as the major fatty acids. The percentages of these major fatty acids were similar in both fractions. However, the ratio of 18:2 / 18:0 in the lower fraction was higher than that in the upper fraction. Phospholipids were the major lipid class in both fractions (80% of the total lipids). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the major phospholipids (80% of the total phospholipids). The principal poly- saturated fatty acids of the hamster spermatozoa were found in only PC and PE. The ratio of each class phospholipid was similar in both fraction but the total amount of phospholipids in the upper frac- tion was 1.4-fold of that of the lower fraction. The ratio sterol/ phospholipids was 0.17 in both fractions. The change of phospholipids content may reflect on the difference of their density and relate to the process of sperm maturation. Biochemistry BI 51 UPTAKE OF *°V-LABELED VANADIUM COMPOUNDS BY THE ASCIDIAN BLOOD CELLS. : Prater: PeNGechibata.. Ni egerenmerne and T.Ido Biol. Inst., Fac. of Sci., Toyama Univ., Toyama, ar ac. Agr., and 3Cyclotron RI CTR, Tohoku Univ., Sendai, Japan. We have recently reported that among several types of ascidian blood cells, the signet ring cell is the very vanadocyte, that involves in the accumu- lation of vanadium ion, by means of Ficoll density gradient and neutron activation analysis (Michibata ee al, |. Exp. Zool... 1987). In the present experiment, influx of **V-labeled compounds into the blood cells was examined. The blood cells of Ascidia ahodori were incubated with *8\V_vanadate (V) or -vanadyl (IV) ions.in the buffer solution. At time intervals, the cell suspension was loaded onto silicone oil in 1.5 ml microtube and then the cell pellet was recovered by centrifugation. The radioactivity of the pellet was measured by y-coun- ter. Consequently, it became clear that the vanadyl ion (IV) was incorporated faster into the blood cells than the vanadate ion (V) and the influx was satu- rated 2 hours after the incubation. This result indi- cates that the ascidian blood cells have higher affini- ty for vanadium ion in IV oxidation state than that in V oxidation state. Moreover, to clarify which blood cells incorpo- rate selectively *°V-vanadyl ion, the blood cells incu- bated with *°V for 2 hours were fractioned by the Ficoll gradient technique. The result shows that only the signet ring cells incorporate selectively *®V-label- ed vanadyl ion. BI 52 CORRELATION BETWEEN VANADIUM IONS AND TUNICHROME IN THE ASCIDIAN BLOOD CELLS. H.Michibata Biol. Inst., Fac. of Sci., Toyama Univ.,foyama, Japan. Recently, Nakanishi's group isolated a_ tuni- chrome from the ascidian blood cells and determined the chemical structure (Bruening et al., J. Am. Chem. Soc., 1985). Based on the results that the crude tunichromes had an ability to reduce V(V) to V(IV) and existed in the morula cells which had been thought to be the vanadocytes, they have believed that the isolated tunichrome acted as a reductant of vanadium ions when the ascidian blood cells ac- cumulated vanadium from seawater. However, we verified that the morula cells con- tained no vanadium, while the signet ring cells, which contained no tunichromes, accumulated a large a- mount of vanadium ions. Moreover, upon species survey of the tunichrome, it became clear that some vanadium-rich ascidianms had no tunichromes, con- versely, some iron-rich ascidians contained the tuni- chromes (Oltz, Ph.D. thesis, Columbia Univ., 1987). The role of the tunichromes in the accumulation of vanadium in the ascidian blood cells is, therefore, skeptical. Independently, we isolated a vanadium binding substance from the ascidian blood cells, which sub- stance was colorless and could maintain vanadium ions in vanadyl form (IV). We suppose that the VBS acts as a carrier of vanadium ions in the ascidian blood cells. Further study to verify this function is in progress. 1019 BI 53 EXTRACTION OF VANADIUM BINDING SUBSTANCE AND CHEMICAL NATURE OF ITS MODEL COMPLEX. T.Miyamoto , H.Michibata!, T.Numakunai~ and I H.Sakurai Biol. Inst., Fac. of Sci., Toyama Univ., Toyama, Asamushi Mar. Biol. St., Tohoku Univ., Aomori and Fac. of Pharmaceut., Univ. of Tokushima, Tokushima, Japan We reported previously that a vanadium binding substance (VBS) was purified from the blood cells of Ascidia sydneiensis samea. The VBS has an ability to maintain vanadium ion in a reducing form (VO(IV)), contains a reducing sugar and has an apparent affini- ty for exogenous vanadium ions in IV and V oxidation states (Michibata et al., Biochem. Biophys. Res. Com.., 1986). In further study, the similar substance has been extracted from the blood cells of A. ahodori, sug- gesting a possibility that the VBS exists in the blood cells of every ascidians that contain a large amount of vanadium. In order to know the chemical nature of the VBS, using a glucose 1l-phosphate as a representative of reducing sugars, glucose 1-phosphate-vanadyl sul- fate (IV) and -sodium orthovanadate (V) complexes were prepared. When those complexes were loaded onto SE-Cellulose, the former was eluted in the same fractions with the VBS and the maximal UV absorb- ance and ESR parameters observed in this complex were very similar with those in the VBS. The latter also exhibited smaller ESR signals, indicating that the vanadate (V) was partially reduced to IV oxidation state by the complexing with the glucose 1-phosphate. BI 54 COMPARISON OF VERTEBRATE CARDIAC MUSCLE PROTEINS BETWEEN ATRIA AND VENTRICLES M.Oh-Ishi and T.Hirabayashi. Inst. of ILO 5 SSiLop Wisalyo Git WS Eloi, IWoyeeelo pWORMe Metropolitan Univ., Tokyo There has been no attempt to obtain collagen molecules from spieces of sea urchin in an amount which allows chemical and biological characterizations. Sea urchin collagens were extracted and chemi- cally purified from this extract by a salting-out with 10% NaCl, followed by a precipitation with 0.0/2M Na HPO, in) “a neutral pH. The purified collagen was completely digestible with a non-specific protease-free bacterial collagenase. The collagens thus obtained were similar to those of manmals in respects to a SDS-PAGE pattern and amino acid compositions. The collagens of sea urchin have a smaller value. for a specific optical rotation (-310-) and a lower denaturing temperature (23.12 @) ‘than! those’ of manmals) (—400° and 39s Sacer respectively). Immunobloting analyses showed that collagens of sea urchin did not cross-react with anti serums against collagens of type I,I1I,III, IV and V of manmals. BI 56 PROPERTIES OF Ca2+-BINDING 15K PROTEIN LOCALIZED IN THE MITOTIC APPARATUS OF SEA URCHIN EGG H. Hosoya!, 1. Mabuchi2, S. Takagi’, 0. Iwaasa ANG Ke M@mn Sink S- LD ej9 to Ultrastruc. Res., Tokyo Metro. Inst. Med. Sci., Tokyo, Dept. Cell Biol., Natl. Inst. Basic Biol., Okazaki, 2Dept. Biol., Col. Arts and Sci., Univ. Tokyo, Tokyo, and SDD 5IIOlds WAGs Mele, Wola Wiis » Sendai. We have already reported about Ca2t- binding 15K molecular weight protein, which is found to localize in the mitotic apparatus of sea urchin egg, Hemicentrotus pulcherrimus, during mitosis and to play an important role in cell division through the experiment using antibody raised against the 15K protein. In order to clarify the properties of this protein, we studied the effect of the 15K protein on the polymerization of cytoskeletal proteins such as actin and tubulin. The 15K protein inhibited actin polymerization in the presence of Ca2?+., The polymerization of microtubule proteins was inhibited in the absence of Ca2t+t, but was not inhibited in the presence of Ca2+. With tubulin further purified and free from MAPs, the 15K protein had no effect on tubulin polymerizarion both in the presence and absence of Ca2+. BI 5/7 IMMUNOCYTOCHEMICAL LOCALIZATION OF 260K PROTEIN IN TRITON-TREATED CORTEX OF SEA URCHIN EGG. T.Yoshigaki, S.Maekawa and H.Sakai. Dept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo 113. An actin binding protein having an ap- parent molecular weight of 260,000 (260K protein) is purified from the cortex frac- tion of sea urchin eggs. It causes actin filaments to bundle in vitro. Immunoblotting analysis showed that the 260K protein is different from porcine brain fordrin and bovine smooth muscle fil- amin. Several lines of evidences including those from rotary shadowed images indicated clear differences between the 260K protein and sea urchin spectrin. Immunofluorescence microscopy of isolat- ed cortices was performed using monospecif- ic antibodies to the 260K protein. The staining showed the localization of the 260- K protein in fertilization cone, microvilli and cleavage furrow. In unfertilized and fertilized eggs, the staining pattern was well correlated with that of actin filaments visualized by rhodaminyl-phalloidin. The localization of the 260K protein in the iso- lated cortices was investigated more pre- cisely by immunogold staining method. The high density of gold particles was observed just beneath the plasma membrane and along the fibrous structure inside protrusions which seemed to be microvilli. BI 58 PROLIFERATION OF C6 GLIOMA CELLS TO FORM CONDENSED OR LINEAR COLONIES, AND THEIR DIFFERENTIATION BY cAMP ANALOGUE. T. Kobayashi. Department of Biochemistry, Jikei University School of Medicine, Tokyo It is well known that confluent C6 glioma cells alter their morphology from epitherial type to that of astroglia. They usually made round and condensed colonies, though they sometimes happened to make linear colonies presumably because their cell division occurred in one direction and cells were chained in line. To make the above story simple and reproducible, C6 cells were passed many generations and mutants were then screened with respect to morphologies of colonies. Isolated subclones were classified into four groups: type C (Condensed), type S (Slashed), type L (Linear) and type D (Dispersed). All of them expressed differentiated morphologies like astroglial cells especially when they were confluent and trerated with cAMP analogue. Each morphology, however, differed in detail. Type C cells resembled protoplasmic astrocytes bearing spiny processes, while type S cells resembled fibrous astrocytes. Type L cells were bipolar and type D cells were mixture of bipolar, unipolar and fibrous astrocytes. I expect that these subclonal cell types reflect several aspects of glial cell differentiation. Biochemistry 1021 BI 59 AMIDASE ACTIVITIES OF ASCIDIAN (HALOCYNTHIA RORETZI) HEMOLYMPH TOWARD PEPTIDYL-MCAS. K. Tanaka and F. Shishikura, Dept. Biology, Nihon Univ. Sch. Med., Tokyo. Amidase activities of hemolymph plasma of the ascidian, H. roretzi, were tested by various peptidyl-4- methyl-coumaryl-amides (PEPLIDES INSTITULE, INC., Osaka) used as substrates for proteolytic enzyme assays of human blood plasma and other sources. Hemo- lymph was collcted into aseptic plastic tubes and centrifuged at 3000rpm for 5min. The plasma was stored at -80°C. The reac- tion mixture (1ml) conta; ning plasma (50- (COWL), Sissies (Sx Owes) SMS OO wal)) Eyal 50mM Tris-HCl buffered Pantin's ASW(pH8) waS incubated at 25°C for 0 - 120min. The reaction was terminated by addition of 173% AcOH (2ml). The AMC released was measured fluorometrically at Ex.380nm and Em.460nm. The plasma showed amidase activities toward ten substrates out of 26; strongly toward Boc-L-G-R-MCA (substrate for horseshoe crab clotting enzyme, HCC), moderately toward Boc-F-S-R-MCA , Boc-V-P-R-MCA and Boc-V-L- K-MCA, but scarcely toward substrates for KK, Urok, chymotrypsin and cathepsin. The optimum pH was 8. The activity for HCC was increased by the pretreatment of plasma with pieces of tunic which can induce ag- gregation of hemolymph cells. The pre- treatment over 30min, however, decreased the effect. These results suggested that ascidian hemolymph plasma involved a contact-induced enzymatic cascade as well as mammalian blood plasma. BI 60 PURIFICATION OF AMIDOLYTIC ENZYMES FROM THE ASCIDIAN, HALOCYNTHIA RORETZI, HEMOLYMPH PLASMA. F. Shishikura and K. Tanaka. Dept. of Biol. Nihon Univ. Sch. of Med., Tokyo. ee In a previous work we have shown that H. roretzi hemolymph plasma was highly susceptible toward Boc-Leu-Gly-Arg-MCA which was recently introduced asa substrate for the horseshoe crab clotting enzyme. In this report we developed a purifica- tion procedure of amidolytic enzymes toward Boc-Leu-Gly—-Arg-MCA from H. roretzi plasma, including 80% saturated ammonium sulfate fractionation, gel filtration on Toyopearl HW-65F, chromatofocusing on a Mono P column (Pharmacia), affinity chromatography on a p-aminobenzamidine coupled column (ABA-5PW, Toyo-Soda) and a repeated gel filtration on Superose 12 (Pharmacia). Three active enzyme fractions, designat- ed A, B, and C, were obtained from the first gel filtration and only A was used for the further purification. The isolated enzyme was demonstrated to be homogeneous judged from gel filtration and SDS-polyacrylamide gel electrophoresis. An apparent molecular weight of active enzyme was about 40,000 dalton and its iso- electric point was 5.5-5.6 based on Mono P chromatography. Bai OCCURRENCE OF POLYAMINE OXIDASE AND n&- ACETYLSPERMIDINE-DEACETYLATING ACTIVITY IN CARRLSH TESSUESK T. Kumazawa and O. Suzuki. Department of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu. Polyamine oxidase (PAQ) and acetyl- spermidine deacetylase are considered to play a role for polyamine re-utilization after their acetylation in mammalian tissues. In the present study, we have found the presence of PAO and N8-acetylsperm- idine-deacetylating activity in tissues of the catfish, Parasilurus asotus. PAO activity was highest in the intestine, followed by the liver, kidney and brain with Nl-acetylspermine as sub- strate, Substrate specificity was tested for the intestinal enzyme. and the highest activity was found with N!-acetylspermine, followed by Nl-acetylspermidine and N!,nl2- diacetylspermine. The optimal pH and apparent Km value for N!-acetylspermine were 8.3 and 19.6 pM ‘for the intestinal enzyme, respectively. Memeo yale pen mnicdiinesdeacesynlaeaine ac-— tivity was highest in the kidney, followed by EaS Tee So UME: liver, skin and brain with !4c-n -acetylspermidine as substrate. The optimal pH and apparent Km value were 7.9 and 89.6 pM for the kidney enzyme, respectively. These results suggest the presence of a system of polyamine re-utilization after their acetylation also in catfish tissues. BI 62 STRUCTURE OF THE PROCESSING SITE OF RAT LIVER CATHEPSIN D. See Yonezawa DEDE. Ol) ZOOM-t id Cam Orn So CHiaE, Hokkaido Univ., Sapporo. The mature form of cathepsin D is gener- ally thought to be a “two-chain" structure of the light and heavy chains, which are produced by proteolytic cleavage of asingle chain. We examined the structure around the processing site of the single chain form of rat liver cathepsin D and compared it with the known structures of cathepsins D from other mammals. When carboxymethylated rat liver cathepsin D was digested with trypsin, followed by Con A-Sepharose 4B chromatography and gel filtration on Sepha- dex G50, two tryptic glycopeptides were obtained, one of which was homologous to a C-terminal segment of porcine cathepsin D. Two chymotryptic peptides which included the connecting region between the light and heavy chains were obtained from BrCN-treated enzyme by a procedure which involves diges- tion with chymotrypsin, gel filtration on Sephadex G50 and HPLC separation of peptides. A part of the identified rat sequence around the processing site is shown below, in com- parison with the known sequences of porcine (P), human(H) and bovine(B) cathepsins D. (shown in single letters) Rat: GSGSLSGYLSQDTVSVPCK S T LGGIKVE ROO OO QGDDOC OOD SOOOO 97 nO 5 ip) : ’"ESGSLSGYLSQDTVSVPSN 2??? VGGIKVE H : GSGSLSGYLSQDTVSVPCQSASSASALGGVKVE B GSGSLSGYLSQDTVSVPCNPS S S SPGGVTVQ c—— leeinashin H-chain-~? 1022 Biochemistry Bile os WHY DOES THE APHID rRNA LACK THE HIDDEN BREAK ? K.Ogino, H.Eda and H.Ishikawa. Dept.of Biol. GOs Nees & Sere, Uinadye O21 VOSO, WOMO. Due to the hidden break the 28S rRNAs of insects and most protostomes are separated into the two parts. We had found that the break is created by excising RNA fragments with length of several tens of nucleotide ICON Gl CSiwieelasn JORWE Ose lao inweneil eirems- Gript to leave the gap) im the sequence: Among insects aphid species are distinct from the other insects in that their rRNAs invariably lack the hidden break. To know what makes this difference, we con- structed an aphid genomic library and studied the structure of aphid rDNA with respect to the region corresponding to the gap which the other insect rRNAs have in common. Sequence comparison revealed that while the other rRNAs are supposed to form an AU=rich loop) im that sregton, the sconmes > ponding loop of the aphid rRNA is not AU- rich. Nor does the loop of the aphid rRNA contain the UAAU tract that can be a Signal for the introduction of the hidden break, suggesting that in this particular region the aphid rRNA resembles those of deutero- stomes which do not contain the hidden bReake BI 64 AN INTRACELLULAR SYMBTONT OF THE PEA APHID HAS HOST GENE-LIKE REPETITIVE SEQUENCES IN THE GENOMIC DNA. So Sene@ euel Vi USlanicenvel, Wee Oi Biol, , Colle Maes G SGiia, Umawe@xr TOMO, LOO. While intracellular symbionts of the pea aphid had been well studied from the struc- tural and nutritional viewpoints, they were seldommchanactenmzed: asm cenecllceehemenicrsy In particular, nothing has been known about the host-symbiont interaction at the genom- icy Te wielleyAsact hievEeingst Site peut Omsieuid vaste gene expression of the intracellular sym- biont, we characterized its genomic organ- ization. Since we noticed that the isolated DNA of the symbiont contains aphid rDNA- like SECUENES, WE COmSiscvicrecd 2) wenomLE library of the symbiont using EMBL4 as a vector. By screening the) dibrary, with cloned aphid rDNA fragments as probes, we picked up several clones containing sequen- ce homologous to the host sequences. One of the clones was mapped and analyzed extens- ively. Dot-blot hybridization revealed that the cloned sequence occurs about 20 to 30 times in the symbiont genome. Southern blot hybridization suggested that most of the sequences are tandemly arranged in the genome. Since the sequence is highly homologous to those of the host and other eukaryotic rDNA it cannot be the symbiont's own rDNA but likely the sequence which had been trans- KOw~wOG eC CaS MOST WiiSSCC. BI 65 TESTIS-SPECIFIC g-HYDROXY ACID OXIDIZING ENZYME OF DROSOPHILA VIRILIS Y.Taka and T.Aotsuka. Dept. of Biology, Fac. of Science, Tokyo Metropolitan Univ., Tokyo. We investigated g-hydroxy acid oxidizing enzymes of Drosophila virilis with electro- phoresis. When homogenates of Drosophila vVirilis were run on polyacrylamide gels and stained with the reaction mixture of lactate, PMS and NBT, two major isozymes were detected. The one (isozyme A) was found in both sexes, but the other (isozyme B) was found only in males. We performed dis- sections in order to determine the locali- zation of the isozymes. Then, isozyme B existed only in testis. Isozyme B could also be visualized with the reaction mixture of lactate, peroxidase and dianisidine. In anaerobic condition, however, no band appeared with this system. These results suggested that, in the oxidation of lactate by isozyme B, O02 is the electron acceptor and H202 is prodused. Examination of substrate specificity on the polyacrylamide gels showed that isozyme B oxidizes a variety of a-hydroxy acids. Genetic analysises indicated that isozyme A and isozyme B are encoded by different loci on differnt chromosomes. BI 66 ROLE OF CYCLIC GMP IN HORMONAL CONTROL MECHANISM OF CRAYFISH, PROCAMBARUS CLARKII M.Shibata, K.Tozawa, T.Ozakil And T. Ohoka Dep cf Bilol., Hac. of Scei-, Tokyo jMetwo— pol. Univ., Tokyo and !Natl.Inst. for Physi- ol. Sci., Okazaki. A prominent increase of cGMP level was found in crayfish heart and intestine, after administration of CHH(crustacean hyperglyce- mic hormone) fraction of sinus gland in an in vitro experiment. The increased cGMP level was 15 fold in intestine and 40 fold in heart, but cAMP level almost unchanged. Membrane bound and hormone sensitive guanyl- ate cyclase was prepared from heart and in- testine of destalked animal, but the sensi- tivity was rapidly lost after 5 min incuba- tion at 30° as well as the cyclase activity decreased to 1/6 of initial level. CHH fraction showed a stimulatory effect on spontaneous, periodical movement of in- testine and an inhibitory effect on that of heart. These effects were mimicked by cGMP derivatives, dibutyryl cGMP and 8-bromo cGMP, not by dibutyryl cAMP. To investigate the molecular mechanism of hormone action, phos- phorylated proteins were analysed by SDS- PAGE and autoradiography. After incubation of tissue homogenate or membrane fraction in presence of [\y-32P]ATP, several radioactive protein bands were detected. More than 3 bands were hormone dependent and required calcium ion for phosphorylation. Two pro- tein bands from membrane were found to be phosphorylated in presence of cGMP. Biochemistry 1023 BI 6/7 RELATIONSHIP BETWEEN NEURAL PROCESS FORMA- TION AND GANGLIOSIDES IN RAT PRIMARY CUL- TURE NEURONS. 1 1 il M.Ogiso M.Ohta ,S.Hirgno MaNoguchaiss ns Ke Kajiwara , and E.Sugaya . Dept. of Physi- Ole LohowUnivie Sch. Of Med.) Tokyo. “bept- of Physiol., Kanagawa Dental Coll., Yoko- suka. In cerebral cortex from fetal rat, the physiological roles of gangliosides, which are enriched in neural tissues, were exa- mined in primary culture using serum-free medium. Colchicine and cytochalasin B were added to the culture to inhibit process formation. Ganglioside composition during the formation of neural processes was ana- lyzed by extraction in chroloform-methanol, DEAE-Sephadex column chromatography, and HPTLC separation. In colchicine-treated celllsy, GD ganglioside significantly decreased ang process formation was inhi- bited at 10 M. Cytochalasin B caused loop- ing of neural processes and disappearance of GM, ganglioside. In comparison with data obtained using medium containing 15% fetal calf serum, some differences in ganglioside composition were observed. The differences May suggest cell-to-cell interaction bet- ween neurons and glial cells at the gangli- oside level on the cell surface. BI 68 EFFECTS OF METHYLMERCURY ON CELLULAR ACTIVITIES IN TISSUES OF RAT AND HAMSTER. S.Omata, T.Hiranuma, K.Hirasawa and H.Sugano. Dept. of Biochem., Fac. of Sci., Niigata Univ., Niigata. The uptake by and elimination of Hg from tissues of the hamster after administration of MeHg were very rapid compared with the rat, and the brain/blood Hg ratio of the hamster was much higher than that of the rat. These results prompted us to study differ- ences in effects of MeHg on cellular activities of these animals. Among several neurotransmitter enzy- mes examined, only the activity of glutamic acid de- carboxylase in the hamster brain showed a signific- ant decrease at the symptomatic period(14-day). Lipid peroxidation in the hamster brain was increa- sed significantly over the period of 5-14 days, and in the rat brain it showed a significant increase only at the symptomatic period. In the sciatic ner- ves of the symptomatic animals, activities of chol- ine acetyltransferase and acetylchokinesterase were reduced in the rat to the extent larger than in the hamster. Decrease in the activity of GSH peroxid- ase in the hamster brain was greater than that in the rat at 14-day, while GSH S-transferase activity were increased in both brains of two species. In vivo protein synthesis was significantly reduced in the brain regions of the rat at latent and symptom- atic periods, while that of the hamster showed a minor change. Similar difference in MeHg effects between rat and hamster was observed in in vitro amino acid incorporation experiments with the brain post-mitochondrial supernatant fractions obtained from these animal species. BI 69 A ere ataaNe ARR ca STERN RE PHOSPHATASES IN THE MIDGUT OF SILKWORM, BOMBYX MORI, M.Azuma, N,Okada, H.Yamamoto and M.Eguchi. Lab.of Appl,Genet. Dept.of Appl.Biol., Kyoto Inst.of Techn., Kyoto, We have purified the membrane-bound(m- P) and soluble ALP(s-ALP) from the alimen- tary canal(midgut) of silkworms and some properties were investigated. Purification was successfully achieved by introducing the affinity chromatography (Con A-Sepha- rose 4B for m-ALP; 4-aminobenzyl phosphonic as ligands for s-ALP). Both ALPs were homo- genous as judged by polyacrylamide gel electrophoresis(PAGE), They were found to be similar Mr=68,000 in gel filtration and a Single subunit_in denaturing SDS-PAGE with Mr=60,000. They also have similar amino acid compositions and pI(4,8), The pH optima of both ALPs were shown to lie at 10,8(m-ALP) and 9.7(s-ALP), and the m-ALP was extremely stable in pH 10-12 accords with the physiological milieu in Bombyx midgut lumen, The antibody against m-ALP recognized m- ALP but not s-ALP. Rocket-immunoassay show- ed that m-ALP distributed similar levels along the length of midgut except for the most anterior portion. Although we have not determined the distribution of s-ALP immu- nologically, 70% of its activity existed in the posterior midgut. These results suggest m-ALP and s-ALP are distinct isozymes and undertook different roles. BI 70 ATP-DEPENDENT PROTEASE IN BOVINE ADRENAL CORTEX. S.Watabe, H.Kouyama, K.Hara, S.Tatunami, K.Shino and N.Yago. Radioisotope Res. Inst., St. Marianna Univ. Sch. Med., Kawasaki. Mitochondria contain a protease which requires ATP for its catalytic activity. ATP-Dependent pro- tease 1S most abundant in adrenal cortex and we purified the enzyme to homogeneity from bovine adre- nocortical mitochondria (S.Watabe & T.Kimura (1985) J.Biol.Chem. 260 14498). The purified enzyme cata- lyzes protein-dependent ATP hydrolysis as well as ATP-dependent proteolysis. ATP Hydrolysis, however, is not a requisite of peptide bond hydrolysis, Since nonhydrolyzable ATP analogs support peptide degradation. Large proteins, on the other hand, were not degraded in the presence of ATP analogs. Degradation of low concentration of labeled proteins /peptides was enhanced by unlabeled proteins/pep- tides, suggesting that substrate proteins/peptides were allosteric effectors. Activation of proteo- lysis by peptides was observed only in the presence of ATP but not in the presence of ATP analogs. Mitochondria contain both activators and sub- strates for ATP-dependent protease. The protease efficiently degraded a mitochondrial protein. of 27,000 M.W. and some other proteins, while most of mitochondrial proteins were remarkably resistant against digestion with mitochondrial ATP-dependnet protease in the presence of ATP. Fractionation of mitochondrial extract with a Sephadex G-100 column revealed that the 27K protein was eluted at two dif- ferent positions, one at void volume and the other at M.W. 30 - 40K. The protein may be one of natural substrates for the protease and might be involved in some regulatory mechanisms. 1024 Biochemistry Bilal Purification of metalloendoprotease from sea urchin sperm. C.Ohtaka, K.Akasaka and H.Shimada (Zool. Ensite, Hace Sicika ,Unuiv.) Lokyo7, Lokyo)) Se VOUS DS we reported the presence of zn“+-dependent and histone H1-specific metalloendoprotease in sea urchin sperm. In this paper, we describe the prelimina- ry result on purification of this pro- tease. Mature male gonads (frozen, 2.6kg) of sea urchin (Hemicentrotus pulcherrimus) were homogenized in Tris buffer containing 1% Triton X-100. The extract was subjected to three cycles of DEAE cellulose column chromatography. The enzyme activity was eluted from the last DEAE cellulose co- lumn by 10-130 mM NaCl. The active frac- tions were concentrated by 80%-saturated ammonium sulfate, dialysed against 20 mM Tris buffer, and applied to the gel fil- tration through Sepharose CL-6B. Perhaps because of the low ionic strength, the enzyme became aggregated during dialysis with the significant loss of the activi- ty, and eluted from the column as two distict peaks at the high molecular weight regions. SDS-PAGE analysis of this peak fraction revealed the presence of three distinct bands at 40-45 KDa which seemed to correspond to this protease. The protease thus purified preferentially hydrolyzed histone H1, and jts activity was clearly stimulated by Zn“*. BI 72 GROWTH ASSOCIATED HISTONE KINASE OF REGENERATING RAT LIVER NUCLEI. Mo /NSElELS! Dao Ore Witole, Wate, imgste5 Or Radiological Sci., Chiba-shi. Liver histone Hl is phosphorylated at about 24 h after partial hepatectomy, when the DNA synthesis starts. xX irradiation prior to partial hepatectomy inhibits the phosphorylation. Thus, phosphorylation of histone Hl will be related with DNA synthesis. To know the enzymes responsible for the phosphorylation, changes in the nuclear protein kinase activities were examined. The cAMP-dependent protein kinase activity “changedt trom 15) toy 24) h after hepatectomy. But the deviations of the data were so large that the change was not significant statistically. The effect of X irradiation was also non-significant. On the other hand, cAMP-independent kinase (non-A kinase) activity increased from 18 to 24 h after hepatectomy. The increase was not observed in the irradiated nuclei. The non-A kinase activity was not inhibited by isoquinolinesulfonamide compounds, H-7 and HA1004, but inhibited by quercetin. These results indicated that this enzyme would the growth-associated histone’ kinase. However, this enzyme preferred casein as a substrate to histone Hl. The casein kinase activity was inhibited by heparin only partially and it was rather high in the irradiated nuclei. The nuclei may contain a casein kinase (NII) as well. Biles PURIFICATION OF LDH-A and B ISOZYMES FROM THE MEDAKA, ORYZIAS LATIPES. Wig Selsalil 7 Mo IshyOClo=Itaceicldlal , Io ucla , and K. Yamagami . Life Sci. Inst., Sophia Winstw75, MlOlxe) elaicl IWsw75 Jeol, Were, Ilinsic. IRACILOIL 5 SCEILo , Caalloe, Lactate dehydrogenase (LDH; EC1.1.1.27) of medaka is known to consist of A, A', B and C subunits, the genetic and develop- mental expression of which was studied in inbred and outbred fish. Recently, LDH-Bg was preliminarily purified from outbred medaka and some of its properties were studied (Ohyama et al., 1986). We further purified and characterized LDH-Aq and Bg of medaka muscle. Crude muscle extracts were fractionated by an affinity chromatography using Blue- Separose CL-4B. Ba was obtained by eluting with 1 mM NAD-3 mM lactate asa major component. By the following elution using 1 mM NADH, Aq was obtained with other proteins. Each of them was further purified on an HPLC system using DEAE- silica column. The molecular weight (mw) of native Aq and B4 was 117 kd. Mws of the subunits A and B were 27.0 kd and 27.5 kd, respec- tively. The optimal pHs of Aq and Ba in reductive reaction were 6.3-6.5 and 6.4- OriGy, respectively. Substrate-inhibition was observed like LDHs of other animals. The Km values of Aq and Ba were 0.62 mM and 0.18 mM, respectively. BI 74 Purification and Properties of Arginine Kinase Y.Yazawa, Dept. of Nutritional Ph- ysiol., Hokkaido Univ. of Education at Asahikawa, Asahikawa, Hokkaido. Arginine kinase(AK) was purified from smooth muscle of sea-cucumber with ammon- ium sulfate fractionation, DEAE-Toyopearl column chromatography, gel filtration, and hydroxylapatite column chromatography. The preparation appeared to be homogene-— ous on SDS-PAGE. The M.W. of 43K was est- imated from SDS-PAGE and 86K was estimat- ed from gel filtration under physiologic-=- al conditions. According to results of amino acid analysis, sea-cucumber AK con- tained 378 amino acids. The pH-activity curve for the forward reaction showed op- timum at pH 5.0 and the specific activity was 93 Umol Arg/mg. min. In the reverse reaction, a plateau was shown between pH8.5 and 9.6. and the specific activities were 1 Umol Pi mg. min. The highest acti- vation of AK by bivalent cation was obse- D) rved in tne presence of Me” *. Biochemistry 1025 BI 75 DECREASE OF SERUM ARGININE CONC. IN TUMOR -BEARING ANIMALS -Nishikawa Ss. Kusunoki! and H.Namiki2. Life Scie =: Inst., Advance Co., Ltd., Hokyouand =Dept. of Biol., Sch. of Educ, Waseda Univ., Tokyo. We have reported that serum arginine almost undetectably disappeared after partial hepatectomy of rats. Further study demonstrated that the phenominon was due to the elevation of arginase activity in the serum brought out from the regenarating liver. Carcinoma is resemble to regenerating liver in terms of being proliferating. We, therefore, made an attempt to measure the enzyme activity in tumor-bearing animals. Significant elevation of the enzyme activity or decrease of arginine conc. was observed as well in sera of animals (mice and rats) bearing cancer of lung, liver or breast. In case of human, Significant increase of enzyme activity was also noticed in that of patiants bearing same kinds of cancer. Postoperative observation showed that the elevated activity returned to the normal level within a month. Whether the arginase originates in the tumor itself or in its peripheral tissues which has been dameged by its proliferation is not yet clarified. Increase of the enzyme activity may, however, be useful as a marker for cancer or hepatopath. BI 76 HEPARIN-BINDING PROPERTY AND A NEW METHOD FOR PURIFICATION OF A CELL- ADHESIVE GLYCOPROTEIN VITRONECTIN. M. Hayashi!. T. Yatohgo2, H. Kashiwagi?. and M. Izumi!. ‘Dept. of Biol., Ochano- mizu Univ., Tokyo, and 2Inst. Clin. Med., Tsukuba Univ., Ibaraki. Vitronectin (serum spreading HAGLOn, O-PLOvteln) is "a cel l—adhesive glycoprotein in human plasma and connec- tive tissues. Heparin-binding activity of purified vitronectin can be activated by 8 M urea, 6 M guanidine-HCl,. or heat- Mmigeatee G0 eC for Somin ENayashi et_al.. Jo) brechem. -98, 1135 1985) 3) Barnes ‘et deeds prob. Chem. “260, 9117 Cu9s5)1. We found that fresh human plasma con- tained a few % of endogeneous heparin- binding form of vitronectin. Therefore. the heparin-binding vitronectin seems to be a functionally and structurally dis- tinet form of vitronectin. Furthermore, we developed a strik- ingly simple method by which vitronectin was purified from human plasma/serum. Heparin-binding activity of vitronectin in serum was also activated by 8 M urea. The activated vitronectin was purified approximately 250-fold by heparin affin- ity chromatography. This procedure pro- duced 3 - 6 mg pure vitronectin from 100 ml of human serum within 2 days. BI 77 ISOLAT1O0N AND COMPARATIVE ANALYSIS OF LECTINS FROM ALBUMEN GLANDS (A. G. )OF LAND SNAILS. THREE STRAINS OF EUHADRA. O. kheda and ff: broue, pepe. Bro... SRPOKYVo. (Gakwgieha Lint vexhokyvo.-. ReIcie eis rom Oo an snails, Euhadra peliomphata, E. subnimbosa and E. quaesita in feeding, were isolated and Studied biochemically. These tslee tins sagg it riraited Yon dey human A type erythrocyte. The agorutinaviion waseiinhib ite diebyiD= GalNAc and D-GICNAc. Each isolation of three lectins was carried out using affinity choromatography with D- GI c¢NAc-— sepharose 4B. On O° Farrell”~s two-dimensional poly- acrylamide gel electrophoresis (2D-— PAGE), the lectins were composed of two groups of polypeptide spots. They showed charge isomers, which electric points were around 5, and Nay Ory Giroups-vshadsabaurt Bis skKeost morecular weight. These spots were not shown in the 2D—PAGE patterns Oa7A.- Gs ne hlsbie Gina, tio neasoOe hb hia. bo see amount, Of AKEGe inn senueAceGawas SUGGEes ted “lo. Valin ed VWilt heeseason. Otherwise they were shown in that Of ECOG Ss Ov skh arts tenes Kec iimussiunh «Asie. was suggested to migrate to egg. In immunological studies , antibody which raised against isolated lectins “rom. three 100k seem to be cross- linked much more than the smaller ones, but the reaction is not strictly specific for some limited proteins. A DT-containing peptide was isolated from the Pronase digest of NFM and its amino acid composition was determined as (DT/Asx,/ Ser /Glu/Pro,/Gly/Tyr). Another fluorescent substance, Unidentified yet, may be the third crosslinking amino acid. DB 18 FERTILIZATION-INDUCED CHANGE OF THE RESPIRA- TORY RATE IN RED AND WHITE HALVES OF SEA URECHIS EGGS. E. Tazawal, A. Fujiwara and I. Yasumasu@, IBiol. Inst., Yokohama City Univ., Yokohama and “Dept. of Biol., Waseda Univ., Tokyo. Red and white halves of sea urchin eggs obtained by centrifugation exhibited drama- tic change in the respiratory rate following fertilization in the same manner as in normal eggs. CN -insensitive respiration, observed for several minutes after fertili- zation was higher in its rate in red halves than in white halves. CN -insensitive res- piratory systems probably localized in some organellae. The rate of CN -sensitive respi- ration in unfertilized white halves was higher than in fertilized red ones, whereas fertilized red halves exhibited markedly higher respiratory rate than in fertilized white halves. Amount of mitochondria was assumed to be lower in white halves than in red ones, on the basis of the rate of res- piration stimulated by TMPD and DNP. Mito- chondrial Ca**t pool size in red halves was markedly higher than in white halves. Mito Ghondrraly Cacm pool size in red and white halves, as well as normal eggs decreased following fertilization. Mitochondria iso- lated from fertilized and unfertilized eggs, which exhibited the same rate of respiration to each other. have been reported to exhibi- te low respiratory rate when they were char- ged with Ca, Ca release from mitochondria probably contribute to fertilization-induced activation of mitochondrial respiration. 1032 Developmental Biology DB 19 INTERNAL MOTION OF DNA IN MALE PRONUCLETI OF SEA URCHIN EGGS BY FLUORESCENCE ANISOTROPY UNDER THE MICROSCOPE K.Hirano, Hamamatsu Photonics, Tsukuba. Change in the internal motion of DNA in the male pronuclei was investigated by measuring stationary fluorescence iso- tropy under the microscope equipped with many filters. Sea urchin eggs and sperm were stained with 10 pM Hoechst 33342 in tha sea water. Fluorescence intensities polarized parallel and perpendicular to the direction of polarized excitation light were measured by the ultra-high sensitive TV camera and the image proc- essor with rotating the polarizing filter. Fluorescence anisotropy of sperm nulcei on the slide glass was 0.351+0.029 and that of male pronuclei in the fertilized eggs was reduced to 0.340+0.030 at 5 min after insemination. About that time the pronuclei grew spherical. Thereafter pro- nuclei continued swelling gradually, but the value of anisotropy was maintained up to 10 min (0.339+0.013). The value was increased and reached to 0.358+0.009 at 15 min, then returned to the value of 10 min-pronuclei. The pronuclear fusion occurred within this period of time. These results show that the degree of the internal motion of DNA molecules in the male pronuclei is settled in spite of the increasing volume, and that DNA temporarily grow ridid during the pro- nuclear fusion. DB 20 MONOCLONAL ANTIBODIES AGAINST PROTEIN COM- PONENTS IN PARTIALLY PURIFIED MICROTUBULE- ORGANIZING GRANULE (MTOG) FRACTION. K.Ohta, M.Toriyama, S.Maekawa, S.Endo and H.Sakai. Dept. of Biophys. and Biochem., HaCiau) Otten Cala MU EAvee Od WhOK VOM mEhOksy.o) We previously showed that protein com- ponents competent to form asters in vitro could be fractionated from isolated mito- tic apparatus (MA) and whole eggs. We pre- pared monoclonal antibodies against the protein components in partially purified MTOG fraction from whole eggs which was obtained by phosphocellulose column chro- matography. Eight positive clones were screened by immunoblottings. Four clones (HP1,2,5,6) reacted with the major 51-kD protein specifically. Localizations in MA of the antigens for these 4 clones corres- poned to that of the 51-kD protein which was previously reported. HP1 and 2 reacted with the homologous protein species in MAs isolated from several kinds of sea urchin eggs. Molecular weights of these immuno- reactive species were close to 51,000. Other 4 clones generated antibodies against the minor components in the partially pu- rified MTOG fraction. Molecular weights of the antigens were 63,000 (HP3,8), 140,000 (HP4), 100,000;~110,000 (HP7). The 140-kD protein was shown to localize in MA. Microinjection experiments using HP series are in progress to study func- tions of the antigens. ID)s} Zl ASTER FORMATION INDUCED BY ENDOTHERMICAL POLYMERIZATION M. Yamanaka’, F. Tamaki2 and S. Ishizaka2. > Dept. Oh VE hhysS Cis ew Niaiehyosnie a Uineianvae ee) ayaon IMB, © Bi@gil, Seis, Wmv, of Teukuba, Ibaraki. AD padi vot -astiens ais) efonmed m= siinmthte time course between mitosis and cytokine- sis. The position where the cleavage furrow appears is determined by the boun- dary of these asters. Thus, unequality of cell division depends on the relative sizes of the two asters. The principal component of the astral rays is tubulin which polymerizes endo- thermically (in a homogeneous system). We attempted to induce aster formation endothermically in a designed temperature gradient. Tubulin was extracted from porcine brain by Shelanski's method. Joule's heat was generated at the fine tip of a glass microelectrode in a cold solution of tubu- IL atin, The electric current and the tempe- SEEVlIES Oi SMS eEMDwilin SGOlwtciom were seg wilecacd CO COMECoOl Eme region tnat exceeds the critical temperature. The polymerizing process was observed by a polarizing microscope. As a result, astral rays extended radially from the microelectrode tip. When the supply of Joule’s heat was stopped, the astral rays depolymerized. Two sets of astral rays were formed from the tips of two microelectrodes. DB 22 SPIRAL ARRAYS OF MICROTUBULES IN SEA URCHIN EGGS. A. HANAYAMA, and T. MIKI-NOUMURA. Dept.of biology, Ochanomizu Univ. Tokyo. Spiral arrays of mictorubules were obsereved in fertilized eggs of sea urchin; Strongyrocentrotus purpuratus by P.Harris in 1979 and T.E.Schroeder in 1985. We induced the spiral MTs in eggs of sea urchins (H. pulcherrimus, A.manni) with hexylene glycole S.W. (HG-SW).HG has been known as a MT inducing and stabilizing agent.To identify MTs, we stained the eggs with antitubulin-antibody using in- direct immunofluorescence technique. When eggs were exposed to 4% HG-SW after fertilization, radially oriented MTS appeared in the subcortical region of eggs and persisted for more than Ih. In 0.75% to 1.0% HG-SW, the spiral MTs appeared at pronuclear fusion and disa— ppeared at streak stage. The eggs divided into two cells normally.The spiral MTs were not obsereved in blastmeres after two cell stage. Immunofluorescent micro— graphs revealed the three dimendional structure, in which MTs arranged spirally around an axis. Although we tried to find some relations between the spiral axis and the animal-vegetal axis, we were un-— able to find any relation. The spiral MTs were also observed in Ca-ionophore-acti-— vated eggs, but not in ammonia-activated eggs. From these results, we concluded that HG inereased tubulin polymerization to form a similar structure to the spiral arrays of MTs in eggs, induced by temper— ature in S. purpuratus.The appearance and dissappearance Of spiral MTs seem to be programed in the subcortical region of eggs and triggerd by fertilization or activation by calcium. Developmental Biology 1033 DB 23 THE CYTOSKELETON AT THE 4TH CLEAVAGE OF SEA URCHIN EMBRYOS. M. Abe and I. Uemura. Dep. of Biology, Tokyo Metropolitan Univ., Tokyo. At the 4th cleavage of sea urchin em- bryos, the vegetal cells undertake unequal cleavage resulting a macromere and a mi- cromere. Dan found that the interphase nucleus with a tiny aster at its front side of the vegetal blastomere moves to- ward the vegetal pole and forms mitotic apparatus there. The mechanism of this nuclearmovement has not beencleared. So far we identified only one centriole around the moving nucleus by the trans- mission electron microscopy. The mitotic apparatus at the 4th cleavage was hardly observed unless isolated in the microtuble preserving solution. By improving microtuble-preserving and fixing method, we succeeded to observe the mitotic apparatus of the 4th cleavage by immuno- fluorescent microscopy, using anti-tubulin monoclonal antibody. We found two centro- somes at the both sides of the interphase nucleus in each blastomeres of the 8 cells embryo, when the nuclei of the- vegetal cells had not moved yet. Tanaka reported the inhibition of nucleus movement and unequal cleavage caused by cytochalas@in B treatment (Tanaka 1978), Suggesting the svaitalk solle of actin filament in nuclear movement. We had been attempting to retain , and succeeded to observe the actin filaments near the cleavage furrow at the ist cleavage by immuno-electron microscopy. DB 24 STRUCTURAL ANALYSIS OF THE CONTRACTILE RING OF THE SEA,URCHIN EGG. 9 S.Yonemura , Sc USURTEET ST Sel suledseel and La MEVOCIGlOT € Dept. of Biology, Collgge of APesmandaeSct.) .uniIv. oF hokyo, Tokyo, Dept. of Ultrastructural Research, The Tokyo Metropolitan Institute of Medical Science, Tokyo. The contractile ring of the sea urchin by electron microscopy. Cortices of the eggs were isolated on a substratum at telophase maintaining the ultrastructural integrity of the contractile ring. When the section was cut’ tangentially through the contractile ring, highly oriented microfilaments were clearly identified. Some microfilaments were observed to be randomly oriented. The HMM- Sil decoration experiment revealed that these microfilaments are composed of actin and that antiparallel actin filaments were involved in the ring. Each actin filament was seen to originate from granular structures closely associated with the plasma membrane. Between adjacent actin filaments, thin cross-—linkers were occasionally observed. The contractile ring in this preparation was characterized by thick filaments about 12-18nm in diameter, suggesting that myosin molecules were organized into long filamentous structures. These results are disscussed with special reference to the molecular organization of the contractile ring. DB 25 CONTRACTION WAVE ON THE CELL CORTEX OF THE GRASSHOPPER NEUROBLASTS DURING MITOSIS. K.Kawamura. Biology, Rakuno Gakuen Univ., Ebetsu, Hokkaido. Grasshopper neuroblasts divide unequally with a definite polarity to produce a large daughter neuroblast and a small ganglion cell. During prometaphase and metaphase, the spindle located in the cap cell side, thereafter shifting to the ganglion cell Side during anaphase. Therefore, unequal division of the neuroblast along with the cap cell-ganglion cell axis is accomplished by sewor factors; tthe: direction of Tehe Spund le[axis and the |) Shuttanoqueot athe Spindle body. The present study deals with cortical movements of neuroblasts treated with Cytochalasin B to analyse the relation between mitosis and cytokinesis. Cortical movements along nuclear axis or Spindle axis were recorded by projecting the 16mm cine-film of dividing neuroblasts through a narrow slit onto a sheet of photographic paper moving at a constant Speed. In Cytochalasin B-treated neuro- blasts, the periodical contraction wave commenced on the cortex of one pole, and then spread toward opposite pole. The periodicity of cortical movements of the neuroblast seems to be well coincided with mitotic events. Polarity of unequal division may be maintained principally by thie Taction of cell cortex pehroughout Mivoieleceyeles DB 26 ELECTRON MICROSCOPIC STUDIES ON UNEQUAL DIVISION OF GRASSHOPPER NEUROBLASTS N.Yamashiki. Biology, Rakuno Gakuen Univ., Ebetsu, Hokkaido. Grasshopper neuroblasts, which repeat unequal division with a definite polarity, have a contact with cap cells (cc) on one Side of the surface, and with ganglion cells (gc) on the opposite side. The pre- WLOUS EENCIZ OM ENE WihteicaSierwcGewisee Oz dividing neuroblasts showed the electron dense layer beneath the cell membrane on cc-side from late prophase till middle anaphase. The layer, however, was rather indistinct with the routine method of TEM. In the present study, neuroblasts were immersed in glutaraldehyde solution con- taining 0.02% saponin and then treated with tannic acid. The cell membrane on cc-side was well preserved, although that on gc- side was perforated. In the dense layer observed clearly on cc-side during meta- phase and early anaphase, some actin-like filaments ran parallel to the cell surface. At middle anaphase, the cell membrane in all regions was. perforated. As the dense layer on cc-side became also discontinuous, another dense layer with perforations ap- peared on the equator. At early telophase the dense layer in the furrow region became distinct, The cc-=side cortex seems ,to be more resistant to saponin than the gc-side, and the resistability changes as mitosis proceeds. The dense layer which appeared in the equator shortly before cytokinesis may be derived from cc-side cortex. 1034 Developmental Biology DB 27 LOCALIZATION OF ACTIN NETWORKS DURING EARLY DEVELOPMENT OF TUBIFEX EMBRYOS [> Siasimivu, AOGL., Waste, PacCoor SClemee, Hokkaido University, Sapporo 060, Japan. In precleavage zygotes of Tubifex, actin filaments segregate to the animal and vege- tal poles forming the polar actin filament networks (AFNs). In this study the fate of the polar AFNs during early development of Tubifex embryos has been followed using rhodamine-phalloidin. During the first two cleavages, which are unequal and meridional, the polar AFNs are retained at the regions of cells corresponding to the polles of the precleavage zygote; thereby, they are segr- egated to the CD-cell at the 2-cell stage then to the D-cell at the 4-cell stage. As the mitotic apparatus forms in the D-cell, however, the vegetal polar AFN translocates toward the animal pole of the cell where the mitotic apparatus is located and unites with the animal polar AFN there. This re- distribution of the AFNs is impaired by colchicine-treatment, suggesting the invol- WEMNEING Oi MELCPOTVIOUIES 5 WinEiee@euriceie, iwlae unified AFN is found to be associated with nuclear regions of the macromeres of the D-cell line, and finally partitioned to the teloblst-precursors 2d and 4d and an endo- dermal cell 4D. Cytothalasin B-experiments indicate that the AFNs play a cytoskeletal role in generating and maintaining the spa- tial organization of the cytoplasm which gives rise to the intracellular localiza- tion of the cytoplasm and the mitotic appa- ratus orientations. DB 28 THE POPULATION OF GERM CELLS IN THE DEVELOPING QUAIL Y. Araki. N. Yamamoto. Dept. of Physiol {oii Unie Nel Osi Mach hi The numbers of germ cells were estimat ed in the testes and ovaries of embryos from fourth to fifteenth days of incuba- tion. Serial sections on the gonad were examined under a magnification of x400, and germ cells situated in the cortex and medulla counted in every tenth section In order to estimate the total number of germ cells. the counts were simply multi plied by 10. Moreover the proportion of germ cell numbers to volumes of developing gonad(10 *mm*) was estimated and compared with that of male and female, That of the left medulla or right entire gonad in the female was about 52 or 18 cells on the fourth day. about 18 or 8 cells on the ninth day and about 4 or 3 cells on the fifteenth day and showed a decrease against that in the male (about 33 or 18 cells on the fourth day. about 45 or 28 cells ninth day and about 37 or 39 cells on the fifteen th day). On the other hand. that of the left cortex in the female gonad was about 18 cells on the fourth day. about 50 cells on the ninth and fiteenth days. and was similar to that of the male (11 cells on the fourth day. 39 cells on the ninth day and about 54 cells on the fifteenth day) These results indicate that the medulla in the female was morphologically regressed but the medulla in the male developed into a testis and the left cortexes developed into an ovary in the female and temporarilly developed into an ovary-like structure in the male DB 29 INCREASED CELL PROLIFERATION IN THE PLA- CODE OF UROPYGIAL RUDIMENT OF THE QUAIL EMBRYO Ve Fukui. Dep. Biol., Medical College, Tokyo. Tokyo Women’s In early morphogenesis of feathers and scales of avian embryos epidermal placodes are formed and DNA synthesis ceases for a certain period in the placodes. The uropy- gial gland is also one of the epidermal derivatives in birds. In Japanese quail (Coturnix coturnix japonica) embryos epi- dermal basal cells of the uropygial region remain cuboidal until day 7 of incuba- tion. At day 8 they begin to elongate, become columnar, and develop placode-like structure at day 9. To examine the proliferative activity of the basal cells, we recorded positions of °%H-thymidine-labeled epidermal basal nuclei in ARG serial sections using a digitizer connected to a personal comput- er The distribution pattern of labeled nuclei was reconstituted and displayed on CRT as two-dimensional computer graphics. Columnarization of cuboidal basal cells was confirmed in three-dimensional recon- stitution of the same serial sections. Unlike the feather and scale placodes, the uropygial placode possesses no resting period of DNA synthesis. Labeling index of the uropygial placode increased consi- derably compared with that of nonplacode epidermis. No dermal condensation was observed under the uropygial placode. DB 30 THE EFFECT OF HALOTHANE AND ENFLUREN GAS ON THE HATCHED CHICKEN, — ON THE RECOVERY OF LIVER DAMAGE BY SHOSAIKOTO — H. HASEGAWA, H. TANAKA and K. NONOYAMA. Dept. of Biol. Aichi Univ. of Education, Kariya. The authors previously investigated on the influ- ence of halothane gas to the hatched chicken on the relationship between the concentration of the gas and the degree of growth inhibition, the variation of serum LDH and the ultramicrostructure of the chicken liver. In this experiment, the authors studied on the clinical condition of the anesthetic gas ( halothane, 1 % and enfluren, 2 % ) to the hatched chicken. The variation of serum GOT and GPT ( by NADH-UV absorption method ), d-GTP ( by p- Nitroanilid substrate colorimetric method ), LDH pattern and scanning electron microscopic observati- on of liver tissue were investigated on the chicken. The chickens treated with halothane and enfluren gas for 100 hours and after that fed normally with- out anesthetic gas were all died between 8-10 days and their body weight recoveries were not recognized. The serum GOT value of the anesthetized hatched chicken with halothane and enfluren for 100 hours was 67.0-68.4 IU/L unit respectively ( control, 340). GOT values of the chicken of anesthetized and Sho- saikoto-administered were 653-610, 5th day; 625-413, 12th day; 401-376, 22th day respectively. Serum LDH patterns were varied from control to anesthetic gas treatment as follows: control LDH 1, 76.8; 2, 13.0; 3, 5.8; 4, 7.1; 5, 3.4: halothane and enflu- ren treatment for 100 hours LDH i, 37.3-36.2; 2, UPS HU GS8 Si USo SilO)5'53 45 Sos-SaSs OS, 24,226.98 Shosaikoto administration after 22th day LDH 1, MAK SaS7o6R 25 IGcSal4io2s 8, Vo@—Goil8 45 Gs9=7o08 Sy 4.9-5.1 respectively. Developmental Biology DB 31 STRUCTURE AND EXPRESSION OF EMBRYONIC CHICKEN PEPSINOGEN. K. Hayashi, S. Yasugi and T. Mizuno. ZGoh inst. Fac... of Sci... Univ. of Tokyo, Tokyo. Chicken proventricular mesenchyme is known to be able to induce production of an embryonic pepsinogen (ECPg) in proven- tricular and gizzard endoderms and hypo- blast, but not in small intestinal and allantoic endoderms, though they form complex proventriculus-type glands. In the present study, we examined the trans- ecription of ECPg-mRNA in these endoderms by Northern hybridization with ECPg-cDNA as a prove. ECPg-mRNA was transcribed specificaly in embryonic proventriculi in certain period of normal development (maximum at day 15). When the esophagus, proventriculus and gizzard endoderms were recombined and cultured with the proven- tricular mesenchyme, they equally ex- hited ECPg-mRNA. In contrast, the small intestinal and allantoic endoderms did not express ECPg-mRNA in the same experimental conditions, though the proventricular morphogenesis occurred. ECPg-gene was isolated from chicken genomic library and its structure was determined. This will be a useful tool for study what occurs in the process of ECPg induction and why the competence for ECPg expression is differ- ent among various kinds of endoderm. DB 32 PROGRAMMED CELL DEATH AND CELL CYCLE DURING CHICK LIMB MORPHOGENESIS. S. Toné, S. Tanaka and Y. Kato. Dept. of Dev. Biol., Mitsubishi-Kasei Inst. of Life Sci., Machida Reet Sewell maknown sathatw anterdigital: mesenchymal cells are programmed to die during normal limb development of amniote. We have previously shown that (1) Pre- sumptive dead cells were withdrawn from cell cycle and dead ca. 20 hrs. after last DNA synthesis. (2) Administration of BrdU, a thymidine analog at this critical last S phase suppressed the programmed cell death. These results suggest the presence of death-related gene(s). What triggers programmed cell death? In order to delineate the program for cell death, especially early phase, at first we investigated cell kinetics of presumptive dead cells. Interdigital cells at various develop- mental stages were pulse labelled in ovo with BrdU, immunohistochemically stained with anti-BrdU antibody (for detection of S cell) and simultaneously stained with DAPI (for DNA amount), and analysed by cytofluorometry. Individual cells can be assigned into several cell populations in terms of amount of DNA synthesis and DNA amount. These analyses revealed that pre- sumptive dead cells were withdrawn at Gp») phase from cell cycle and gradually loose their genomic DNA, suggesting that cell death may be caused by a failure of transition from Gy to M phase. DB 33 THE GRADIENT OF REACTIVITY TO THE GROWTH PROMOTING ACTIVITY OF ZPA EXISTS IN THE CHICK LIMB BUD H. Aono and H2 ide Biol- inst.,. Tohoku Univ., Sendai. The growth promoting activity of ZPA (the zone of polarizing activity) was examined using in vitro coculture system. The limb bud posterior tissue fragments (containing ZPA) showed stronger growth effect on limb bud meso- dermal cells of anterior region than on those of posterior region, and there existed a gradient of reactivity along the anteroposterior axis. Other frag- ments (anterior, distal or proximal) promotedcell growthinposition-non- specific manner. To compare with the mitogen(s) which could be thought to be secreted by posterior fragment, growth promoting activities of FGF, EGF and insulin were tested in the same culture conditions. FGF showed position-depend- ent growth promotion which is similar to Ehat Yor ©ZPA "while: EGE? and amsulan promoted growth in the cells of any regions to the same extent. Serum-rich medium (10%FCS) also promoted cell growth but the promotion was observed only in the cell culture of central axial region. These results suggest that the cells of posterior region secrete FGF-like growth factor(s), which con- trols normal limb development and experimental duplicate formation. DB 34 LIMB BUD SPECIFIC PROTEIN IS SYNTHESIZED IN EARLY CHICK EMBRYO S.Nashimiya land > Heides | Brols inst, Tohoku Univ., Sendai. From the comparison of protein composition between limb bud and trunk ( as a control ) of chick embryo, a limb bud specific protein was found. Both limb bud and trunk tissues from stages 21-27 embryos were respectively homogenized and fractioned Dy Cente raistuigaikt aon. Nuclear and mitochondrial fraction and microsomal fraction obtained were dissolved in lysis solution and resolved by two- dimensional gel electrophoresis. proteins in gels were visualized by silver staining. Among protein spots from microsomal fraction, there was a spot specific to limb bud. It appeared at stages 22-23 and disappeared at stages 26-27. It was moderately basic protein and its relative molecular mass was about 130kD. There was no limb bud specific spot from the nuclear and mitochondrial fraction. Comparisons were also made between proximal and distal regions and between anterior and posterior regions within a limb bud. But no significant differences were found in any of these comparisons. 1035 1036 Developmental Biology DB 35 CHARACTERIZATION OF THE AV-1 ANTIGEN WHICH SHOWS POSITION SPECIFIC EXPRESSION IN CHICK LIMB _ BUDS. 2 1 1 T,.Momoi“~ and H.—Ide . Biol. K.Ohsugi ’ inst. TohokulUnaw., soendany, Natl. Inst. for NeuroScience., Tokyo. The AV-1 antigen shows position specific expression in the mesoderm of stage m 9-28 chack limbebudisemlmusizage 2S wing buds, the AV-1 antigen expresses in the restricted regions between the radius and ulna, and between the anterior two metacarpals. Here, we characterized the AV-1 antigen molecule. The results of cell fractionation, immuno-blot analysis, indirect immuno-staining and WGA-agarose column chromatography revealed that the AV-1 antigen was a glycoprotein of molecular weight 130 k dalton and was localized on the plasma membrane. The antigenicity of the AV-1 antigen was stable against collagenase or some endo- glycosidase treatments but was not stable against heat (80°C) or some protease treatments. This indicated that the AV-1 antigenicity was due to the protein portion and not the glycoconjugate portion. DB 36 DISTRIBUTION OF MELANOBLASTS IN EPIDERMIS OF CHICK EMBRYO : AN ANALYSIS’ WITH MONOCLONAL ANTIBODY SPECIFIC FOR CHICKEN MELANOBLASTS. K. Kitamura and M. Sezaki. Dept. of Dev. Biol., Mitsubishi-Kasei Institute of Life Sciences, Machida. A monoclonal antibody (III2A6) raised in mouse in response to homogenates of dorsal skin of chick embryo was found to exhibit a unique reactivity towards the retinal pigment epithelium of the eye and the cultured neural crest-derived cells. III2A6 strongly reacted with the melano- blasts with premature melanosome rather than melanocytes with mature melanosome. Melanoblasts from other avian, such as quail, showed no staining with III2A6. Immunoblot analysis showed that III2A6 culture supernatant strongly reacted with a polypeptide of 28KDa in addition to those of 23 and 78KDa. Temporo-spatial appearance of III2A6- positive cells in dorsal region was studied using immunofluorescence assays. These cells: were firstly detected in the overlayer region of neural tube of stage 22, migrated into superficial dermis and homed into epidermis of shoulder part of EnUNnK Of sitagen 23% In the subsequent stages, the regional homing to epidermis of these cells was noted (1) in pteryla and apterium and (2) in feather rudiment. III2A6 is thought to be useful tool for investigation of differentiation and migration of melanoblast. DB 3/ INDUCTION OF FUNCTIONAL DIFFERENTIATION IN CHICK STOMACH EPITHELIUM BY REAGGREGATED PROVENTRICULAR MESENCHYMAL CELLS IN VITRO K. Takiguchi. Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo. We developed in vitro organ culture system for embryonic chick proventriculus to express pepsinogen. Explants were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% COQ). In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was expressed in 6-day proventricular rudiment after 3 days of cultivation. Epithelial-mesenchymal recombination experiments showed that 6-day oesophageal, proventricular and gizzard epithelia express pepsinogen when recombined and cultured with the proventricular mesenchyme, while 6-day small intestinal epithelium does not. These results were consistent with the results obtained by grafting on the chorioallantoic membrane and showed that the culture conditions are permissive for pepsinogen expression. Both 6-day proventricular and gizzard epithelia expressed pepsinogen when recombined and cultured with reaggregated proventricular mesenchymal cells. These results indicate that the reaggregated proventricular mesenchymal cells retains inductive potency for pepsinogen expression of stomach epithelia. DB 38 RE-ORGANIZATION OF F-ACTIN AND CELL POLARITIES IN ESOPHAGEAL EPITHELIUM OF THE CHICK EMBRYO. T.Fujimoto, R.Murakami and I.Yamaoka. Bilolks nist, Hace. SCisy,.) Vamacuchndm Uneavaen, Yamaguchi. The lumen of esophagus of the chick embryo closes at 5-6 days of incubation and re-opens at 6-8 days. The morphology of the epithelium changes from pseudo- stratified epithelium to compact mass of cells, then to simple columnar epithelium. Changes in the distribution of F-actin and ultrastructures associated with the mor- phogenesis of the epithelium were studied by use of rhodamine-phalloidin and TEM. In the open area, F-actin was intensely detected just under the cell membrane near the apical junctional areas of the epithelial cells. When the lumen became closed, F-actin was scarecely found in the epithelial cell mass, and the polarities of epithelial cells except the basal cells became disorganized. Re-opening of the lumen was initiated as the appearance of several intercellular spaces in the cell mass. F-actin reappeared around the Spaces. Apical junctions also formed. The basal-apical polarities of the epithe- lial cells were restored around the spaces. The spaces fused or enlarged to form the lumen. These results suggest that the re- organization of the F-actin and apical junctional complex is essential for the establishment of the lumen. Developmental Biology 1037 DB 39 DIFFERENTIATION OF THE EPITHELIUM LINING GIZZARD-DUODENAL JUNCTION OF THE CHICK EMBRYO. S.Matsushita. Dept. of Biol., Tokyo Women’s Medical College, Tokyo. The differentiation of the epithelium lining the junctional region of gizzard(G) and duodenum(D) of the chick embryo was studied. SEM observation showed that from 10 days of incubation villus formation proceeds in D and G-D boundary can be noticed at the point where D leaves G. Mucus secretory activity of G epithelium, as revealed by alcian blue-staining in paraffin sections and phosphotungstic acid(PTA)-stained gran- ules in ultrathin sections, was prominent at 6 days and later. PTA-stained granules were found also in the anterior and middle region of D at 6 and 8 days. At 10 days, epithelial cells with the granules in D were reduced in number, and at later days they were only occasionally found. The immunofluorescence study with the affinity- purified anti-sucrase antibodies showed that the sucrase appeared only in D from 10 days. Thus, at early stages the anterior and middle part of D contains epithelial cells with G-like differentiation, which are almost lost when intestinal (D-like) differentiation manifests in D. Immuno- electron microscopy of chorioallantoic grafts of 6-day G-D fragment revealed that there exist some cells with both G- and D- like differentiation characters(PTA-stained granules and sucrase) in G-D boundary. DB 40 TROPOMYOSIN ISOFORMS IN CHICKEN GIZZARD SMOOTH MUSCLE. Mantosioy ail: 5, T.Ishimoda-Takagi* and T.Hirabayashi' init Ome uO. 8Sci. Unaiv. Of Tsukuba, Ibaraki, *Dept. of Biol., Tokyo Gakugei Univ., Tokyo. In the chicken gizzard smooth muscles of 7,10,14 day embryos we found four high- and six low-molecular-type tropomyosin(TM) isoforms in addition to the &- and 4- ones. These proteins were identified as TM by (1) LS OS 1S Gis is ake Hj oOabianer (2) anomalous electrophoretic mobility in the presence of urea, (3) stability against heat treatment, (4) inclusion in Bailey's tropomyosnin PadeE Ton (>) soandumg jaba lity. io smusicile AGE EAMG (6) 1 ChOSS reactivity, with antisera against TMs from chicken gizzard, skeletal muscle and brain. im ElNne differentiation of gizzard smooth muscle, the ratio of accumulated TM to ¢-actin was found to be reasonably constant after hatching, while it appeared to be lower than expected during the embryonic deveropment (7-14 days). The relative anoumes sof. the asoforms | found ~ ain | thas experiments at the earlier stages were predominant enough to bring up the ratio to f-actin to those observed at the stages after hatching. Mhepeac oOlonacdsisn’ ait e accumulation of actin and TM was expected even at the embryonic stages, just as observed after hatching. DB 41 COORDINATE ACCUMULATION OF TROPONIN SUB- UNITS IN CHICKEN BREAST MUSCLE. M. Kawabata and T. Hirabayashi. INNES Che Biol {scr , Univ. of Tsukuba, Tbaraki. The accumulation of troponin subunits in developing chicken breast muscle was determined by two-dimensional gel electrophoresis and an image analyzing system. All troponin T isoforms, including those hidden behind creatin kinase, were detected on the two-dimensional pattern by the addition of 6M urea in the second dimension SDS=polyacrylamide gel electrophoresis. These troponin T isoforms were classified into four types in develop- mental order by the isoelectric point and molecular weight: leg-muscle type (L), neonatal breast-muscle type (BN), young chicken breast-muscle type (BC) and adult breast-muscle type (BA). L-, BN-, and BC- type troponin Ts were transiently expressed at specific ranges of developmental stages. BYASIEVjOOQ jicieGjOO Malin W, ieWtieSie COcCeGiEe@E AE around 7th day after hatching, was retained in adult. Three kinds of individual varia- tions were detected in the way of BA-type expression, while troponin C and troponin I gave no change during development or among individuals. Quantitative analysis of two- dimensional patterns of troponin subunits showed a moderate coordination among the three components all through postnatal development, when total amount of all the types of troponin T was considered. DB 42 LOCALIZATION OF AN EMBRYO-SPECIFIC MYOSIN LIGHT CHAIN (L-23) IN SMOOTH, CARDIAC, AND SKELETAL MUSCLE CELLS IN VITRO. Y. Ogasawara, K. Takaoka and T. Obinata. DEE OL WiC, Gniwos: Wiasiws5 5 Cao. A novel myosin light chain (MLC), L-23, exists in various muscles in chick embryo and also in nervous tissues. As a first step to clarify the role of L-23, we examined its location in cultured muscle cells by an immunofluorescence method. We prepared two monoclonal antibodies (McAb), EL-64 & EL-49, with L-23 as an immunogen; the former was specific for L-23, while the latter crossreacted with alkaline MLCs of smooth, cardiac, and skeletal muscles, and of nonmuscle cells. The McAbs were applied to cultured gizzard, ventricular, and pectoralis muscle cells. In the case of gizzard, both EL-64 and EL-49 stained stress fiber-like structures. Both McAbs stained brightly young cardiac myocytes to give striations. When the McAbs were applied to skeletal muscle cells, EL-64 stained myoblasts and young myotubes without giving any particular structures, although EL-49 gave a typical striated pattern. Cardiac myocytes and skeletal myotubes cultured for about a week were hardly stained with EL-64. Thus, the assembly of L-23 into the structures differed among the various muscles. We assume that the difference in affinity between L-23 and various myosin heavy chains is responsible for such phenomena. 1038 Developmental Biology DB 43 DISASSEMBLY AND REASSEMBLY OF MYOFIBRILS DURING DISSOCIATION AND REASSOCIATION OF CARDIAC MUSCLE CELLS M. Vagily sli Abe) Sulsukaitalandils Obimaital Previous investigation has shown that when muscle tissue was dissociated with trypsin, myofibrils are disassembled, but they were recovered without protein synthesis as muscle cells reaggregated (Fischman 1970). In the present study, we re-examined myofibrillar disassembly and reassembly during dissociation anda reassociation of chicken embryonic heart cells as to 1) how cell adheion affects the initiation of myofibrillar assembly and 2) whether depolymerization of actin filaments into G-actin is caused during disassembly of myofibrils. By means of electron microscopy, we observed that when cell aggregates were formed in rotating cultures, assembly of myofibrils was initiated mostly at the site of cellular adhesion, suggesting that cell adhesion Site can be a trigger for the assembly. Amount of G-actin as well as total actin in the cells were measured before and after dissociation of the tissues and during cell aggregation by DNase I inhibition assay. Either total actin or G-actin scarcely differed in amount during disassembly of myofibrils. However, free actin filaments in the cytoplasm increased significantly. Therefore, we suggest that actin filaments were not depolymerized but become free from Z-line during disassembly of myofibrils. DB 44 CYTODIFFERENTIATION OF THE PROSTATE-LIKE GLANDULAR EPITHELIUM INDUCED FROM URINARY BLADDER EPITHELIUM OF THE ADULT RAT. N. Suematsu, Zool. Inst. Fac. of Sci. Univ. of Tokyo, Tokyo. Urinary bladder epithelium of the adult rat formed prostate-like glands, when com-— bined with fetal urogenital sinus mesen— chyme and cultured beneath the renal cap- sule of male rat hosts. With the progres— sion of the gland formation, the bladder epithelium lost its alkaline phosphatase activity and antigenicity against anti- functional bladder epithelium—antiserun. Like the normal prostate, the induced epi- thelium expressed acid phosphatase, non- specific esterase and antigenicity against anti-human prostatic acid phosphatase-— antiserum, but did not show androgen re- ceptors nor an antigen against anti-4week— ventral prostate epithelium—antiserum. The SDS-PAGE patterns of proteins in the glan- dular epithelium induced from the urinary bladder epithelium and in the normal uri- nary bladder and prostatic epithelia revealed that the induced gland loses some bands identified in the bladder epithelium and became to express other bands similar to but not identical with those of the nor- mal prostatic epithelium, suggesting that the induced gland does not differentiate as completely as the normal prostate. It is possible that this is linked with the ab- sence of androgen receptors in the induced glandular epithelium. DB 45 LOCALIZATION AND CHARACTERISTICS OF MICROTUBULES, IN MATURE MYQTUBES IN CULTURE. O54 Saaicoa 4 7 Wo Ooltmatea 5 iMOOl, LHS. , IVor ene Seal Gp Winalya Che Wel aio, Wel aixe) ein! Dept On BL Ola ChavbaUnkivin aCe In the previous report, we have shown that microtubules are the cytoskeletal element which is most reponsible for elongation of developing myotubes. In this study, the distribution of microtubules in well-developed myotubes were examined by an indirect immunofluorescent microscopy. When mature myotubes were reacted with anti-tubulin antibody, fluorescence was observed with striations along myofibrils as well as with filamentous structures parallel to the longitudinal axis of the cells. Similar striation patterns were also observed when myotubes were treated with the antibody to MAP2. Microtubulular fraction was isolated from chicken embryonic muscles in the presence of taxol and the constituents of muscle microtubules were examined by SDS-PAGE and immunobotting. We detected a MAP2-like peptide which was 300 kDa in size and reacted with anti-MAP2 antibody in addition to tubulin. Treatment of myotubes with either colcemid or taxol caused local accumulation of actin which led to modulation of myofibrillar structures. From these observations, we suggest that microtubules exist in association with myofibrils and play some role in supporting the myofibrillar structures. DB 46 THE CYTOSKELETAL SYSTEMS AND LOCOMOTION OF EPITHELIAL CELLS—A POSSIBLE ROLE OF MICRO-— TUBULES. S.Takeuchi. Zool.Inst.,Fac.Sci.,Univ. Tokyo, Tokyo. In order to know the role of microtubule during locomotion, the epithelial cells in culture were treated with colcemid, and the effects of it were examined with the methods as follows: phase contrast micro- scopy, interference reflexion microscopy(I RM), secondary immunofluorescence method, and cytochemistry. The epithelial sheets were completely inhibited to spread along the surface of Millipore filter(pore size, 0O.3yum), when cultured for 96hr on Wolff—-Haffen's medium containing colcemid(>0.05x%g/ml). The mar— ginal cells of epithelium explanted on the glass surface, started to retract, when cultured more than 40 min after transferr- ed into a medium(L— 15, 20% horse serum) containing colcemid(0.05yg/ml). The loss of focal and close contacts under the lead- ing lamella was confirmed with IRM. In the cells, no fibrous structures were recog-— nized either with anti-tubulin anti-—body or with rhodamine phalloidin, suggesting not only microtubules but F-actin were dis- integrated with colcemid. From these, microtubules were supposed to be responsible not only for mechanical maintenance of the leading lamella but for the organization of focal contacts from which the microfilament bundles were re- ported to elongate(Fiichtbauer et al.1983). Developmental Biology 1039 DB 47 RECONSTITUTION OF HYDRA DISSOCIATED CELL-- REEXAMINATION OF EXPERIMENTAL METHODS AND ABOUT INITIATION OF HEAD FORMATION DURING RECONSTITUTION. K.Noda and C.Kanai. Lab of Electron micro- scopy , Tokyo Metropolitan Inst. of Gerontol. The effect on reconstitution of aggre- gates of dilution ratio of dissociation medium was examined in P. robusta. M-29 solution (Its components were reported in 1986) is able to be diluted to 20% with 1mM TES buffer for reconstitution to perfect form in aggregates larger than 300p. Compo- nents and their ratio of M-29 are the same as those of the culture solution for indi- viduals. Difference in ratio of Ca ion in reconstitution medium is influential for survival ratio in aggregate smaller than 250m during reconstitution. Hours required for tentacle bud forma- tion in reconstitution were compared with those of regeneration of tissue fragments with two cell layers and mesoglea. As a result, it is suggested that aggregates larger than 400p can be initiated for head formation within 6-8hrs after the begin- ning of reconstitution. The mesoglea is not found even under electron microscope in 6-8 h-aggregates, which have partially arranged endodermal epithelial cells along the ecto- dermal epithelial cells covered on the entire surface of aggregates. These phenomena are discussed briefly in contrast to those of embryogenesis. DB 48 NERVE NET FORMATION IN REGENERATING TISSUES OF HYDRA I. PATTERN FORMATION OF RFamide-LIKE IMMUNOREACTIVE NERVE NET. O.Koizumi & Y. Nakashima, Physiol. Lab., Fukuoka Women's Univ., Fukuoka. The pattern formation of hypostomal nerve net was examined in regenerating head of hydra. Using a whole mount technique with indirect immunofluo- rescence, the spatial pattern of neurons showing RFamide-like immunoreactivity (RLI) was visualized. An antiserum to RFamide binds to two subsets of neurons in head: sensory cells of the apex of the hypostome and the ganglion cells of the lower hypostome and the tentacle. During initial stages of head-regeneration, only the ganglion cells arose at the regenerating tip. At later stages, the sensory cells began to appeare at the apex of the dome and at the same time the ganglion cells disappeared from the apex and relocated at the side of the dome close to the location of the tentacle ring. Head-regeneration deficient mutant, reg-16, shows very poor and delayed appearance of RLI+ sensory cells during head regeneration, while the appearance of RLI+ ganglion cells are normal. These results and the behavior af tentacle specific antigen visualized by a nongelonal antabody, LSl9.) support. ‘the two part pattern during head regeneration" (Bede et als, 19865 1987). DB 49 THE DEFORMATION AND POLYMORPHISM OF POLYPS FORMED BY CONTACT BETWEEN TWO SPECIES OF HYDROZOAN'S COLONIES. H.Namikawa and Y.Kakinuma. Dept. of Biol., Fac. of Sci., Kagoshima Univ., Kagoshima. Two species of hydrozoans, Stylactis sp. and Leuckartiara sp., were transplanted to One petri dish and observed the influence of mutual interference on the formation of colonies of both species. After the contact of two colonies. Regardless of deformation, both species still coexised on the same petri dish. At the contacted part of stolons of two species the ectoderm tissues became thick, and the number of nematocysts of two species was increased. The nematocysts formed a line in the stolon; each nematocyst facing to each one of the other species. The regression and regeneration of tissues were observed between 24 and 48 hours after the discharge of nematocysts. Fertheremore the elongating stolons of two species contained each other. In the case of Stylactis sp., polyp budding form polyps and abnormal develop- ment of tentacles were also founed. When the amount of food for colonies were reduced to a half, the diffrentiation of finger like polyps gave rise and polymorphism of polyps was also observed at the contacted part of stolons of both species. DB 50 ELECTRON MICROSCOPIC STUDIES OF THE REDIFFERENTI- ATION OF COPULATORY APPARATUS FROM THE DEDIFFEREN- TIATED CELLS ORIGINATED IN THE OLD COPULATORY APPARATUS IN A FRESHWATER PLANARTAN, BDELLOCEPHALA BRUNNEA. M. Kudoh and W. Teshirogi. Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. The regression of the copulatory apparatus of planarians is caused by the excision of tne regen- erating head-blastema at an intervai of three days after decapitation. In this case, the copulatory apparatus disintegrated to cell clusters, then dissociated to neoblast-like cells and finally transfonned into ordinary mesenchymal cells in 60 days after decapitation. When a head of the worm which has regressed the copulatory apparatus was regenerated by discontin- uance of excision of the head-blastema, a new cop- ulatory apparatus was redifferentiated from the dedifferentiated ceils, that is, the neoblasi-like cells reaggregated to clusters and then differen- tiated to penis and other parts of the copulatory apparatus. In this redifferentiation of the penis, new secretary epithelial cells were made from dedif- ferentiated celis originating in the old penis and the same holds good for nerves and muscles, How- ever, ii was not clear whether the neoblast-like cells are multipotent cells or totipotent cells. This problem is yet to be soived in the future. 1040 Developmental Biology DB 51 WOUND CLOSURE IN PLANARIAN EPIDERMIS. I1. SCANNING ELECTRON MICROSCOPIC EXAMINATION OF THE PROCESS OF EPITHELIZATION. So disjass, Waly, Ceili Sei,, Cem, MES, ILAO6, Fukushima Med. Col., Fukushima. The process of epidermal wound healing in planarians has not yet been fully un- derstood, several different hypothetical views being prevalent. This study con- sists of SEM examination applied to D. ja- ponica regenerants in various stages of the healing (O min. to 40 hrs. postamputa- tion). In order to avoid agonal distor- tion of specimens during fixation, they were quickly fixed with sulphuric acid (1.2 sec.). Owing to the initial wound contraction (for 10 min. after amputation) many epidermal rugae develop around the wound, giving rise to a special rim of confused array of epidermal cells. Cell crowding in the rim seems to stimulate migratory activities of epidermal cells toward the wound. Two steps of epidermal migration could be distinguished: 1) Ini- tially formed marginal aggregates of cells, freed from the basement membrane, extend locally across the wound to form a very thin incomplete provisional covering called "epithelioid" (30 to 60 min. post- amputation). 2) The ensuing steady ad- vance of migration, probably due to a total sliding mechanism of the epidermis, results in a swallow of the wound, entire- ly replacing the degenerating epithelioid (24 to 40 hrs. postamputation). DB 52 THE INSINKING PROCESSES OF NUCLEI OF PHA- RYNGEAL EPITHELIAL CELLS IN PHARYNX REGE- NERATION OF PLANARIAN, DUGESIA JAPONICA. Bo ABEL 5 WEj5 Oar WalOllo 4 KAME WEVA Macs Wins, » Ishikawa. The insinking processes of nuclei of pharyngeal epithelial cells were observed in the pharynx regeneration in the head piece cut transversely in prepharyngeal region of planarian, Dugesia japonica, and the following results were obtained. (1) About 6 days after the cut, the nuclei of the outermost epithelial cells of pharyn- geal rudiment begin to sink into underlying muscle cell layers. (2)Several basal nucleie as well as cytoplasmic projections seem as if they were trying to find suitable spaces between the muscle cells. After finding the most suitable space for sinking, the cells retract the other projections and concentrate on one projection with which to penetrate the muscle cell layers. (3)In the cytoplasm of insinking cells, numerous free ribosomes, several chromatoid bodies, many mitochondria(Mt.), rod-shaped bodies, rER, Golgi complexes, a few microtubules, small vesicles and glycogen particles were ob- served. (4)Around the insinking nucleus several Mt. were present. This suggests that much of the energy needed for insink- ans abs SHU yodstecl ly MiE>> (SD)Ome Ox jelne acl vantages of insinking of the cell nucleus seems to be that apical cytoplasm can ex- tend much longer without the nucleus than with the nucleus when the pharynx is pro- truded to take the food. DB 53 HISTOCOMPATIRILITY IN TWO SPECIES OF FRESH- WATER SPONGES, EPHYDATIA MULLERI AND EPHYDATIA JAPONICA. Y. Watanabe and Y. Yanagihara. Dept. of Biol., Ochanomizu Univ., Tokyo. The taxonomic position of two freshwater sponges, E.mulleri and E.japonica has been identified by the roughness of their skele- WL SjoLCVlLOS, 2o6oS55 Was Oise Mas awouyzia. Spiny megascleres and the latter has smooth ones. Because of skeletal variations be- tween two species, there have been discus- sed that E.japonica should be a variety of E.mulleri and synonymize with the latter. By the use of histocompatibility between these two species, we aimed to approach this problem outside spicular morphology. We examined the fusibility in the sponges derived from gemmules of 18 specimen from 6 localities. In two specimens with similar Spicule composition in the same population, they were each other compatible fusible; that in ones with different spicules, they did not fuse. Cytotoxic rejection reactions occured even between the sponges in the same population. On the other hand, the sponges with similar spicular composition from different locality, were found to be incompatible. These results showed that the morpholog- ical variations of megascleres may be influ- enced to some extent by environmental fac- tors, though it is rather considered to be dominated genetically. Another approaches should be required to make sure that the incompatibility is interspecific rejection on anitirals peci mac One. DB 54 DEVELOPMENT OF COELOMIC CELLS, GAMETOGENE- SIS AND GONADOGENESIS IN ASCIDIA AHODORT. Y.M.Sugino & M.Ishikawa. DSHe5 Cie Biol, BEGoa. Ore Calo pF Ehime Univ., Matsuyama. The differentiation of coelomic cells, gametogenesis, and the organogenesis of the ovary in the ascidian Ascidia ahodori were studied by light microscopy. Paraffin sections stained by the azan method showed that the adults have at least 6 types ,of coelomic cells: the lymphocyte-like, blue, purple, brown- vacuolated, single-vacuolated, and red morula cells. The lymphocyte-like cells are already observed in the swimming larva. All other kinds of the cells appear 20-43 days after metamorphosis. The single-vacuolated cells, which seem to be the so-called signet ring cells, devel- op later than the other vacuolated cells. Early oocytes appear in the 43-day individual whereas spermatogenic cells are detected initially in the 80-day individ- ual. Both mature eggs and spermatozoa, however, become observed simultaneously in the 100-day individual. Formation process of female reproductive organs is divided into the following 3 steps. When the Ovarian primordium becomes observed first, it shows a hollow epithelial structure composed of an oogenetic and a non- oogenetic part. The ciliated epithelium of the ovarian epithelium differentiates in the 60-day individual. Finally, forma- Exon wot ithe OvaAlduct als compiletedt a nis ehe 100-day individual. Developmental Biology 1041 DB 55 REINITIATION OF MIOSIS IN ASCIDIAN EGG BY THE INJECTION OF CALCIUM ION. N. Sensui. Zoological Inst., Faculty of Saami Unive of Tokyo, Tokyo. a In Ciona savignyi, an injection of CaClg (more than 20mM, 65pl) into unfertilized egg induced the deformation of the egg and the lst polar body formation like a fertilized egg. These changes were also induced by the injection of Ca*+-EGTA. However, the injection of KCl, NaCl or MgClg did not induce these changes. In the egg injected with CaClg, microvilli elongation and aggregation were observed as seen in the normal fertilized egg. In the eggs injected with EGTA (200mM, 65pl), these changes were suppressed for 60 min after insemination, then the deformation occurred slowly and abnormal- iyoebuen che ist polar “body was’ not formed. Most of the eggs injected with CaClo did not form the 2nd polar body after the lst polar body formation. In these eggs, the 2nd polar body formation was often induced by insemination or the treatment with Ca2+-ionophore. These results suggest that the deformation and the lst polar body formation need Ca2t and that the Ca@+ plays an important role in the changes after the fertiliza- tion and also that there are at least two steps in the miotic process of the ascidian egg. DB 56 SELF-FERTILITY OF THE ASCIDIAN, CIONA SAVIGNYL. lie Numakunail, Z. Hoshino” and M. Hoshi>. 1Mar. Biol state.) Hac. of Sex. Tohoku Univ.;, 2Dept. of Baolembacemohelduc. iwate Unive. slabs of Broil: Fac. of Sci. Tokyo Inst. of Technol. In the species C. savignyi, which spawns at dawn, self—-fertility of gonad eggs, oviduct eggs and eggs released under natural light cycle was surveyed. To observe self-fertilization of the re- leased eggs two methods were used: (1) spawning in running sea water and catching the eggs in a trap, (2) spawning in a glass container. In the glass container a high percentage of self—fertilization was observed, whereas in the trap almost all the eggs were not self—fertilized. Each animal showed a different rate of self-fertilization from day to day. In the glass container, embryos at different developmental stages were observed. When the ani- mals with great self-fertility were transferred to a trap in running sea water, almost all the eggs were found unfertilized. To get gonad eggs, C. sa- vignyi gonads were dissected, and fully matured eggs were selected. Fertilization was confirmed by observing cleavage 1 hour, 2 hours and 3 hours af- ter insemination. Newly collected animals showed a small percentage of self-fertilization in both go- nad eggs and oviduct eggs. When animals were kept in the laboratory, especially under continuous light, almost all the gonad eggs and oviduct eggs were self—fertilized. The first cleavage was ob- served 1 hour later than in cross—fertilization. The self—fertility of C. savignyi is affecte more easily by conditions under which the animal is kept. DB 5/ FORMATION OF PIGMENT BODIES IN THE BRAIN VESICLE OF AN ASCIDIAN EMBRYO, STYELA PLICATEA CLESUERUR). H. Ohtsuki and M. Yoshida. Biol. Inst., Fac. of Education, Oita University, Oita. Formation of pigment boidies in otolith and ocellus of the developing ascidian embryos was investigated by whole mount method (Whittaker, 1973) and with serial paraffin sections (2um thick). Two pigment bodies, which were nearly shper- ical and about the identical size (3.5m, diam- eter), appeared simultaneously in the presump- tive otolith cell and in the ocellus pigment pak at 5.5 to 6 hr after fertilization (23-24 ° ( : The pigment body in the otolith continued to increase its size during development, until the hatching stage (10 hr after fertilization) and became ovoid form (13um and 11.5ym, axis length). It was composed of two blocks (ventral! and dorsal) at the time of its appearance. At the swimming larval stage, one of the two blocks of the otolith pigment body looked like concave lens and the other, convex one. The ocellus pigment body grew up by 6 hr after fertilization (5.5um, diameter) and it retained its shperical form, from the stage of first visible indication of it to the swimming larval stage. It was encircled with a small vesicle at postero-dorsal wall of the brain vesicle in the swimming larvae. DB 58 INSULIN-LIKE SUBSTANCES MAY BE A GROWTH FACTOR OF STEM CELLS IN ASCIDIANS. K. Kawamura and M. Nakauchi. Dept. of Biola Hace LOrm SCier KOGhia Unie nhOChte. In the polystyelid ascidian, Polyandrocarpa misakiensis, hemoblasts are the multipotent stem cell, 4-5ym in diameter, that partic- ipates in the formation of several organ rudiments during palleal budding. In this work, we examined how the dynamics of stem cell population were regulated in the proc- ess of ascidian blastogenesis. Insulin pre- pared from the bovine pancreas (ljyg, 0.lpg, 0.0lpg/ml) could enhance the mitotic activ- ity of 1.5-day- or 2-day-old buds two or three folds as much as that of controls. The promotive effect of insulin on DNA syn- thesis first appeared on the mesenchymal blood cells, exclusively the hemoblasts and large lymphocytes, at 30 hours of bud de- velopment. Double staining of sectioned buds with insulin-FITC and DAPI (DNA-spe- cific fluorescent dye) showed that insulin bound only to the periphery of the nucleus of hemoblasts. Anti-human insulin antibody prepared from guinea pig cross-reacted with the large vacuoles of morula cells in the blood and the inner-most layer of the tunic. Histological studies showed that the morula cells underwent autolysis at the earliest stage of bud development. These results strongly suggest that insulin-like growth factor(s) derived from the morula cells operate on the hemoblasts, and then the activated hemoblasts are assigned to several organ rudiments, contributing to the asex- ual development of ascidians. 1042 Developmental Biology DB 59 CLONING OF cDNA AS A PROBE FOR ANALYSING MOLECULAR MECHANISMS INVOLVED IN EPIDERMAL CELL DIFFERENTIATION IN THE ASCIDIAN EMBRYO. K.Takamura, K.Makabe, T.Nishikata and N. Satoh. Dept. of Zool., Kyoto Univ., Kyoto. In order to study molecular mechanisms involved in epidermal cell differentiation in ascidian embryos, we produced several monoclonal antibodies which specifically recognized epidermal cells of Halocynthia roretzi embryos (Nishikata et al., Dev.Biol 121, 408-416, 1987). Among them, the Epi- 2 antigen, about 100kd in MW, first appears at the tailbud stage and is present until at least the swimming larval stage. Studies with inhibitors of protein and RNA synthesis suggested that an activation of the gene encoding the antigenic polypeptide occurs at the late gastrula stage. In this study, we first produced cDNA library against poly(A) RNA isolated from the tailbud embryos. The cDNAS were inserted into LacZ promotor region of an expression vector Agtll. Induction of the expression by IPTG resulted in synthesis of proteins encoded by cDNAs. Proteins pro- duced were blotted onto nitrocellulose mem- branes, on which the proteins were cross- reacted with the Epi-2 SELB OGY- Reacted clones were visualized with S3°-labeled second antibody. Thus, we screened a cDNA of about 1lkb in length encoding the epider- mis specific antigen. The cDNA was re- cloned by inserting into plasmid with T7 promotor, and the antisense RNA was select- ed as the molecular probe. DB 60 RECONSTRUCTION OF THE ROWS OF STIGMATA DURING REGENERATION IN THE COMPOUND ASCI- DIAN, POLYANDROCARPA MISAKIENSIS. Y. Taneda and C. Ono. Fac. Educ., Yoko- hama Natl. Univ., Yokohama. In ascidians, the rows of stigmata is approximately constant in number among the species. The compound ascidian, Polyandrocarpa misakiensis has a capacity to regenerate whole animal from a frag- ment. We revealed that the number of the rows of stigmata in the regenerated returned to the original number in intact zooid. Thus, we studied how to return the rows. Two modes of increasing pattern were observed. In one case, the rows of stigmata were once disappeared. New rows were produced afterwords. In another case, the increasing of the rows was caused by addition of the rows without disappearance of them. When a zooid was cut into two parts leaving them on a slide, they usually fused together into single zooid. In some cases, an excess sperture was pro- duced at the fused area. We revealed that the fused zooid had more rows than control and new rows were produced on the branchial wall near the fused area. Not only the reconstruction of the rows in the regenerated but also the increasing of them and the formation of excess aper- ture in the fused supports the existence of positional information along antero- posterior axis of the zooid. DB 61 STRUCTURES OF COLONIAL MARGIN IN ASCIDIANS INVOLVED IN SELF-NONSELF RECOGNITION. E.Hirose, K.Hashimoto*, and H.Watanabe. Shimoda Mar. Res. Ctr., Univ. of Tukuba, Shimoda and *Meiji Inst. of Health Science, Odawara. Six species of botryllid ascidians, Botryllus scalaris, B. primigenus, Botrylloides simodensis, B. lentus, B. fuscus, and B. violaceus, were used. For SEM observations, interior surface of colonial margin was exposed by following methods: materials embedded in paraffin were cut into pieces, deparaffinized, and dried at critical point. Styrene resin was also used for embedding medium. There were more than two types of test cells in the tunic. "Amoeboid test cell" was common to all species. "Granular test cell" was seen only in the species, in which the allogeneic recognition took place in the sub-cuticular region, but not in the other species. Thus, the "granular test cell" might have an important role for allo-recognition. The external surface of the tunic was composed of cuticule. There were minute protrusions on the cuticule. Since the density of the protrusions decreased toward the edge of the colonial margin, the cuticle might grow at the edge of the colonial margin. Each species has’ the different shape of the protrusion and the difference might be related to xenogeneic recognition. DB 62 THE MUTANT GENE ter CAUSES GERM CELL DEFI- CIENCY, NOT ACCOMPANIED BY TESTICULAR TER- ATOCARCINOGENESIS, IN A C57BL/6J GENETIC BACKGROUND IN MICE. M.Noguchil, H.Kato2, K.Moriwaki3, and T. Nosuciicone. Aol, lunsie, MAG, OF S@W-, Shizuoka Univ., Shizuoka, 2Central Inst.for Exp.Animals, Kawasaki, 3Natl. Inst. of Genetics, Mishima, “Deceased Nov. 24, 1983. A recessive mutant gene ter causes germ cell deficiency, accompanied by a high incidence of testicular teratomas in 129/Sv -ter strain mice (T.Noguchi & M.Noguchi, 1985). In order to examine the influence of a genetic background on the ter action, we have backcrossed animals carrying the mutation to C57BL/6J for N1-N8 generations. We found that germ cell deficiency occurred in 20-29% of offspring from N1-N8(+/ter) x 129/Sv-ter(+/ter), which would be the ter/ ter genotype, but that testicular terato- carcinogenesis did not occur in ter/ter males after the 5th backcross. Also, we analysed biochemical markers produced by the alleles at 9 loci on 7 chromosomes, which differ between 129/Sv-ter and C57BL/ 6J strains, in N3-N8 generations. All of these alleles in N5-N8 animals were found to be those of C57BL/6J. Thus, it is concluded that this introduction of the ter gene onto the C57BL/6J background causes germ cell deficiency in homozygoutes and that gene(s) other than ter in 129/Sv axe involved in determining susceptibility to testicular teratocarcinogenesis. Developmental Biology 1043 DB 63 FUNCTIONAL MORPHOLOGY OF EPITHELIAL CELLS RELATING TO SPERM STORAGE IN THE JAPANESE HOUSE-DWELLING BAT, PIPISTRELLUS ABRAMUS. PoaMocss ands fA Uchida. Zool. Lab.” Fac. of Agr. Kyushu Univ., Fukuoka. Spermatozoa were stored in both the uterus and the uterotubal junction (UTJ), and there was no infiltration of polymor- Phonuclear leucocytes into both parts. The heads of spermatozoa were in close contact with epithelial microvilli covered by developed fuzz filaments in the uterus, while they were lodged in indentations of the non-ciliated epithelial cells in the UTJ. Although the endometrial epithelial cells did not endocytose cationized ferri- tins as well as dead spermatozoa, in the UTJ the ferritins were concentrated within endocytotic vesicles and vacuoles of the epithelial cells. The phenomenon seems to explain the mechanism of epithelial engulf- ment of spermatozoa in the UTJ. The epithelial cells of the uterus and UTJ showed both alkaline phosphatase and glucose-6-phosphatase activities; they con- tained secretory granules which seem to play a role in antisepsis of dead spermato- zoa and seminal plasma remaining in the uterus. Thus, spermatozoa appear to be provided with nutritive substances from the epithe- lial cells and to be alive under suitable conditions, kept safely by the action of the secretory material from the epithelial cells in the uterus and kept clearly by the epithelial engulfment in the UTJ. DB 64 DISTRIBUTION AND DIRECTION OF THE SITE IN MITOSIS IN THE SEMINIFEROUS TUBULE OF THE IMMATURE MOUSE. M. Chiba. Dept. of Biol., Fukushima Med. Coll. Fukushima. pe Early indications of the spermatogenic waves in the mouse seminiferous tubules were sought. Whole testes from 9-day-old mice were fixed in Bouin's, embedded in paraffin and cut serially at 10 pm. The Mallory's stain was used. Two-dimensional reconstruction maps were made through pho- tomicrographs. The sites in spermatogonial mitoses were recorded along the length of the tubules. The types of sprematogonia were not identified. Other reconstruction maps were made to show the zigzag tubules as if they had been extended. Examination of the reconstructed tubules showed that the extent of the site in spermatogonial mataphase varied considerably.When a mark was allotted to the center of each site and the distance between the adjacent marks was measured, the average length was calculated to be 0.6 mm. Prophase spermato- gonia were often seen near the site. On examination, such spermatogonia were situ- ated mostly in the distal position. Loca- tion of the centers in prophase revealed that they were distributed more regularly along the tubule with the average interval of 1.2 mm. These findings may indicate that in the mouse the spermatogenic waves are taking place 9 days after birth. DB 65 SURFACE PROTEIN ON ROUND SPERVATIDS FROM RAT TESTES. M.NAKAVLRA, R.MATSUDA* ,S.OKINAGA & K.ARAI. Dept. of OB/GYN, Sch. of Med., Teikyo Univ. Tokyo 173 and *Dept. of Biol., Tokyo Metropolitan Univ. Tokyo 158. Spermatids undergo drastic morphological chan- ges and mature to be sperm. In this process, mR\WA synthesis is active. However, there are few reports concerning the analysis of mRNA dependent protein synthesis in spermatids. This study was performed to analyze membrane bound proteins sensitive to actinomycin D. Purified population of rat spermatids was obta- ined using unit gravity sedimentation on 2-4% linear gradient of BSA in PBS. Cells were incubated at 32°C with [3°S]methionine and actinomycin D. Plasma men- branes were isolated by centrifugation on disconti- nuous gradients and SDS-PAGE in one and two dimen- tions was performed. For autography, gels were dried under vacuum and films were exposed to gels. A labelled major protein of spermatids in the membrane bound proteins had a molecular weight of 24K. Actinonycin D (30 ug/ml) had no effect on newly synthesized proteins of the cytosol, while it inhibited the labelling of the membrane proteins significantly. Membrane bound 24K protein was ext- racted with 2 cholic acid. Elution profile of 24K protein fron Sephadex G-200 colum showed that this protein came off with larger proteins. This protein might play an important role in differentiation of round spermatids to sperm. DB 66 Analysis of the mechanism of zona reaction in the mammalian egg IV. T. Oikawa*, Joe Fukui* and Osamu Ikeda**, *; DRB Center, Yamagata 990, Japan, **; Tokyo Univ. of Arts and Sci., Tokyo 184, Japan Very recently,during examination of the biochemical changes of zona pellucida be- fore and after fertilization, we found a phenomenon that two kinds of lectins (BS- 1 and BS-2) can discriminate the zona pe- llucida of unfertilized egg from that of fertilized one. So, for the purpose to know whether this phenomenon can be in- duced by cortical granule material (CGM) or not, we did an experiment to examine whether it is possible to induce the zona reaction-like changes on the zona pellucida of unfertilized egg by pre- treatment with A23187 or not. As the re- sult, it became clear that, if we arti- fitially activate unfertilized egg with A23187, we can induce the zona reaction- like biochemical changes on the zona pe- llucida of unfertilized egg. These data suggest that the biochemical differences detected with BS-1 and BS-2 lectins on the zonae pellucidae of unfertilized and fer- tilized eggs refrect the zona reaction it- self which was induced by CGM at the time of fertilization. On the basis of the data obtained here, the mechanism of the zona reaction in the mammalian egg was discussed. 1044 Developmental Biology DB 6/ INITIATION OF ANAPHASE CHROMOSOME MOVEMENT IN MOUSE UNFERTILIZED OOCYTES BY MEANS OF MICROINJECTION Y. Hamaguchi. Biol. Lab., Tokyo Inst. of Technol., Tokyo. When two kinds of calcium buffers that contained EGTA and EDTAOH (N-hydroxyethyl- ethylenediamine-N,N',N'-triacetic acid) were injected into the oocytes obtained from hormonally superovulated ICR mice, cortical granules disappeared at Ca concen- tration of >1 pM. Anaphase chromosome movement was observed at >4 pM in 96% at 37°C within 60 minafter injectionas reported at fifty sixth annual meeting of this society. Moreover, when inositol 1,4,5-trisphosphate (IP3) (0.2-20 iM) were injected into the oocytes, anaphase took place ina manner dependent on IP concentration, but 20 pM IP3 solution containing 50 mM EGTA did not initiate anaphase when injected. However, when EDTAOH solutions (10-200 mM) were injected with or without 50 mM EGTA, anaphase also took place in a manner dependent on EDTAOH concentration. In this case, however, cortical granules of EDTAOH-injected oocytes appeared as before, when observed by differential interference microscopy, and the zona pellucida was dissolved as that of uninjected oocytes when treated with 0.1% chymotrypsin, which indicates that exocytosis did not take place. Therefore, it seems likely that EDTAOH initiates anaphase chromosome movement in Ca-independent manner. DB 68 PERIOD OF ACTION OF MOUSE BLASTOCYST HATCHING ENZYME AND ITS HISTOCHEMICAL LOCALIZATION IN EMBRYOS H. Sawada!, K. Yamazaki?, E. Hojo?, Y. Kato’, T. Someno?, and M. Hoshi? (*Biol. Lab., Fac. of Sci., Tokyo Inst. of Technol., Tokyo, ?Mitsubishi-Kasei Inst. of Life Sci., Tokyo, *Res. Labs., Pharmaceut. Grp., Nippon Kayaku Co., Tokyo ) We previously reported that a trypsin-like pro- tease participates in hatching of mouse blastocyst by examining the effects of various protease inhibitors on hatching. In the present study, we attempted to demon- strate the period of action of hatching enzyme (HE: trypsin-like enzyme) and the localization of HE. Embryos were obtained from pregnant ICR mice at 88 h after hCG administration, and was cultured at 37 °C in a-MEM containing 3 mg/ml BSA in the pre- sence or absence of 0.1 mM TLCK at an appropriate time. The HE in embryos were stained at 37 °C for 3 h by using Z-Gly-Gly-Arg-MNA and Fast Blue B. The period of action of HE was estimated by examining the effects of timing of TLCK treatment on hatching. The results suggest that the HE plays a role at 80-95 h after hCG treatment. 4 h or 10 h pulse treatments of TLCK were not sufficient to block hatching. Histochemical study demonstrated that the HE was localized in trophectoderm but not restricted to mural trophectoderm as reported by Perona and Wassarman (1986). In our procedure, the embryos were not stained in the absence of substrate or in the pre- sence of TLCK and leupeptin, potent inhibitors against hatching, suggesting the specific staining of HE. Contrary, in their procedure, the embryos were stained similarly even in the absence of substrate or in the presence of the inhibitors. DB 69 BISMUTH STAINING OF PREIMPLANTATION MOUSE EMBRYOS. I.Takeuchi and Y.Takeuchi. Aichi Prefec. Colony, Inst. Dev. Res., Kasugai, and Gifu Col. Med. Technol., Seki. The changes in the distribution of the structures contrasted with bismuth stain- ing following glutaraldehyde fixation (Locke and Huie, 1977) were examined in the preimplantation mouse embryos. In the 1-cell embryos, the fibrillar centers (FC) located at the periphery of condensed nucleoli, and the perichromatin granules (PG) somewhat distributed in the nucleoplasm, were stained. In the nuclei of 2-cell embryos, PG increased in number, and numerous interchromatin granules (IG), well stained with bismuth, appeared in the nucleoplasm. In the cytoplasm of 2-cell embryos, the onset of appearance of bismuth-stained crystalloid inclusions was noted. In the 4-cell embryos, the dense fibrillar components (DFC) and granular components (GC) emerged around the con- densed nucleoli, and both nucleolar com- ponents greatly increased in amount in the subsequent 8-cell and blastocyst-stage embryos, while the condensed nucleoli tended to be fragmented and disappear. In the nucleoli from the 4-cell embryos on- ward, bismuth deposited in the FC and the adjoining DFC. Cytoplasmic crystalloid inclusions increased in number along with the process of embryonic development. DB 70 REGULATORY FUNCTIONS OF ADENOSINE 3',5'- CYCLIC MONOPHOSPHATE IN 1-METHYLADENINE PRODUCTION BY STARFISH FOLLICLE CELLS. *M. Mita, *N. Ueta and **Y. Nagahama. *Dept. of Biochem., Teikyo Univ. Sch. of Med., Tokyo, **Lab. of Reprod. Biol., Nat. Dnsit. Lorn Basic Bil nOkaizalkane Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1- MeAde) produced by ovarian follicle cells under the influence of gonad-stimulating substance (GSS). The present study was conduced to examine the importance of cyclic AMP in the GSS-induced increase in 1-MeAde production by starfish, Asterina pectinifera, follicle cell suspensions. GSS treatment significantly stimulated 1-MeAde production during 1-2 hr incubation. The treatment also caused a 4 to 5-fold increase in intracellular levels of cyclic AMP, but not cyclic GMP. GSS is required at all times to maintain elevated levels cyclic AMP. 3-Isobutyl-1-methyl-= xanthine, a phosphodiesterase inhibitor, stimulated both 1-MeAde and cyclic AMP production in a dose-dependent manner. Other substances, such as concanavalin A and proteases, which are known to induce 1- MeAde production in starfish follicle cells, also stimulated cyclic AMP production. Thus, in all cases examined, the induction in 1-MeAde production parallels the elevation in cyclic AMP levels. It is concluded that cyclic AMP mediates the action of GSS upon 1-MeAde production in starfish follicle cells. Developmental Biology 1045 DB 71 AN INCREASE IN OOCYTE CYCLIC AMP LEVEL INITIATES OOCYTE MATURATION IN THE BRIT- TLE-STAR AMPHIPHOLIS KOCHII. MeaviandchitateZOoolmmsnste,eracw Of Sicaye, Hokkaido Univ., Sapporo. Maturation of the brittle-star oocytes was induced by an external application of CAMP (10 mM), an inhibitor of cyclic nu- cleotide phosphodiesterase (25 mM theo- phylline, 25 mM caffeine) or an activator of adenylate cyclase (100 pM forskolin, 0.6 uM cholera toxin). Experiments in which the oocytes were treated with for- skolin or theophylline for various periods of time, demonstrated that there was a positive correlation between the oocyte CAMP level measured by radioimmunoassay and the extent of germinal vesicle break- down (GVBD) induced in each treatment: Both increased as the treatment period became longer and about three-fold in- crease in cAMP level induced 50% GVBD. Oocyte maturation was also induced by microinjection of cAMP (5.7 pM/oocyte) or the catalytic subunit (0.7 uM/oocyte) of cAMP-dependent protein kinase (A kinase) into the immature oocytes. Microinjection of heat-stable inhibitor protein (0.5 pM/oocyte) of A kinase inhibited cAMP- induced oocyte maturation. These results indicate that an increase in oocyte cAMP level initiates maturation of the brittle-star oocytes, probably via activation of A kinase. DB 72 POSSIBLE INACTIVATION OF MATURATION-SPECIF- IC PROTEIN KINASE IN STARFISH OOCYTES BY Ca-ACTIVATED PHOSPHOPROTEIN PHOSPHATASE. K.Sano, Marine Biological Station, Hokkaido University, Akkeshi, Hokkaido In Asterina pectinifera, maturation-spe- cific protein kinase (M-sp PK), which is specifically observed in maturing oocytes (K.Sano,1985,Develop. Growth & Differ.,27, 263-275), is shown to be inactivated by | adding Ca to the supernatant of maturing QOCITESS (IX Seine, LOY psllontcls p29, 392) 5 auto) clarify the mechanism of inactivation by Ca, effects of various enzyme inhibitors were examined. Inactivation by Ca was sup- pressed by the inhibitors of phosphoprotein phosphatase such as glycerol-2-phosphate and pyrophosphate, suggesting that M-sp PK is inactivated by a Ca-activated phospho- protein phosphatase which is present in Maturing oocytes in addition to -SH oxida- tion. Next, we have characterized protein kinase activity which is activated by add- ing dithiothreitol to the supernatant of immature oocytes (1987,ibid.). The newly activated kinase was found to be Ca-, cAMP- independent and sensitive to iodoacetamide, using histone Hl as substrate. This indi- cates that the newly activated kinase is the M-sp PK. The inactivation of M-sp PK by two types of mechanisms suggests that inactive form of M-sp PK may be activated by some unknown mechanism such as phospho- rylation in addition to the activation by =S='S= ic@ Sil ieeeinGslieiom., Swjysyooieeecl Joyy Grant-in-Aid for Sci. Res. (61480024). DB 73 A MONOCLONAL ANTIBODY TO 56- AND 58-KILO- DALTON-POLYPEPTIDES PRESENT IN THE EGG OF THE STARFISH, ASTERINA PECTINIFERA. M. Kataoka, S. Yajima and S. Ikegami, Dept. Applied. Biochem., Fac. Applied Biological Science, Hiroshima Univ., Fukuyama. In order to prepare a monoclonal anti- body that reacts specifically with compo- nents of eggs and embryos of the starfish Asterina pectinifera, Balb/c mice were immunized with a 150-200 mM NaCl eluate of a DEAE-cellulose column that had been loaded with an egg extract. Spleen cells were removed from the mice and fused with myeloma SP-2 cells. Hybridoma cells that secreted immunoglobulin G reacting spec- ifically with the polypeptides with molec Ular weights of 56,000 and 58,000 were selected. The immunoglobulin G reacted to extracts of eggs, morulae, gastrulae and Ovaries, but not with those of testes, pyrolic cacea, body walls and tube-feet. The immunoglobulin G did not reacted with extracts of eggs of the starfish Asterias amurensis or sea urchin Hemicentrotus piilichemriinviss SUggestung: thake ri ise @dthnes: specific. This work was supported in part by a grant from the Nissan Science Foundation. DB 74 DISULFIDE-REDUCING AGENTS INCREASE 1-METHYL ADENINE-BINDING TO CORTICES ISOLATED FROM STARFISH OOCYTES. M. Yoshikuni!, K. Ishikawa’, & Y. Nagahama’ Mh. CE IRGjorcOcle “ iENL@IlLe; Wevely itingte, Foie Balssicu Bole mOkaz akisn = DeptwoOte Boles Hace of Sci., Shizuoka Univ., Shizuoka. a4 Several disulfide-reducing agents, such as dithiothreitol, 2,3-dimercapto-1-propanol, cysteine ethyl ester and cysteine methyl ester, have previously been reported to induce oocyte maturation in starfish, Asterina pectinifera. In the present study we found that these agents enhanced the effectiveness of 1-methyladenine (1-MeAde) to induce oocyte maturation. This enhance- ment occurred at relatively low concentra- tions where these agents by themselves were ineffective in inducing oocyte maturation. Pretreatment of oocytes with these agents shortened the hormone-dependent period for 1-MeAde-induced oocyte maturation. Treat- ment of oocyte cortices with the agents caused a marked (about 2-fold) increase in specific [?H]1-MeAde binding. The binding increased directly in relation to the potency of the agents in enhancing 1-MeAde action. Further analysis of this binding by the Scatchard plots indicated that dithiothreitol increased the Bmax without affecting the affinity of 1-MeAde binding. Thieisicn resupstis ssitnonglywsuggest | tihait disulfide-reducing agents enhance the maturational action of 1-MeAde by increas- ing the number of 1-MeAde binding sites in oocyte cortices. 1046 Developmental Biology DB 75 ARACHIDONIC ACID-INDUCED INHIBITION ON THE 1-METHYLADENINE-INDUCED OOCYTE MATURATION IN ASTERINA PECTINIFERA. H. Tosuji and T. Nakazawa. Dept. of Biol., Fac. of Sci., Toho Univ., Funabashi. In Asterina pectinifera, we reported that the arachidonic acid-induced oxygen consumption was exhibited (Zool. Sci. 3, LOS 7/}) 6 This oxygen consumption existed in particlar fraction of the oocyte homogenate, and was inhibited by lipoxygenase inhibitors. The arachidonic acid-induced oxygen consumption was also found in Asterias amurensis. lin VAs Syeceeieg wie arachidonic acid-induced oocyte maturation was found and this arachidonic acid- induced oocyte maturation was inhibited by arachidonic acid cascade inhibitors. In Asterina, the oocyte maturation is not induced by arachidonic acid (Meijer et al, 1984), on the contrary, arachidonic acid inhibited 1l-methyladenine-induced oocyte maturation. This inhibition depended the pre-treatment time of arachidonic acid, and showed competitive inhibition to 1- methyladenine. But on the 1-methyladenine recepter of oocyte, the binding of 1- methyladenine was not affected by the concentration of arachidonic acid. IL te suggests that exogenous arachidonic acid is oxydized and its product which might induce oocyte maturation in Asterias, inhibit the 1-methyladenine-induced oocyte Maturation competitively in Asterina pectinifera. DB 76 CHANGES IN ISOELECTRIC POINT OF TUBULIN DURING STARFISH OOCYTE MATURATION N. Hosoyal, H. Shirai2, H. Hosoya4, Y. Nagahama2 and H. Mohril. IDept. Biol., Colle Aiwts and! Sicis,) Uniivn Lokyo; MWokyol; 2Dept. Reprod. Biol., Natl. Inst. Basic Biol., Okazaki, and %3tokyo Metro. Inisit. Med. Sci., Tokyo. Microtubule structures in starfish oocytes ( Asterina pectinifera ) exhibited remarkable changes during maturation process induced by l-methyl adenine ( Shirai et al, in preparation ). In order to analyze these changes biochemically we checked the changes in isoelelctric point ( pI ) of tubulin molecules. The changes in pI of tubulin were detected using isoelectric focusing, immunostaining and two-dimensional gel electrophoresis. @-tubulin in maturing oocytes shifted to more acidic two isoforms at around germinal vesicle break- down, returned again to the original ( immature ) ones after first polar body emission, SubSequently shifted to acidic ones during the process of second polar body emission, and finally returned to the original ones. On the other hand, pl of B -tubulin did not change remarkably. When maturation was inhibited by 6- dimethyl amino purine, one of the inhibitors of phosphorylation, the changes in pI of @-tubulin were inhibited. From these results, the changes in pI seem to couple tightly to maturation process. DB 77 INHIBITION OF MOUSE OOCYTE MATURATION BY A SYNTHETIC PROTEASE INHIBITOR, TLCK. Naohiro Hashimoto*, Takeo Kishimoto**, and Yoshitaka Nagahama**. *Mitsubishi-Kasei Inst.Life Sci., Machida and Dev.Reprod. Biol.Centr., Yamagata, **Dep.Dev.Biol., Natl.Inst.Basic Biol., Okazaki. Involvement of proteases in mouse oocyte maturation was studied using a synthetic protease inhibitor, Na-tosyl-L-lysine chloromethyl ketone (TLCK). When isolated follicles were cultured with both 1 ug/ml ovine LH and TLCK (0.02-0.5 mM) for up to 6 h, LH-induced germinal vesicle breakdown (GVBD) was blocked to 2% by 0.5 mM TLCK. However, TLCK did not affect LH-induced elevation of follicle cell cAMP. Thus, follicle cells seem to respond to LH nor- mally, suggesting that TLCK affects oo- cytes. Then denuded (cumulus-free) oo- cytes were liberated from follicles after preincubation with 0.5 mM TLCK for 3 h and further incubated with TLCK. Spontaneous GVBD in these oocytes was completely blocked throughout 5 h of incubation. When follicles were preincubated with TLCK for 1 h, GVBD at 5 h after liberation was decreased to 66% in denuded oocytes where- as 20% in cumulus-enclosed oocytes. It is likely that this enhancement of inhibition in cumulus-enclosed oocytes is due to facilitated transport of TLCK from cumulus cells into oocytes through gap junctions. The results suggest that a trypsin-type protease in oocytes plays a regulatory role in mouse meiotic maturation. DB 78 WRINKLED BLASTULA OF THE SEA-STAR, ASTERINA MINOR: BEHAVIOR OF BLASTOMERES M. Murase, M. Komatsu and C. Oguro Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan The wrinkled blastula stage has been reported in a number of asteroid species during normal develop- ment. However, nothing has been known on the details of the behavior of blastomeres during the wrinkled blastula stage. In the present study, behavior of blastomeres was observed with a SEM and vital staining of the embryo. Before the commencement of wrinkling, blastomeres on the surface of the blastula were stained by immersing the embryo into 0.05% neutral red solution. By this treatment, blastomeres situ- ated in the blastocoel were not stained. When fine furrows appeared, 10 hours after ferti- lization, unstained blastomeres were located at the bottom of the furrows. At the most wrinkled stage, cell masses composed of stained blastomeres were separated by cell masses of unstained blasto- meres which were mostly composing egression tracts. After the recovery from the wrinkling, the embryo became a coeloblastula. The blastoderm of this blastula showed a mosaic pattern which was composed of stained and unstained cell masses. When the embryo became a brachiolaria, the surface of the embryo still retained a mosaic pattern. These results may indicate that the wrinkled blastula stage in this species is a process of cell rearrangement for the appropriate distribution of the blastomeres which were situated on the sur- face and in the blastocoel of the blastula before the wrinkling. Developmental Biology 1047 DB 79 SCANNING ELECTRON MICROSCOPIC OBSERVATIONS ON SDHEMVITELLARTA: OF THE BRITTLE, STAR, OPHIOPLOCUS JAPONICUS M. Komatsu, M. Murase, T. Wada and C. Oguro Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan It was observed with a light microscope that the larva of Ophioplocus japonicus is Beueetiobias (ohosakuy et, ad«,.1 981)... Four ciliary bands appear in the vitellaria, 3.5 days after fertilization, when tube feet emerged on each hydrocoel lobe. In the present study, the embryos from the gastrula to the juvenile through the metamorphosis were observed with a SEM, Surface of the early gastrula was uniformly Seucccd wi the Cilia, Z20,hours, after ,.fertil- iatetone hour hours later, ciliary bands were not observed in the late gastrula. One and half days after fertilization, the presence of ciliary bands was first recog- MNazed in the early vitellariae. They were furnished with 5 hydrocoel lobes with rudi- Nemes sonehe wtube: feet... Three and, halt days after fertilization, vitellariae had 4 definite ciliary bands. Just before the commencement of the absorption of the lar- Val peste.» Gtlia on the ciliary bands. rapid- ly decreased in number and length. Five day Sicmeet heme hli zation,» no, trace) of, cil - lary bands was observed in the metamorpho- Sing larva with the reduced larval part. The present study shows that ciliary bands are formed in the early vitellaria, not like reported previously. DB 380 EFFECTS OF INHIBITORS OF RNA SYNTHESIS ON EMBRYONIC DEVELOPMENT OF THE STARFISH ASTERINA PECTINIFERA. H. Isomura, N. Tsuchimori, N. Itoh and Saetkegama 3 Applied. Biochem., Fac. Ap- plied Biological Science, Hiroshima Univ., Fukuyama, Hiroshima. The rate of RNA synthesis is low during the morula stage and increases sharply at the beginning of blastulation. When fer- tulazed eggs of the starfish, Asterina peseinubena, were cultured in artificial sea water containing formycin at a concen- tration of 25 ug/ml, RNA synthesis of the embryos was inhibited by approximately 40 % at 6 hr after fertilization and the de- velopment of the embryos stopped at the early blastula stage with the cell number OF L-OZ8: he CoMmesnEes Cr UTP elnel Gilet the formycin-treated embryo were low as compared with those of control embryo whereas the contents of ATP and GTP was the same for both types of embryo. When cytidine (100 ug/ml) and uridine (100 uUg/ ml) were introduced into a culture of fertilized eggs which were placed in ar- tificial sea water containing 25 ug/ml of formycin, they were able to develop to the late blastula stage, suggesting that the inhibition of RNA synthesis by formy- Cin is due to the inhibition of biosyn- Ehesis of CTP and UTP. This work was supported in part by a a grant from Nissan Science Foundation. DB 81 EPITHELIAL CELLS OF THE STARFISH EMBRYO OPEN AND CLOSE THE SEPTATE JUNCTION IN RESPONSE TO SOLUTES PRESENT OUTSIDE THE BODY Nips DAN-SOHKAWA!, Cr KANAI7 and K. NODA? Dept. of Biol., Osaka City University, 2Tokyo Metropolitan Inst. of Gerontol. a I vt en lem Ve encour aa Seema hans pole! Narre MEN AY Septate junction and tight junction are classified as impermeable junctions, whose primary function is considered to prevent water and solutes to permeate freely into the body cavity by way of intercellular spaces. It is known, how- ever, that in some vertebrate epithelia water and soluble materials are allowed to permeate through paracellular pathways when certain concentrations of low molecular weight substances, such as NaCl, urea and glycine, are present outside the body. We show, in this study, that this paracellular transepithelial permeation also takes place in the body wall epi- thelium of the starfish embryo. What is more, three additional conditions to the apical hypertonicity, mentioned above, were found to evoke the paracellular permeation in this simple epithelium, namely Ca*-free environment, 0°C and pH statuses over 9. Macromolecules, such as FITC-labeled IgG and dextrans, were introduced into the blastocoel by taking advantage of the inflowing water to visualize the para- cellular permeation. DB 82 BLASTOCOEL FORMATION IN THE MONOLAYERED CELL SHEET OF SEA URCHIN EMBRYOS. Y.D. Noda, *Y. Nakajima, and *M. Ikeda. Dept. of Biol., Ehime Univ., Matsuyama. and *Dept. of Biol., Keio Univ., Yokohama. In the 8- or 16-cell stage embryo of the sea urchin, Mespilia globulus, the blastomeres begin ixo) transform into flattened cell shape when the eggs removed fertilization membrane were cultured in Ca- deficient sea water (Nakajima & Noda '85). These flattened cells divide vertically to the substratum and form the monolayered cell sheet. Microvilli occur only on the free surface of the flattened cell. The surface facing the substratum is smooth. The cilium of the blastula stage cell is formed on the free surface. When normal embryos develope into a blastula, monolayered cell sheet embryos begin to make a sphere (i.e.,blastocoel) between the cell sheet and the glass substratum. Starfish eggs of denuded fertilization membrane develope into a monolayered cell sheet. Microvilli are made on the cell surface facing the substratum and the free edges of the sheets begin to curl upward after 9th cleavage, then their sheets become a blastula-like structure (Dan-Sohkawa, '76, Kadokawa et al., '86). The cell sheets of sea urchin in Ca- deficient sea water do not occur ‘closing movement' of starfish sheet. But, some sheets form a hollow sphere surrounding a center of blastocoel, and become a blastula- like structure. 1048 Developmental Biology DB 83 CELL CYCLE STUDY OF MICROMERES IN THE EMBRYOS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS. S.Tanakal, K.Nakajima and K.Dan2. lnab. of Develop. Biol. Mitsubishi-Kasei IS, Ore Ihskiee Seabs / Machida-shi, 2Misaki Marine Biological Station, Miura-shi To investigate cell cycle kinetics of micromeres of a sea urchin embryo, a meth- od was developed so as to dissociate and prepare every cell onto a glass slide ob- tained from one embryo, from which the fertilization membrane was removed. At early 8 cells stage, embryos were pulse labeled(30min)with 5-bromodeoxyuridine (BrdU) which was detected immunocytochemi- cally using anti-BrdU antibody. Cell cy- cle related parameters such as DNA con- tent, total protein content and BrdU a- mounts were successsively measured with specified cells utilizing a system com- posing of a microfluorometer, TV cameras, video recorders and microcomputers. Small micromeres were clearly identified by scattered plots of two parameters of BrdU /protein and protein of each cell from one embryo. A parameter of DNA /protein was also found to correlate well with that of BrdU/protein and be useful in identifying small micromeres. According to these methods, it was suggested that small mi- cromeres progressed only one cell cycle of about 4h during a period ranging from 32 cells stage through about 250 cells stage. DB 84 INTRACELLULAR caZt POOLS RELATING TO GROWTH OF SPICULES AND PSEUDOPODIA IN MICROMERE-DERIVED CELLS OF SEA INOS N K.Mitsunaga, , -Shinohara’, Y.Fijono“~ and I.Yasumasu . Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo and “Dept. of Pharmacol., Teikyo Univ. Sch. of Med., Tokyo. —— Relation of intracellular Caz pools to growth of spicules and pseudopodia was studied using Ca“* transport inhibitors. Spicule formation in the cultured micromere-derived cells was inhibited by ssthelieyciar whe red and verapamil. Mitochondri- al Ca“* pool was reduced by these compounds at concentrations effective to block spicule sO Wen ea Ci, though extra- mitochondrial Ca** pool was enlarged by ruthenium red and was diminished by verapamil. Ruthenium red also induced excess drowsy of pseudopodia and stimulated P incorporation into protein, though verapamil inhibited both of them. Their rates correlated with extra- mitochondrial Ca“* pool size. The growth of pseudopodia and activity of C-kinase in micromere-derived cells were inhibited by H-7 but were not by HA1004. pes results suggest that mitochon- dizials Cao pool as amdiispenisalbilier stor spicule formation. Growth of pseudopodia in spicule forming cells requires protein phosphorylation catalyzed by C-kinase which is) regulated by) Gach in extra— mitochondrial pool or cytosolic free CaZt, DB 85 IN VITRO FORMATION OF SEA URCHIN SPICULES IN SEA WATER CONTAINING BLASTOCOELIC FLUID M.Kiyomoto and J.Tsukahara. Dept. of BiO@lsf WAGs Oe Selo, KalcGoslaiwe Wins, , Kagoshima. Okazaki('75) reported that descendants of isolated micromeres of sea urchin embryo formed larval spicules when they were cultured in sea water containing horse serum. If they were cultured in sea water without horse serum they couldn't form spicules. Under the normal development descendants of micromeres become primary mesenchyme cells and form a larval spicule in the blastocoel of sea urchin embryo. So some facters necessary for spicule formation may exist in blastcoel. In the present study isolated micromeres were cultured in sea water containing blastcoelic fluid (BCF) obteined from centrifuged embryos. The descendants of micromeres could form spicules in this medium. The activity of spicule formation was higher in the BCF obteined from mesenchyme blastula, lower from gastula and not detected from early blastula. Such active substances were non-dialyzable and lost their activity after tripsin treatment. The cross experiments between H. pulcherrimus and P. depressus have shown that such substantces are not species specific. DB 386 ELECTROPHORETIC ANALYSIS OF COLLAGEN-LIKE PROTEIN IN WATER-SOLUBLE EXTRACELLULAR MATRIX OF SEA URCHIN EMBRYO. HAZIME MIZOGUCHI. Div. of Biol., Jun. Col. of Rissno Univ. Saitama. To investigate whether extracellular matrix of sea urchin embryo contains col- lagen, electrophoretic analysis of water- soluble extracellular matrix of the embryo was performed with sodium dodecyl sulfate polyacryamide gel electrophresis. Water soluble extracellular matrix (WSE) was obtained as follows. Embryos were dis- sociated with the medium ( sea water con- taining glycine-EDTA ), which was then centrifuged at 3,000 rpm for 5 min.) The supernatant was dialyzed against distlled water. WSE was digested with pepsin over night at 4°C followed by coilagenase over inalelone Ets BIC, eine joreaiieel c@ie IL min. Sam- plies thus obtained were electrophoresed on 7.5% Or 15% sodium dodecyl sulfate poiyacrylamide gels at constant current. Gels were observed after silver staining. Collagen-like protein was observed in WSE obtained from gastrulae and plutei. It was however not detected in that of 16 cell embryos and blastulae. These results suggest that collagen- like protein is contained in the extra- cellular matrix of sea urchin embryos after gastrula stage. Developmental Biology 1049 DB 8/ EXPRESSION OF A PRIMARY MESENCHYME CELL SURFACE SPECIFIC GLYCOPROTEIN, msp 130, AND ITS POTENTIAL ROLE ON THE CELL MIGRATION. H. Katow, Biology Laboratory, Rikkyo Univ., Tokyo. A monoclonal antibody, B2C2, raised against sugar moieties of sea urchin primary mesenchyme cell (PMC) surface specific glycoprotein, msp 130 (Anstrom, et aly 07, Development, in press) has been utilized for immunohistological studies. The antibody was also applied for examinat- ions on the role of the glycoprotein during PMC migration in blastulae of the sea urchin, Clypeaster japonicus. Indirect immunohistologies indicated that 1) the antibody binds only to the PMCs ingressed, but not to those in the embryos raised 2) in sulfate-deficient sea water, 3) with tunicamycin, or 4) with monensin. In those embryos the PMCs were formed but their migration did not occur. Transmission electron microscopies indicated that in monensin treated embryos the transitional vesicles of Golgi complex were abnormally inflated, which suggested that the cyto- plasmic organ is associated with intra- cellular transportation of msp 130 to the cell sustace. In vitro PMC migration studies conducted in mixtures of fibro- nectin which promotes PMC migration (Katow tedayvasmie OD), J.C .B) and the antibody indicated moderate to rather weak inhibi- BORyNCEEeece Of B2G2. B2C2 has been kindly provided by Dr.R. Raff, Indiana University, USA. DB 88 EXPRESSION OF PRIMARY MESENCHYME CELL (PMC) LINEAGE SPECIFIC ANTIGEN DURING DEVELOPMENT OF SEA URCHIN EMBRYO (II). K. Shimizu®, H. Katow* and R. Matsuda°. °Dept. Of Biol. Tokyo Metropolitan Univ. and *Lab. of Biol, Rikkyo Univ., Tokyo. PMC specific antigens have been studied by using monoclonal antibodies, P4 (Shimizu , et al. Develop. Growth and Differ. 29:389 eG) manda BAC 2Zai(kaundliv provided by, Die. Re A. Raff; Leaf et al. Develop. Biol. 121:29- 40, 1987). P4 and B2C2 reacted with the same cell surface glycoproteins. From pep- tide mapping analysis, it was suggested that both antibodies recognized the same N- linked carbohydrate (CHO) chain. During de- velopmnt, the P4 epitope appeared earlier than the B2C2 epitope. At the vegital plate stage of Clypeaster japonicus, presumptive PMCs already reacted with P4, however, PMCs did not react with B2C2 until they com- pleted ingression. Besides, embryos of Anthocidaris crassipina grown in sulfate- free sea water reacted only with P4, but not B2C2. Tnese results suggest that P4 and B2C2 epitopes localize at different regions SEe@ene same CHO chain. Biological function of P4 antigen was also examined. PMCs of C. japonicus embryos were obtained by Cyto- chalasin B treatment and were cultured in the presence or absence of P4. P4 inhibited PMC migration. This result indicates that the P4 antigen may play an important role LOR MaAgTratory activity of PMC. (Supported by grant-in-aid from the Ministry of Educa- tion, Science and Culture of Japan.) DB 89 RELATIONSHIP BETWEEN PRIMARY MESENCHYME CELL BEHAVIOR AND THE SUBSTRATUM OF THE SEA URCHIN EMBRYOS TREATED WITH LITHIUM IONS. S. Amemiya. Misaki Marine Biological Station, University of Tokyo, Kanagawa. SS PS ae Aa SN ie ee ee In the normal development of sea urchin embryos, primary mesenchyme cells accumulate ina ring-like pattern between the equator and vegetal pole after migrating along the basal lamina in the blastocoel. The author previously reported the basal lamina to consist of a network of fibrils and amorphous materials situated in the spaces among the fibrils. Some of the fibrils projecting from the basal lamina and lying alongside the ectodermal wall in the blastocoel may possibly constitute the substratum for primary mesenchyme cell migration. In this report, the extracellular matrix involved in the formation of the primary mesenchyme cell pattern was examined. Scanning electron microscopical observation revealed extracellular matrix fibrils to be well developed around this pattern. In embryos treated with lithium ions, the pattern formed in the animal hemisphere where extracellular matrix fibrils developed well. Spicules formed in mesenchymal aggregates that had accumulated in the animal hemisphere, thus indicating that the functions of primary mesenchyme cells are Maintained essentially in embryos treated Waltela Ibsliclnalwil somes, DB 90 SEA URCHIN DNA POLYMERASE Y: INTRACELLULAR LOCALIZATION AND CHANGES IN ACTIVITY DURING EMBRYOGENESIS. M.Shioda. Dept. of Physiol.Chem. & Nutri., Fac. of Med., Univ. of Tokyo, Tokyo. DNA polymerase y is known to participate in mitochondrial DNA replication. In the present study, intracellular localization and changes in activity of the enzyme were examined in sea urchin (Hemicentrotus pul- cherrimus) eggs and embryos. On different- ial and isopycnic centrifugation of egg or embryo homogenate, bulk of DNA polymerase y activity recovered in the 10,000g precipit- ate and equlibrated at buoyant density of 1.216 g/cm3, respectively. These results indicate that DNA polymerase y is localized predominantly in mitochondria. The enzyme activity per embryo remained constant throughout the egg, cleavage and early blastula stages, thereafter, it began to increase. The amount of mitochondrial DNA per embryo also began to increase from cleavage or early blastula stage, suggesting that mitochondrial DNA replication begins to occur from this stage. The enzyme activity per mitochondrial DNA remained almost con- stant during embryogenesis. These results indicate that mitochondria contain a const- ant amount of DNA polymerase y regardless of whether mitochondrial DNA replication occurs or not. This suggest that different regulation mechanisms operate in mitochond- rial and nuclear DNA replication as to the amount of replicative enzyme. 1050 Developmental Biology DB 91 OCCURRENCE OF CASEIN KINASE TYPE PROTEIN KINASE IN THE CHROMATIN FROM SEA URCHIN EMBRYOS. Y.Masuyama, M.Motojima, and I.Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. Cyclic nucleotides-independent and polyamines-enhanced protein kinase was estimated in isolated chromatin from sea urchin embryos. Spermine (5mM) enhanced by about 2 holds the rate of phosphorylation of endogenous proteins by protein kinase reaction. this enzyme activity was relatively high in embryos at the morula stage and the gastrula stage, and was quite low at the swimming blastula stage. In extract of nuclei with 0.4M NaCl, protein kinase activity , which phosphorylated casein and phosvitin but not histone 2b, was found and was activated by spermine. Heparin did not influence this enzyme activity in the isolated chromatin, but inhibited it in the extract. Isolated nuclei having been extracted with 0.4M ygCl hardly exhibited Spermine-stimulated P incorporation into endogenous proteins. A sharp peak of this enzyme activity was obtained by a casein- phosvitin-Sepharose column chromatography of this 0.4 M NaCl extract. Ion exchange chromatography by Mono Q (Pharmacia FPLC system) exibited two peaks of casein Kinase activities. These probably correspond to casein kinase 1 and 2, respectively. DB 92 METHYLATION OF NUCLEIC ACID IN SEA URCHIN EMBRYOS DURING EARLY DEVELOPMENT. M.FUJII, K.FUSHIMI and I.YASUMASU. Dept. of Biol. Sch. of Educ. Waseda Univ., Tokyo. CTS aT Da wT LY, BG ER oa a Tad TET | OL, PR ea The rate of the incorporation of [7H- methyl] group from [?H-methyljmethionine into the nucleic acid of the sea urchin embryos changed during eariy development. The rate of the incorporation into DNA up to 6hr after fertilization, the cleavage stage, was remarkably and at 24hr, the gastruia stage, was slightly high. The rate was low at l1Zhr, the blastura stage, also low after 36hr, the prism and pluteus stage. The rate of [? H-methy1] incorpola- tion into RNA was undetectable up to 8hr, and thereafter it was substantially in- creased, the highest rate was found at 36hr. These rates of [?7H-methy1] incorpora- tion were decreased by addition of 5'-iso- buthy1-5'-deoxyadenosin, an analogue of S-adenosyl-methionine. This compound re- suited in abnormal deveiopment of sea urchin embryos. The profile of the change in incorporation of [?H-methyljgroup from S-adenosyl- (? H-methyljmethionine into. DNA was essentiaily similar to that of [?H- methyi]Jincotporation into DNA from [?H- methyl]methionine in embryos. But the in- corporation in isolated nuclei at 6hr was almost the same as at 18hr. Labelled DNA in nuclei was isolated, digested with EcoRI and analysed by 1l%agarose gel electrophoresis. Pattern of %H-radioactiv- ity at 6hr evidently differ from that at U8hr. DB 93 Onset of arylsulfatase gene expression and DNA methylation of sea urchin (H. pulcherrimus) embryos ois) K. AKASAKA, H. SASAKI and H. SHIMADA ZANOLG MMNSIEG p HAC, Silo, Wintlys Oi Wolkwo, Tokyo. In the sea urchin (H. pulcherrimus) embryo, the abundance of arylsulfatase mRNA decreased until early blastula stage (9h after fert. 15 C) and began to increase reaching about 1000-fold of that in prism stage, and then decreased again. The extent of DNA methylation of the embryos was estimated by HapII and MspI digestion method. The second cytosine residues within the CCGG recognition sites of the bulk DNA were significantly methylated (CG methylation) through the development up to prism stage. Degree of DNA methylation was especially high at prism stage. The extent of methylation of aryl- sulfatase gene was estimated by Southern Jere Zalenoin uSing arylsulfatase 32p_cDNA as a probe. Arylsulfatase DNA became highly methylated (CG methylation) after fertilization, demethylated at the onsetof thegene expression (early blastula), and highly methylated again with the cessation of the gene expression of this gene (prism embryo). DB 94 FORMATION AND DIFFERENTIATION OF GONADAL MEDULLA IN RANA NIGROMACULATA AND RHACOPHORUS ARBOREUS , Akihiko Tanimura and Hisaaki Iwasawa. Biol. Inst., Niigata Univ., Niigata. Gonadal development was observed in R. nigromaculata and Rh. arboreus in light and electron microscopy. Concerning the mode of gonadal sex differentiation, the former is a direct differentiated type and the latter is a semidifferentiated type. In both species, the gonadal medulla derived from the epithelial cells of the rudimentary gonad, and the medulla was iin GOMEaACE Wiligla cli® CORES. IEF Rie nigromaculata, the medullary cells had a cytoplasm rich in organella, and were in close contact with each other. The mass of medullary cells was covered with a continuous basal lamina. On the other hand, in Rh. arboreus, “the” basal™ lamina covering the mass of medullary cells was fragmentary, and the contact among the medullary cells was rather loose. In both species, the dorsal portion of the medulla was elongated, extending to the mesonephric region, and differentiated into an efferent duct in male specimens. The medulla developed to the testicular rete in male specimens, and to the inner epithelium of the ovarian cavity in female ones. Developmental Biology 1051 DB 95 THE POSITIONAL EFFECT OF PRESUMPTIVE PRI- MORDIAL GERM CELLS (PPGCS) ON THEIR DIF- FERENTIATION INTO PGCS IN XENOPUS LAEVIS. K. Ikenishi and Y. Tsuzaki Dept. of Biol., Fac. of Sci., Osaka City Univ., Osaka. In order to know whether the location of "germ plasm"-bearing cells [presumptive primordial germ cells (pPGCs)] is crucial for their,differentiation into PGCs in Xenopus, ~H-thymidine labeled pPGCs were implanted into the anterior or posterior halves of the endoderm in unlabeled host neurulae. Labeled PGCs in the genital ridges of experimental tadpoles were in- vestigated by autoradiography. The proportion of tadpoles with labeled PGCs was significantly higher when labeled pPGCs were implanted into the posterior en- doderm (p-tadpoles) than when labeled pPGCs were implanted into anterior endoderm (a-tadpoles). The average number of la- beled PGCs in the p-tadpoles was much high- er than that in the a-tadpoles. Besides, labeled PGCs in the p-tadpoles were found to be distributed throughout the genital ridges while those in the a-tadpoles were localized in the anterior part of the ridges. These facts indicate that the location of pPGCs in the endoderm affects their successful migration into the genital ridges. Therefore, it is reasonable to think that not only the presence of the "germ plasm" but also the proper location in endoderm are prerequisite to PGC differ- entiation of the germ line cells. DB 96 MORPHOGENESIS OF THE GONADAL ANLAGE IN THE LIZARD EMBRYOS (TAKYDROMUS TACHYDROMOIDES ) iawkaeewep. Ot BVols.Kac. ot Educ., Tokyo Gakugei Univ., Tokyo The differentiation of the gonadal an- lage was examined by light and electron microscopy in Takydromus embryos incubated at 25°C. In three day old embryos a proli- feration of the coelomic epithelium which consisted of "dark" and "light" cells, was observed on the ventromedial surfaces of the mesonephros. This proliferation occurre: especially rapid in the laterally placed nephrostomal. area of the developing geni- tal ridge. At the same time a cellular mass was segregated from the mesonephric blas- tema. In slightly older embryos the medu- llary cords approached the neighbouring mesonephric corpuscle and joined at the external border of Bowman’s capsule and at the developing interrenal blastema. The medullary cords were composed of flattened Udare sand “Wight celi's* wath elliptical large nuclei and moderately developed rough endoplasmic reticulum. These flattened cells were very Similar to the cells of germinal epithelium in the ultrastructural appearance. These results suggest that the cells of the medullary cords mostly derived from the germinal epithelium and the epi- thelium of the juxta-gonadic coelom in the undifferentiated gonad, although some cells may be derived from the mesonephric blastema. DB 9/ GONADAL SEX DIFFERENTIATION AND CHANGES IN THE NUMBER OF GERM CELLS IN ONYCHODAC- TYLUS JAPONICUS. Takashi Nomura and Hisaaki Iwasawa. BBLOWLG JASE G pF INGLIC EINER, Winsiity 5 Nala eice) 6 Changes in the number of germ cells (nGC) in the course of gonadal sex differentiation were examined in O. japonicus. In both male and female specimens, there was a high positive correlation between gonadal size and snout-vent length (SVL). The mode of sex differentiation of this species belongs to a differentiated type. Gonadal sex differentiation was recognized in some specimens in stage 67 (SVL 19-22mm). At this time, nGC in sexually indifferent gonads was 400-600. In the female specimens in this stage, secondary gonia appeared, and then increased in number. Therefore, nGC became more numerous in the ovaries than in the testes. Simultaneously with the appearance of auxocytes, the individual variation in nGC became greater. In female specimens having 39mm or larger SVL, nGC ranged 2000-10000. Nevertheless, there was found a correlation between nGC and SVL in the female specimens. And there was a positive correlation between the diameter of auxocytes and SVL. In male specimens, on the other hand, there was a positive correlation between nGC and SVL, and nGC reached about 6000 in the metamorphosing larvae having a 40mm SVL (st.71). DB 98 ROLE OF PHOSPHORYLATION IN THE ACTIVITY OF YEAST MATURATION-PROMOTING FACTOR (MPF). K.Tachibana’?, N.Yanagishima~* and Tekishimotols “Deptasof Devi. —Bilol., Natl: Inst. Basic Biol., Okazaki and *Dept. of Biol., Fac. of Sci., Nagoya Univ., Nagoya. We examined the effect of the presence of phosphorylated groups on the meiosis- reinitiating activity of yeast MPF which was extracted from cdc20 mutant cells of Saccharomyces cerevisiae synchronized at M- phasee iinmconerasiimtomthe (cumnrent assumption that MPF is a phosphoprotein stabilized by phosphorylated small molecules such as ®-glycerophosphate and ATP, yeast MPF was still active after the treatment with alkaline phosphatase when injected into immature Xenopus oocytes. Further, even when treated with ATP-Y-S, yeast MPF lost its activity after the removal of ATP-Y-S. Thus, MPF need not necessarily be in a phosphorylated form in order to be active. On the other hand, the meiosis-reinitiating activity was detected when both yeast MPF and the phosphorylated small molecules were present simultaneously and almost undetectable when either of them was present alone. Such complementary effect was also observed when injection of yeast MPF was followed by that of the phosphorylated small molecules. Thus, the phosphorylated small molecules seem to have another effect, such as preventing the activity of phosphatases that might be present in recipient Xenopus oocytes rather than stabilizing the MPF molecule itself. 1052 Developmental Biology DB 99 HOW IS THE FLAGELLAR LENGTH OF MATURE SPERM DETERMINED ? I. COMPARISON BETWEEN NEWT AND XENOPUS IN VITRO. Gunna) Goals Woe, Wejoes OF BiOls, Pac. of Science, Kumamoto Univ., Kumamoto. We have developed cell culture systems which support meiotic divisions and early, mid-spermiogenesis in Cynops pyrrhogaster and Xenopus laevils|\(ine Ww jReve Cy es loomigs7)- In order to study how the flagellar length of mature sperm is determined, we compared the flagellar growth in spermatids between newt and Xenopus; the flagellar length of newt is more than 10 times as that of Xenopus. The dissociated newt primary spermato- cytes were embedded in collagen matrix and those from Xenopus were inoculated in ei coated dishes at cell density of 2- Z x ILO ay) dish. The cells were cultured at 22°C in L- 15 medium containing no serum. Some primary spermatocytes were marked. At regular intervals after second meiotic divisions, 2 dishes were fixed and the flagellar length in spermatids were measured. ineXenopus, pene rlagel lasane teased. smn length up to 27ym in average by the 6th day and stopped the growth. In contrast, in newt, flagella elongated to about 110yum and did not increase the length after the 10th day. 10yg/ml actinomycin D had no effect on flagellar elongation in either animal. But, 10M cycloheximide inhibited the flagellar growth by 80-90% in both animals. These results indicate that the flagellar elonga- tion is under the translational control. DB 100 ACROSOME FORMATION IN NORMAL AND FUSED SPERMATIDS FROM XENOPUS LAEVIS AND THE EFFECT OF CYCLOHEXIMIDE(CH) IN VITRO. Sa Asakura sandy Sha Abe Dept. NOL bio lear Fac. of Science, Kumamoto Univ., Kumamoto. In order to study the mechanism of acrosome formation, we observed the process of acrosome formation in vitro in normal and artificially fused spermatids and examined the effect of CH. Dissociated primary spermatocytes were inoculated in pLL-coated dishes and cultured in L-15 medium without serum at 22°C. Cell fusion was induced between spermatids derived from single primary or secondary spermatocytes by treatment with hypotonic media for 5-10 min. Both in normal and fused spermatids, a number of small vacuoles exist just after the second division. These vacuoles in- creased in size while decreased in number and finally formed a single large acrosome within 30 hr. 10pM CH inhibited the forma- tion of acrosomes completely both in normal and fused spermatids. When CH was washed off, acrosomes reformed. The sensitive pened) tonCH was tnittiale wy hw ateter =the second division. Neither cytochalasin B nor colchicine inhibited acrosome formation in normal or fused spermatids. When spermatids were fused after an acrosome was formed in each spermatid, two acrosomes fused to form a Single larger acrosome. These results indicate that acrosomes are formed by fusion of small vacuoles and that some proteins synthesized within spermatids are responsible for the fusion of vacuoles. DB 101 ISOLATION OF ACROSOME REACTION INDUCING SUBSTANCE FROM AMPHIBIAN OVIDUCT. NEP MOBIMLuvENGL 5” IDS G5 Ol aol s, MAC, of Gen, CUGos 5 Galicun Waligs 5 Calieu. An acrosome reaction inducing substance (ARIS) was isolated from the pars recta portion of the oviduct of the frog, Rana Japonica, Vlhe “ARS walsh tele sit lob aemecimals secretory granules and then fractionated on a Sepharose 6B column after removal of the granules’? limiting membranes. The ARIS has a molecular weight of 195 kD in a reduced condition... he) activaltva om mriae ARIS was assayed as follows. Coelomic eggs treated with both the ARIS and sper- matozoa together showed a dilation of vitelline coats, whereas eggs treated with either one) of them alone did not. | Eilec= tron microscopic observation disclosed vesicles in the most affected part of the vitelline coats which might be the remains of acrosome-reacted spermatozoa. When Spermatozoa were treated directly with the ARIS, membrane vesicles, presumably com- posed of plasma and acrosomal membranes, appeared on the surface of the spermatozoa. Immunofluorescent staining using an anti- serum raised against the ARIS in the rat Showed) Uhe localization joe he yARmSm ann mtaae cells of pars recta and in the vilteliine COL Ov UceieaiaSe Sees, Walls amolies tat during the ovulation the ARIS is deposited in the vitelline coat where the acrosome Peaecitwon oceurs jan Mormady encitaelsanalelorae DB 102 BINDING PROPERTIES OF ACROSOME-REACTED TOAD SPERM K.Takamune and Ch.Katagiri Zool. Inst., Fac. of Science, Hokkaido Univ., Sapporo. Treatment of fresh sperm of Bufo japonicus with FITC-conjugated peanut ag- glutinin (PNA) was found to provide a reliable measure of acrosome-reacted sperm. This was based on the specific binding of PNA to the inner acrosomal membrane (IAM) as defined both by the electron microscopical localization of Ferri tin PNAS on Ene) LAMPands wes icompeier. tive inhibition by galactose. The sperm treated with the oviducal pars recta secretory granules (PRGs) were not stained with FITC-PNA, although they had evidently been acrosome-reacted. However, a high proportion of sperm treated with PRG showed a binding of FITC-labeled PRG also to IAM, and the surface of mid-piece and tail. Analyses of the similar binding pattern of sperm with various FITC-labeled substances revealed that PRG, vitelline coats (VCs) of uterine and fertilized eggs, and VCs of coelomic eggs pretreated with PRG showed a significantly higher binding than the background exhibited by PNA and coelomic egg VCs. These special binding properties of IAM provide a good means of elucidating the molecular mecha- nism of acrosome reaction and its induc- tion by oviducal pars recta substances. Developmental Biology 1053 DB 103 CHARACTERIZATION OF VITELLINE COAT LYSIN FROM TOAD SPERM. H. Yamasaki and Ch. Katagiri. Zool. TiaGit.; PASS Oe SSiep ilOrsieelsCloy Wraaywya- Sapporo. A mild sonication supernatant of sperm of the toad, Bufo japonicus, can easily digest the vitelline coat (VC) of uterine eggs, hardly the VC of coelomic eggs, but mot the VEC of activated eggs. Both this VC lysis and fertilization were inhibited in the presence of Boc-Gln-Arg- Arg-MCA, suggesting the involvement of proteases in fertilization process. Starting from sonication supernatant, a strong VC lysin possessing the hydrolytic activity on Boc-Gln-Arg-Arg-MCA was purified by anion exchange chromatography (Mono Q HR5/5) and gel-filtration (Superose 12 HR 10/30). The activity of purified lysin was inhibited by such serine protease inhibitors, as DFP, SBTI and p-APMSF, but not by chymostatin, E-64 and EGTA. The molecular weight of VC lysin was estimated to be 32K on the basis of the fluorographic image of ~H- DFP-binding. The VC lysin was most active at pH 7.0-7.6 and under the lower ionic strength equivalent to fresh water. These results suggest that the hydrolytic activity studied here represents the VC lysin of Bufo sperm that is involved in fertilization by digesting VC of uterine eggs. DB 104 EXPRESSION OF THE MURINE DIHYDROFOLATE REDUCTASE GENE IN XENOPUS LAEVIS OOCYTES INT (CWiL WWIRE s CeOGa Neen Ol, Kee Mon samcdrSi.. i kegama:, Dept. Applied. Biochem., Fac. Applied Biological Science, Hiroshima Univ., Fukuyama, Hiroshima. When 4 ng of plasmid pSV2-dhfr which contains the simian virus 40 early pro- moter upstream to murine dihydrofolate reductase cDNA was microinjected into the germinal vesicle of a Xenopus laevis oocyte, 3%S-labeled murine dihydrofolate reductase was produced during a 48-hr culture in the OR-2 medium containing [3>S]methionine. On the other hand, the production of the reductase was low and not consistent in oocytes the germinal vesicle of which was microinjected with 4 ng of plasmids containing the adeno- virus major late promoter upstream to murine dihydrofolate reductase cDNA. These results suggest that the simian virus 40 early promoter is more efficient than the adenovirus major late promoter in the transcriptional machinery in the germinal vesicle of the toad. DB 105 DIFFERENTIATION OF VITELLINE ENVELOPE (VE) IN GROWING OOCYTES OF XENOPUS LAEVIS, STUDIED BY ANTI-VE SERUM. nat S.Yamaguchil, J.L.Hedrick2, Ch.Katagiri!. 7AOXG) I. ele WAGs SCikop WOKE LCS Whaaly7s Sapporo, Dept. Biochem. and Biophys., Wmailwo Cali, Dawils, WaoSaAy Differentiation of vitelline envelope in growing oocytes of Xenopus was studied using the rabbit antiserum specific to VE components. Light- and electrone micros- copic observations, employing FITC-labeled antiserum and IgG-conjugated colloidal gold respectively, revealed that the VE antigens were first detectable throughout the cytoplasm of the previtellogenic QOCVvVESS Ate DUMOMe’S Sic, LU, localize preferentially in the periphery of oocytes at st. Il, £ollowed by decrease in the oocyte cytoplasm at later stages. The same antigens localized outside the OOGYECSME Mrs, ait, Watelsits sf Masiipateheis, increased its thickness sharply during st. LESUULUL Aine GCGieaclwaililsy icowaseel Sic, WW—\Wil, The antigens were frequently observed in and around the highly extended oocyte microvilli during late st. I-IiI, but were MEVEr cLOMMGE iam FOlilinelse Cells ae all stages observed. Western blot analyses employing each 10 oocytes revealed that the extracellular VE components appeared first at st. II, whereas the intracellular antigens detected from st. I on contained unique components which are distinct from those in the VE after secretion. DB 106 SCANNING ELECTRON MICROSCOPIC STUDIES OF THE PRIMITIVE STREAK AND MIGRATING MESO- DERMAL CELLS OF THE MOUSE EMBRYO. K.Hashimoto and N.Nakatsuji. Div. of Dev. alos 7 W@algal INS s Ose lelSeliliclay Steal pp Oleleieleel « We observed the ingression and migration of mesodermal cells(MCs) from the primi- tive streak of the embryonic ectoderm in early primitive-streak-stage(6.5-7.0 d)ICR MOUSE SwaAvOSa Was seslsessic Salen Opg ieloKS\ ali GieSSsiGn C1 ene MCS sin Ged-G5 7/5 Cl EGiloiayos was disruption of the epithelial ectoderm and underlying basal lamina(BL). Cells at the periphery of this region spread on the BL and extended many long filopodia to the BL, while those in the central and anterior parts of the primitive streak had many blebs. Later, a few long slender filopodia grew out from each bleb toward the basal surface of the visceral endoderm layer. We saw several MCs on the BL migrated away from the periphery of the mesodermal cell layer where cells kept contact with each other. These cells were spread on the BL, and having a large cell process with long filopodia extending from the tip. These individually migrating cells and MCs at the periphery protruded many long filo- podia toward the direction of migration. The filopodia attached to the BL or a meshwork of the extracellular fibrils present on the BL. Thus, extracellular matrix seems to play an important role in the migration of MCs. 1054 Developmental Biology DB 10/7 CLONAL ANALYSIS OF KIDNEY AND LIVER TISSUES USING BEIGE MOUSE AGGREGATION CHIMAERAS. N.Nakatsuji. Div. of Developmental Biology , Meiji Institute of Health Science, Odawara. Anomalous giant granules of beige (bg) mice have been used as a cell marker in the study of cell lineages of mast cells. Such giant granules are known to exist in other tissues including kidney proximal tubules and liver parenchymal cells. We found that these granules give yellow or orange auto- fluorescence when fixed with formaldehyde. Thus, they can be used as a convenient cell marker recognizable in serial paraffin sections without need of any histochemical processing. Chimaeric mice were produced by aggrega- tion of 8-cell stage embryos of beige (C57BL - bg/bg, Jackson Laboratories) and A/J strain mice. Kidney and liver tissues were fixed in 10% Formalin, dehydrated through ethanol - buthanol series, embedded in paraffin, and serially sectioned at 10um of thickness. After removal of paraffin with xylene, sections were mounted in glycerin and examined with a fluorescence microscope. In the liver tissue, there were patches of beige parenchimal cells with very complicated irregular shapes. In the kidney cortex, the boundary of a patch usually coincided with the tubule struc- ture. Beige cells occupied either whole cross sections of the proximal tubules or only part of them, thus showing polyclonal OnAgain) Of ja jeubwlies DB 108 LONG-TERM RETENTION OF LABELED DNA STRAND IN MOUSE SKIN FOLLICULAR MELANOBLASTS WwoMoieakicel, SjlOieiMeElnal,; IcINeEO@,; WolxoOSlaslCle- Dept. of Biol., Coll. of Gen. Educ., Osaka Univ., Osaka. A hypothetical mechanism for selective segregation of DNA strand at cell division in stem cell populations has been proposed bya Camianisi. It has been assumed that UV inactivation of melanoblasts having 5- bromodeoxyuridine-labelled DNA (BrdU-DNA) may result in the depigmentation of hair during the regeneration of hair follicles. The long-term retention of BrdU-DNA in follicular melanoblasts was examined to test the hypothesis. BrdU injected was incorporated into DNA during stimulated juaG baie SiceieslOin O12 ~MeEIlAinO@lolages Asie ie eis plucking. UW Ligne ceOm ain Wi 2O Soho lamp was directed on the back of mice at 6 Oe 12 WEEISS GicieSie WieClW ins) SCieLOing The proportion of white to fully pigmented hair was estimated during 7 to 16 weeks after UV irradiation. In UV-irradiated mice, the white hair ratio was 20%, and finally reached 50%, but in non-irradiated or no-BrdU mice, the ratio was less than 1%. In mice which were plucked 3 times at intervals of 3 weeks after BrdU injection and irradiated with UV, the white hair ratio was 208. BrdU-DNA in the cell nucleus was examined by the immunoperoxi- dase staining using a mouse monoclonal antibody against BrdU. Cells containing BrdU-DNA could be detected in follicular cells up to 9 weeks after BrdU injection. DB 109 VIDEO-IMAGE ANALYSIS OF COAT-COLOR PAT- TERNS IN ARTIFICIALLY-PRODUCED CHIMERIC MICE BY MEANS OF A PERSONAL COMPUTER- BASED Sauls Co RMaelaiis Zool. MInst., Faculty of Science, Univ. of) folsvzor Tokyo. A personal computer-based program package, VIAS.CM (Video Image Analysis System for Chimeras and Mosaics ), developed by the author is particularly suited for the quantita- tive analysis of the sampled video-images of the coat color patterns on the pelts of the artificially-produced chimeric mice. No similar system has been des- cribed in the literature. It was shown that the use of VIAS.CM, or an equivalent system, is indispensable for the analysis of coat color patterns due to complex phenotypic expression of hair pigmen- tation, such as those created on the coat of BALB/c -- C3H/HeJ mice. In VIAS.CM, it is possible to estimate the relative content of eumelanin and pheomelanin differentially, and calculate the mixing ratios between BALB/c and C3H/HeJ compo- nents. From the results of grey value profiles taken along mid-dorsal axis, it wa susggested that number of melanoblast clones in mice is probably much larger than so far proposed and 22 in terms of mimimum recognizable stripes ( 28 after correction for the random clumping ) unilaterally in the trunk region. DB 110 Morphological studies on Purkinje cells in the neo- and archicerebellum of rats. H.Keino, E.Aoki and S.Kashiwamata. Dept. Perinatol. Inst. Dev. Res., Aichi. i It is generally known that the neo- cerebellum is severely damaged by various E\CSaics SwiCle ENS} loyal aLaliaibioalin GinGl WaiewS, lowic the archicerebellum is scarcely affected. We previously showed histochemically that Purkinje cells in lobule VII contain more acidic materials than those in lobule X. In this study, ultrastructural observa- tions were done on Purkinje cells of adult and neonatal (7-day-old) Sprague-Dawley strain Gunn rats. Purkinje cells of new- borns showed less organelles than those of adult rats. Packed cisternae of the smooth ER, some of which were tightly associated with mitochondria, were conspicuous in Punkinwe welts amy Wobillcis 7 laa amnici aaron adult rats, while the cisternae of rough ER, which were arranged in parallel with the nuclear membrane, were markedly less in the former loble than in the latter. Well-developed Golgi apparatus were recog- nized in Purkinje cells in both lobules of adult and neonatal rats: No striking mor- phological differences of Golgi apparatus were observed between the neo- and archi- cerebellum, but the number was found more in lobule VII than in lobule X. The well- developed Golgi apparatus of Purkinje GELS ain LOluUle WL may ALSO medicaice higher activity of the membrane synthesis (neonatal rats) and turnover (adult rats) compared with those in lobule X. Developmental Biology 1055 DB 111 INTERACTION BETWEEN MELANOCYTE AND KERA- TINOCYTE FROM GENETICALLY DIFFERENT EPI- DERMIS OF NEONATAL HUMAN FORESKINS. T. Hirobel, E. Flynn2 and G. Szabo2. DiveOeesToOles, Natl. mnst. Radiol Sea., Chiba and Lab. Elect. Microsc., Harvard Sch. Dent. Med., Boston, USA Pure black (Negroid) and white (Cauca- soid) melanocyte cultures in melanocyte- growth-factor-supplemented medium were co-cultured with pure black and white keratinocyte cultures in Minimum Essential Medium containing 10 % fetal bovine serum, and pigment donation was observed with light and electron microscopes. Melanosome donation from black melano- cytes to white keratinocytes was observed to have a similar tendency to that of black melanocyte donation to black kerati- nocytes. Similarly, pigment donation from white melanocytes to black keratinocytes was observed to have the same manner as donation from white melanocytes to white keratinocytes. This suggests that pigment donation occurs between melanocytes and keratinocytes derived from genetically different human foreskins. White melanocytes co-cultured with black keratinocytes became more dendritic and more pigmented than the control group. The result suggests that some substance produced by black keratinocytes is involved in regulating melanogenesis of white melanocytes. DB 112 TISSUE MOVEMENT SPECIFIC TO THE MESENCHYME WHICH CAN INDUCE BRANCHING MORPHOGENESIS OF MOUSE SALIVARY EPITHELIUM. Hr Nogawa! and Y. Nakanishi 1Biol. Lab., Coll.,of Arts and Sci., Chiba Univ., Chiba and Dept. of Chem., Fac. of Sci., Nagoya Univ., Nagoya. We studied two behavioral activities of mesenchymes from mouse embryonic subman— dibular gland, lung, stomach, mandible and skin, comparing with their ability to induce submandibular epithelium to branch. Submandibular epithelium branched well in recombination with submandibular or lung mesenchyme, less well with stomach mesen- chyme and never with mandibular or dermal mesenchyme. Studies of the first behav-— ioral activity of contraction of collagen gels revealed that all the five kinds of mesenchymal cells were able to contract collagen gels in various degrees and that dermal cells with no branch-inducing abil- ity had the highest gel-contracting activ— ity among then. In studies of the second activity of separation of plastic beads placed in a mesenchymal mass, submandibu- lar or lung mesenchyme separated the beads well and the other mesenchymes separated the beads in a degree corresponding to their branch-inducing activities. hme lapse cinematography showed that the sepa-— ration of beads was caused by.the flowing movement of the mesenchymal tissue, suggesting that this movement may have an important role in bundling of collagen fibres and widening of clefts. DBe iS DIFFERENTIATION OF EPITHELIAL CELLS IN THE GOLDEN HAMSTER OVIDUCT H.Abe and T.Oikawa , Developmental and Reproductive Biology Center, Yamagata The oviduct epithelium of the golden hamster consists of two kinds of cells, ciliated cells and secretory cells. The ultrastructural changes in the process of the differentiation of the two cells in postnatal hamster oviduct were investigated by means of electron microscopy. in thes sepachel um soneestine ampulla of newborn hamster, differentiated ciliated cells or secretory cells were not identified. In these undifferentiated cells, free ribosomes were wel] developed, but rough endoplasmic reticulum (RER) and Golgi complex were undeveloped. Ciliated ceils i were first identitted on 4.5 “days. They gradually increased in number until 12.5 days, then increased at an increasing rate UE ial 2107. 5a dialy’S.. In contrast, nonciliated cells of 12.5-day-old hamster contained well-developed RER and Golgi complex. These cells began together’ the production of secrtory granules from 15.5 days. The number of these cells was Constant] 1 rom oo. tOeZ2o.b. dayser Nosh com the epithelial cells differentiated into either wel iated ‘celiise on secretpmymee celels bys Z0co days, Dhese results: anditcate that ciltvated cells besin to. diftterenttare son Aon dayse al ters bith wand! 1h daysmaslaltker: nonciliated cells begin the production of secretory granules. DB 114 DEVELOPMENT OF COLLAGENOUS MATRIX IN THE FIBROCARTILAGE OF THE OS PENIS OF THE RAT. R.Murakami, K.Miyake and I.Yamaoka. Briolky inst, aikaG. 1oCiy,. Wamague hws Ummrsvier, Yamaguchi. Distal segment of the os penis of the rat is a fibrocartilage whose development is dependent on androgens. Collagenous components in the fibrocartilage of the rat were analyzed by SDS-PAGE, immunoblot and immunofluorescence using anti-types I and II collagen sera. Major collagens in the fibrocartilage were types I and II. Type I collagen appeared in the extracellular matrix of immature cartilage at about 1 week. Type II collagen appeared at about 4 weeks surrounding mature chondrocytes which developed as small clusters in the distal segment of the os penis. Both types of collagens co-existed in the cartilage matrix in adult males. When the rudiments of the distal segment in rat fetuses were cultured in vitro in the presence of testosterone, fibrocartilage containing both types I and II collagens developed. In the absence of testosterone, only a loose connective tissue positive for anti-type I collagen serum developed. These results suggest that androgen is necessary at least for the synthesis of type II collagen in the fibrocartilage of the oS penis of the rat. 1056 Developmental Biology DB 115 THE EFFECT OF 12-O-TETRADECANOYL-—PHORBOL- 13-ACETATE (TPA) ON THE DIFFERENTIATION OF THE MOUSE NEURAL CREST CELLS IN VITRO. S.Tanaka’, Koleo> ame Wo Walkeucinit > 5 UBaell Inst., Tohoku Univ., Sendai. “WEE of Biol., Col. of Gen. Educ., Osaka Univ., Toyonaka. In the mouse neural crest cell culture, only crest cells associated with epithe- lial sheet from the explanted neural tube differentiate into melanocytes. When the explanted neural tubes were removed in the early phase of the culture period, however , the differentiation into melanocytes never occurred in spite of the appearance of adrenergic neurons (Ito and Takeuchi, 1984). When the tumor promoter TPA, which has been shown to influence cell differentia- tion in several in vitro systems (Sieber- Blum and Sieber,1981; Weston et. al.,1986) , was added to the culture medium, the differentiation of the mouse neural crest cells into fully developed melanocytes was observed. On the other hand, a-MSH, which induces the differentiation of melano- blasts into mature melanocytes, did not induced the differentiation of neural crest cells into melanocytes. This result seems to indicate that TPA promotes the differentiation of the mouse neural crest cells into melanocytes. DB 116 EFFECT OF 5-AZACYTIDINE ON RAT EMBRYONIC DEVELOPMENT IN VITRO. M. Matsuda, R. Kimura and R. Shoji. Dept. Embryol., Inst. Dev. Res., Aichi Prefec. Colony, Kasugai, Japan. Rat embryos (day 9.5 of gestation) were cult nied’ ini valtaom sions = 4:8) hiaon rotating bottles containing rat serum with or without 5-azacytidine (0.2 ug/ml) and assessed for embryonic growth and development at regular intervals. After 48 h in culture in rat serum, development of the embryos was at a rate almost identical to that seen in vivo 11.5-day embryos. When the embryos were cultured for 48 h in rat serum with 5-azacytidine, their heads were smaller compared with control embryos and cephalic neural folds remained open, although they closed after 33 h in control culture. However, somite number, crown-rump length and morpho- logical features except head were almost the same as control embryos. Histologi- cal observations of the abnormal embryos showed that unfused neural folds at the hindbrain, an abnormal elevation of neural folds at the midbrain and the lack of the apices of the forebrain. These embryos also showed the abnormal migration of neuroepithelial cells. The abnormal cell migration may cause the irregular elevation of neural folds, which prevent the fusion of neural folds. DB 117 FOFMATION OF ELASTIC FIBERS BENEATH MOUSE ORAL EPITHELIUM IN VITRO AND IN VIVO. T.Yamaai, Dept. ‘of Orall Anat®,\ Okayama Univ. Dent. Sch., Okayama. The formation of the elastic fibers in the mouse oral proper layer was examined on in vitro and in vivo by the method of the light microscopy. The upper jaws from 17-day fe- tuses of C3H mice which placed in milli-cell culture cups were cultured in DM-160 medium with 10% fetal calf serum for 7 to 14 days. The elastic fibers were stained by resorcin- fuchsin. The evidence for the elastic fibers was found in the upper jaws from 17 days of preg- nancy and indicated quite clear by a week af- ter birth. However, the elastic fibers were appeared in the only buccal side of the dent- al lamina beneath the oral epithelium. On WINS Ollie Ineiael) Ela Eimowinw Cit Wie Glasbie i= bers in the cultured upper jaw on the 7th day was already more abundant than it in vivo. In addition they were existed on the buccal and palatal sides of the dental lami- na. There were many types of cells on the surface of the oral epithelium of the cul- tured upper jaws. Since they were arranged in aS a mosaic, it seemed no specific ar- rangement from place to place of the surface of the oral epithelium as compared with that of in vivo. The cultured upper jaws adhered to the surface of the milli pore filter and were maintained under normal condition. DB 118 SPICULE FORMATION IN SEA URCHIN EMBRYOS KEPT IN Cl -DEFICIENT ARTIFICIAL SEA WATERS y. Fujino!, k. Mitsunaga2, A. Fujiwara2 and I. Yasumasu2 Ipept. of Pharmacol., MEALS) Winsiyc Selon, Cie Med., Tokyo and 2Dept. of Biol., Sch. of Educ., Waseda Univ., ‘lokyo In artificial sea waters (ASW's) in which Ci concentrations were reduced to 30 3 of normal concentration in ASW by its re- placement with Br , Cl0,, NO; and HCOO , and to 70 % by its substitution with ace- tate, propionate and BrO7, early develop- ment of sea urchin embryos was apparently normal and developed to quasi-normai plutei with spicules. The replacements of more than 80 % of Ci amount with the former gruop of anions, made spicule rods somewhat poor and the substitutions of more than 40 3 Cl amount with the latter anions strongly biocked spicule rod formation. In embryos kept in the former Cli deficient ASW's, anions replaced with Cl” were transported into embryos in relation to the rate of spicule tormation and the transports of these anions were inhibited by nifedipine or D{LDS, which biocked formation of spic- ules. In embryos kept in the latter ASW's, the rates of uptake of these anions, with which Cl7 in ASW's was substituted, were quite poor and did not relate with the rate of Ca** uptake. Uptake of these anions was not affected by nitedipine and DIDS. These suggest that Ca’* uptake through Ca’* chan- nels is coupled with co-transport of anions through anion channels. Developmental Biology 1057 DB 119 SPECIFIC BINDING OF LECTINS WITH THE CHROMATIN OF SEA URCHIN EMBRYOS DURING THE COURSE OF DEVELOPMENT. S.Kinoshita!, K.Yoshii“~ and Y.Tonegawa?>. Dept. of Biol., Saitama Medical School, Saitama, “Biol. Lab., St. ee Univ., Sch. of Med., Kawasaki and ~Dept. of Regulation Biol., Saitama Univ., Urawa. Embryonic cells of sea urchin were made permeable by treating with glycerol for the purpose of allowing penetration of lectins into the cell, and it was found that some lectins showed notable affinity with the nucleus. Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins, and the amount of lectins bound with the chromatin was measured by fluoro- metry. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) Extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) Very low level of binding was main- tained throughout all stages, and no Particular change was observed at any stage of development (WGA, SBA and UEA-I). DB 120 AP4A-HYDROLYZING ENZYMES IN SEA URCHIN EMBRYOS M.Morioka & H.Shimada (Zool.Inst., Fac.Sci. Univ.Tokyo, Tokyo) Based on our findings that the level of soluble AP4A drastically decreased at the onset of S phase with the concomitant inc- rease its binding to the nuclear matrix, we suggested that AP4A is involved in the ini- tiation of S phase in sea urchin embryos. However, the amount of AP4A being bound to the nuclear matrix was not large enough to explain the fate of AP4A disappeared from the soluble fraction at the onset of §S phase. In this paper, we report some features of the AP4A-hydrolyzing enzymes in sea urchin embryos. The embryo contains several enzymes which hydrolyze AP4A to ATP and AMP but not to ADP. One of these enzymes was partially purified by DEAE-cellulose column chromato- graphy, Sephadex G-75 gel filtaration and AMP-Sepharose affinity chromatography. The molecular weight of the enzyme was 20,000 and the Km value for AP4A was 3uM. The enzyme required Mgt* for the maximal acti- vity, while it showed little activity in the presence of EDTA. The activity was completely inhibited by ZnCl2 and markedly inhibited by CaCl2. AP4A-hydrolyzing acti- vity was very low in the unfertilized egg. Tough the enzyme became activated immedi- ately after fertilization, the activity did not change corresponding to a cell cycle. DB 121 DIFFERENTIATION OF SEROTONERGIC GANGLION CELLS IN SEA URCHIN LARVAE, MESPILIA GLOBULUS. Y. NAKAJIMA. Dept. of Biology, Keio Univ., Yokohama. a ee This work was undertaken in order to as- certain the relationship between’ the pre- oral serotonergic nerve of pluteus and the cells composing the apical plate with long cilia, which is supposed to function as a sensory organ in blastula and gastrula. Localization of serotonergic cells in ani- malized blastulae induced by the treatment with Evans Blue, vegetalized embryos pro- duced by LiCl, and open and flattened em- bryos made by the treatment with Ca-defi- cient sea water were examined by indirect immunofluorescence microscopy. In the animalized 24 h-old embryo, sero- tonergic cells appeared not in apical zone but in the second quarter of animal-vegetal axis. In later stages, numerous serotoner- gic cells and cell processes became visible mainly in this zone. Serotonergic cells were not detected in vegetalized exogas— trulae. In "open" embryos, cellular dif- ferentiation proceeded normally after the transfer into sea water, and serotonergic cells and cell processes appeared as a ringlet in the epithelium around the tip of archenteron. These findings indicate that serotoner- gic preoral ganglion cells of echinopluteus derive from the ectodermal epithelium sur- rounding the apical plate cells. DB 122 Crt ANALYSIS OF POLY(A)*RNA POPULATION IN VEGETALIZED SEA URCHIN EMBRYOS. M. Komukai and I. Yasumasu Dept- Of Biol., Sch. of Educ., Waseda Univ., Tokyo. The change in the poly(A)*RNA population between Li*-treated and normal sea urchin embryos was analyzed by cDNA-RNA hybridization. Poly(A)*RNA was isolated from gastrulae or 24 hr vegetalized embryos which were treated by 60 mM LiCl <= 3 hr (from 16 cell to 64 cell stage). H-labeled cDNA was synthesized in vitro using AMV reverse transcriptase with poly(A)*RNA from yederal "aed embryos as a template. This H-cDNA had a specific FadLoacta vaibyiotsl 6c +107 Sdpm/ugs rhe cDNA was hybridized with a large excess of poly(A)*RNA from either vegetalized or normal embryos (RNA/cDNA>1.1x10~), and the extent of the hybrids was detemined by a S1 nuclease assay. The reaction occurred over 3 orders of Crt. Plateau value of the fraction of S1 nuclease-resisitant cDNA hybridized with gastrula poly(A)*RNA was substantially lower than that with its own vegetalized poly(A)*RNA. This subtracted value indicates the qualitative difference between normal and vegetalized poly(A)*RNA population. 1058 Developmental Biology DB 123 EFFECT OF APHIDICOLIN ON THE GASTRULATION OF SEA URCHIN EMBRYOS H. Fujisawa and S. Amemiya. Fac. of Educ., Saitama Univ., Urawa, and Misaki Marine iOQlowiCeail SieeeloOm, Bees O% Selo, Wily. of Tokyo, Miura. In sea urchin embryos, we noticed that the rate of increase in cell number at the time of archenteron invagination was generally higher than that during the preceding swimming blastula stage. We also found that the cell number of embryos animalized with Zn ion was larger than that of the embryos vegetalized with Li ion. From these facts we postulated that in normal embryos, partial cell prolifera- tion in the animal hemisphere contributes the invagination of the archenteron. In order to test this, we observed the effect of aphidicolin, an inhibitor of DNA alpha- polymerase, on gastrulation. The sea urchins used in this experiment were Hemi- centrotus pulcherrimus and Clypeaster japonicus. Aphidicolin ( 0.2 pg/ml sea water ) was added to the embryos at_ the mid-blastula stage. The drug effectively suppressed cell proliferation from that stage, although the embryos were able to form the archenteron. This result shows that the increase in cell number occurring at the onset of gastrulation does not play any role in the invagination of the arch- enteron. DB 124 IONIC EFFECT ON THE REVERSAL FORMATION OF CILIA IN THE ISOLATED GUT OF SEA URCHIN EMBRYOS. H.Suzukawa & M.Ishikawa , Dept.of Biol., Fac.of Sci.,Ehime University, Matsuyama. It has been reported by Amemiya(’'79) that when isolated guts of sea urchin embr- yos were incubated in a normal sea water for several hours, cilia were recognized on the opposite cell surface to their original growing positions after closure of the bla- stopore. In the present study, effects of several ions and ion-transport blockers on reversal formation of cilia in the isolated gut were investigated. 2+ and ait was found that free from Ca Mg in the culture medium does not influe- nce on cilium formation. Verapamil (Ca2t+ bl- ocker) and ouabain(Nat+-K*tATPase blocker) not inhibit cilium formation. Defect of Nat and ethacrinic acid(Cl~,HCO3~ blocker) inh- ibit elongation of the cilium after initial appearance of it. Reversal formation of the cilium does not occur in the K*t-free medium or allyl isochiocyanate(Ht-Kt blocker), but when the isolated gut is removed into the above conditional medium soon before initi- al appearance of the cilium, elongation of the cilium is not inhibited. From above mentioned results, it may be suggested that existence of Kt in the cult- ure medium is necessary for reversal forma- tion of cilia in the isolated gut. DB 125 PURIFICATION OF EXOGASTRULA-INDUCING PEPTIDES IN EMBRYOS OF THE SEA URCHIN, ANTHOCIDARIS CRASSISPINA. K.Ishihara, Y.Tonegawa, T.Yoshizumi and T. Suyemitsu. Dept. of Regulation Biology, ICS KONE Seiko 7 Seliliczbivel Wiel, , Wieewvel - We tried to purify the exogastrula- inducing factors from supernatant of the embryo homogenate of the sea urchin, Antho- Cidaris crassispina. Successive colume chromatography with DEAE-cellulose, Sepha- dex G-100 and G-50 revealed only one frac- tion which was active for inducing exo- gastrula. The fraction was separated by CM- sepharose CL-6B column into 8 fractions among which 3 fractions of Fr.D, Fr.E-2 and Fr.VI were biologically active. SDS-poly- acrylamide gel electrophoresis of these fractions showed the presence of a single band in Fr.D and Fr.VI, and two bands in Pr.E>2 with MW. of 4 ,.800),. 7, 200,60 5500mand 8,300, respectively. Reverse phase HPLC of these fractions gave also a single peak from Fr.D and Fr.VI, and two peaks which were eluted separately from Fr.E-2. These fractions were named as peptide A, D, B and C, and analyzed the amino acid composition. N-terminal amino acid sequences were found to be Asp-Ser- for peptides A and B, and to be Asp-Thr- for peptides C and D by direct Edman degrada- tion. C-terminal sequences were found to be -Thr-Glx for peptide A, -Glx-Thr for pep- tides B and C, and -Ser-Ala for peptide D by the amino acid analyses of hydrolyzed peptides with carboxypeptidase Y. DB 126 THE PRIMARY STRUCTURES OF THE EXOGASTRULA- INDUCING PEPTIDES IN EMBRYOS OF THE SEA URCHIN, ANTHOCIDARIS CRASSISPINA. T.Suyemitsu, Y.Tonegawa, S.Noguchi and kK. Ishihara. Dept. of Regulation Biology, Fac. of Sci., Saitama Univ., Urawa. The complete amino acid sequences of the exogastrula-inducing peptides A, C and D in embryos of the sea urchin, Anthocidaris crassispina were determined. The homogenous peptides were cleaved with lysine endo- peptidase or arginine endopeptidase. The resulting peptides were purified by reverse phase HPLC. Sequencing of the fragments was achieved with automated Edman degradation. The exogastrula-inducing peptides A, C and D were composed of 52, 58 and 53 amino acid residues and their molecular weights were calculated to be 5,754, 6,463 and 5,735, respectively. The sequence of the peptide A was AspSerValTyrGlinCysAsnArgAsp ThrAsnSerCysAspGlyPheGlyLysCysGluLysSerThr PheGlyArgThrThrGlyGinTyriIleCysAsnCysAspAsp GlyTyrArgAsnAsnAlaTyrGlyGlyCysSerProArgThr GluOH. The sequence of the peptide C was AspThrLysGlyGlyCysGluArgAlaThrAsnAsnCysAsn GlyHisGlyAspCysValGLuGlyArgTrpGlyGlinTyrTyr CysLysCysThrLeuProTyrArgValGlyGlySerGluSer SerCysTyrMetProLysAspLysGluGluAspValGlulle GluThrOH. The sequence of the peptide D was AspThrValAlaArgCysGluArgAspThrLysAsnCysAsp GlyHisGlyThrCysGlnLeuSerThrPheGlyArgArgThr GlyGlnTyrileCysPheCysAspAlaGlyTyrArgLysPro AsnSerTyrGlyGlyCysSerProSerSerAlaOH. Developmental Biology 1059 DB 127 STIMULATION OF PROTEIN SYNTHESIS IN THE MITOCHONDRIA OF SEA URCHIN AND STARFISH BEFORE GASTRULATION. T.Kawashima and T.Nakazawa. Dept. of Biol.,Fac. of Sci.,Toho Univ.,Funabashi. a a A transient increase in protein synthesis in vitro was observed at the mesenchyme blastula stage of sea urchin and starfish embryos. This stimulated activity was inhibited by chloramphenicol but not by cycloheximide, and was localized in the mitochondrial fraction. Reconstituting experiments in which poly U-dependent protein synthesis was carried out showed the mitochondrial peptide elongatlon sfactor to be essential for increasing the protein synthetic activity in mesenchyme blastula, but aminoacyl tRNA synthetase and ribosome fraction containing initiation factor not to be involved in this increase.To clarify the nature of protein synthesized in the mitochondrial fraction of starfish embryos, [-H]-amino acid-incorporated protein was purified with column chromatograhy. Properties of the synthetic protein will be examined in future. DB 128 CORRELATION BETWEEN PROTEIN PHOSPHORYLA- TION AND DIFFERENTIATION IN SEA URCHIN EMBRYOS. 1 1 S.Shinohara , M.Motojima,, K,Mitsunaga , Y.Fujino and I.Yasumasu . Dept. of iol., Sch. of Educ., Waseda Univ. and Dept. of Pharmacol., Teikyo Univ. Sch. of Med., Tokyo. H-7, an inhibitor of C-kinase, inhibited morphogenesis, especially spicule formation, in sea urchin embryos and pseudopodial cable growth, in which spicule rods were to be produced, in cultured micromere-derived cells. HA1004, an inhibitor of A-kinase, did not block them. Pi incorporation into proteins was markedly inhibited by H-7 but was not by HA1004 both in embryos and in cultured cells. These results suggest that spicule formation, especially outgrowth of the cables, is probably supported by protein phosphorylation mediated by C-kinase. In embryos, which were introduced to H-7 and HA1004 at 16 hr after fertilization, proteins were analyzed by SDS-PAGE at the gastrula and the pluteus corresponding stages. The abnormal embryos kept with H-7 in a period between the mesenchyme blastula and the pluteus equivalent stage contained 41 K protein, which was found in blastulae and was absent in plutei. P incorporation into 43, 41, 28 and 25 K proteins was somewhat inhibited by H-7 but was not by HA1004. DBY 129 CATHEPSIN B ACTIVITY IN SEA URCHIN EGGS AND EMBRYOS. Yoo Okada andimu YOKOtale » Boles mabe Anche: Pref. Univ., Nagoya. Cathepsin B activity was studied in sea urchin eggs and embryos. Cathepsin B was purified approximately 70-fold from un- fertilized eggs of Hemtcentrotus pulcherri- mus by ammonium sulfate fractionation, col- umn chromatography on CM-cellulose and gel filtration through Sephadex G-100 with a recovery of 4%. Gel filtration gave a mo- lecular weight more than 200,000. It is as- sumed that cathepsin B may be in the form of complex with other proteins. The enzyme showed the highest activity arround pH 5.5 to carbobenzoxy-Arg-Arg-methylcoumary lami- de, and Km to the same substrate was esti- mated to be 0.01 mM at pH 5.5. Localization of cathepsin B in unferti- lized eggs and the activity of cathepsin B in the embryo during development was inves- tigated in Anthoctdaris crasstspina. Nine- ty-eight percents of the activity in the egg were detected in the nuclear and mito- chondrial fractions. This result suggests that cathepsin B may be distributed mainly in the yolk granules. During the embryoge- nesis, remarkable changes in the activity of cathepsin B, which is assumed to partic- ipate in the disintegration of a major yolk protein, were not observed. DB 130 PROPERTIES OF THE CONTRACTION-INDUCING FACTOR IN THE ABORAL INTESTINE OF THE SEA URCHIN AND ITS MONOCLONAL ANTIBODY. N. Takahashi!, N. Sato2s M, Takahashi! and K. Kikuchi2, Marine Biomed. Inst. and Dept. of Pathology, Sapporo Med. Coll., Sapporo. The characteristics of gonadal contrac- tion-inducing factor(s) (CF) in the aboral intestine of the sea urchin Strongylocentro- tus intermedius were determined on the basis of measurements of gonad responses to CF under various conditions. CF was found to show heat stability and resistance to- ward several kinds of proteases. Its mo- lecular weight was within the range of 2000 to 4000. The monoclonal antibody (MoAb) of this factor was obtained from hybridomas secret- ing it, by the mechanical response of gonad to mixture of CF with the MoAb, the enzyme- linked immnosoluvent assay (ELISA) and the avidin-biotin method (ABC). The spleen cells of BALB/c mice immunized two times with 0.5 ml supernatant (40,000 rpm) from aboral intestine were hybridized with NS-1, a hypoxanthine phosphoribosyltransferase- deficient myeloma line of BALB/c mice. The hybridomas responsing to CF by the ELISA and the ABC method and inhibiting gonadal contraction were selected for the MoAb of CF. The MoAb was designated as "#11-B-2". We are presently working to establishing the MoAb.of the receptor of CF. 1060 Developmental Biology DB 131 OOGENESIS AND TIMING OF COPULATION DURING FEEDING IN THE ADULT TICK, HAEMAPHYSALIS LONGICORNIS. Y.Yano, T.Mori, S.Shiraishi and T.A.Uchida. “ZOO > Ibeio, BAO Or ABien , IMiyusinw Winitwo » Fukuoka. In the unfed stage, the ovarian oocytes in the late stage of prophase of the meiosis I were connected with each other through intercellular bridges; some oval mitochondria were present in groups in the scanty cytoplasm. At 4-day phase in the feeding stage, the oocyte protruded into the haemocoel increasing its cytoplasmic volume and was attached to the ovarian wall by the funicle cells originated from the Ovarian epithelium. The microvilli of both the oocyte and the funicle cell developed well on the boundary zone between them, and many mitochondria became scattered in the egg cytoplasm. Myoepithelial cells with contractile filaments were recognized and appeared to be derived from the ovarian epithelium. Copulation seemed to occur after the above differentiation of the epithelial cells. At the engorged and detached stage, vitellogenesis and egg- shell synthesis have almost completed in the oocyte with polarity. Because of the invagination between the adjacent funicle cell membranes, it may be easy for the oocyte to fall into the ovarian lumen. Spermatozoa found in the lumen were in close contact with the microvilli of the Ovarian epithelial cells containing a large quantity of rER, or lodged in inden- tations of the funicle cell membranes. DB 132 REPEATED REGENERATION OF THE COCKROACH LEGS. A. Tanaka, M. Ohtake-Hashiguchi and E. OME, “DE Ows Ost IiOlZ, WEOGs Oil Cio, INebeel Women's Univ., Nara. GERMAN Two series of experiments were repeated-regeneration Caiionlteck Ole Ga Caen LOIS, jWhalcls Eelael Jouligchllog O22 jooOwa SOxnos during the entire postembryonic develop- MEINE o One was autotomy at trochantero- femoral articulation; the other was amputation from basal coxa. iia © lorEla GOCrilMie rcs, ONG “OO “wae Six Jess was operated before the regeneration critical INSioak@el sigl wae Sie Wnseeleo iit ey PE EeMmeicense appeared in the following instar, it was repeatedly operated; if not, operation was postponed until a regenerate appeared. In autotomy regenerates repeatedly appeared almost every instar until the adult stage, while in amputation no regenerates were present just after postoperative molts. Regenerated femur and tibia developed well after autotomy, whereas those after amputation were considerably smaller. All the regenerated legs had tetramerous tarsi. Regenerated legs from autotomized stumps showed incomplete homoeotic MOAN PAMGLOMN AS wo Whe lalinol or IeeSc Regenerates from foreleg stumps’ showed intermediate tibia/femur ratios between normal foreleg and midleg, and those from the hindlegs, intermediate between normal hindleg and midleg. Possible modifiers were suggested for the homoeotic’ and tetramerous regeneration. DB 133 REGENERATION OF ANTENNAE IN Riptortus clavatus (HETEROPTERA) IN RELATION TO AMPUTATION LEVELS AT THE FIRST INSTAR K.IKEDA and H.NUMATA. Dept. of Biol., Fac. of Sci., Osaka City Univ. Osaka. The structure of regenerated antennae of adults was observed with a scanning electron microscope after the amputation at various sites at the first instar. The excessive growth of each segment depended on the length of the distal segment of the remaining antenna (RDS). If RDS was longer than a certain length, the more distal the segment was, the more the excessive growth was. Moreover, the structures peculiar to the normal distal segment, e.g. sensory hairs, grooved pegs, appeared on RDS which grew most excessively. If RDS was shorter than the critical length, however, the second distal segment grew most excessively, and the structures peculiar to the normal distal segment appeared on both RDS and the distal part of the second distal segment. When a whole antenna was removed, the antenna was never regenerated. After the amputation at the first (proximal) or the second segment, a new segment often arose from RDS. When the amputation was at the proximal part of the third segment, anew segment sometimes arose from the second distal segment. DB 134 EXPERIMENTAL EGG ACTIVATION IN THE SAWFLY, ATHALIA ROSAE (TENTHREDINIDAE, HYMENOPTERA) M. Sawa and K. Oishi. Div. of Sci. of Bio- logical Resourses, Grad. School of Sci. and Technol. ,Kobe Univ., Kobe. Mature unfertilized eggs (ripe oocytes) dissected from ovaries of the turnip sawfly (Athalia rosae), if soaked in distilled wa- ter, are activated and initiate parthenoge- netic development (Naito 1982). In the present study, we examined the effects of various treatments on egg activation. Ef- fect of osmorality was examined by dis~— secting and maintaining the ripe oocytes in in NaCl, KCl, CaCl,, MgCl,, and sucrose at various concentrations. Frequency of acti- vated eggs, determined at blastoderm stage, decreased with increasing concentrations of the solutions. Egg activation was prevented at about 300 mOsm in CaCl,, MgCl,, and su- crose. NaCl, however, inhibited egg acti- vation completely at 200 mOsm, while KCl did so only at 600 mOsm. Effect of pH was examined using 0.15M phosphate buffer. Most eggs were activated at pH 5.0, but none at 7.0 and 9.0. Finally effects of various other treatments were examined. Ripe oo- cytes were dissected out, kept in NaCl (300 mOsm), and variously treated: Brief (3 min) desiccation (up to 70% activated), passage through a narrow capillary tubing (up to 34 %), puncture with a sharp glass needle (up to 50%). Altogether the present results ap- pear to indicate the involvement of the egg surface membrane potential in the egg acti- vation in this system. Developmental Biology 1061 DB 135 THREE STORAGE PROTEINS IN THE COMMON CUT- WORM, SPODOPTERA LITURA. Se LoyOubabweNematol se bntomol.) Fac. Agricul., Saga University, Saga. In the common cutworm, Spodoptera litura, whole of two proteins named SL-1 and SL-2, and part of one protein named SL-3 were found to be sequestered into the fat body from the haemolymph during the pharate pupal stage.SL-1 and SL-2 appeared in the haemolymph for the first time in the early last larval instar and their appearance was blocked by juvenile hormone application at this stage. While SL-3 increased during larval-larval ecdysis in the penultimate larval instar and the juvenile hormone treatment could not inhibit its rapid increase in the haemolymph in the last larval instar. All of these proteins decreased in the body during the pharate adult development and disappeared shortly after the adult emergence. The properties of these proteins were similar , all of which had molecular weight of around 500,000 and were composed of sub- unit of around 80,000. SL-1l and SL-2 were similar in the amino acid composition, but differed from SL-3, which contained excep- tionally high level of phenylalanine and tyrosine and resembled calliphorin in the amino acid composition. From these resurts, it was concluded that SL-1l and SL-2 were pupal storage proteins and SL-3 was arylphorin. DB 136 HISTOLOGICAL STUDIES ON THE G. PROSTATICA OF THE SILKMOTH, BOMBYX MORI. H. KASUGA, M.OSANAI and T.AIGAKI. DEOI5 Ole BLOM LOO Meticop. nse. Geront),, Lokyo). The secretion of glandula (g.) prostatica contains an endopeptidase, initiatorin, which causes in the spermatophore the mo- tility acquisition of apyrene sperm, the dissociation of eupyrene sperm, the activa- tion of a carboxypeptidase producing free Arg, and the Arg degradation cascade for yielding energy. G. prostatica is distin- guished to four parts from the external genitalia to g. alba due to histological differences of the exocrine cells and their SSCHSEIOMAS, 15 A ieldilin Clete CGoOMmswsieinae Cre flat epithelial cells which is surrounded by a spincter. 2. A layer of exocrine cells projected to the tube, making many folds in it. At the lower part, there is a valve shuting in the newly emerged adult to avoid an outflow of secretions. 3. An exo- crine gland consisting of a cubical epithe- lium of three cell-layers. Secretion con- tains heterogeneously neutral fat, droplet of acidy fat and PAS-positive material. 4. An exocrine gland of a cylindrical epithe- lium. Cells contain much large and small secretion glanules stained brown and dark green with toluidine blue, respectively. Neutral fat and a material unstained with any dyes are found in this secretion. At the ejaculation, cytoplasm in the glandular epithelium cells is also excreted in part and the intracellular granules diappear. DB 13/7 A histological study of capsules during pupal-adult metamorphosis in Samia cynthia igal@aiyaal 5 S.TAKAHASHI, G.ENOMOTO and Y NAKAJIMA. Dept. of Biol. Nara Women's Univ. Nara. Changes of capsules taking place during pupal-adult metamorphosis were studied in Samia cynthia ricini. Pieces of cellulose acetate membrane were implanted into pupae within 10 hr after ecdysis. Hemocytes reacted rapidly against CEA and formed thick capsule around it. Capsules, 24 hr after implantation, consisted of an inner layer of necrotic cells nearest the melanized surface of implant and an outer layer of numerous flattened cells. When the insect entered the period of apolysis (70Ehis y olla!) large, basophilic cells appeared between the two layers. These cells also surrounded the implant and finally formed an epitheliumlike mono-layer. There was a secretion which covered the whole inside of the layer. In the outside of the layer, flattened cells disappeared for a time , but accumulated again in later stages of pharate adult. Origin and nature of cells of the newly formed layer are unknown. In conclusion, during pupal-adult metamorphosis, hemocytic capsules having formed in an early pupal stage were decomposed like other larval tissues, anda turnover of capsule cells occurred. DB 136 EVEN DISTRIBUTION TOR SS CALES MOTHERS CELIES iN THE PUPAL WING DISC OF SMALL WHITE CABBAGE BUDE REG SPE RUS mA AER. Atos hatdaland #Ke Aoki iiasienSicil lnisitt-nESioi- hia Univ., Tokyo. ne te We studied the even distribution pattern of one of the two kinds of scale mother eells, termed Si, in the pupal wing ‘dase of Pieris. During the phase S1's were dif- ferentiated, right wing discs were examin- ed with scanning electron microscope, while whole mounts of left wing discs were Stained by Feulgen reaction and examined with optical microscope. Tin® wesuliis aie as E@llows, Piles, Sil’s are differentiated from the homogeneous epidermal cells of the wing disc without cell divisions. Second, the number of S1's increases and finally S1's are separated by one or two undifferentiated cell(s). Third, S1 does not divide during that pha- se, while some undifferentiated epidermal cells divide in the definite area of the wing disc. Forth, the distribution pattern of S1 is the same between in the areas with cell divisions and without cell divisions. All the results can be accounted for by assuming that alll vundifferentrvated cellls are going to give rise to S1's and the earlier differentiated S1's inhibit the adjacent undifferentiated cells to give PLGO EO Si So 1062 Developmental Biology DB 139 MUTATIONS AFFECTING NUCLEAR MIGRATION AND F-ACTIN ORGANIZATION IN DROSOPHILA EARLY EMBRYOS. K. Hatanaka and M. Okada. Inst. Beyoplie Sci., Univ. of Tsukuba, Ibaraki. Maternal effect mutations, N26, N441 and par share a similar phenotype showing defective nuclear migration with little effect on synchrony in cleavage mitosis. A phenocopy of Ehesie mutants can be induced by treatment of wild-type embryos with cytochalasins immediately after egg laying. In addition to abnormal nuclear migration, these mutants showed cleavage- stage specific defects in F-actin organization: formation of aggregates in the cortical F-actin meshwork, and retarded 1G 16 Glin S al 1 al © im Ly BMoaee nim Gla Sic ie dl Jo Wic a © in inside the yolk mass(Zool.Sci.,2,952,1985). Distribution of F-actin was normal after the syncytial blastoderm. N26 and N441 were located at 10A6-B3 and 11A7-B9, respectively. Both Aig weElic cicOm EAL knowin Acti LO@iu, Phenotypes of hemizygous mutants showed that N441 and par were amorphic and N26 was hypomorphic. A double mutant of N441 and N26 showed an extreme phenotype with completely spherical nuclear distribution at a late cleavage stage, suggesting that N441* and N26* contribute independently and cooperatively to normal F-actin organization, which is required for normal nuclear migration. DB 140 EFFECTS OF GENE DOSAGE ALTERATION ON RIBOSOMAL PROTEIN EXPRESSION IN DROSOPHILA MELANOGASTER. H.B.Tamate’?, M.Rosbash*, and M.Jacobs- Lorena*. lYamagata Univ.Sch.Med. *Case Western Reserve Univ. *Brandeis Univ. USA To study the regulatory mechanism for the coordinate synthesis of ribosomal proteins (r-proteins), the effect of altered gene dosage on the r-protein expression was analyzed in transgenic flies carrying extra copies of r-protein 49 gene. The abundance of the rp49 mRNA in ovaries was quantitated by RNA blot analysis. The same blots were also hybridized with other r-protein DNA probes. The rp49 mRNA was overrepresented in most of the transgenic lines, while the abundance ot other r- protein mRNAS remained unaltered. Despite the larger difference in rp49 mRNA content, the proportion of the rp49 mRNA that is associated with polysomes did not differ in control and transgenic flies. There was no apparent difference in 2D-gel pattern of newly synthesized r-proteins in transgenic and control flies. These results suggest that the rp49 expression is controlled mainly at post-translational level, probably at protein turnover. DB 141 HOMOLOGY TO A MITOCHONDRIAL LARGE rRNA OF THE poly(A) RNA RESPONSIBLE FOR POLE CELL FORMATION IN DROSOPHILA S.Kobayashi and M.Okada. Inst. Biol. Sci., Univ. of Tsukuba., Ibaraki. Pole cells, determined for germ line, AdiaS leSveEneSec ZeOM Oeming oy ww irradiation at the posterior pole of cleavage embryos. Injection of poly(A)+RNA extracted from cleavage embryos restores EIN joe SWI ieee aioe: elo laliesy tO wiy= igiacioli Cesc!) SinisavOos, ScieSeimding a Cin A library, prepared from poly(A)*+RNA of cleavage embryos, we isolated a single CDNA clone (pDE20.6) of the RNA with pole cell inducing ability. A computer analysis of the nucleotide sequence revealed that hie TeDNA as, haghl ys homologous omtehie mitochondrial large rRNA (1rRNA) gene of Drosophila. No nuclear gene homologous to the cDNA was detected by the Southern blot analysis. In cleavage embryos, the poly(A)+RNA complementary to the pDE20.6 CDNA was found dominantly in post-mito- chondrial fraction (P3 fraction) but less in mitochondrial fraction (P2fraction). DS CiecaCecioOn imelucecd AlmoOSte MO MiCO= chondria. After the pole cell formation stage, pDE20.6 poly(A)tRNA in P3 fraction CMe Gecl Neo, MAUS -OS3S level of tine cleavage embryos. These results indicates that poly(A)*RNA transcribed from the lrRNA gene is transferred to the cytosol to play a role in pole cell formation, and degraded after pole cell formation. DB 142 SOMATIC CMI, SWB wi IE PAN WIGS I IN DROSOPHILA EMBRYOS. F, Maruo*, M. Kurabayashi and M. Okada. HiASiIC5 BWiaOll, SCi5, UWaslyo Ot Weulxwloa. We have generated monoclonal antibodies (MAbs) against Drosophila ovaries to find developmentally regulated molecules during embryogenesis. One of the MAbs, E7-10, detected the antigen that is produced in nurse cells, shifted to the oocyte, and differentially distributed in early embryo cells. The distribution of the antigen was surveyed immunocytochemically with the MAb on polyester wax sections of embryos and first instar larvae. The antigen was found throughout cytoplasm of freshly laid eggs, later gathered in cytoplasm of every energid. At the blastoderm stage this antigen accumulated in the cytoplasm of the basal region of the somatic cells, and some antigen was also seen in the surface region of the central yolk mass. In the blastoderm and organogenesis stages, the antigen was found in all somatic cells, and then it gradually decreased from cyto- plasm of all somatic cells to disappear by the end of embryogenesis . The antigen WAS Mesbicliaie sO wince! alin joole Cells Moi im primordial germ cells in the embryonic and larval gonads. The MAb bound to a single band of MW 46 Kd in the immunoblot analy- sis. These observations indicate that during embryogenesis a common molecule is present in all somatic cells but not in germ line cells. Developmental Biology 1063 DB 143 EFFECT OF COLCHICINE ON CLEAVAGE OF AMPHI- BIAN EGGS 9 A. Yomota and.T. Sawai’ Dept. of Biol. lpac. of Sci. Fac. of Gen. Ed. Univ. of Yamagata., Yamagata ~ It is known that the cleavage plane is determined by some stimulus transmitted to the cortex through astral rays of mitotic apparatus. In this experiment, we examined the timing of determination of the cleavage plane in Cynops and Xenopus eggs by injec-— ting colchicine at various times before the onset of cleavage. When colchicine was injected into eggs more than 10 min before the start of cleav- age in Cynops and more than 3 min before that in Xenopus, no furrow was formed. Ten min in Cynops eggs and 3 min in Sas ones Boreas noaded ton0. LOs an Det tila unit and the telophase of nuclear division. In both species, when the colchicine injec- tion was made within 0.10D, the cleavage furrow was formed on the animal hemisphere but not on the vegital one. Colchicine in- jection at various times after the furrow appearance caused a cleavage arrest in various degrees. These results indicate that, in the ani- mal half, the cleavage plane of amphibian eggs would be determined by colchicine sen- sitive factor about 0.10D before the cleav- age, and that the similar factor would be also take part in the furrow formation in the vegital half. DB 144 ANALYSIS OF STAGE-SPECIFIC EFFECTS OF Li ON AMPHIBIAN MORPHOGENESIS. Y. Yamaguchi & A. Shinagawa, Dept. Biol., Fac. Sci., Yamagata Univ. Yamagata 990. Lit+ is known to have teratogenic effects on amphibian morphogenesis. However, there is no information about precise relations between the timing of Li+ treatment and the consequent malformation excepting some studies suggesting the stage-specificity of the teratogenic response by the embryo to Lit treatment. This study attempts to analyze precisely these relations by EA a relatively high concentration (0.4M) of Li+ on Xenopus embryos for a short period (5 min) at various stages up to the neurula stage. Embryos treated at a stage from 16= cell to mid-blastula showed a syndrome of malformation as if dorsalized while those treated at a stage from mid-blastula to late gastrula showed another syndrome of malformation as if ventralized. These two syndromes of malformation was most efficiently induced by treatment at morula to early blastula stages and that at a mid-blastula stage, respectively. Neither treatment before the 16-cell stage nor that after the late gastrula stage induced significant malformation. Thus, there are two distinct periods at which a Xenopus embryo is sensitive to Li ions and the way of its response to Lit is quite different. DB 145 MECHANISMS IN A XENOPUS EGG WHICH DETERMINE THE ORIGINATING SITE OF SURFACE CONTRACTION WAVES. A. Shinagawal, S. Konnol2, Y. Yoshimoto34, Y. Hiramoto34. 1Dep. Biol., Fac. Sci., Yamagata Univ., 2Devl. & Rep. Biol Cent., Yamagata, 3Div. Cell Prolif., Natl. Int. Basic Biol., Okazaki, 4Dep. Biol., Broadcast Univ., Chiba. The originating site of SCWs is examined in Xenopus eggs after various treatments such as colchicine-injection, egg-inclination and enucleation. SCWs originate (1) at the sperm entrance point in fertilized eggs, regardless of the egg orientation, when injected with colchi- eine at O72=0-3, (2) at the uppermost site in fertilized eggs, regardless of the egg orientation, when injected at 0.8-0.9, (3) at the animal pole in activated eggs, regardless of the egg orientation, when injected at 0.2-0.3, (4) at the animal pole in the normally oriented activated eggs but often at the uppermost site as well as at the animal pole in the 90°-rotated activated eggs when injected at 0.8-0.9, and (5) at the uppermost site in enucleated eggs, regardless of above treatments. De Sais concluded from these results that the originating site of SCWs is determined by such cytoplasmic factors as have tendency to concentrate primarily around the male nucleus and secondarily around the female nucleus but around the uppermost region of the egg when no nucleus is present. DB 146 VITELLINE ENVELOPE FORMATION IN URODELE OOCYTE. Ko Onacake, Ko Morieand io. Oh7tte: «aDe pine wor Biol vac. OF Cin, Vanagra ba jUM vier. Yamagata 990, Japan. The vitelline envelope (VE) of anuran eggs has an important roles of polyspermy block after fertilization, but little is known about the function of urodele VE. In this study, we examined the VE forma- tion of the newt, Cynops pyrrhogaster, during oogenesis with the immunohisto- chemical (indirect method) and histochemi- cal (PAS-staining) methods. Developmental stages of oocytes were identified accord- ing to Dumont (1972). The anti-VE sera were obtained from an injecting rabbits of isolated VEs from matured eggs. Anti- sera were absorbed with homogenates of the VE-free matured eggs. In stage II, immunofluorescence was first observed in some large vesicles of cytoplasm. In late stage II-stage III, the vitelline envelope began to form, and immunofluorescence was first observed on the surface of the oocyte as isolated patches. In stage IV, the VE formed a continuous layer over the surface of the oocyte. The oocyte of stage I and the follicle cells were not stained with FITC- labelled antibody. The same results were obtained from PAS-staining. It is suggested that the vitelline envelope of the newt were formed mainly with the products from oocyte. 1064 Developmental Biology DB 147 PORLAR BODY FORMATION IN XENOPUS EGGS. We, Koyocel, IDWejres Oi IBIOl, 7 WUT Ox Sei. , Kagoshima Univ., Kagoshima. Xenopus laevis eggs dejellyed with thio-— glycollate and electrically activated were used at 20 C. In eggs fixed with Zenker fluid and stained with Feulgen, the second meiosis was at metaphase, anaphse, and telophase, respectively, at 0-6, 8-15, and 20 min after activation, and the first sign of polar body formation was found as a minute protrusion of the surface close to a spindle pole at 4 min. Eggs without their membrane were suspended between Steinberg solution and undiluted Percoll, and observed from side with a phase micro-— scope.(1) an extruded and constricted cy- toplasmic mass was not spherical. The mass became spherical after separation from the egg surface. (2)the separated second polar body and often the first remained there by some holding structure. This peculiar de- vice may be related for amphibian eggs to rotate following activation. Colchicine injection after activation resuited in suppression of the polar extrusion. Dipp- ing of activated eggs into a 10uU9 /mcyto- chalasin-containing medium had no effect on the polar body formation, presumably because of little permeation of cytochala-— sin into the egg. Polar bodies were formed in eggs part of whose cytoplasm was remov- ed by its outflow produced by rupture of the the vegetal egg surface, or by its suction with a micropipette, suggesting that polar extrusion does not always ne- cessitate the inner pressure originating from egg-surface tension. DB 148 A POSITIVE FERTILIZATION POTENTIAL IN THE SALAMANDER, HYNOBIUS NEBULOSUS EGG. Wo Iwa@>s Bios Wnsics 5 Paes Sie, Vemacucinit Univ., Yamaguchi. In order to determine what kind of mecha- nisms of polyspermy block is operating in the salamander, Hynobius nebulosus egg, the electrical responses at fertilization and the behavior of accessory sperm nuclei in the egg cytoplasm were investigated. The unfertilized eggs had a membrane potential of about -10 mV. The eggs elicited a rapidly-rising positive fertili- zation potential of about +40 mV. The positive potential lasted more than 30 min. All the eggs exhibited monospermy and developed normally. The same positive activation potential was produced by pricking, electric shock, or ionophore A23187. The positive potential was reduced to about -20 mV by 100 mM choline-Cl in the external medium. When the partially- jellied eggs were inseminated, 3-4 sperm entered each egg. The eggs underwent a multipolar cleavage and abnormal develop- ment. The accessory sperm nuclei never degenerated at all in the egg cytoplasm and formed extra bipolar spindles. These indicate the primitive Hynobius eggs lack an intracellular polyspermy block, but the positive fertilization potential may contribute to a fast block to polyspermy. Thus, the Hynobius eggs exhibit an evolutionally intermediate state. between anurans and higher urodeles. DB 149 AN INHIBITION OF CYTOKINESIS IN NEWT'S EGGS WITH WGA TREATMENT. T. ESAKA. Dept. of Biol., Science Education Inst. of Osaka Pref., Osaka. Eggs of Cynops pyrrhogaster deprived of jelly and vitellin membrane were treated with WGA(100 pg in 1mlof modified Steinberg's solution : 0.34 % NaCl, 0.0053 KCl, 0.008 % Ca(NO3)5°4H 50, 0.0025 & MgSO,4°7H50, and 0.0061 % phenol red, buffered to pH 7.2 with 0.05 % HEPES and NaOH) before first cleavage. In the eggs, furrow formation was initiated normally at each cycle of cleavage, but cytokinesis was inhibited. Rhodamine conjugated WGA seemed to bind uniformly to the egg surface, which is confirmed witha fluorescence microscope, while injected WGA was not effective to provoke the inhibition of cytokinesis. Time-lapse VTR and 16 mm cinematograph showed that the appearance of surface contraction waves were not inhibited. The study with serial paraffine sections and with TEM proved that the division of nucleus and appearance of diastema were normal, and the egg surface was uniformly coated with WGA about 0.1 pm in thickness. To identify for the WGA layer, ferritin conjugated WGA was used. DB 150 REGIONAL CHARACTERISTICS IN PROTEIN PATTERN OF 8-CELL STAGE OF X. LAEVIS. A. Osada and A. S. Suzuki. Dept. of Biol. Fac. of Gen. Edu. Kumamoto Univ. Kumamoto. Regional pattern of proteins in 8-cell stage of X. laevis was analyzed by two- dimensional — SDS-polyacrylamide gel electrophoresis. The blastomeres of 8- celal stage were separated into four groupso animal-dorsal(AD), animal- ventral(AV), vegetal-dorsal(VD), and vegetal-ventral(VV). Each group was separately sonicated in 70% ethanol- detergent buffer solution and centrifuged ere = IA OOWC, sk@ie IS insling Wns SUjosiemeicames were applied to 2D SDS-PAGE. Each gel was stained by highly sensitive silver stain method. we could found regional differences in several spots on each gel. Six spots were Significantly different between AV and VV, five spots were also different between AD and VD. However, only two spots were different between AD and AV, VD and VV, respectively. These differences were qualitative or quantitative. The present experiment suggested the possibility that there are animal-specific and vegetal- specific proteins, because two spots were observed only in animal half and one spot was only in vegetal half. These results suggest that there is already regional OOS NSE OIE joreCeSslin jOeNEESicioy alin J-CSILIL stage. Developmental Biology 1065 DB 151 IMMUNOBLOTTING DETECTION OF MYOSIN TO MUSCLE DIFFERENTIATION CAUSED BY MESODERMAL INDUCING SUBSTANCES. M.Asashima, K.Shimada and H.Nakano. Dept. of Biol.,Yokohama City Univ., Kanazawa-ku, Yokohama 236. Mesodermal inducing substances (F1) purified from carp swim bladder were used as the inductor in this experiment. Fl induces mesodermal tissues such as muscle and notochord in the ectoderm of early gastrula. To detect and analyse myosin molecule to muscle differentiation, immunoblotting method (western blotting) was employed. Following culture days (0,5,10 and 15 days) of the explants with or without the inducer, the samples were extracted with lysis buffer, and run on 7.5% polyacrylamide gel. At this time silver staining was also carried out to compare with the immumoblotting bands. The ectoderm only explants without inducer could not detect any myosin specific bands in both immunoblotting and silver staining in 15 days culture. But the explants with inducer after 10 days culture had recognized clear specific band at the high molecular(M.W. about 220K) which is corresponded to standard myosin band. This myosin specific band could observed in the explants from 10 days culture, but not 0 and 5 days culture explants. These immunoblotting data are discussed with the histological H-E staining observation and immunohistochemical study. DB 152 MONOCLONAL ANTIBODY PRODUCTION AGAINST THE 38-KD PROTEIN LACKING IN XENOPUS MUTANT EMBRYOS. Y.Tsuzaki and K.Ikenishi, Dept. of Biol., Hac mon Iocan, Osaka City Univ. , Osaka. A maternal-effect mutant female in Xenopus (designated as no. 65) whose off- Spring always arrested the development at gastrulation was incidentally found (sub- mitted). 2D-gel analysis revealed that an acidic, 38-kD protein which was always present in the wild-type unfertilized eggs was absent in the counterparts of female noe 05s" [tis known in Ambystoma mexicanum that the mutant embryos from o/o female which also showed the developmental arrest at gastrulation could develop beyond gas- trulation when:a protein fraction from the wild-type eggs of embryos was micro- injected (Briggs & Justus, 1968). In order to know whether this is the case in the female no. 65, it is necessary to obtain sufficient amount of the 38-kD protein, which is not denatured, for micro- injection. So, we tried to make monoclonal antibody against the protein extracted from polyacrylamide gels. Culture supernatants of the hybridoma cell lines were primarily screened by ELISA with the extracted pro- tein, and further examined with western blotting of 2D-gel electrophoresis of pro- tein samples from the homogenate of wild- type, 2-cell eggs. The cloning of some cell lines which were positive in the blotting is now in progress. DB 153 THE NEURAL TRANSMISSION OF NEURAL INDUCING SIGNAL EVOKED BY NEURALIZED CELL. N.Sasaki, K.Yamazaki-Yamamoto, T.Kameda §& K.Shimizu, Lab.of Biol. ,Div.of Ghakdi Educ: ; Seinan-Gakuin Univ. ,Fukuoka During the morphogenesis of amphibian em- bryo,the neural plate is formed in the ecto- derm by the underlaying chordamesoderm. In this study, we showed the neuralized cells induced by the chordamesoderm obtained the potency to produce a neuro-inducing signal (homoiogenetic one), and this signal trans- mitted within ectodermal cell layer played an important role in the spatial extension of the neural plate of embryo. Followings were the experiments; (1) In chimera experiment with Hynobius and Cynops,young ectoderm was implanted into an area of neural plate of early neurula, The ectoderm cells were transformed into neural EQUUS (2) An isolated neural plate could induce neural tissues in young ectoderm. On the other hand, the myotome of neurula, by which the implanted ectoderm in (1) was underlaid had no ability for provoking neural diff- erentiation in the ectoderm. (3) The pieces of presumptive ectoderm of Cynops gastrula were transplanted into a ront and adjoining lateral side of the neu- ral plate of Cynops neurula. These ectoderm cells were transformed into neural ones. In the contrast,those of ectoderm transplanted into abdorminal area could not differentiate UMNIEO) INEwheel CGSlls 5 DB 154 ISOLATION OF NEURAL RECEPTOR PROTEIN ON THE INNER SURFACE OF THE COMPETENT ECTODERM OF, CYNOPS GASTRULA. M.R.M. DIA4 rKy TAKESHIMA, K. TAKATA™ and N. TAKAHASHI. Dept. of iO, BAGG CE SCGils, Nagoya Winsli7. Radio- isotope Ctr., Nagoya Univ. and Nagoya City Univ. Sch. of Med., Nagoya. Results of previous studies have pointed to the specificity by which neural inducers like Con A and the living organizer react with glycoproteins on the inner surface of the competent ectoderm to bring about neu- ral formation. This finding has led us to suspect the presence of receptor proteins which can be isolated and identified. Asa preliminary, therefore, affinity between Con A-Sepharose beads and the inner surface of the ectodermal explants reacted together for 30 min and 24 hrs was observed by SEM. Intense affinity was manifested by skirts, filopodia, lobopodia, blebs, stalks and half-sunken beads. Con A-Sepharose bead- bound ectodermal explants were treated with deoxycholate and DNAse, and centrifuged in Percoll solution. The isolated bead frac- tion was then washed with 0.5M a-methyl- mMannoside and the eluate radioiodinated and ran in SDS PAGE. Two major bands, 80kd and 50kd, and a few minor bands were observed. To determine whether any of these bands turly corresponds to neural receptor pro- teins, monoclonal antibodies have been raised against the surface antigens of the competent ectoderm. 1066 Developmental Biology DB 155 ANALYSIS OF PROTEIN INVOLVED IN NEURAL IN- DUCTION BY CON A IN THE | NEWT ECTODERMAL EXPLANT. pie TAKABATAKE , A. MUTQ f o: OSA- WA , K. TAKESHIMA™ and K. TAKATA . Dept. f Biol., Fac. of Sci., Nagoya Univ. and Radioisotope Ctr., Nagoya Univ., Nagoya. Treatment of the presumptive ectoderm of newt early gastrula with Con A brings about neural differentiation. In the absence of Con A, it differentiates into epidermis. Using this as a model for neural induction, O'Farrell 2-D-electrophoresis was done to detect proteins appearing in neural or epi- dermal differentiation. Also, in order to compare these with proteins in normal deve- lopment, proteins of the neural plate and epidermis were analyzed. The ectodermal explants were treated with S-Met, cultured for 3 hrs and the labeled protein samples were used for elec- trophoresis. Following O'Farrell's method (1977), the acidic and basic sides were ran separately. In the acidic side, spots which specifically accompany neural induc- tion were not observed, but one spot, which disappeared in the presence of neural in- duction, and some spots specifically accom- panying epidermal development were observed In the basic side, induced ectoderm showed one strongly stained spot. To determine the time of appearance of the spots, the culturing time after ectoderm excision was prolonged. Some differneces were observed in the Con A-treated and untreated explants with respect to time. DB 156 EFFECTS OF PROTEIN-SYNTHESIS INHIBITORS AND TUNICAMYCIN ON NEURAL-INDUCING ACTIVITY OF NEWLY-MESODERMALIZED ECTODERM. J.Matsuda and A.S.Suzuki. Dept.of Biol., Fac.of Gen. Edu., Kumamoto Univ., Kumamoto. Presumptive ectoderm of Cynops gastrula was mesodermalized by swimbladder of Carassius sp. The newly-mesodermalized ectoderm(nME) acquired very strong neural- inducing capacity. In the present study, nMEs were treated with cychloheximide(0.5yg/ml), puromycin(20yg/ml), and tunicamycin(1-5yg/ml1 ) OS ena Sh Neural-inducing capacity of cycloheximide- treated nME decreased one-seventh of the control, puromycin-treated nME was one-tenth of the control. Tunicamycin-treated nME also showed lower neural-inducing capacity: one- tenth of the control in the case of 5ug/ml. It seems to be concluded that neural-inducing Capacity of the newly-mesodermalized ectoderm is coupled with protein synthesis. It is also suggested that cell surface(sugar-containing complexes) of the nME cells plays a important role in neural-inducing activity as same as in reacting activity of the competent ectoderm cells. Changes in protein pattern of the newly- mesodermalized ectoderm was also analyzed by two-dimensional polyacrylamide gel electro- phoresis. We could observed the qualitative and quantitative differences in several spots between nME and control ectoderm within 24 hrs. DB 15/ DISTRIBUTION OF TRANSPLANTED X. BOREALIS ANIMAL-CAP CELLS ONTO PROSPECTIVE NEURAL REGIONS OF X. LAEVIS GASTRULA. A. S. Suzuki and K. Harada. Dept. of Biol. Fac. of Gen. Edu. Kumamoto Univ. Kumamoto. Small pieces(0.2x0.3mm) of the animal cap of X. borealis gastrula(stage 10) were transplanted onto various regions of the noninvoluting marginal zone of albino xX. laevis gastrula(stage 10). Distribution of the donor cells were analyzed by quina- crine fluorescence staining. Transplanted pieces on the dorsal median plane extended remarkably in the animal-vegetal direction during gastrula- tion and finally formed a ventral side of the central nervous system(CNS), particu- larly of the anterior part. Both dorsal- lateral and lateral regions converged toward the dorsal midline, extending in the animal-vegetal direction. The former constituted a lateral side of the anterior CNS and the latter formed a lateral side of the posterior CNS. Lateral-ventral and ventral regions remarkably converged to- ward the constricting blastopore and the epidermis surrounding the anus consisted of their descendant cells. The present data suggest that the prospective CNS lies as a belt-shaped area in the noninvoluting marginal zone of early gastrula and the limiting line of the prospective CNS is between 0.6mm and 0.7mm above the blasto- pore on the dorsal side. DB 158 EFFECT OF ANTIBIOTICS ON PH OF NEWT BLASTO- COEL FLUID AND ANALYSIS OF THE CONTENTS OF THE FLUID T. Asao. Biol. Lab. Sch. Med. St. Marianna Univ. Kawasaki As we have already reported blastocoel fluid of newt gastrula is fairly alkaline. In order to know the main cause that induced such a phenomenum and to analyze the role of the alkalinity of blastocoel fluid in an em- bryonic development, inhibition experiments using several antibiotics were performed. Antimycin A stopped the development and also lowered the pH of the fluid to 7.6. Calcium ionophore inhibited invagination but gave hardly effects in the change of pH. Cyclo- heximide inhibited the neural plate forma- tion. Abnormal effects were not observed with the treatment of ouabain, monensin and malonic acids. Quantitative analysis of in- organic ions of the fluid showed that only the Ca ion contents of antimycin A-treated gastrula was twice larger than that of con- trol. The alkalinity of blastocoel fluid may be related to the energy consuming Ca pump- ing action. On the other hand, the organic substances of the fluid were qualitatively estimated by thin layer chromatography and SDS polyacrylamide gel electrophoresis. Small ninhydrin reactive substances were de- tected in thin layer chromatography and a low molecular protein (about 26 k dalton) was clearly found in polyacrylamide gel electrophoresis. The electrophoretic band pattern showed that several silver stained proteins were contained in the flmid. Developmental Biology 1067 DB 159 ALKALINE PHOSPHATASE ACTIVITY DURING DEVE- LOPMENT IN THE BULLFROG Me kashiwaG item es liabe fon Amphibian) Baolks, HaG mom SGae, | haroshama Unive, Hiroshima. The change of alkaline phosphatase (Alp) activity during development in various organs of Rana catesbeiana was examined by acrylamide gel electrophoresis and spectro- photometer. Alp was extracted from the Pancreases, livers, kidneys and intestines of embryos at five Shumway's stages, tad- poles at 13 Tayler and Kollros' stages and juvenile frogs. The results showed that Alp activities greatly differed with organs and developmental stages. In the pancreas, a light band appeared at stage III and existed in all subsequent stages. In the liver, a wide band which had the same mo- bility as that of the pancreas appeared at stage III, disappeared by one week after completion of metamorphosis, and was three weeks later changed for another more cathodic band. In the kidney, a single band appeared at stage III and remained in subsequent stages. In the intestine, the first band appeared at stage 25, became a major band at stage III, and then disap- peared at stage XxX. A minor band with faster mobility appeared during stages VII to XIV. A new band which was often accom- panied by one minor band with slow mobility appeared at stage XXV and remained there- aktes. “Alp activity in all organs except the liver increased rapidly at late tadpole stages, decreased temporarily during meta- morphosis and increased again after comple- tion of metamorphosis. DB 160 PIGMENT CELL DIFFERENTIATION IN CYTO- CHALASIN-TREATED EXPLANTS OF XENOPUS EMBRYO. Het Cen STO ee inst.) LLOhoku Unive, Sendaa To make clear the distribution pattern of presumptive pigment cells in Xenopus embryos, neural tubes of tail bud embryos and whole dorsal region of gastrula- neurula embryos were isolated and cultured in a medium containing cytochalasin B, in which morphogenetic movement such as neural tube formation or neural crest cell migration was blocked and the cells differentiated in their original positions. Two types of pigment cells, melanophores and xanthophores differentiated in the explants. In the neural tube explants, melanophores and xanthophores differenti- ated only on the dorsal surface, the neural crest region. In the explants of whole dorsal region, the melanophores differenti- ated in the trunk neural fold region, and first aligned in four rows running parallel with anteroposterior axis. The xantho- phores also differentiated in the neural fold region. Both pigment cells differen- tiated also in the isolated dorsal ectoderm sheets of late gastrula embryos. These results suggest that the commitment of the differentiation pathwat to the pigment cells begins at late gastrula stages and the committed cells differentiate to the melanophores and xanthophores in situ in the explants without morphogenetic movements. DB 161 ONTOGENY OF LEUKOCYTES IN LARVAL XENOPUS AS STUDIED BY MONOCLONAL ANTIBODY. He. Ohainata, «Ss. hoch iivader and .ch: Katagiri, ZOO INS t.5 Fac. Of Sci,e Hokkando. Unive The monoclonal antibody (mAb) reactive to all types of leukocytes was developed for studying the chronological appearance of leukocytes in Xenopus laevis. The mAb-positive cells were first detectable around st.35/36 mostly in the mesenchymal tissues throughout the body and blood vessels, but few in the ventral blood island where the red blood Celis SCRBCs)) “had “been differentia t ime Sic sit. 26. = Ine thie alliivielss et hes po:seisternvwe celiissappeared dnethe il tiwernea teste 3 71/ 3ee and accumulated significantly in the sub- CAMSUIair PEZTOM rrOmM Sw.47 On. niet ne prospective mesonephric region, the aggregations of non-lymphoid leukocytes were first detectable in the mesenchyme surrounded by the Wolffian duct, dorsal SOrEG, ANG VENA CAVA, WETOrEG ihe Turse appearance of lymphocytes in the thymus Qt Stof7/, In lareir Se@ges., ENe poste we cells were apparently localized along the medial wall of the mesomephros. Thymocytes became positively stained after st.46-47, following the appearance of strongly-positive dendritic cells at st.44/45 thymus rudiments. At st.49, many leukocytes were detected in both red and white pulps in the spleen. DB 162 EFFECTS OF TRIIODOTHYRONINE ON THE EPIDER- MAL CELL DEATH OF THE ANURAN TADPOLES. A. Nishikawa and K. Yoshizato. Department .of Biology, Faculty of Science, Tokyo Metropolitan University, Tokyo. “In order to know the mechanism of cell death induced by thyroid hormone at meta- morphosis, we cultured the tail epidermal cells (T) and the back skin epidermal cells (B) of Rana catesbeiana tadpoles and compared the effects of triiodothyronine (T3) between T and B on (1) DNA synthesis, (2)keratinization and (3)protein synthesis, (1) Imunofluorescent micrographs using anti-BrdU showed that T3 (10-8m) decrease the number of cells in the S-phase in T JOWNE INGE ai I> alyovli@eiesine; ielMelie Wey Success the progression of T into the S-phase. (2) The increase of transglutaminase activity and the number of SDS-insoluble cells (keratinized cells) were promoted by E3 (10-8mM) more markedly in T than B. (3) Two-dimensional electrophorogrammes of the extracts of T and B labelled with 35S5-methionine revealed more than 100 pro- tein spots. The extent of staining in the 30 spots was changed by the treatment with , (10-8mM). The fourteen spots of them were found only in T, the eight spots were found more intensely in T than in B, and the rest were found equally in T and B. From these results, thyroid hormone induced changes in protein synthesis, which might be involved in the regulation of cell cycle in T and the promotion of keratinization of T and B. 1068 Developmental Biology DB 163 LOCALIZATION OF LECTIN-BINDING SITES IN ANURAN LARVAL SKIN. T. Kinoshita, H. Takahama and F. Sasaki. DAoe, Oi WiOls, SEaool Ox Wein, Mec, Tsurumi Univ., Yokohama. Localization of lectin-binding sites in tail skin of Rana japonica tadpole was examined at pre- (st. X) and climactic metamorphosis (st. XXIII). Binding sites of lectins were explored using MPA and GS-1 directly labeled with FITC for light MN CHSOISIC OD, van\Gi wast hyaconslouidaliqoidiations electron microscopy. In tail skin at st. A WUMN Joo wlincl Oimils7 OQ Ajilcal Cells Oz epidermis. in weet Sleitim ie Sieo XCM, however, MPA bound not only to apical epidermal cells but also to basal cells, especially to degenerating epidermal cells. Furthermore, MPA bound to dermal collagen layers. GS-1 also bound to apical epidermal cells at st. X. The number of binding sites of GS-1 to apical cells of epidermis decreased at st. XXIII. At the same time, GS-1 INOQwincl EO macrophages which had increased in number from st. X through XXIII within epidermis and dermis. Apparently it was not to fibroblast-like cells which phagocytosed degrading dermal collagen fibers. These results suggest that MPA and GS-1 are useful to detect qualitative differences among cells and extracellular matrixes in anuran larval skin during metamorphosis. DB 164 ULTRASTRUCTURAL CHANGES IN INTESTINAL CONNECTIVE TISSUE OF XENOPUS LAEVIS DURING METAMORPHOSIS. A. Ishizuya-Oka and A. Shimozawa. Dept. of Anat., Dokkyo Univ., Sch. of Med., Tochigi. We studied by transmission electron microscopy the development of intestinal connective tissue of Xenopus laevis tadpoles from stage 50 to 66 (Nieuwkoop & Faber), to clarify ultrastructural relations between the intestinal epithelium and the connective tissue during metamor- phosis. Throughout the larval period to stage 60, the connective tissue consists of immature fibroblasts surrounded by a sparse extracellular matrix. At the beginning of the transition from larval to adult epithe- lial form around stage 60, extensive changes are observed in the connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in density. Through gaps in the thickened basal lamina, frequent cell contacts between the epithelium and the connective tissue are established. Thereafter, with the progression of fold formation, the fibroblasts around the trough of the fold become aligned parallel to the curvature of the epithelium. These observations indicate that the developmental changes in the connective tissue are closely related spatio- temporarily to the epithelial transition during the metamorphic climax. DB 165 CHANGES OF HEART BEATS DURING NORMAL EMBRYONIC, DEVELOPMENT AND ORGAN CULTURE M. Taguchi M.Uemura“~ and M.Asashima. Dept.of Biology, Yokohama City Univ. Yoko- hama 236.“Med.Sci.,Dept.of Ped.Teikyo Univ. ,ltabash-ku,Tokyo 173 Heart beat rates in normal development and organ culture were investigated using the material of newt. Heart rate at 20°C was assayed before and after metamorphosis. In normal development,heart beats began from st.33 (32beats/min) and its rate went up to st.52 (80beats/min). Then its rate went down to st.60 (55beats/min). Heart beats rate was temperature sensitive showing the linear equation between 4°C to 30°C. These rates were depend on their developmental stages. On the other hand the beat rate of organ cultured hearts was depend on starting stage and there culture days. In organ culture started from st.33 its beating rate showed 22beats/min in one day,and then its rate showed the maximum peak after 54-58 days showing 65beats/min. After this culture period the rate went down following culture days. In culture started from st.47,the peak of their heart rate showed after 14-21 days,but in cultur- ed heart from st.58 it had no peak after 70 days. The heart beat rate in organ culture was also showed tempereture sensitive between 4°C and 30°C. These organ culture data of heart beats seem to reflect the resemble states of the heart in normal embryonic development. DB 166 DEVELOPMENT OF THE WATER TRANSPORTING SKIN OF TREEFROGS DURING THE METAMORPHOSIS. Y. Kamishima, and H. Nakashima. Depivemos aL Oto, WACGULG>, Oi SCiloy Okayama Univ. Okayama 700 Water intake in japanese treefrogs was mainly carried out through the ventral pelvic skin and was positively controlled by a neural agent through an adrenergic B- receptor. However, this integumental water transport was observed only in adult frogs and neural agents failed to facilitate the function in premetamorphic larvae. In aquatic larvae, no morphological differ- ences were observed between dorsal and ventral epidermal cells. At the metamor- phic climax when disorganization and re- formation of the integument took place, the pelvic skin differenciated into a charateristic one in which epidermal cells showed microvillus-like processes, and wider interspaces. The outermost layer of - pelvic epidermal cells also showed charac-— teristic features with dense granules and perforated cytoplasm during this period. The dorsal skin lacked all these features even after the metamorphosis. When the skin is activated to permeate the water, outermost cells of the epi- dermis took strongly metachromasic dyes. These cells showed strong cytoplasmic density and appeared as masses of inter- mingled long processes which were formed during the metamorphosis and a character- istic feature of the ventral pelvic skin. Developmental Biology 1069 DB 16/7 FREQUENT OCCURRENCE OF RENAL TUMORS IN AMS, HYBRIDS BETWEEN BUFO JAPONICUS AND B.RADDEI M.Nishioka and Y.Kondo. Lab. for Amphibian Biol.,Fac.of Sci.,Hiroshima Univ. ,Hiroshima Tt was remarkable that most of the hybrids between a female Bufo j. japonicus and a male B. raddei died of renal tumors. While 73(11.6%) of the normally cleaved eggs ob- tained in 1982 became young toads,many of them died by 1987. When the cause of death was examine in 27 toads, it was found that 22 suffered from renal tumors. These toads consisted of 10 females, nine males and three young ones of unkown sex. Eight, one, twelve and one died in 1982, 1983, 1984 and 1986, respectively. Of the other five dead toads, one was a five-year-old male which suffered from a pancreatic tumor, and four were yearlings in which the cause of death was undetermined. Eleven other toads are now living. One of them appeared to have a tumor in the body cavity from examination by touch, while the other ten seemed to be Spllneakehye) im the control series, mone Sec eapont cus ‘and —B.yradden "diedof tumors during the five years 1982)- 1987. When the renal tumors of the dead toads were histologically examined, it was found that some mesonephric tubules became first abnormal and were filled with pathologic cells. Hydronephrosis and necrosis occur- ped ame the morbid portion, whereas the Malpighian bodies remained almost normal Oneal ew Mey. In the hybrids between Bufo red@en Qincl B. fa gaieyaien Zains Cie 15 Walies(Cl Sy, tumors occurred very rarely in the pancreas or Bidder's organ. DB 168 ABUNDANT TUMORS WHICH RECENTLY OCCURRED IN THE LABORATORY FOR AMPHIBIAN. M. Nishioka and H. Ueda Lab. for Amphibian Biol.,Fac.of Sci.,Hiroshima Univ.,Hiroshima It was a wonder that several kinds of tumors were suddenly detected in 1982 and widespread in the following years in the anurans kept in our laboratory. When dead or dying anurans were examined at ages of one to 11 years, a specific tumor was found in a definite kind of anurans. Of the toads which were produced in 1980 and died by 1987, 46(17.6%) of the 262 female hybrids between Bufo japonicus from Japan or China Zin! ” [3}5) [abe @ one Bo YiriGd1 Ss from Europe suffered from ovarian tumors, while 9(2.0%) of the 440 male hybrids suffered mostly from tumors in the Bidder's organ. At present, 45 (28.0%) of the IO 9 ibaly suing, hybrids suffer from abdominal tumors. On the other side, many pancreatic tumors and a few others occurred in Rana plancyi, R. nigromaculata, their inter- and intra- specific hybrids produced in 1980. Of the 487 dead frogs examined during 7 years from IDEA qeqQ WNIT WOW AOS Ves) SueseSieecl sero Weve— ious tumors. Tumors were also detected in 80 dead frogs of the hybrids, backcrosses and allopolyploids produced in 1975-1984 except 1980 among five species and two sub- species of pond frogs. Fifty-seven of them died of pancreatic tumors, while the re- Maining 23 suffered from ovarian, renal, hepatic and some other tumors. It was re- markable that a highly contagious disease occurred in 1985 and spread thereafter in our laboratory. DB 169 SPERMATOGENESIS OF HALICHONDRIA OKADATI K.Tanaka-Ichihara’ and Y.Watanabe2. ist.) OL eComprEehen. Mede Sci. , 0 (SChi.) Of Med., Fujita-Gakuen Health Univ., Aichi. DEI4 Oi IO, Wee, one SSi4, OelasinoniaLrv Univ. Tokyo. Spermatogenesis of H.okadai was exa- mined by light and electron microscopy. At the outset of spermatogenesis, number of choanocyte chambers suddenly decreased. Simultaneously, many amoeboid cells be- came sighted in the mesohyl. These cells, uniflagellated and with nucleo- lated nuclei, were observed only in the early stage of spermatogenesis. They differentiated to primary spermatocytes, and entered into the meiosis retaining flagella. Primary spermatocytes were enclosed by a single layer of flattened cells. Thus, Spermatic cysts were formed. Two meiotic divisions to secondary sper- matocytes and spermatids almost synchro- nously ocurred in a spermatic cysts. Four spermatids derived from a single spermatocyte were connected each other through their cytoplasmic bridges. Each mature spermatozoon contained a condensed nucleus) {or 135 mein diameter, ay jring— shaped MELCCEINoNClicilOm, el joculic Cit Ceao elles, El sllereSil yd Cpe 20) jodi alin IWeveyejicln, and an acrosome-like structure comprising several tens of rods of 44 nm in diameter and 300 nm in length. In summary, spermatozoa were generated from flagellated amoeboid cells that were thought to be originated from choanocytes. DB 170 ROLES OF MAJOR ACROSOMAL PROTEINS OF ABALONE SPERM IN THE VITELLINE COAT LYSIS. N. Usuil and K. Haino-Fukushima2. lDdept. of Anat., Teikyo Univ. Sch. of Med. and 2Dept. Git WsiLO@l, 5 WAS. OH Slo, Wolav~o@ Mertemojooilarieein Univ., Tokyo. Major soluble components released from the acrosomal vesicles during the acrosome reaction of the abalone dHaltotits discus sperm are two proteins of molecular weights 15,500 and 20,000. Crude acrosomal contents and a sample which contained predominantly 15.5K protein possessed lytic activity on the vitelline coat (VC) ('84). To elucidate role(s) of each protein in the lysis of the VC, 4H. diseus oocytes were treated with various mixtures of the acro- somal proteins; (a) 20K and a trace amount Oie INS Sip (lo) Abe Six eile, (ey) Ake Elieie and (d) a loll mixtune of both proteins. the mixtures of both proteins (a, d) induced swelling of the VC but each protein alone (b, c) was incapable of dissolving the VC. Thin-sectioning revealed that 15.5K protein could not induce any morphological change in the VC, whereas the surface electron- dense layer of the VC was dissolved by 20K DEOteimM and chat, che Wve mixture |(d)y led to the lysis of the surface layer and extreme loosening of the VC main part,i.e., a feltwork of fine filaments. These observations indicate that the 20K protein contained in the anterior half of the acrosomal vesicle first dissolves the outer layer so that the posteriorly-located 15.5K protein acts on the inner main part. 1070 Developmental Biology DB 1/1 A PROTEIN COMPONENT OF THE "TRUNCATED CONE" IN JAPANESE ABALONE SPERM Y.Shiroya, S.Maekawa* and Y.T.Sakai. Biol. Lab., Wayo Women's Univ., Chiba. *Dept. of RIO owe3e S Item. 5, WEGo Oi Sis. p, WimIKyo Oi Tokyo, Tokyo. We have previously reported that the unique structure lying at the acrosomal apex in abalone ( Haliotis discus ) sperm named the "truncated cone" transforms into the cylindrical structure during the acro- SOM icearciento ((sCo>5 Ao, Zp IUVG4 jj. Was present study deals with characterization of the protein components of this structure by immunological technique. The truncated cones were isolated from abalone sperm by treating sperm heads with a Nonidet P-40 solution followed by dis- continuous sucrose gradient centrifugation. After treating with Ca’t-free sea water, Ehe) ansolubiley = sractton Orn themteruncaced cone was enriched in proteins of 60, 86 and 200kD, as determined by SDS-PAGE. Indirect immunofluorescence staining using affinity- purified monospecific antibodies against 60, 86 and 200kD proteins showed that only the anti-60kD protein antibody stained the apex of the acrosomal vesicle. Tine wexcihor Sive localization of the 60kD protein to the truncated cone of both the isolated apical half of the acrosome and acrosome- reacted sperm was clearly confirmed by immunoelectron microscopy. These results indicate that the 60kD protein is a compo- nent Of the ECruncated) cone” DB 172 Determination of the Structure of Lysin Binding Sites on Vitelline Coat: Fractionation of Solubilized Vitelline Coat M.Shitara & K.Haino-Fukushima. Dep.of Biol. Fac.of Sci.,Tokyo Metropolitan Univ. , Tokyo Vitelline coat lysin of marine Mollusca, Tegula pfeifferi irreversibly binds to the vitelline coat (VC) ,bringing about the lysis of VC.VC of this species is composed of sulfated-glycoprotein and is solubiliz-— ed by alkaline degradation. We reported previously that the solubilized VC (sol.VC) also bears LBS nearly equivalent to those hOUNGI An: snitacteViCE In the present study,we found that Pronase digestion of sol.VC totally destroys LBS. Amino acid analysis indicated that residues including Val,Phe,Leu,Ile,and Lys are absent from the Pronase digested WE, We Also tOwAGl Wiens TOCA loss Or IWS on sol.VC is caused by cleavage of N-glyco Sidic bond. Deglycosylation of sol.VC by TFMS, which leaves N-glycosidic bond un- affected, failed to show any remarkable effect on LBS. On the other hand, the 90K fragment was recovered when sol.VC was degraded in succession by V8 protease digestion, serial periodate oxidation,and TFMS deglycosylation. This fragment contains only 2 LBS despite the presence of 75 N-glycosidic bonds. From these results,it is evident that LBS requires not only N-glycosidic bond but certain amino acid sequence to be recogonized by Lysin. DB 173 ROLE OF DIFFERENT KINDS OF ZONA PELLUCIDA ON THE ACROSOME REACTION OF GOLDEN HAMSTER SPERM TOZOA. N.Uto!, R.Yanagimachi?. 1lDept. of Biol. Hamamatsu Univ. Schocl of Med., Hamamatsu, Univ. of Hawaii School of Med., Honolulu, Hawaii, U.S.A. The occurence of hamster acrosome reac— tion on zona in cumulus free eggs was studied. Different types of egg were inseminated with preincubated hamster Spermatozoa and then the sperm acrosome reaction on zona of them was investigated. The zona of homologous hamster eggs were most effective on hamster spermatozoa. In younger oocytes, the inducing ability of their zona were relatively accomplished State. In pronuclear stage, the zona of them did not loss the ability so much in Spite of post zona reaction. When hamster living eggs were treated with salt-solution or fixatives, the ratio of acrosome reac- tion on zona in these eggs decreased a little but retained in spite of some de- naturalization with reagents. Furthermore, heterogenous kinds of egg i.e. mouse, rat, guinea pig, hen and human eggs were compared. The zona of human egg induced the acrosome reaction well. On the other hand, in zona of other kind eggs, we hardly could observe the spermatozoa that underwent not only acrosome reaction but penetration into zona. In these studies it was suggested that the characteristics of hamster zona is dif- ferent relatively from other kindsof animals. The prcper itself was discussed everywhere. DB 174 EGG JELLY COMPONENTS RESPONSIBLE FOR THE INDUCTION OF ACROSOME REACTION IN THE STARFISH ASTERINA PECTINIFERA. %, Oldie, I AMenNe > 5 T. Matsui and Mo IlOGloa 4 “IGLOS. IbaAldS yp FACS Cie SE do Tokyo) inisita of Technol, loky oman cles Dayz. of Biomed. Polymer Sci., Inst. for Compre. Med. Sci., Fujita-Gakuen Health Univ., Toyoake. ARIS, a high-molecular weight sul- fated glycoprotein in the egg jelly, requires diffusible jelly components for inducing the acrosome reaction (AR) ina starfish, Asterias amurensis that belongs to the Forcipulata. Similarly, both ARIS and a diffusible fraction (Mg) of the jelly were proved essential for the induction of AR in an- other species of starfish belonging to the Spinulosa, Asterina pectinifera. The AR was induced also by combinations of the calcium ionophore A23187 and either monen- SJalial ©jie Mg, or of ARIS and NH,Cl. Meg in- creased the internal pH of sperm monitored by using a fluorescent probe, 9-aminoacri- dine. Both NH,Cl and monensin are known to increase the internal pH. Thus ARIS pre- sumably has a function in stimulating Ca**-uptake into sperm in Asterina as known in Asterias. The basic mechanisms underlying AR induction by the egg jelly appear common at least to the two species of starfish so far studied and may be very similar throughout the Class, Asteroidea. Developmental Biology 1071 DB 1/5 EFFECTS ON THE SPERM OF EGG JELLY COMPO- NENTS RESPONSIBLE FOR INDUCING THE ACRO- SOME REACTION IN THE STARFISH, ASTERINA ESOT EEHERA 5 5 Ts Matsuz Pe LomAIMNca IO ea Oleastian sand M. Hoshi“. Div. of Biomed. Polymer Sci., Inst. for Compre. Med. Seika Fujita-Gakuen Health Univ., Toyoake and “Biol. Lab., HoeceOrescig, Tokyo Inst. of Technol..,, Tokyo. Ce Soe) A high-molecular weight sulfated glycoprotein and diffusible substance(s) in the egg jelly were essential for the induction of acrosome reaction (AR) in the starfish Asterina pectinifera as known in Asterias amurensis. Either component did not induce the acrosome reaction by itself, but within a few minutes it made sperm species-specifically unable to undergo the AR in response to the egg jelly. The AR was not induced in suck sperm by increas- ing the external Ca * concentration or pH. Similarly, if the sperm was incubated gs short as a minute with egg jelly in Ca“*- deficient seawater, they never underwent the AR even after calcium was sufficiently supplemented. A combination of calcium ionophore A23187 and monensin, however, did induce the AR even in thus pretreated sperm. These results suggest that the jelly components modulate the transmembrane control systems simultaneously, transient- ly and irreversibly ending up in the acro- some reaction. DB 176 INITIATION OF SPERM HISTONE DEGRADATION BY EGG JELLY COMPONENTS IN THE STARFISH, ASTERINA PECTINIFERA. MwAManoOn Vea Okita, amd Ms Hosa: Bi@Gl, LelOo, FaCe Gi SSile, WOMWO WinSice sO Technol., Tokyo. Egg jelly induces two distinct sperm reactions, the acrosome reaction and histone degradation in the starfish, Asterina pectinifera. We have studied whether the jelly components required for these reactions are the same or not. A glycoprotein (ARIS) and a diffusible fraction of the jelly were required for inducing histone degradation as well as acrosome reaction. Both reactions were susceptible to calcium channel antago- nists, verapamil and diltiazem. Once SG were treated with the egg jelly in Gap Geemsea waten, they ilbecame unabye to undergo these reactions even after Ca“t was fortified. Calcium ionophore A23187 did not induce either reaction. However, histone degradation without acrosome re- action was induced by treating sperm with a low concentration of egg jelly, or monensin, or simply lowering the concen- tration of Na* in sea water to 50 mM or less. Thus jelly components required for the two reactions are the same or very simi- lar, but the mechanisms underlying these reactions seem different at least partly. DBRT, FURTHER STUDIES ON ARTIFICIAL PARTHENOGENE- SIS OF STARFISH EGGS PRODUCED BY SUPPRES- SION OF POLAR BODY FORMATION. C. Saitoh”, S. Washitani-Nemoto , and S. Nemoto . Tateyama Marine Lab., Ochanomizu Univ., Tateyama, and “Lab., of Biol., Hitotsubashi Univ., Tokyo, Japan. As we previously reported (Biol. Bull., "84), caffeine produced parthenogenetic development of starfish eggs by two-step actions, i.e. the activation of the eggs and the inhibition of polar body (PB) for- mation. To know the role of these two steps in production of parthenogenesis, we separated the steps by using two drugs; calcium ionophore for egg activation, and cytochalasin’ Be (Cyt. |B) for. they inhwb1 elon of PB formation. The drugs were applied to Asterina pectinifera eggs at some stages during meiosis. Cleavages did not occur in eggs treated with the ionophore alone, which formed 2 PBs and showed monoaster cycling only. Additional treatments with Cyt. B caused cleavages in more than 80% of the eggs that failed to form either both lst and 2nd PB or 2nd PB. The embryos derived from both types of eggs developed as tetraploids like caffeine-induced embryos. SDS, inhibiting PB formation, induced cleavages in eggs without ferti- lization membrane formation. These results indicate that the inhibition of polar body formation is prerequisite for production of parthenogenetic development in starfish eggs. DB 178 RELATION BETWEEN CLEAVAGE INDUCED BY _ IN- HIBITION OF POLAR BODY FORMATION AND NUM- BER OF CENTRIOLES, IN PARTHENOGENET ICALLY ACTIVATED STARFISH EGGS. K. H. Kato!,A. Hino, S. W. Nemoto%and S. Nemoto4 Coll.Gen. Educ. ,Nagoya City Univ. ,Nagoya, “Fac. Sci. , Nagoya Univ. , 3Lab. Biol. ,Hitotu- bashi Univ. , *Fac. Sci. Ochanomizu Univ. Stariisiy OOCVUCS Chin DO ArrNiINCIAlIY AC> tivated by caffeine, during the course. of maturation division induced by 1—MeAde When caffeine EPOAvI@ne WAG CARRi@cl Om SO AS TO inhibit the formation of polar body(s), a high percentage of cleavage and parthenogenetic development was attained (Obata and Nemoto, 1984). Minair@ 1S 2 poss sibility that the number of centrioles remaining in the egg has relation to the capacity of eggs to cleave. In this study, we examined with TEM the number of centri- OES, tm VAS ASTOR AL MATUTATIOM civisSioms and at parthenogenetically induced mito- tic divisions. In parthenogenetically de- veloped embryos, it was confirmed by con- secutive serial sections, that there was a pair of centrioles iin Am AStOR O71 the third division. The centriole number in EIN ASEOr Of ENG LU MAEMAENOM GiwiStom was two(€a pair) and that of the second was one. In normally fertilized eggs, there was A PATE Of CEMEFIOIES tm Che ASU@r OF ElNe second division. A schematic diagram on the number of centrioles and their dupli-— cation timing during meiosis Ainel imi wos s in normal fertilization and parthenogene— sis was presented. 1072 Developmental Biology DB 179 MASS ISOLATION OF GERMINAL VESICLES FROM STARFISH OOCYTES. 2 }. Hashimoto , K. Yamamoto and S. Nemoto Tateyama Marine Lab., Ochanomizu Univ., Tateyamnay, ang sDepit. Of PBHIOM) a bac. Om Gem HOG 7 Sasewl Winsiyo 7 Gaievl, wWeyoeiina Germinal vesicles (GV) of primary oocy- tes contain some factors necessary for the completion of meiosis and development. To know the character of the factors, we developed a simple isolation procedure for GVs by using fully grown oocytes of the starfish, Asterina pectinifera. Oocytes were treated with pronase to re- move the vitelline coat, and then with cytochalasin B to decrease the rigidity of cell surface. The oocytes were then centrifuged on a discontinuous sucrose- density gradient. GVs and enucleated frag- ments came to rest in separate layers according to their density. The isolated GVs were fairly uniform in size and morphorogy, and surrounded with a very thin layer of cytoplasm, in which micro- tubules were observed. The GVs isolated by this procedure appeared morphologically very Similar to those in the oocytes. When the content of the isolated GVs was introduced into enucleated fragments by microinjection, cyclic appearance of male pronuclei was observed. 1 DB 180 SPECIES SPECIFICITY OF FACTORS IN THE GERMINAL VESICLE REQUIRED FOR SPERM PRONUCLEUS FORMATION IN STARFISH OOCYTES K. Yamamoto , N. Hashimoto“ and S. Nemoto Dept. of Biol., Fac._of General Edu., Gifu Univ., Gifu and “Tateyama Marine Lab., Ochanomizu Univ., Chiba. The transformation of the sperm nuclei into the male pronuclei is known to be dependent on the germinal vesicle (GV) material in most animal eggs including starfish. In the present study we examined whether GV material exhibits any species specificity on the activity of inducing sperm-pronucleus formation using three different starfish. Inseminated nonnucleate fragments obtained from immature Asterina pectinifera oocytes were injected with GV contents of either Astropecten scoparius or Astropecten polyacanthus oocytes. Following application of 1-methyladenine (1-MA) the fragments were examined with Nomarski optics. Sperm pronuclei developed in these fragments about 3 hrs after 1-MA treatment, suggesting that the factors in GV have Similar nature in Asteroidea. Furthermore, our preliminary experiments in which nonnucleate fragments of starfish (A. pectinifera) oocytes were injected with GV contents of sea cucumber (Holothuria hilla) oocytes suggest the factors in GV have no specificity among echinoderms. DB 181 SUCCESSION OF THREE DISTINCT LEVELS IN THE CAPACITY OF FORMING FERTILIZATION ENVELOPE DURING MATURATION OF STARFISH EGGS. Ko Clarallogy, Euncl Mle IlO@Slna> WalOll> aldo, PAS. OQ SCio, WOKVO Liasicg OF. Weclmol,, WOkVvo- When oocytes of Asterina pectinifera were treated with calcium ionophore A23187 before 1-methyladenine (1-MA) addition (A phase), fertilization envelope (FE) was formed near the surface of oocytes. But between 1-MA addition and germinal vesicle break down (GVBD)(B phase), A23187-induced FE formation was inhibited. After GVBD (C phase), this inhibition was canceled and a fully elevated FE was formed by A23187. Thus we raised a question whether the phase-dependent difference is due to differences in intracellular calcium con- centration (Ca;) after A23187 treatment. To answer this question, Ca. was fixed by the microinjection of calcium buffers. Fixation of Ca. at 2.8uM resulted similarly as A23187 treatments. Electron microscopic observation showed that exo- cytosis of cortical granules was blocked only in A23187-treated oocytes of B phase. These results indicate that an increase in Ca. is not necessarily sufficient for inducing the exocytosis as believed gener- ally. There seems to be a post-calcium- increase step(s) that is modulated on the course of maturation. DB 182 ELECTROPHYSIOLOGICAL PROPERTIES AND EFFECTS OF SH-BLOCKING REAGENT ON THE CAPACITY FOR FERTILIZATION MEMBRANE FORMATION IN STARFISH OOCYTES. H. Nakamura. Akkeshi Mar. Biol. Stat. Hokkaido Univ., Akkeshi, Hokkaido Immature starfish oocytes have been re- ported not to form fertilization membrane ( F.M.) upon insemination under natural con- ditions. However, since we have observed that immature starfish oocytes occasionally formed F.M. upon insemination, differences of the electrophysiological properties ( Elec. Prop.) between F.M.-forming oocytes and none-forming oocytes were investigated using Asterina pectinifera. Effects of SH- blocking reagent on the capacity of the oo- cytes for F.M. formation was also examined. F.M.-none-forming oocytes showed a constant Elec. Prop. On the other hand, F.M.-forming oocytes showed four different types of Elec. Prop. When F.M.-forming oocytes were treated with iodoacetamide, the capacity for F.M. formation was abolished. Although the Elec. Prop. of the treated oocytes changed to those of another type, there seemed to be no direct correlation between the type of Elec. Prop. and capacity for F.M. formation. These results suggest that the acquisi- tion of the capacity for F.M. formation in oocytes may be correlated with the increase in SH-groups in their surface area, and that the Elec. Prop. of immature oocyte can be shifted from one type to another by ox- idizing surface SH-groups. Developmental Biology 1073 DB 183 ARTIFICIAL INDUCTION OF MEIOSIS-RESUMPTION IN OYSTER OOCYTES. K. Osanai, K. Kyozuka and R. Kuraishi. Mar. Biol. Stn., Tohoku Univ., Aomori. EOE ————————————————————e——e Crassostrea gigas oocytes obtained by dissecting the ovary were at the germinal MeScHuchecnstages. “Che oocytes undergo germinal vesicle breakdown in sea water containing serotonin (5-hydroxytrypt- aneney, but are arrested at the first metaphase of meiosis. The present examination showed that alkaline sea water (pH 9.3) induced the resumption of meiosis from the first metaphase. Alkaline sea water containing calcium ionophore A23187 (Cad, "025 =1:0 pM) was more effective than Pi iecHEScCdmwatereor Calvin pH 8-3) sea water, and induced meiotic changes from the first metaphase to the pronuclear stage within 1 hr. When metaphase I oocytes were incubated in alkaline sea water containing 5 or 10 pM Cal (Alkaline Cal), the oocytes began to extrude the first polar body and the second polar body about 15 min and 35 min after incubation, respectively. After the incubation for 60 min, some oocytes formed the polar lobe. Alkaline CaI seems to induce also mitotic change following the meiosis. The present result suggests that the successive changes of meiosis require the rise of pH and the increase of calcium ions in oocytes. DB 184 EFFECTS OF LIGHT ON OOCYTE MATURATION AND SPAWNING IN AMPHIOXUS, Branchiostoma belcheri tsintauense. T. Watanabe!, M. Yoshida! , and H. Shirai. Mar. Biol. Stn. Okayama Univ. Ushimadocho, Okugun, Okayama and *Lab. Reprod. Biol. Natl. Inst. for Basic Biol. Myodaijicho, Okazaki, Aichi. According to Wu, Institute of Oceanology, Academia Sinica, China, adult amphioxus spawns mature gametes in laboratory after sunset when a large number (about 2,000) of animals are kept in a tank with airation. Such activities usually last for about 1 hr and are observed intermittently through the breeding season (June to August). The time-dependent activity infers in- volvement of a light-dependent mechanism in the reproductive behavior, particularly the timing of oocyte maturation and egg dis- charge. The present experiments showed that mature oocytes were detected only in afternoon and spawning occurred 1-1.5 hr after sunset. Day length-prolongation by artificial illumination shifted the spawn- ing time accordingly, but its shortening by early darkening did not. We conclude that the spawning time is controlled at least by two factors; one is darkening, an external factor which direct- ly triggers mature egg-discharge and the other is susceptibility to darkness, an intrinsic factor which gradually develops during the course of day in mature animals. DB 185 PURIFICATION OF TWO KINDS OF SIALOGLYCOPRO- TEINS FROM MATURE CARP EGGS AND THEIR IMMU- NOHISTOCHEMICAL LOCALIZATION. S.Kudo', S.Inoue?,and Y.Inoue*. ‘Dept. of Anatomy, Gunma Univ. School of Medicine, *School of Pharmac. Sci., Showa Univ. and ? Dept. of Biophs. Biochem., Univ. of Tokyo Mature carp eggs crushed were extracted with a solution of 0.8% NaCl. The mixture was filtered through gauze and the filtrate was centrifuged at 5,000 rpm for 30 min.The supernatant was mixed with 1 vol. of 90% phenol, and this mixture was stirred at room temperature for several hours. The aqueous phase was separated by centrifuga- tion for 15 min and dialyzed against dis- tilled water, followed by lyophilization. The partially purified material containing Sialoglycoproteins (SGPs) and phosvitin was applied to a DEAE-Sephadex A-25 column pre- equilibrated with 0.01 M Tris-HCl buffer(pH 8.0) and eluted with a linear gradient of NaCl (0.1-0.5 M). The SGP peak was then subjected to Sephacryl S-200 chromatography following pre-equilibration with the same buffer, in order to separate SGPs from phosvitin, and eluted with 0.1 M NaCl in the same buffer, to obtain two kinds of SGPs (4-1 and 4-3). SGP 4-1 contained much more sialic acid than SGP 4-3 and aggluti- nated fish sperm, whereas SGP 4-3 did not. Application of antibodies against SGP 4-1 and SGP 4-3 revealed that they were immuno- histochemically localized in the cortical alveoli of carp eggs. DB 186 ELECTRON MICROSCOPIC OBSERVATION OF MICRO- PYLAR CELLS IN THE OVARIAN FOLLICLES OF MEDAKA, Orizias latipes. S.Nakashima and T.Iwamatsu. Dept. of Biol., Aicha Univ. Of Educ., Kariya The formation and morphological changes of micropylar cells during the course of vitellogenesis and maturation of oocytes of Medaka, Orizias latipes were examined with electron microscope. The micropylar cell was distinguished from neighboring cells as a large mushroom-shaped cell and was stained with methylene blue slightly deep blue compared with other follicle cells. In previtellogenic stage, this large cell stained with methylene blue was not observed and all cells in the follicle layer showed the same stainability. The special large cell was observed in the stage which yolk vesicles appear. In this stage a bundle of tonofilaments extending from desmosomes was observed in the cyto- plasm of both micropylar cell and neighbor- ing cells. Bundles of the tonofilaments were gradually developed in the micropylar cell with the progress of vitellogenesis and maturation of the oocyte. On the con- trary, these filaments gradually dimin- ished in the other follicle cells. The apical part of the cytoplasmic process with meandering tonofilaments extended in the cortical region of the oocyte. The main cell body contains well developed Golgi bodies, tonofilaments and rough endoplasmic reticulum. 1074 Developmental Biology DB 18/7 FERTILIZATION POTENTIAL OF THE MEDAKA (ORYZIAS LATIPES) EGG. S.Ito and K.Shimamoto. Dept of Biol., Fac. of Sci., Kumamoto Univ., Kumamoto. Fertilization of the medaka egg in 103% Ringer, generates a depolarization of a few mV just before the appearance of achar- acteristically longer hyperpolarization. The depolarization appears to result from a nonspecific leak triggered by sperm-egg fusion; and the amplitude of the depolari-. zation is reported to be independent of {ca (Nuccitelli, 1980). We have investigated the ionic dependence of this depolarization An initial small depolarization (3-4mV; duration, 5-8 sec) is followed by a rising phase of a spike-like depolarization rang- ing from 10-60 mV when recordings are made in appropriate Ca*t concentrations (1-18 mM) dissolved in isotonic sucrose(210 mM) solution. The amplitude of this spike- like depolarization is proportional to log Jes anging from 0.33-18 mM. Calcium antagonist, i.e. 10mM cobalt or 10pg/m1 verapamil dissolved in 10% Ringers do not block the depolarization. We conclude that the spike-like depolarization of the medaka egg is dependent on (ca** however the mechanism underlying the genesis of the depolarization may be different from the generation of Ca-action potentials in excitable tissues. DB 188 PROPERTIES OF FERTILIZATION POTENTIAL IN THE MEDAKA, ORIZIAS LATIPES EGGS. Me Nakayal ander lwalOribniop en em Site, Fac. Sci., Yamaguchi Univ., Yamaguchi. Upon fertilization of Oryzias latipes eggs, cortical alveoli breakdown (CABD) is preceded by a fertilization potential (FP) that has a small depolarization (DP) followed by a large hyperpolarization (HP). To investigate the relationship between the CABD and the FP, the centrifuged eggs (450 g, 10 min) were inseminated in 10% Ringer. The magunitude of DP in the eggs whose cortical alveoli (CA) had been accu- mulated at vegetal pole (VP eggs) were smaller than accumulated at animal pole (AP eggs). The time lapsed from the onset of DP to the peak of HP in the VP eggs was longer than in the AP eggs. These indicate a close correlation between the CABD and (ee Isl Ole ieloys JEP ili, Welova. ie@iciewilsgacl apf. Upon activation by pricking or Ca-iono- phore A23187, the eggs elicited an activa- tion potential similar to the FP in 1.8 mM CaCl (resting potential; about -60 mV), but the DP was not detected in 10% Ringer (resting potential; about -10 mV). The DP as well as the HP is caused by the ionic channels on the egg-membrane, but the appearance of DP is voltage-dependent. When the naked eggs were inseminated in Ringer, short-lived recurring DPs (1-2 mV) were observed, besides the DP followed by the HP of the FP. These step-like DPs may correlated with sperm-egg collisions. DB 189 2+ ON THE INITIATION TIME OF Ca~" RELEASE AND EXOCYTOSIS BY MICROINJECTION OF VARIOYS AGENTS IN THE MEDAKA EGG. Tex Iwamatsul, Y. Yoshimoto“, K. Onitake3 and H. Hira- moto. IDept. Biol., Aichi Univ., Kariya,“Biol. Lab. Univ. Air, Chiba, and 3Dept. Biol., Fac. Sci., Yama— gata Univ., Yamagata. The real initiation time of Cat release from cytoplasmic stores and exocytosis of cortical alveoli in Oryzias latipes eggs were examined by microin jec- tion of inositol 1,4,5-triphosphate (IP3), ionophore A23187, Ca2+, cGMP and GIP under Ca2+-free conditions. Intracellular release of Ca‘t+ by microinjection was analysed by measuring luminescence of aequorin loaded in unfertilized eggs. Microinjection gf IP3 or A23187 could rapidly induce a propagative Ca‘*treléase with— out delay, while microinjection of Ca** or cGMP could induce it with a time lag. In these microinjections, the Ca** release commenced at the cytoplasmic region close to the egg surface. Furthermore, a propagative exocytosis was rapidly triggered by Ca*t- or IP3- microinjection, but slowly by cGMP- or GTP-microin-— jection. However, cAMP- and ATP-microinjections that triggered no Ca“trelease, failed to induce exocytosis. Cyclic GMP or GIP failed to induce exocytosis, when was simultaneously ULCOP MN JSCES with Co¢+. Unlike cGMP and GTP, IP3 and Ca“* could induce exocytosis even in the presence of Co2+. The inhibitory effect of Co*+ was also observed on exocytosis by sperm stimulation. These resylts in the medaka egg suggest that the cytoplasmic Ca“*+ and GIP induce indirectly Ca“? re- lease from cytoplasmic stores,probably via a_membrane factor such as IP3, and that both IP3 and Ca¢t act to the same process of exocytosis, different from that as cGMP and GTP act. DB 190 IMMUNOCYTOCHEMICAL ANALYSIS OF THE EGG ENVELOPE FORMATION OF MEDAKA. T. S. Hamazaki, I. Iuchi and kK. Yamagami. ImieS SChlL — WMaSies p Sophia Univ. Lokyvo- The egg envelope (chorion) of Oryzias latipes has a liver-derived constituent. This substance is produced in the liver under the influence of estrogen, and incorporated finally into the oocyte chorion from the inside. In the present study, an indirect immunocytochemical observation of the Ovary using anti-chorion glycoprotein antibody and protein A-colloidal gold particle revealed that a high immunore- activity was present in the inner layer (IL) of the oocyte chorion, but not in the outer layer (OL) of chorion, the chorionic fibrils, the follicle cells, nor the theca cells. Moreover, the dense cored vesicles (DCV) showed no reactivity, which were considered to contain the precursor sub- stance of the IL, and appeared in the cytoplasm of the oocyte only the initial phase of the IL formation. However, the vesicles with an intermediate electron density, occurring throughout the period of the IL formation, showed a little immunoreactivity. Thus, the possibilities arise that the OL of medaka egg envelope has two sublayers, inner one of which is made up of the DCV, and the IL is made up of the liver-derived constituent together with a few other constituents which might be synthesized in the oocyte itself. Developmental Biology, Endocrinology 1075 DB 191 SOME PROPERTIES OF HATCHING ENZYME AS EXAMINED BY MONOCLONAL ANTIBODIES. Si Yasunasu ese Katowe,) | rh. duchicl and Pee Vanagania! bake. Scio Inst..; Sepbwaeunive, = Natl. "inst Health Jpn, Tokyo. Two enzymes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), have been considered to participate in digesting the chorion cooperatively at the hatching time of medaka embryos (Yasumasu et al., 1987). We obtained various clones producing the monoclonal antibodies (MAbs) against either LCE or HCE. Tested by the immuno- blotting method, anti-LCE-MAbs were found to be specific for LCE, while anti-HCE- MAbs were not completely specific for HCE but a ite ee Gross iGreacerlver, to), Ch. Competition experiments showed that anti- HCE-MAbs could be classified into two groups in respect to their recognition sites. One of anti-HCE-MAbs (7-E-2) inhibited BN SmChOmVolyilCe accel vailby, Of JHEE but not its caseinolytic (proteolytic) activity. In addition, 7-E-2 inhibited also the association of HCE with chorion. These results suggest that the binding action and the proteolytic (catalytic) activity were segregated and that a step of its Ioskiavelat inj EC) Claverealyol —jorealo@ye” EC) hydrolyzing it may be involved in chorion digesting process. DB 192 MORPHOLOGICAL OBSERVATION ON THE REGENERATING SCALES IN THE SKIN OF FLOUNDER, N.Makino! and S.Kikuchi®, /Dept.of Biol., Fac. of Sci., Chiba Univ., Chiba and 2Kominato Lab., Fac. of Sci., Chiba Univ., Awa-gun. “ In the flounder, the skin of both body sides differs not only pigmentation but also the scale shape. Color anomalies occur frequently in hatch- ery-reared flounder. Including color anomalies, ctenoid scales are observed in the melanized skin and cycloid scales in the non-melanized skin. Present work deals with regenerating scales in the melanized and non-melanized skin of flounder, Paralichthys olivaceus. Operation was performed surgecal removement of the skin. Scales were formed in parallel with regeneration of the skin in both sides. Reformation of melanophores in ocular side was observed 10-20 days after operation. On the tip of regenerating scale, formation of ctenium be- gan, and they grew gradually into ctenoid scales. In blind side cteium formation did not occur in regenerating scales and they grew into the cycloid scales. Also in the regeneration, formation of Ctenoid scale was observed only in theskin in which melanophores well develop. DB 193 MORPHOLOGICAL OBSERVATION ON AUTO-TRANS- PLANTED SCALES IN THE FLOUNDER. S.Kikuchil, N.Makino2 and H.Nakamura?, lkominato Lab., Fac. of Sci., Chiba Univ., Awa-gun, 2Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba and 3Dept. of Anat., Dokky6 Univ. Sch. of Med., Shimotsuga-gun. In the flounder, Paralichthys olivaceus, there are two types of scales, ctenoid in melanized black region and cycloid in non- melanized white region of the skin. In the present work auto-transplantation of scale was performed in order to examine effect of melanophore on the formation of ctenoid scale. Transplantation was performed as follows: Scales were pulled out and used as transplants. An isolated scale with a frag- ment of the skin was inserted into empty scale pocket which was made by removement of pre-existed scale before transplantation. In surrowndings of the blind side, melano- phores proliferated and formed in crowds small black spot. After 3 months grafted ctenoid scale barely stayed under suppres- sion of regenerating scale. Formation of ctenium did not found in the natural and regenerating cycloid scales in the mela- nized region. In transplantation of cycloid scales in ocular side, transplants were gradually restroyed in parallel with forma- tion of regenerating ctenoid scales. In the present work formation of ctenium on the natural and the transplanted scales did not OCCuIE EN 1 THE ROLE OF CALCITONIN IN THE GOLDFISH Y. Sasayama, F. Mizutani and C. Oguro. Fac. of Sci., Toyama Univ., Toyama In teleosts, the role of calcitonin (CT) in Ca metabolism has not been clari- fied. In the present study, at first, the effect of CT on the serum Ca level was examined in the goldfish. Synthetic salmon CT was administered (100mU- or 3U/100g bw) directly to the arterial bulb. However, the serum Ca level did not show any changes during 24 hr. Second- ly, effects of CT on other aspects in Ca metabolism than the serum Ca level was’ studied. Young goldfish was’ kept ine waterme,Containimag 4 5Cam (l20)= pCi/l)) for 17 days. They were injected intraperi- toneally with the extract of the ultimo- branchial gland (UBE) of the adult gold- fish (1/2 gland/individual) for 5 times during this experiment. In the result, in UBE-injected group, 45Ca activity of scales, otoliths and other hard tissues was higher than that in the control. It was noted that the radioactivity of gall bladder in the experimental group is extremely higher than that in the control. In feces, the value of the former was higher than that of the latter, too. These facts suggest that CT functions to accelerate the accu- mulation of Ca into hard tissues. and the excretion of Ca into alimentary tract via gall bladder, although CT does not seem to participate in the serum Ca con- eIEOMe 1076 Endocrinology EM 2 MINERAL CONCENTRATIONS OF BODY FLUIDS IN CYCLOSTOMES AND CARTILAGINOUS' FISHES Y. Sasayama, N. Suzuki and C. Oguro. Fac. of Sci., Toyama Univ., Toyama Mechanisms of the excretion of salts via alimentary tract has not been clari- fied so far in cyclostomes and cartilagi- nous fishes. In the present study, mineral concentrations of the serum, the bile and the fluid stagnated in alimentary tract were determined in 2 species of cyclostomes and in 4 species of cartilagi- nous fishes. Those are all sea-water fishes. The values obtained were compared with those determined in bony fishes which we examined previously. In bony fishes, minerals in the fluid stagnated in alimentary tract show a constant tendency of descent or ascent with going down to the end of the tract. In cyclostomes, however, it became clear that mineral levels do not change through- out the tract, except for Pi. In addition, it was noted that in the lamprey Na is already diluted at stomach to 1/22 of the sea-water level. Ca and Mg were con- centrated to 2 times. It is well Known that cartilaginous fishes do not drink sea-water. Therefore, the importance of alimentary tract on the mineral excre- tion has been neglected so far. However, present study revealed that handlings of minerals in the tract are different from species to species. Furthermore, it was known that in the bile Cl level is fairly higher than that in bony fishes. EN 3 EFFECTS OF ADRENALECTOMY ON SERUM Na, K AND GLUCOSE CONCENTRATIONS IN THE SNAKE, RHABDOPHIS TIGRINUS. C. Oguro, E. Ohba and Y. Sasayama Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan Function of the adrenal gland in the reptile has not been completely clarified. One of the main reasons is that the surgical removal of the adrenal gland is very difficult because of the intimate relation of the adrenal tissue to the kidney and large blood vessels. However, a technique for complete adrenalectomy without defect in kidney function was developed in snakes in this laboratory. Using this technique, effects of adrenalectomy on serum Na, K and glucose concentrations were studied in the snake, Rhabdophis tigrinus tigrinus. Ten days after adrenalectomy, serum K concent- ration was increased by 35% (16.7222.3 mg/100 m1) and serum glucose concentration was decreased by 68% (125939 mg/100 ml). But, serum Na concentra- tion (400 mg/100 m1) was not changed. Therefore, it was concluded that the adrenal gland of R. t. tigrinus functions to maintain serum glucose concentration at a normal level and to suppress the increase of serum K concentration. However, the adrenal gland does not seem to have an important role in the control of serum Na con- centration. The similar results have been reported in two species of lizards, Trachysaurus rugosus and Vara- nus gouldii. Taking these facts into account, it is highly probable that in squamate reptiles the adrenal gland has a major role in the control of serum K and glucose concentrations but has very minor role in the control of serum Na concentration. EN 4 RELEASE OF CALCITONIN FROM THE ULTIMOBRANCHIAL GLAND OF THE BULLFROG IN VITRO C. Oguro, T. Ishijima and Y. Sasayama Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan a It has been known that calcitonin (CT) from the ultimobranchial gland (UB) has a role in the regu- lation of serum Ca concentration in anuran amphibi- ans. However, nothing is known on the regulatory mechanism of CT secretion from the UB in amphibi- ans. It was reported that secretion of CT in vitro from the rat thyroid C cells is enhanced by a high Ca concentration in the culture medium. In the present study, isolated UB of the bullfrog were cultured for 72 hours in various Ca concent- rations. CT released into culture media and con- tained in the UB after the culture were determined in rat bioassay. Four kinds of culture media were used; [1] frog saline (Ca, 1.1 mM), [2] 2X Ca frog saline (Ca, 2.2 mM), [3] 3X Ca frog saline (Ca, 3.3 mM), [4] phosphate added frog saline (Ca, 1.1 mM; Pi, 1.45 mM). Every culture media contained 0.1% glu- cose. Every culture media and the UB after the culture showed hypocalcemic potency when given to rats. The highest activity was obtained in medium [3]. Medium [2] showed the next highest followed by medium [1]. Presence of phosphorus seems to inhibit the release of CT from the UB in this culture condition, since the hypocalcemic potency of medium [4] was lower than that of other media. From the results obtained, it was concluded that isolated UB of Rana catesbeiana releases CT into culture media and the release is promoted by a high Ca concentration in the medium. EN 5 EFFECTS OF PARATHYROIDECTOMY AND ULTIMO- BRANCHIALECTOMY ON THE SERUM Pi CONCENT- RATION IN BULLFROG TADPOLES Y. Sasayama, M. Kiyozuka and C. Oguro. Fac. of Sci., Toyama Univ., Toyama It has been known that in mammals parathyroid hormone and calcitonin affect not only Ca but also Pi metabolism. There- fore, it has been suggested that these calcemic hormones may have emerged for the regulation of Pi rather than for Ca. In lower vertebrates, however, the relationship between these hormones’ and the serum Pi level has not been clarified so far. In the present study, effects of parathyroidectomy and ultimobranchial- ectomy on the serum Pi level were studied in the bullfrog tadpole. Parathyroidectomy and the sham-opera- tion brought about significant rises in the serum Pi level 7 days after. When comparison was made in those values be- tween the both groups, the level of the former tended to be higher than that of the latter. This result shows’ that parathyroid gland may be concerned with the serum Pi regulation, although the degree of the participation is small. Also in ultimobranchialectomy and the sham-operation, serum Pi level increa- sed significantly 7 days after. Between the both groups, however, there was no significant difference. This result sug- gests that ultimobranchial gland does not participate in the control of the serum Pi concentration. Endocrinology EN 6 EFFECT OF PARATHYROID EXTRACT ON CULTURED BONE OF CYNOPS PYRRHOGASTER. C. Oguro, Y. Minamimura and Y. Sasayama. Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan. In urodelan amphibians, parathyroid gland (PT) plays a role in the maintenance of normal serum Ca concentration in species belonging to phyletically higher groups. Thus, it was reported that in Cynops pyrrhogaster PT is one of the major organs for the maintenance of serum Ca concentration. On the bases of the results in preliminary ex- periments, it was suggested that the target organ of PT is the bone. In the present study, the effect of bullfrog parathyroid extract (PTE) on the cultured bone of C. pyrrhogaster was observed by the 4Ca release from the bone which had been labelled with *Ca. The incubation was done at 20°C. The rate of *Ca release to the media was 17.3% at the end of incubation. This value was lower than the values reported in fetal bones of mammal and bird. Femur and humerus obtained from the #Ca labelled individuals were incubated in media with or without PTE. The #Ca release in the PTE containing media was higher than that from the control media in every sampling time(4,8,12,24 and 48 hrs). The difference between experimental and control groups was statistically significant(P<0.05). 4#5Ca release from the dead bone was unstable and thus the results noted above were noticed to be physiologi- cal. From these results, it is concluded that in C. pyrrhogaster parathyroid hormone exerts its effect directry to the bone and promotes the release of Ca to the blood for the maintenance of serum Ca concentration normal. EN 7 SEASONAL CHANGES OF PLASMA CA, MG AND INORGANIC PHOSPHORUS LEVELS IN MALE SNAKES. M.Yoshihara’, M.Uchiyama , T.Murakamil, H.Yoshizawa* and C.Oguro’. Dept. of Oral Physiol., Sch. of Dentistry at Niigata, The Nippon Dental Univ., Niigata, Dept. of Oral Histol., Matsumoto Dental Coll., Shiojiri, Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. There is no information currently in the literature concerning possible seasonal fluctua- tions of plasma Ca levels in male reptiles. In the present study, seasonal changes of plasma Ca, Mg and inorganic phosphorus (Pi) levels of the rat snake, Elaphe quadrivirgata, were investigated. Snakes used were sexually mature males and they were bled 5-14 days after capture. Blood samples obtained in December and February were obtained from artificially hibernating snakes maintained in an incubator at 5°C from early November. Plasma Ca levels in pre- and post-hibernating (October, November and April) and hibernating (December and February) seasons were significantly lower (8%) than those in the active season (June-September, 3.57 + 0.02 mM/1, n=167). On the contrary, plasma Mg levels in pre- and post-hibernating and hiber- nating seasons were significantly higher (12 and 55%) than those in the active season (1.57 + 0.03 mM/1, n=105). Plasma Pi level in pre- and post hibernating seasons was significantly lower (17%) and that during hibernation was significantly higher (17%) than that in the active season (1.23 + 0.03 mM/1, n=98). These results indicate that in E. quadrivirgata seasonal changes occur in plasma Ca, Mg and Pi levels. 1077 EN 8 DEVELOPMENT AND SALINITY TOLERANCE IN TADPOLES OF THE CRAB-EATING FROG. M.Uchiyama!, T.Murakami! , C.Wakasugi2 and S.Sudara?. !Dept. of Oral Physiol., The Nippon Dental Univ., Niigata.,2Dept. of Histol., The Nippon Dental Univ., Niigata and*Dept. of Marine Science, Chulalongkorn Univ., Bangkok, Thailand. It has been reported that the tadpoles of the crab-eating frog, Rana cancrivora, inhabit brackish environment. The present study was performed to clarify physiologi- cal mechanism of their great tolerance for salinity. For this purpose, ontogeny, salinity tolerance and histology were in- vestigated. The rate of embryonic develop- ment was fairly rapid (96 hrs) in 10% sea water at about 25° C. Tadpoles before the limb bud stages were able to survive in media up to 40% sea water but died within 8 hrs after being placed acutely in 502% sea water or more high salinity. On the other hand, tadpoles (stages I-XVIIT) adapted in higher salinities when ac- climatization was carried out in steps by changing the salinity 10-20% every 2-7 days. In the histological examinations, hypertrophy of the epidermis was demon- strated in the tadpoles adapted to the higher salinities although no salt gland was observed. Eosinophilic cells were observed in the internal gills of tadpoles adapted to 10-100% sea water. These structures might be useful for the adapta- tion to the environment of various salinities. EN 9 SUPPRESSION OF LH RELEASE BY SHORT DAYS. M. Wada Depirs (Geni Educat,) —Loky.o = Medes Demi Univ., Ichikawa, Chiba In my previous paper, I reported that a pattern of LH release changed during early photostimulation from "episodic" to "constant" release in Japanese quail and that short days were not enough to Suppress LH release after the release pat- EeOrEn aus Tonce changed to = cons tami release. To test an effect of short days on LH secretion, one to 7 photopulse were given to quail kept under 8L16D. One hour photopulses at the photosensitive phase induced sustained LH secretion; plasma concentration of LH maintained increased levels even after the pulses being no more delivered. Plasma LH concentrations were tended to decrease, however, by 20 days after the initial treatment in the quail given 1, 2, or 3 pulses each day. On the other hand, LH concentrations in the quail delivered 5 or 7 pulses were not decreased to the initial basal LH level by 20 days after the treatment. The increased LH levels were around 1 ng/ml, which were lower than those in long day control birds. The cloacal protrusion of these birds maintained a developed state even in the short days. Another experiment indi- cated that low temperature is also required for short days to express full suppression on LH secretion. 1078 Endocrinology EN 10 EFFECTS OF LONG-DAY PHOTOPERIODS ON CHANGES OF PLASMA GONADOTROPINS AND SEX STEROIDS IN JAPANESE . COMMON PHEASANTS DURING BREEDING SEASON H. Sakai, M. Sato, S. Wakabayashi, K. Hirano , Y. Oono! , and M. Kasugal, Dept. of Biol., Nihon Univ. School of Dentistry, Tokyo, and !Saitama Prefectu- ral Poultry Experiment Station, Saitama. Eight-month old pheasants were reared under long-day photoperiods (16L:8D) from February to study the relationship of changes in plasma gonadotropins and sex steroid hormones in wild birds. Male LH and female progesterone were already high in February both in long-day and natural photoperiod groups, and no significant difference was observed between them. On the other hand, egg laying and increase in plasma levels of male FSH, female LH, FSH and estrogen by long-day photo stimulation preceded natural photoperiod group by 4 to 8 weeks with different features of change. Testosterone levels of male groups increased for the same period, but their levels during breeding period were different. This study showed that long-day photo stimulation preceding the breeding period hasten hormone secretion but not necessarily the hormonal changes observed in natural condition. These results suggest that further study of the gonadal responsibilities will be needed to account for these results. EN 11 EFFECT OF PROLACTIN ON WATER DRIVE SYNDROME OF THE SALAMANDER HYNOBIUS NIGRESCENS. Masato Hasumi and Hisaaki Iwasawa. Biol. Inst., Niigata Univ., Niigata. Hibernating adult males of Hynobius nigrescens, which were bred outdoors throughout the year, were divided into 8 groups (initial control, intact cont., saline, 5 IU PRL injection for 12 days, 50 YW 12 Cews, SW 2O cays, SO mW 2O ces, 5S IU 20 days in a poolless container) just before the breeding season. These animals were injected intraperitoneally with ovine prolactin (PRL) every other day, and the position of the salamanders, on land or in water, was examined every 2-6 hours. The salamanders were observed to wander back and forth between water and land up to whole submergence. The specific gravity of the whole body was greater than unity in all the individuals irrespective of the treatments, and no significant difference was found among the groups. The connective tissue inside the skin was tight in PRL-injected animals kept in a poolless container. Cornified epidermis and mucous glands filled with mucus were seen in intact animals (initial control), whereas these structures were not seen in any in the PRL-injected groups. In the field, the mucous glands of some males shortly after migration into a breeding pond were filled with mucus yet, but little mucus was seen in the glands of typical aquatic-phase males. Ni SEASONAL VARIATION OF THE PROLACTIN (PRL) EFFECT ON ACTIVE Na TRANSPORT ACROSS AMPHIBIAN SKIN. M. TAKADA, Dept. of Physiol., Saitama Med. Sch., Moroyama, Saitama a Ie ENA ile The long-term effect of PRL. Long- term application of PRL, i.e., 20 ug/g body weight injected into autumn newts (Cynops pyrrhogaster) every other day for 2 weeks, induced an increase in resistance to active Na current (Rnya), and a resulting decrease in the poten- tial difference (PD) and the short cir- cuit current (SCC), but barely changed the electromotive force of the active Na current (Ena)- Such long-term applica- tion of PRL to spring newts induced a decrease in PD and SCC but exerted no effect on the Enya and Rna- 2s The short-term effect of PRL. Ten ug/ml of PRL was applied to the dermal side of isolated skin of the tadpole (Rana catesbeiana) during metamorphosis. PRL induced a transient increase in SCC across the skin of both spring and autumn tadpoles. Percent increase in scc of the skin of spring tadpoles was larger than that of autumn ones in Cl- Ringer's condition. By contrast, the PRL effect was only slightly or not at all present in the skin of both spring and autumn tadpoles in SO4-Ringer's solution. EN 13 FACILITATION OF MASCULINE SEXUAL BEHAVIOR BY SYNTHETIC INHIBITOR OF SEROTONIN IN SEPTAL LESIONED MALE RATS. MG IOC, YN5 Sidalinocle, IK. Yamanouchil, and Y. Awan Dept. Psychol., Fac. Letters, Gakushuin univ., 1) Dept. Human Sci., Fac. Human Sci., Waseda univ., and 2) Dept. of Anat., Juntendo univ. Sch. Med., Tokyo. Previously, we reported the suppres- sion of masculine sexual behavior following lesioning in the medial preoptic or lateral septal area (MPOL and LSL) in male rats. In this study, we examined the effect of a synthetic inhibitor of serotonin, p-chloro- phenylalanine (PCPA), on masculine sexual behavior in these rats. Orchidectomized rats received either bilateral LSL or bilateral MPOL. Three weeks later, all animals were treated with testosterone(T) by implantation of Silastic tubings. Observations of masculine behavior were carried out 10 and 20 days after T im- plantation. Each observation was followed by 4 daily injections of PCPA (100mg/Kg) or saline. Saline-treated males with LSL or MPOL showed significantly less masculine behavior than sham controls. Administration of PCPA markedly facilitated masculine behavior in control and LSL males. However, PCPA failed to increase masculine behavior in MPOL males. The results suggest that the lateral septum has facilitatory influences upon a regulating system of masculine be- havior which may be inhibited by serotoner- gic neurons, while the medial preoptic area may be more important for masculine behav- LOO 6 Endocrinology EN 14 EFFECT OF ANDROGEN ON EARLY POSTNATAL DEVELOPMENT OF THE MEDIAL PREOPTIC NUCLEUS(MPN) IN THE RAT. S. Murakami! s. [to@and Y. Arai! Depts of Anatt and Ob-Gyn*, Juntendo Univ. Sch. of Med. Tokyo. The medial preoptic nucleus(MPN) is an intensely stained neuron group in the rostroventral periventricular gray of the preoptic area. Its volume is significantly preater in females than in males. This sex difference is dependent on neonatal androgen. Treatment of female rats with 50 pg of testosterone propionate(TP) for 5 days from the day of birth effectively reduced the nuclear volume to the level just comparable to that of normal males. In order to elucidate the possible mecha- nisms involved in the volume reduction of the MPN following neonatal androgen treat-— ment, the number of degenerating cells in the MPN during early postnatal period was examined aA females and TP-treated females. At postnatal days 7 and 10, TP- treated females had significantly more pyenotie cells in the MPN than females. Furthermore, most: of the pycnotic cells were distributed in the peripheral zone of the MPN. These results suggest that neo- natal androgen may promote the loss of MPN neurons during developmental stages, and this may account for the reduction of the volume of the MPN by neonatal treatment Want =EPy. EN 15 STRUCTURAL RELATIONSHIPS OF HYPOPHYSIAL VASCULARIZATION AND ADENOHY POPHYSIAL CYTOLOGY IN THE SNAKE, ELAPHE QUADRIVIRGATA I.Koshimizu and Y.Oota. soils AbNeeo, HAC eOme Cl.) onl zZuoka Unive, , Shizuoka. The vascular supply of hypophysis and distribution of the adenohypophys- ial cells were studied in the snake. The median eminence iS supplied by a very dense primary capillary plexus deriving from branches of the infun- dibular arteries. The primary capil- lary plexus converge into several large portal vessels. The pars distalis (PD) receives its blood supply via _ the portal vessels. In the PD, the portal vessels give off the sinusoid system which has intimate contact with the PD cells. Histologically, the PD can be divisible into cephalic and caudal lobes, which are marked by differential distribution of glandular cells. Immu- nohistochemically, the prolactin- immunoreactive cells are found exclu- sively in the cephalic lobe, whereas the growth hormone-immunoreactive cells are distributed in the caudal lobe. Structurally, the "anterior group” of portal vessels is mainly supplied in the cephalic lobe, whereas the posteri- or group of portal vessels enters the caudal lobe. 1079 EN 16 AN ELECTRON MICROSCOPE STUDY OF THE PARS TUBERALIS OF THE TURTLE, GEOCLEMYS REEVESIT Y.OotaWand K.Miyata. Biol: Inst., ‘Fac. of Sci., Shizuoka’ Univ., Shizuoka: ee ee AT ee ea. eel ae The morphology and cytophysiology of the pars tuberalis (PT) were studied in the turtle. The PT is well developed and can be divisible into three parts juxta™ neural “PTY (3juxP?) >; “the portal zone and the PT interna (PTint). The cells of the juxPT spread over the ven- tral surface of the median eminence. In the portal zone, the PT cells enve- lope the portal vessels connecting the median eminence and the pars distalis. The Pfint forms ~a’ continuous” tissue to the pars distalis. Most “om “they Pr cells are chromophobic nature, however, a small number of cells showing posi- tive immunoreaction to thyrotropin- antisera are demonstrated in the juxPT. Similarly, cells showing positive immunoreaction to follicle stimulating hormone-antisera are identified in the [PALI ONE Wath ther electron microscopy, the “PT cell's can” be “readily classified into two types secretory cells and rOIsaouilae Cells. Although there is some morphological variability, charac-— WSIS Sig 1S secretory cells containing electron-dense granules are demon- Strated in the juxPT and PTint. EN 17 EFFECTS OF LHRH ON EEG ACTIVITY AND PLASMA ANDROGEN LEVELS IN JAPANESE TOADS. Y. Fujita and A. Urano. Dept. of Regul. Biol., Eac. -of Sci. , Saitama Univ.s, Urawa: A series of our studies has suggested that initiation of reproductive behavior in hibernating toads is controlled by var- ious neurohormones, such as LHRH and TRH. We showed that systemically applied LHRH enhanced amplitudes of EEGs in the toads. In this study, we measured changes in the plasma androgen levels after LHRH adminis- tration to examine whether EEG enhancement is induced by direct central action of LHRH or indirect action through the hypo- thalamo-hypophysial-gonadal axis. Animals were immobilized by pith, and were cannu- lated through the femoral artery and vein for a intravenous application of LHRH and collection of plasma samples 2-36 hr after the LHRH administration, respectively. Intravenous LHRH at dose levels of 10 and 100 g that could arouse toad EEG activity did not elevate the plasma andro- gen levels. Moreover, the increases in androgen levels by 4-hr continuous LHRH infusion did not temporally coincide with the EEG enhancement in the same indivi- duals. Intraventricularly applied LHRH yielded a similar result. These results indicate that LHRH may act directly on the brain and enhance its electrical activity. 1080 Endocrinology EN 18 HYPOTHALAMIC LHRH CONTENT FOLLOWING NOR- EPINEPHRINE INFUSION INTO CEREBRAL VENTRI- CLE IN aI GINA REIL Se ESTROGENIZED RATS. . Aihara’, S. Hayashi“ and K.Wakabaygshi i Dept.Biol., Toho Univ., Funabashi, Dept. Anat/Embryol., Tokyo Met.Inst.Neurosci., Fuchu & ~Inst.Endocr.,Gunma Univ.,Maebashi. Administration of 10 pg estradiol benzo- ate (EB) for 10 consecutive days to newborn female rats of the Sprague-Dawley strain induced extreme reduction in gonadotropic activity in adulthood. However, the hypo- thalamic LHRH content of these rats was higher than that in intact cycling rats. In this study, we examined the effects of intracerebroventricular (ICV) infusion of norepinephrine (NE) on hypothalamic LHRH content. Animals were ovariectomized and received ICV cannulation at 70 days of age. They received subcutaneous injection of 30 pg EB 2 weeks later. Two days later, they were killed 6 min after ICV infusion of 100 pg NE. The hypothalamus was frozen immedi- ately. The LHRH content in the frontal slices of 400 pm thickness was measured by RIA. Two clear peaks in the rostro-caudal LHRH distribution were detected which were divided at the suprachiasmatic nucleus region. In neonatally non-treated rats, the caudal peak was significantly reduced by ICV infusion of NE. In contrast, in neona- tally estrogenized rats, no significant decrease in the peak value of LHRH was observed. These results indicate that neo- natal administration of EB affected the hypothalamic noradrenergic system which is intimately related to LHRH secretion. EN 19 CHARACTERIZATION OF LUTEINIZING HORMONE BINDING INHIBITOR(S) SECRETED FROM CULTURED MOUSE SERTOLI CELLS. M. Takase, K. Tsutsui and S. Kawashima. Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima. We have found and previously reported the secretion of some LH binding inhibi- tor(s) (LH-BI) from mouse Sertoli cells in culture. In order to assess basic features of LH-BI, Sertoli cell-cultured media (SM) were subjected to heating at 100°C for 20 Min, wands ineubation iwalchmn Ey psi (22> mg/ml) sowmnchazcoa ly @20smei/ml) Pee Aste ryneat— ing and trypsinization of SM 1.8-fold and 1.3-fold increases in LH binding, respec- tively, were observed in Leydig cells cul- tured in intact non-treated SM, but the in- creases were not statistically significant. Charcoal treatment of SM induced 4.4-fold increase of LH binding as compared to iNeace SM Gs < O.05)). In order to know whether mouse Sertoli cells in culture secrete LHRH or E2, RIA for LHRH and E?2 in SM was performed. LHRH was undetectable «4.46 pg/ml original. medium) in acetic acid-ethanol extracts from lyophilized SM. Ether extract from SM contained 66.8 pg E2 per ml original medium. These results may suggest that the possible candidate for LH=BI may be steroidal substance; e.g: E2), rather than proteinic substance. EN 20 EFFECTS OF COMPLETE SUPRACHIASMATIC DEAFFERENTATION ON THE POSITIVE FEEDBACK RESPONSE TO ESTROGEN AND PROGESTERONE ON LH RELEASE. K. Nomura, S. Takahashi and S. Kawashima. ZOOM, Insit.) Fac. of Sei. . Hiroshima Umaaver Hiroshima. Estrous cycle and ovulation were pre- vented by complete deafferentation (CSD) of the suprachiasmatic nucleus (SCN) in female rats. In this study, positive feedback ef- fects of estradiol benzoate (EB) or proges- terone (P) on LH release were examined in Ovariectomized CSD rats. In control rats, LH surge was induced by EB or P following EB treatments. In CSD rats, EB failed to rise LH levels. Significant elevation in LH level occurred in CSD rats by P following EB, but the amplitude was’ less than that observed in the controls. However, P-induced LH release was abolished when the lesions by CSD extended further anteriorly to the medial preoptic nucleus. In control rats, LHRH content was significantly greater be- fore EB-induced LH surge than that during the surge in both preoptic-anterior hypo- thalamic (POA-AH) and medial basal hypo- thalamic) (MBH) regions). in) 1¢SD) cats tals difference was not observed, and their LHRH content of POA-AH was greater, but that of MBH was less than those of the controls. These *cesullts) "supeesit that themes Chmarns involved in the regulation of EB-induced LH release rather than P-induced LH release, and in the stimulation of LHRH neurons to release LHRH. EN 21 AGE-RELATED CHANGES IN GROWTH HORMONE BINDING TO THE LIVER OF FEMALE RATS S. Takahashil, S. Kawashima’, J.Meites?. LZool., Insit., ) Baca. of) Scileneebangorsiintme Univ., 2 Dep. of Physiol., Michigan State Univ. , East Lansing, USA. Growth hormone (GH) secretion decreases with aging in male and female rats. Liver is one of the target organs of GH and secretes somatomedin-C (SM-C) in response to GH. Age-related changes in binding of '257_bhovine GH to liver membrane fractions were measured in female Long-Evans rats at PAR 6, 12, and 20 months of age. Specific GH binding was not different between 2 and 6 months of age but it increased signifi- cantly at 12 and 20 months of age. Scatchard analyses showed that the plots were curvilinear and consisted of high- and low-affinity binding sites. The age- related increase in binding sites was mainly due to an increase in the number of low-affinity binding sites. Serum levels of SM-C in 20-month-old rats were about half of those in 6-month-old rats. Twice daily injections of ovine GH (2 mg/kg body weight) for 7 days depressed liver GH binding and increased serum SM-C levels in 19-month-old female rats, but had no effect on GH binding in 2-month-old female rats. These results suggest that the increase in liver GH binding sites and the decrease in SM-C secretion were associated with the decrease in GH secretion in old female rats. Endocrinology 1081 EN 22 THE ROLE OF INTERNALIZATION ON DOWN-REGULA- TION OF FSH RECEPTORS. A. Shimizu, K. Tsutsui and S. Kawashima. ZOO EnSse., Fac. Of Seis, Hiroshima Univ... Hiroshima. The role of internalization of FSH-recep- tor complexes on down-regulation of FSH re- ceptors was investigated in cultured Serto- li cells of C57BL/6NCrj mice. NaN3 was used as an inhibitor of internalization. Sertoli cells were cultured for 8 days or 4 day- plus-4 day combinations in either the fol- lowing media 1) F12/DME (control group), 2) F12/DME, then F12/DME containing 5pg/ml FSH (down-regulated group), 3) F12/DME then F12/DME containing 5yg/ml FSH and 10-2 107-4 or 1073M NaN3, 4) F12/DME containing FSH and NaN3, then F12/DME containing FSH. In all cultures, surface, internalized and degraded radioactivities were measured. The Eaeo cOnscant Of internalization (kj) in control and down-regulated groups was esti- mated. It was shown that surface and inter- nalized radioactivities were decreased by FSH and the decrease was suppressed by co- emrseence Siok NaN3. En contrast, degraded radioactivity was increased by FSH and this increase was also suppressed by NaN3. In addition, k; of down-regulated group was Significantly larger than that of control group (6.94x10-2 vs. 4.11x1072min-1. re- spectively). These results indicate that down-regulation of FSH receptors may be due to the translocation of surface receptors into the cells and asa result the number of available surface receptors is decreased. EN 23 PROPERTIES OF TESTICULAR FSH RECEPTORS OF SHORT-TAILED BANDICOOT RAT,NESOKIA INDICA. K. Tsutsui!, S. Kawashimal, R. Kapania2 , V. Kumar’ and R. N. Saxena “¢. lZool. Misia we ACe Ores SC. , Hiroshima Uaivacu, Hiroshima and DSIES OI ACGOlo, Winl7>o Cie Delhi, India. Basic properties of testicular FSH re- ceptors of short-tailed bandicoot rat, Nesokia indica were characterized in the present study. Specific FSH binding was detectable only in the testis among vari- ous organs, and it reached an equilibrium aster 2 hrvot incubation at: 35 °C... The’ FSH binding was inhibited by mammalian FSH preparations as a function of the concen- tration, but not by highly purified rat LH and PRL. Scatchard plot analyses of FSH binding to the testicular preparations of Nesokia indica showed straight lines, sug- gesting the presence of a single class of binding sites. The dissociation constants (Kds) for FSH of adults of Nesokia indica were 1.416 ( 0.964 - 2.667, 95% confidence interval ) nM in December, 1986 and 1.348 (OB865—277849))) nM im April, 1987. In con- trast, Kds of the albino rat were 0.459 ( 0.357 - 0.641 ) nM at 19 days of age and 0.444 (0.308-0.806) nM at 3 months. These results showed that the specific FSH bind- ing sites were present in the testis of Nesokia indica but the affinity of FSH binding in Nesokia indica was lower than that in the albino rat. EN 24 STIMULATION OF GROWTH OF THE NUCLEAR VOLUME AND NEURONAL PROCESS LENGTH BY ESTRADIOL-178 IN RAT HYPOTHALAMIC AND LIMBIC NUCLEL. M. Uchibori and S. Kawashima*. Suzugamine Women's College and *Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. We have studied the morphometric effects of estradiol-17B8 (E27) on some E2 concentra- ting neural substrates in rats castrated on the day of birth. They were given 10 pg E9 daily for 10 postnatal days and killed on Days 11 and 31. The results were as follows. (1) The ventromedial nucleus(VMN): In males E9 significantly stimulated the growth of nuclear volume on Day 11 but not on Day 31. In females, it significantly increased the volume both.on Days 11 and 31. The total neuronal process length of E2-treated rats was significantly greater than that of con- trols on Days 11 and 31 in males, and on Day 31 in females. (2) The stria terminalis (ST): Stimulation of the growth of nuclear volume by E2 was observed only on Day 31 in males. E2 significantly increased the total neuronal process’ length on Days 11 and 31 in males, but not in females. (3) The su- prachiasmatic nucleus (SCN): The nuclear volume was not significantly different be- tween E2-treated and control rats on Days ll and 31 in both sexes. The total neuronal process length in the SCN was significantly greater in E9-treated. rats of both sexes than controls on Day 31, but not on Day 1l. Thus, the effects of E29 on these morpho logical parameters were evident in the VMN but not so marked in the ST and SCN. EN 25 EFFECTS OF ESTRADIOL-178 ON THE SURVIVAL OF NEURONS FROM NEONATAL RAT HYPOTHALAMUS- PREOPTIC AREA IN PRIMARY CULTURE. K. Takagi!, H. Shimizul, M. Uchibori2, and S. Kawashimal . 1Z00). IMSte, , HAC, OF Sei, lablie@Slostne Winstw and “Suzugamine Women's Coll., Hiroshima. Dissociated cells of neonatal rat hypo- thalamus-preoptic area were grown in pri- mary culture, and effects of estradiol- 17B8(E5) on the survival of neurons were studied. Cells were cultured in culture media with or without E (100 ng/ml), and the number of viable neurons were counted ats ande 9) daysmornculktunens Ene msUtsvsvic al of meurons tended to be greater in Ej9-added cultures than control cultures at all culture ages and in all triplicate observations. In Second series of expri- ments, cytosine-8-p-arabinofuranoside (AraC, 10-5 M/ml) was added to the culture media from 3 days t0\6 days in culture with . or without E92, in order to inhibit the non- neuronal cell proliferation. AraC is known to inhibit mitosis. The survival of neurons was considerably. increased by AraC treat- ment; and effects of E59 cultured with AraC were likewise observed as in cultures without AraC. These results suggest that E» has some stimulatory influence on the neuronal sur- vival, and that the influence is maintained when the non-neuronal cell proliferation is inhibited with apparent elevation of the basal level of the survival. 1082 Endocrinology EN 26 BEHAVIOR OF ARGININE VASOTOCIN-PRODUCING NEURONS OF THE MEDAKA Oryzias latipes IN DIFFERENT ENVIRONMENTAL SALINITY. K.Haruta, T. Yamashita and S.Kawashima. Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima. A wild population of the medaka collected in a pond in Hiroshima City was used. Im- munoreactive arginine vasotocin(AVT) neu- rons and axons were identified by their GeaGeLvity) | GO) anita agence = walsioprelsisnn serum which was proved to crossreact with AVT. Immunoreactive AVT neurons were present in the nucleus preopticus of the hypothalamus. The nucleus can roughly be divided into pars magnocellularis and pars parvocellularis. The number of magnocellu- lar AVT neurons showed an instantaneous changes after transfer from fresh water(FW) to sea water(SW) or from SW to FW. when FW-adapted fish were transferred to SW, The number of these neurons increased 30 minutes after the transfer. Then, the num- ber decreased with the lapse of time. Radi- oimmunoassay of AVT showed that AVT content in the pituitary decreased two hours after the transfer to SW but increased after that approaching the initial control level. On the contrary, when SW-adapted fish were transferred to FW. The number of AVT neu- rons increased one day after the transfer, but it then decreased reaching the level of FW-adapted fish after one week. The present results suggest that AVT is one of the important hormonal factors involved in the osmoregulation of the medaka. EN 2/7 SYNAPTIC PATTERN OF THE MEDIAL PREOPTIC NUCLEUS OF RATS. M.Miyakawa and Y.Arai. Dept. Anat. Juntendo Univ. Sch. Med., Tokyo. Medial preoptic nucleus(MPN) is Jlo- cated along the ventricular wall of the anteroventral part of the preoptic area. The important role in cyclic ovulation and the presence of abundant estrogen receptors have been reported on the MPN. This nucleus is sexually dimorphic being larger in the female than in the male. In the present study, synaptic pattern of the MPN was examined in male, female and neonatally androgen-treated female rats at 780 days of age. The numbers of axo- somatic Synapses, axodendritic shaft syn- apses and axodendritic spine synapses were counted differentially in 9,909 sq.um area of the identical part in each animal. The number of axosomatic syn- apses was small and almost the same in all groups. The numbers of both axo- dendritic shaft and spine synapses were significantly larger in male rats than in females. The synaptic pattern of females treated with 5@yg testosterone propionate for the first 5 days of life was not different from that of intact females. Synapses containing large granular vesi- cles showed Similar pattern of sexual dimorphism. Further analysis is neces- sary to clarify the effect of sex steroid on the differentiation of synaptic pat- tern of the MPN. EN 28 NEUROGENESIS IN THE MEDIAL PREOPTIC NUCLEUS OF THE RAT: BROMODEOXYURIDINE (BRDU)-ANTIBRDU MONOCLONAL ANTIBODY METHOD. M. Nishizuka and Y. Arai, Dept. Anat., Juntendo Univ. Sch. Med., Tokyo. 37.5%). Combined pretreatment with pentobarbitar and metoclopramide showed no anterior pituitary hemorrhage in all mice. While bromocriptine(a dopamine D2 agonist, 5ug/g B.W.) stimulated the incidence of pituitary bleeding 16 out of 20(80%) at a low dose(0.02m1l/g B.W.) that occurred pituitary bleeding only 1 out of 20(5%). Endocrinology 1083 EN 30 AGE-ASSOCIATED CHANGES IN THE DISTRIBUTION OF CYTOCHROME P-450PB IN THE RAT LIVER. T. Machida!, S. Kawashima! , and C. F. A. VanuBezootgen’. ‘Zool, Inst.4 Fac. of Seis’, Hiroshima Univ., Japan and TNO Inst. of Gerontol., The Netherlands. Cytochrome P—-450°°(P=450): iis a family of heme proteins that serve as the terminal oxidase in a mixed-function-oxidase system. Several forms of P-450 isozymes are known Gommspecteneal ly) induced in’ the. liver by the treatment of animals with specific drugs. In the present experiments, distri- bution of a P-450 isozyme induced by pheno- barbital (PB) was immunohistochemically investigated in the liver of the male rats of different ages. In the liver of 3-month- old rats, an intense staining against anti- P-450PB antiserum was invariably observed in the hepatocytes of the perivenous (PV) Zone, whale “cells! in’ the periportal. (PP) zone reacted only slightly to the antise- rum. At this age, difference in the distri- bution of P-450PB between PV and PP cells was statistically significant in all the four lobes of the liver, although the pre- dominant localization of P-450PB in the PV cells was most conspicuous in the caudal liebe. | Prominent localization of P-450PB in the PV hepatocytes became less obvious at 18 months of age and came to be almost undetectable at 30 months. The result dem- onstrated the age-related decline of the heterogeneously distributed P-450PB_ in the liver lobules of male rats. EN 31 EFFECTS OF ABDOMINAL VAGOTOMY ON THE HYPO- THALAMIC AND PITUITARY FUNCTION IN MALE MICE. T.S. Nakazawa, T. Machida and S. Kawashima. Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima. Possible involvement of vagus nerve in the control of hypothalamic and pituitary function in male mice was examined. Male mice of the CD-1 strain were cas- trated and/or abdominal vagotomized at 2 or 8 months of age, and changes in the size of neuronal nuclei in the hypothalamus- preoptic area and plasma levels of gonado- tropins were investigated one week after operation. Bilateral castration or removal of left testis caused a significant reduc- tion in the size of neuronal nuclei of the POA, while removal of right testis failed to affect the size of neuronal nuclei. On the other hand, vagotomy induced a signifi- cant enlargement of neuronal nuclear size of the POA, the VMN and the ARC. Neuronal nuclei of these regions in vagotomized mice exhibited a consistent decrement in their sizes following castration, although vagot- omy-induced enlargement of nuclear size still persisted after castration. Signifi- cant changes were not induced by vagotomy in plasma levels of LH and FSH. The results indicate that the vagus nerve is not modu- lating the effect of castration per se but may have some other intrinsic effects on the hypothalamic control of pituitary func- tion in male mice. EN 32 THE EFFECT OF AN AROMATASE- AND A 5a- REDUCTASE-INHIBITOR UPON THE OCCURRENCE OF POLYOVULAR FOLLICLES, PERSISTENT ANOVULATION, AND PERMANENT VAGINAL STRATIFICATION IN MICE TREATED NEONATALLY WITH TESTOSTERONE. T. Iguchi, R. Todoroki, N. Takasugi and V. Petrow*. Dept. Biol. Yokohama City Univ. Yokohama and *Dept. Physiol. Duke Univ. Med. €tr. North Carolina, U.S.A. Female C57BL/Tw mice were given 5 daily injections of 20ug testosterone (T), 100ng 4-hydroxy-4-androstene-3,17-dione (4-HA), 100ug 6-methylene-4-pregnene-3,20-dione (6-MP), 4-HA+6-MP, T+4-HA, T+6-MP and T+ 4-HA+6-MP starting on the day of birth. The incidence of polyovular follicles (PF) at 30 days was significantly increased by neonatal treatment with T. By contrast, the PF incidence was significantly reduced by injections of 4-HA with T. Neonatally T- or T+6-MP-treated 90-day-old mice had Ovaries without corpora lutea. By contrast, T+4-HA (64%)- and T+4-HA+6-MP (82%)-treated mice had ovaries with corpora lutea. There were no significant differences in the vaginal epithelium of T-treated mice as compared with those of T+4-HA-, T+6-MP- or T+4-HA+6-MP-treated mice. They showed vaginal stratification. The present results indicate that 1) development of PF and persistent anovulation are due to the direct action of estrogen (E) derived from T; 2) T itself can induce ovary-independent vaginal changes, although 5a-reduced androgen and E derived from T seem to be more effective in this regard. EN 33 OCCURRENCE OF HERNIA AND PERMANENT CARTILAGINIFICATION OF PELVIS IN MICE TREATED NEONATALLY WITH TAMOXIFEN S. Irisawa, T. Iguchi and N. Takasugi. Dept.Biol. Yokohama City Univ. Yokohama. Male and female C57BL/Tw mice were given 5 daily injections of 100 ug tamoxifen (Tx) starting on the day of Ijiiceia, BS, 5p 7p Oe LO Cavs oO age. Isilelcleis hernia with or without caecum hernia occured in mice given Tx starting within 5 days. The incidence of hernia in female (100%) and male (50%) mice reached plateau at 90 days. Pubic symphysis in Tx mice was significantly longer than that of the controls. Permanent inhibition of pelvic ossification was found in all Tx mice. Development of pelvis was studied in mice of 18-day-fetus to 540 days, using X-rays and differential staining of cartilage and bone. In control mice, ilium was ossified even in 18-day-fetus. But pubis and ischium showed cartilaginous state until at 30 days. After 30 days, they joined together to form a ossified pelvis. In contrast, ossification of the junctional area of pubis and ischium was permanently inhibited in Tx mice. Permanent cartilagi- nification of pelvis was found in all mice given Tx starting within 10 days of age, but not in mice given Tx after 30 days. Neonatal treatments of mice with clomiphene and nafoxidine induced neither cartilaginification of pelvis nor hernia. These results suggest that inhibition of pelvic ossification is Tx specific effect. 1084 Endocrinology EN 34 CORRELATION BETWEEN PLASMA LEVELS OF TWO GONADOTROPINS AND SEX STEROIDS IN TOADS. M.Itoh, M.Inouve, and S.Ishii. Dept. of Biol., Sch. of Edu., Waseda Univ. Tokyo. We previously reported that plasma go- nadotropin (GTH) levels of Bufo japonicus showed distinct annual changes. Regression analysis revealed that LH had positive and significant correlation with total androgen in males (r=0.95) and with estradiol (E2) in females (r=0.79). We performed more de- tailed survey of plasma GTH and sex steroid levels around the breeding period. Blood samples were collected from toads resting under the ground, migrating to a pond, mov- ing around the pond, swimming in the pond and moving on the ground after breeding. In males, GTHS increased suddenly when they came near the pond. Testosterone (T) and 5a-dihidrotestosterone (DHT) started to increase in advance to the rise of GTHs. Low (r=0.27-0.36) but significant correla- tions were obtained between FSH and T, and two GTHS and DHT. In females, changes of GTHS were similar to those in males. Pro- gesterone (P) showed a rapid increase in paralled with GTHs when toad approached the pond . A rise of E2 preceeded surges of GTHs and P. Correlation analyses revealed that GTHs were positively correlalated with P (r=0.70 and 0.76) but negatively with E2 (r=-0.45 and -0.44). Thus, we could con- firm in the field observation that egg maturation and ovulation are induced by P under the influence of GTH in amphibians. EN 35 PURIFICATION OF GONADOTROPINS OF THE CYNOMOLGOUS MONKEY, MACACA FASICULARIS 1H. Ando, 1S. Ishii and 2T. Yoshida 1 Dept. of Biol. Waseda Univ., Tokyo 160. and 2Tsukuba Primate Center for Medical Science N.I.H., Ibaraki 305. FSH and LH were purified from acetone dried powder of cynomolgous monkey pitui- tary glands by the ammonium sulfate precipitation method and the successive column chromatographies of the Pharmacia FPLC system with Phenyl-Superose, Superose 12 and Mono Q (for FSH) or Mono S (for LH). Gonadotropic activities were monitored by the following two radioreceptor assay systems : one, for FSH activity, using rat testis receptors and radioiodinated rat FSH, and the other, for LH activity, using rat Leydig cell receptors and radioiodi- nated human LH. FSH and LH activities were completely separated into different fractions by the Phenyl-Superose chromato- graphy. Final preparations after Mono Q or Mono S were highly active, as increases in the specific activity from the starting material were 720-fold and 410-fold for FSH and LH, respectively. The specific activi- ties of FSH and LH were 1.9-fold and 4.6- fold higher, respectively, than those respective hormones of rhesus monkey (Yamji et al., 1973; Monroe et al., 1970). By the SDS-PAGE, the final FSH and LH preparations migrated as a diffused band at the position of M.W. 22000. The subunits of FSH and LH seem to have a similar molecular weight. EN 36 BINDING ACTIVITIES OF LUTEINIZING HORMONE- ISOHORMONES IN CYNOMOLGUS MONKEY PITUITA- RIES 3 T.Yoshida!, K.Ohtoh? and F.Cho!. ! Tsukuba Primate Center for Medical Science, N.1I.H. and “The Corporation for Production and Research of Laboratory Primates, Ibaragi. Competitive binding activities of lutei- nizing hormone(LH)-isohormones were studied by the radioreceptor assay using rat Leydig cell fractions as the receptor preparation. Male cynomolgus monkey(Macaca fascicularis) pituitaries were fractionated by isoelec- trofocusing technique. Seven LH-isohor- mones\(pE=(5 Ol 7-55 Sa Ol, Cle Gly Olen Wl OOlns and 11.5) were obtained. The competitive binding activity of each isohormone was compared with that of hCG by the radio- receptor assay using labeled hCG as the radioligand in the presence or absence of 150mM NaCl. In the absence of NaCl, the binding-inhibition curves of hCG and neutral isohormones yielded similar slopes, while alkaline and highly alkaline isohor-=- mones yielded steeper slopes than those of hCG and neutral isohormones. In the presence of NaCl, the binding rates of iso- hormones except one species were consider- ably diminished, but binding rates of hCG and highly alkaline isohormone(pI=11.5) were not affected. From these results, we Suggest the difference of functional states of the hormone-receptor complex between highly alkaline LH-isohormone as well as hCG and other LH-isohormones. EN 3/7 INDUCTION OF GONADOTROPIN SURGE BY STEROID HORMONE IMPLANTATION IN OVARIECTOMIZED GOLDFISH M. Kobayashi, K. Aida, and I. Hanyu. Dept. of Fisheries, Univ. of Tokyo, Tokyo. Female goldfish show a GtH surge at the time of ovulation. The occurrence of this ovulatory GtH surge is considered to be mainly controlled by environmental and physiological cues. The present study examined the involvement of some steroid hormones in relation to the occurrence of this GtH surge. Female goldfish were ovariectomized and testosterone, estradiol, or both steroids were implanted by silastic tubing. As a control, empty tubing was implanted. These fish were kept below 12°C for three months after the implantation, and then the water temperature was raised to 20°C, which is the same method for induction of Spontaneous ovulation in goldfish. After the water temperature was raised, some of the steroid implanted fish, especially in the testosterone implanted group, showed a GtH surge which was quite similar to that of the normal ovulatory GtH surge. Control fish showed no marked change in the plasma GtH levels. These results indicate that the ovarian steroids are one of the important physilogical cues for the GtH surge in goldfish. Endocrinology 1085 EN 38 TWO-CELL TYPE MODEL OF FOLLICULAR ESTRADIOL-178 PRODUCTION IN THE SALMONIDS Y. Nagahama and S. Adachi Babe oL Reprod. Biol., Natl. iInst.. for Basic Biol., Okazaki. Our earlier studies based mainly on the use of in vitro incubation experiments using isolated thecal and granulosa cells of vitellogenic amago salmon, Oncorhynchus rhodurus, led to the proposal of a two-cell type model for the follicular production of estradiol-178. In this model, the thecal cell, under the influence of gonadotropin, secretes aromatizable androgens, mainly testosterone, which are converted to estradiol-178 by granulosa cells. In the present study we have found evidence which indicates that this two-cell type model is applicable to the production of follicular estradiol-178 in four other species of salmonids, the chum salmon (O. masou), the masu salmon (0. masou), the rainbow trout (Salmo gairdneri) and the white-spotted char (Salvelinus leucomaenis). We have also found that the thecal cell from amago salmon and the granulosa cell from rainbow trout could produce the same effect as has been observed using combinations of thecal and granulosa cells from the same species. The reciprocal use of amago salmon granulosa cells and rainbow trout thecal cells is also effective. These findings imply that there may be little species specificity of each of these cells among salmonids. EN 39 ISOLATION OF ALDOSTERONE-RELEASING SUBSTANCE FROM THE BULLFROG ADENOHYPOPHYSES. S.Iwamuro and S.Kikuyama. Dept. Biol., Sch. of Educ., Waseda Univ., Tokyo. We have previously observed that the bullfrog anterior hypophysis contain principle(s) which enhances plasma aldosterone levels in hypophysectomized Xenopus. Attempt was made to isolate the active substance(s) from the bullfrog adenohypophyses by extraction with acid-aceton and separation by C18 SEP-PAK cartridge, Sephadex G-50 column and RP-HPLC. The 4-5 KD fraction obtained by gel filtration chromatography showed a prominent bioactivity. When this fraction was subjected to RP-HPLC as the final step of purification, an active principle emerged as a main peak at a concentration of approximately 50% AcCN. It has a molecular weight of 4,400 as determined by SDS-PAGE. The isoelectric point is 7.9 as determined by analytical electrofocusing. This substance is as potent as porcine ACTH in promoting aldosterone release from the adrenal glands of hypophysectmized Xenopus juveniles. However, the amino acid composition does not resemble that of ACTHS ever known, since it lacks Arg and Tyr which are invariably contained in the amino acid residues of 1-24 ACTH. EN 40 ISOLATION OF NEWT PROLACTIN K. Matsuda, K. Yamamoto and S. Kikuyama. Dept. “GF” Biol., “Seh. “ot Educ-, * Waseda Univ., Tokyo. Attempt was made to isolate prolactin(PRL) from the pituitary gland of the newt, Cynops pyrrhogaster Ppyrrhogaster. PRL- containing fraction was Obtained from the acetone-dried pituitary powder by extraction with acid acetone. Then, the extract was subjected to polyacrylamide gel electrophoresis. PRL band was identified immunologically by Western blotting method using antiserum against bullfrog PRL. PRL was eluted from the fast-moving protein band which was identified as PRL, using an electroeluter. The eluate had a marked bioactivity to stimulate collagen synthesis in the tail fin of bullfrog tadpoles. The PRL fraction thus obtained was subjected to reversed phase HPLC. PRL emerged as the main peak at a concentration of approxi- mately 70% acetonitrile. It gave a single band on SDS-polyacrylamide gel electro- phoresis and had a molecular weight of 23,700. The isoelectric point was 4.3 when determined by electrofocusing. EN 41 CHANGES IN HORMONE LEVELS IN TOAD TADPOLES DURING METAMORPHOSIS K. Niinume’, K. Yamamoto, N. Mamiya, S. Kikuyamal, M. Tagawa’, T. Hirano’ Nept. of Biole.sseh. of Educ., Waseda Univ., Tokyo. ?7Ocean Res. Inst., Tokyo Univ., Tokyo. It has been known that thyroid hormone, aldosterone(ALDO) and prolactin(PRL) are involved in metamorphosis. These hormone levels were measured in Bufo japonicus formosus larvae by radioimmunoassay. Since they are of relatively small size, PRL and ALDO were assayed with pooled plasma samples. Thyroxine(Ts) and triiodothyronine (T3) were extracted from the individual body with methanol, recovery being about 60%. PRL levels elevate as metamorphosis progresses. However, the maximum concentration in toad tadpoles is about one twentieth of that of bullfrog tadpoles. ALDO levels elevate during prometamorphosis, reach maximum at mid-climax and decline at the end of metamorphosis. Both Ts and T3 concentrations elevate during prometamorphosis and reach maximum at climax. Relatively lower levels of PRL may account for the rapid metamorphosis in this species. 1086 Endocrinology EN 42 PLASMA PROLACTIN AND OSMOLALITY IN CRAB- EATING FROGS S. Kikuyama, K. Yamamoto, K. Matsuda, T. Ogasawarat*, S. Hasegawat* and T. Hirano* Dept..of |Bioll.; sch of bide. "5 Wasieda Univ., Tokyo. *Ocean Res. Inst., Tokyo Univ., Tokyo Ne ee Prolactin (PRL) levels in the plasma of marine frogs, Rana cancrivora kept in various concentrations of sea water (SW) were measured by radioimmunoassay using an- tiserum against bullfrog PRL. Osmolality of the plasma of frogs kept in fresh water (FW) was 339+10 mOsm/kg. When the animals were transferred into 50 and 75% SW, it elevated upto 556+2 and 7354+8 mOsm/kg, respectively, which are slightly above the values of environmental SW. It was con- firmed that the elevation of plasma os- molality in the SW-adapted frogs is largely due to the increase in the plasma urea con- centration. Average PRL concentration in the plasma of the FW-adapted frogs was 158+39 ng/ml. It declined to 56+10 and 29+8 ng/ml when put into 50 and 75% SW, respec- tively. Involvement of PRL in osmoregula- tion in this species is suggested. EN 43 PRIMARY STRUCTURE OF BULLFROG GROWTH HORMONE 5 T.Kobayashi!"? S.Kikuyama? A.Yasuda? H.Kawauchi! K.Yamaguchi $ H.Sano! 1)Dept. of Basic Human Sciences, Sch. of Human Sciences, Waseda Univ., Saitama. 2) Dept. Of Biol SChi. — OnmenGucr ma waseda Unaivas Tokyo. 3)Scen. of Fisheries, Kitasato Univ., Iwate. 4)Tokyo Res. Lab., Kyowa Hakko Co. Ltd., Tokyo Growth hormone(GH) was isolated from adenohypophyses of bullfrog, Rana catesbeiana, by acid-acetone extraction, anion-exchange chromatography and reverse-phase high performance liquid chromatography(rp-HPLC). Amino acid composition of bullfrog GH thus obtained resembles other vertebrate GHs, possessing 4 half-cystines. To determine the amino acid sequence of bullfrog GH, purified GH was reduced, carboxymethylated and subsequently cleaved with cyanogen bromide. GH was also digested with enzymes such as lysyl endopeptidase and trypsin. The resulting fragments were separated by rp-HPLC and subjected to sequence analysis by Edman method. Thus, about 140 amino acids sequence of bullfrog GH has been determined. The amino-terminal 40 amino acid residues show 53%, 70%, 50% and 30% sequence identity with human, chicken, eel and salmon, respectively. The carboxy-terminal 40 residues show 53%, 83%, 83%, and 63% sequence identity with human, chicken, eel and salmon, respectively. EN 44 ROLE OF INFUNDIBULAR PRIMORDIUM ON THE DIFFERENTIATION OF PITUITARY CELLS IN TOAD TADPOLES (BUFO JAPONICUS). K. Kawamura and S. Kikuyama, Dept. Biol., Schl. Educ., Waseda Univ., Tokyo 160. ——_—————————— eee Aim of the present study is to see whether the differentiation of pituitary cells is dependent on infundibular primordium. Anterior part of the neural plate ectoderm was surgically removed from open neurulae. In the operated animals, the melanin granules of dermal melanophore were permanently concentrated, showing no background response. These animals completed metamorphosis slightly later than sham-operated control. The brain regions were fixed and stained with specific antisera against adenohypophyseal hormones. In the experimental group, infundibulum was absent, and the epithelial hypophysis was located away from the normal site without morphological contact with the brain tissue. Neither MSH nor ACTH cells were detectable whereas PRL, GH and TSH cells were invariably present in the epithelial hypophysis. These results implicate an important role of the infundibular primordium on the differentiation of pituitary POMC cells. If these cells originate from the stomodeal ectoderm as is described in classical embryology, induction from the brain seems to be necessary for the differentiation of the cells. It is also conceivable that the pituitary POMC cells are derived from the neuroectoderm itself. EN 45 Immunocytochemical study of TSH cells in toad tadpoles during metamorphosis. S. Kurabuchi!, S.Tanaka® and S. Kikuyama?. 'Dept.of Histol., the Nippon Dental Univ., Tokyo. ?Dept. of Morphol., Inst. of Endo- crynol., Gunma Univ., Maebashi. 3Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. TSH producing cells in the pars dis- talis of Bufo japonicus formosus tadpoles at different metamorphic stages were iden- tified by peroxidase-anti-peroxidase meth- od using anti-human TSHB serum. The first sign of immunoreaction appeared in a very cells at stage 35, the reaction being very weak. At stage 40, immunoreactivity and number of TSH-positive cells suddenly in- creased and they were maintained through- out metamorphosis. In tadpoles deprived of thyroid primordium at external gill stage, immunoreactive TSH cells never appeared even after their hatch-mate completed metamorphosis. When thyroid- ectomized tadpoles were artificially meta- morphosed by thyroxine (Ts), considerable number of MTSH-positive cells with an intense immunoreactivity appeared in the pituitary gland. In the pituitary gland which had been autotransplanted to the tail region of the hypophysectomized tad- poles at tail-bud stage, TSH cellsS were abundant when examined at climax. These results suggest that thyroid hormone is necessary for the development of TSH cells. Endocrinology 1087 EN 46 STIMULATION OF DRINKING BY CAPTOPRIL, AN ANGIOTENSIN I -CONVERTING ENZYME INHIBI- TOR, IN THE GOLDFISH, CARASSIUS AURATUS. H. Kobayashi ! and Y. Okawara2, TRes. Lab., Zenyaku Kogyo Co. Ltd., Nerima, Tokyo and 2Dept. Pharmacol., School of Med., Univ. of Calgary, Calgary, Canada. Single intraperitoneal (ip) injections of angiotensin IL (ANG IT ) stimulated water intake for 60 min in a dose re- lated manner in the goldfish, Carassius auratus. Single ip injections of captopril (SQ14225), an angiotensin I -converting enzyme (ACE) inhibitor, at lower doses (0.4 and 4.0 ug/fish) stimulated water in- take for 60 min. This stimulation cannot be ascribed to an increase in levels of plasma ANG I , since ANG I[ (2.2 ypg/fish) did not stimulate drinking in the presence of S$Q14225 (two injections of 9.9 ng /fish). It is suggested that the elevated plasma ANG I concentrations achieved after blockade of ACE was converted into ANG [Il approximately 50 min after SQ14225 injections (4.0 pg/fish), when the in- jected S$Q14225 was effectively metabo- lized. Thus, the newly elevated level of ANG Il may have been responsible for the vigorous drinking. Higher doses of $Q14225 (40 and 200 ng/fish) did not af- fect the water intake for 60 min, indicat- ing that the rate of basal water intake is independent of the renin-angiotensin sys- tem in the goldfish. EN 47 ATRIAL NATRIURETIC PEPTIDE(ANP)-LIKE IMMUNOREACTIVITY OF, FISH HEARTS. H.Uemura’ , M.Naryse , T.Hirohama , S.Naka- mura! and T.Aoto!. !Biol. Lab., Kanagawa Dent. Coll., Yokosuka, “Dept. of Med., Tokyo Women's Coll., Tokyo. Hearts of the banded dogfish (Triakis scyllia), four species of freshwater (FW) teleosts (Cyprinus carpio, Anguilla japonica, Lepomis macrochirus, Channa maculata) ae three species of seawater(SW) teleosts (Hexagrammos otakii, Pagrus major, Trachurus japonicus) were studied ultra- Senuerural ty and immunohistochemically using antiserum against &-human ANP. Immunoreactive ANP(IR-ANP) in heart tissue and plasma was estimated by RIA. ANP-immunostained cardiocytes were found imeboObtmethenatria and ventricles of all species except Hexagrammos, being present in far greater number in the former than latter. In Triakis, Pagrus and Trachurus, very little stained material cou e detected in only a few cardiocytes. These three species and Hexagrammos had fewer electron-dense granules in cardiocytes and a much lesser amount of IR-ANP in the heart tissue compared to the other species. The plasma content of IR-ANP was about 31- 91 and 3-16 pg/ml in the FW and SW species, respectively. From these findings, it thus appears that the overall extent of synthe- sis and release of ANP-like material is greater in the FW species than SW species. EN 48 EFFECTS OF SALINITY ON ANP-LIKE SUBSTANCE AND ULTRASTRUCTURES IN EEL CARDIOCYTES. S. Nakamura, T. Hirohama, H. Uemura and T. Aoto. Biol., Lab., Kanagawa Dent. Coll., Yokosuka. _ Atrial cardiocytes were investigated immunohistochemically, using antiserum against d-human ANP, and ultrastructurally for (Anguilla japonica) adapted to either fresh water or artificial sea water. Dense ANP immunoreactive material (ir-ANP) and numerous secretory granules (about 200 nm) were found situated about the nucleus of cardiocytes in fish adapted to fresh water (control). In fish transferred from fresh water and adapted to sea water for 24 hr, the amount of ir-ANP and number of granules decreased somewhat whereas in fish kept 1n sea water for one week, they resum- ed initial values to some extent. Further- more, in fish transferred to fresh water for 24 hr after having adapted to sea water for one week, these values were recovered to essentially those of the control. From Ehesemnesultce lt ws) evident that the amounts of ir-ANP and number of granules in cardlocytes are closely related, that eel 1r-ANP 1s immunologically related to human ANP, and that ir-ANP in eels plays physio- logically important role in adaptation to change of environmental salinity. EN 49 ANU RIG/A Ly NV-MIB RIEU IEIEG, Pa eMCIEDB, (C/N) )) IL EIA PEPTIDE IN THE CARDIOCYTES OF DEVELOPING AND ADULT TOADS. T. Hirohama, S. Nakamura, H. Uemura and Ee LOEOs: BLO, Labo, Kanagawa Dems Coll. Yokosuka. The ontogeny of ANP-like immunoreactivity and ultrastructures of the cardiocytes in developing and adult toads (Bufo japanicus formosus) were studied, using antiserum against &-human ANP. The number of the electron-dense granules ( about 115 nm ) that could be observed in periphery of the cardiocytes was quite few and all atrial cardiocytes showed weak ANP-immunoreacti- vity in larvae as early as the limb-bud stage. The number of granules increases rapidly during metamorphosis, and could be seen frequently in the atrial cardiocytes of young toads with tail remnants. In adults, ranules of two different sizes ( about 115 nm and 200 nm’) could be seen in atrial cardiocytes. Stronger ANP-immuno- reactivity and a greaternumber of secretory granules were found in the right atrium compared to the left atrium of an adult heart. The numbers of granules in breeding (April) and non-breeding (August) animals did not differ significantly. Throughout the life, only a trace of immunoreactivity was detected in ventricular cardiocytes. These results indicate that atrial cardio- cytes of toads synthesize a peptide immuno- logically related to human ANP and which may be essential to land life. 1088 Endocrinology EN 50 IN VIVO CHARACTERIZATION OF ANGIOTENSIN II RECEPTORS AND CONVERTING ENZYMES IN THE QUAIL AND EEL. Y. Takei. Dept. Physiol., Kitasato Univ. Sch. Med., Sagamihara, Kanagawa 228 As an initial step for comparison of angi omens ns lit S(CANG i) ieee tions and converting enzymes among vertebrates, pressor potency of ANG I's and II's from the rat, fowl and eel, and effects of ANG II competitive inhibitors and converting enzyme tnihwiba toms) Ol pEessomer Feces ot angiotensins, were examined in the guail and eel. Among, all cangaeten si need inns examined, ANG II from the native class of vertebrate (fowl ANG II for quail and eel ANG II for eel) exhibited the greatest pressor potency in the respective species. MMoOme WWE Lil COMmppO@Ewedyve —WiMlllloiicors examined, only [Sar , T1e8} ANG II blocked pressor effects of ANG II in both species. Gimee all ANG NUS ancl Compe@edlierL Ve inhibitors have similar in the rat, ANG II receptors appear to be different in the alien CUAL Ame Gol, Among ally ANG 17s examined, ANG I from the native class again had greatest pressor potency in the quail and eel. Among converting enzyme inhibitors examined, bradykinin potentiator B and SQ14225 blocked the pressor effect of ANG I in the eel, and only SQ14225 was an effective inhibitor in the quail. Since all inhibitors examined are effective in the rat, converting enzymes also may be different in the rat, quail and eel. EN 51 INHIBITION OF HCG-INDUCED OOCYTE MATURA- TION BY PMSF IN INTACT FOLLICLES OF XENOPUS LAEVIS. K. Ikeo and K. Ishikawa, Biological insti- tute, Faculty of Science, Shizuoka Univer- sity, Shizuoka. Human chorionic gonadotropin (HCG) (50 IU per 2 ml medium) induces oocyte matura- tion [designated as germinal vesicle breakdown (GVBD)] in intact follicles of Xenopus laevis. The objective of the pre- sent study was to investigate protease in- volvement in regulating oocyte maturation induced by HCG. Experiments were carried out using intact follicles containing follicle cells and oocytes to assess the effect of phenyl methanesulfonylfluoride (PMSF) as an irre- versible inhibitor of serine protease. When intact follicles were previously ex- posed for 60 min at 21°C to PMSF, PMSF (0.7-1.4 mM) was effective in inhibiting HCG-induced GVBD. Intact follicles devel- oped an insensitivity to PMSF some 4 hours after HCG addition which preceded onset of GVBD by one hour. In addition, PMSF in- hibited to some degree progesterone pro- duction induced by HCG. In this study, it was suggested that serine proteases in intact follicles may play roles in mediating progesterone pro- duction and induction of GVBD by HCG. EN 52 DIFFERENTIAL ONTOGENIC APPEARANCE OF THE SUBUNITS OF GLYCOPROTEIN HORMONES (LH, FSH AND TSH) IN BULLFROG PITUITARY IN RELATION WITH HYPOTHALAMIC RELEASING HORMONES. S.TANAKA, Y.TANIGUCHI, M.K.PARK! AND K.KUROSUMI. Dept. of Morphol. and !Hormone Assay Center, Inst. of Endocrinol., Gunma Univ., Maebashi. The ontogeny of gonadotrophs and thyro- trophs in bullfrog pituitary was demon- strated by the immunocytochemical technique using anti-human TSH8& serum in addition to monoclonal antibodies (MCAs) against bull- frog LH8, FSH§ and their a-subunit. Immuno- reactive (IR) a-subunit and TSHB were ob- served before premetamorphosis (St.24), but IR-FSHB and LH8& were not. IR-FSH8& appeared earlier than IR-LH8. At TK St.XV a few gonadotrophs containing FSH and LH were found in ventrocaudal region. However, most gonadotrophs contained only FSH throughout metamorphosis. The number of IR-a-subunit cells was always higher than the total num- ber of IR-8-subunits cells. The ontogenic study suggested that hypothalamic TRH and LHRH may be related with the appearance of each subunit of glycoprotein hormones. At St.24, IR-TRH was first found in the pars nervosa, the primitive median eminence (ME) and some other parts of the brain. During prometamorphosis, a few TRH fibers termi- nated near some of the pars distalis cells, suggesting direct action of TRH upon these cells. IR-LHRH fibers in the ME were found from TK St.XIII onwards. IR-f-subunits of FSH and LH increased, as the number of IR- LHRH fibers increased (St.XV- XIX). EN 53 EFFECTS OF SERIALLY ADMINISTERED MDP AND URIDINE ON NOCTURNAL SLEEP IN RATS. M.Kimura, K.Honda, Y.Komoda and S.Inoué. IEIONSHE G Med. Dent. Eng., Tokyo Med. Dent. Winans 7 Wolo. The differential compound-dependent sleep- promoting activity is demonstrated by our 10-h intracerebroventricular (1S Chive) nocturnal infusion of muramyl dipeptide (MDP, 2.0 nmol) and uridine (10 pmol) in freely behaving rats. The combination- dependent sleep enhancement is also observed by the 10-h i.c.v. simultaneous infusion of MDP and uridine. A serial i.c.v. infusion of uridine (5 pmol/5h) and MDP (1.0 nmol/5h) resulted in a increase in both slow wave sleep (SWS) and paradoxical sleep (PS) which gradually appeared during the uridine-infusion period, and lasted for a few hours even after the termination of the MDP-infusion. The total amount of SWS and PS was Significantly larger than that of the baseline level. The time-course pattern of the sleep modulation was quite different from iliac alin = tein similar experiment in which uridine was replaced by saline. Thus, the pre-existing uridine modified the effect of MDP. In conclusion, the single, the simultaneous, and the sequencial administrations resulted in the different temporal changes in sleep-waking activity. Sleep seems to be regulated by an interaction of multiple sleep factors. Endocrinology 1089 EN 54 MECHANISMS OF DEVELOPMENT OF UTERINE ADENO- MYOSIS IN MICE T. Mori Zoological Institute, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113 Adenomyosis is an abnormal growth of glands and stroma into and beyond the smooth muscle layers of uterus. I have reported that ectopic pituitary isografting induces an early and high incidence of adenomyosis in mice associated with hyper- prolactinemia. The goal of the present study is to clarify the mechanisms of the development of this uterine lesion. The results showed that spontaneous de- velopment of adenomyosis was significantly suppressed by removal of ovaries between 4 and 10 weeks but not between 10 and 16 weeks of age. It was further found that temporary administration of bromocriptine, a potent suppressor of pituitary prolactin release, between 4 and 11 weeks of age only inhibited spontaneous development of adeno- myosis with age. These results suggest that there is a critical period during youth for the spontaneous development of adenomyosis at advanced age. Furthermore, the results showed some morphological dis- orders of uterus such as glandular hyper- plasia, disintegration of muscle cells, dilation of blood vessels, accumulation of collagen in the musculature on the develop- ment of experimentally induced-adenomyosis. The relevance of these histological findings to the hormonal milieu induced by ectopic pituitary grafting is discussed. EN 55 DIFFERENCES IN FOOD- AND WATER-INTAKES AND GLUCOSE TOLERANCE BETWEEN HIGH AND LOW MAMMARY TUMOR STRAINS OF MICE. S.Ozawa, T.Iguchi, N.Takasugi and H.Nagasawa*. Dept. Biol. Yokohama City Univ. Yokohama and *Dept. Agri. meiji Univ. Kawasaki. We have measured the differences in food- and water-intakes, urination, blood glucose level and glucose tolerance between high(SHN) and low(SLN) mammary tumor virus (MTV)-expressed strains and MTV-unexpressed strains (C57BL, BALB/c and ICR) of mice. Three-month-old male and female mice of the five strains were used for this present study. Food- and water-intakes in male and female SHN mice were significantly higher than those in the corresponding sexes of any strains of mice. Nevertheless, SHN mice were not larger in body weight than SLN mice. Urinary volume was significantly higher in SHN than in SLN strain in both sexes. Blood glucose levels were Significantly higher only in female SHN mice than in other strains. Glucose tolerance in male SHN mice was Significantly lower than in the other strains. These results suggest that SHN mice possess abnormal glucose metabolism. Previous studies demonstrated that there is a close relationship between diabetes and tumor growth. Therefore, it is strongly suggested that) the) hughes anevdence's) of Mammary tumors in SHN mice are ascribed partially, at least, to glucose metabolic abnormality, leading to a diabetic state. EN 56 MAMMARY TUMOR CAUSES HYPERGLYCEMIA IN MICE N.Nishimura, T.Iguchi, and N.Takasugi. Dept.Biol., Yokohama City Univ., Yokohama. ~ Blood glucose levels (BGL) were examined in 7-month-old female SHN mice with spon- taneous mammary tumor (MT). BGL in mice with MT were significantly higher than in mice without MT. BGL increased in propor- tion to the size of MT from 0 to 30 mm in diameter, but decreased when MT reached a diameter bigger than 30 mm. Serum insulin levels in mice with MT were significantly higher than in mice without MT. There were no differences in weights of pancreas and adrenals in mice between the presence and absence of MT. lLangerhans' islets having cells with pycnotic nuclei were found in 5 of 10 mice with MT. No difference was found in occupancy of A (10%) and B cell areas (80%) of normal islets between groups of mice with and without MT. Trans- plantation of MT into male SHN mice raised BGL; mice with MT grafts showed an increase in BGL in proportion to the size of MT grafts. The BGL increase was also found in 10-month-old C57BL female mice with MT grafts. No differences in weights of pancreas and adrenals were found be- tween groups of male SHN and female C57BL mice with and without MT. These findings indicate that 1) there is a correlation between occurrence of MT and hyper- glycemia. 2) blood glucose levels increase in pKROpertion to the size of MT. 3) ~serum insulin levels are markedly high despite the hyperglycemia in female SHN mice with MelvS EN 57 MULLERIAN- INHIBITING ACTIVITY IN CIRCULATION OF QUAIL EMBRYO A. Ohuchi and T. Noumura. Dept. Regul.Biol., Fac. Sci., Saitama Univ., Urawa. We have demonstrated that the Mullerian- inhibiting substance (MIS) in circulation can cause regression of the Miillerian ducts (Md) at a site distant from the testes in the chick embryo (Proce WapaneNcad-. OZR eZorp L980). alent, comparable experiments have been performed in the quail embryo. Two experimental approaches were designed in the quail embryos which were cultivated in the chicken egg shells: briefly, 2-day quail embryo and egg contents were transferred into a half of the chicken egg shell, sealed the opening with plastic wrap, and continued to incubate at 37.6°C. In the first series, two testes from 8-day embryo were grafted onto the chorioallantoic membrane (CAM) of 6-day female embryo. After four days, female host embryos showed signs of regression in their Mds. In the second series, Md from 7-day female embryo was grafted onto CAM of 8-day male embryo. After three days, only the Mds grafted onto male embryos showed clear signs of regression. These results suggest that the circulating levels of MIS in the quail embryo, as well as in the chick embryo, may be effective to cause regression of Mds. 1090 Endocrinology EN 58 IMMUNOCYTOCHEMICAL STUDIES ON THE PAN- CREATIC ENDOCRINE TISSUE IN SOME LOWER VERTEBRATES K.Miyata ; Y.Oota+ and M.Nozaki~. Biol, INSGo5 KECS Cit Chea eo haezwokal UML? 6 5 Shizuoka and Primate Res. Inst., Kyoto Univ., Inuyama. Immunocytochemically, the pancreatic endocrine tissue was studied to con- sider the phylogenetic aspects of cell types and topographical distribution of cells. in WAS joigFSSeing Swwchy, 7 S\eer1es of lower vertebrates including cyclo- stome (Eptatretus burgeri), elasmo- branchs Triakis scyllia), teleosts (Carassius auratus), amphibians (Cynops rrhogaster rrhogaster and Rana tagoi) and reptiles (ae s reevesii and Elaphe quadrivirgata) were exam-— ined. For immunocytochemical study, avidin-biotin peroxidase complex method was used. The following antisera obtained from rabbits were used : anti human glucagon, anti human insulin, anti mammalian somatostatin and anti porcin pancreatic polypeptide (PP). Cells containing PP-immunoreactivities are not revealed in all the animals tested. In Eptatretus "burgeri,” insu— lin- and somatostatin-immunoreactive cells are found. Although there are species differences’ in cell distrib-— Wissen, eho NSeisSie adwucse CEN iweyjoes Bure identified in the rest animals. EN 59 AN IMMUNOCYTOCHEMICAL STUDY ON THE HATSCHEK'S PIT OF THE AMPHIOXUS M. Nozakil, A. Gorbman2, and kK. Miyata3. lprimate Res. Inst., Kyoto Univ., Inuyama, 2Dept. of Zool., Univ. of Washington, Seattle, WA, USA, and 3Dept. of Biol., Shizuoka Univ., Shizuoka. Hatschek's pit of the amphioxus has long been postulated to be the ancestor of the vertebrate pituitary gland, with no con- vincing evidence. In this study, the Hatschek's pit of Branchiostoma belcheri was examined immunocytochemically with regards to a possible hormonal content. Antibodies to various hormonal peptides were used as immunocytochemical probes. Substance P- and Met-enkephalin-positive immunoreactions were observed in distinct cells of the margin parts of the pit, which correspond to the distribution of electron microscopically granulated cells. Faint immunoreactions to anti-human (h)-LHf and anti-h-CG were observed in the deepest part of the pit, but other antibodies to pitui- tary glycoprotein hormones or subunits gave no immunoreaction there (i.e., h-FSH8, h-TSHB, salmon (s)-GTH, s-TSH, carp-GTH). No immunoreactivity was obtained with anti- bodies to hypothalamic hormones (mammalian- LHRH, SRIH-14, AVP), pituitary single hor- mones (h-PRL, h-GH, porcine (p)-ACTH, a-MSH, S-PRL, S-GH), pancreatic hormones (h-insu- Inlin7 IN-CGIVUCACON, jIIDD, Sralmewiliin, Socllweas gon, S-SRIH-25), or brain-gut peptides (p- CCK, p-VIP, neurotensin). EN 60 THE EFFECT OF SALMON MELANOTROPIC HORMONES ON ISOLATED MEDAKA MELANOPHORES. S. Negishil I. Kawazoe?and H. Kawauchi+ Dept. of Biol., Keio Univ., Yokohama, and tLab. of Mol. Endocr., Sch. of Fish. Sci., Kitasato Univ., Sanriku, Iwate. The isolated medaka melanophores, which are light sensitive, are utilized to deter- mine MSH activity. Though the scale bio- assay of Oryzias are rather insensitive to MSH, the isolated melanophores bioassay, which respond to light with pigment disper- sion and dark with aggregation, are remark- ably sensitive to MSH. The dark-asgregated melanophores disperse by ad-MSH I, N-des-Ac- &-MSH I and B-MSH I isolated from chum salmon, despite of complete lack of illumi- nation. The minimum concentration which is required for melanosome dispersion was 10-'5 M &-MSH I, 107!3 M N-des-Ac-&-MSH I and 107!'' mM B-MSH I. Melanophores of most cold- blooded vertebrate, which are sensitive to MSH, respond to the hormone at 107§-107!9 m. Therefore, the isolated medaka melanophores are surprisingly sensitive to A@-MSH. Light sensitive medaka xanthophores also disperse by similar concentration of &-MSH, though the rate of pigment dispersion is 3 times slower than melanophores. Chum salmon en- dorphin I (EP) enhances Oryzias melanophore dispersing activity of B-MSH I about 40%, but does not cause pigment dispersion by itself. The mechanism of the enhancement by EP has been unclear. EP may possibly change the affinity of Q@-MSH to receptors on melanophores. EN 61 EFFECTS OF AGING ON URINARY AND SERUM PARA- METERS FOR RENAL FUNCTION IN THE WISTAR/TW MALE RATS:I. 3- TO 13-MONTHS-OLD. Y. Kobayashi, M. Senoo* and K. Hattori. Dept. of Pharmacol. and *Cent. Res. Lab., Shimane Med. Univ., Izumo. The Wistar/Tw strain male rats_ show polydipsia and polyuria after 16 months of age. Morphological changes in renal glomeruli have reported from 3-months-old males. In the present study, urinary and serum parameters for renal function such as urinary volume, urinary protein concentra- tion, urinary and serum creatinine concen- tration were measured from 3 to 13 months of age. For comparison, the Wistar strain males were used. Urinary volume was smaller in the Wistar/Tw strain through the period and it was significant at 6 months of age. Urinary protein concentration was almost 2- fold in the Wistar/Tw strain through the period and daily excretion of urinary pro- tein was significantly higher at 6 months of age, suggesting the early occurrence of renal failure in the Wistar/Tw strain. Uri- nary creatinine concentration was not dif- ferent. Serum creatinine concentration was significantly lower in the Wistar/Tw strain at 4-months-old. Then, relative glomerular filtration rate was significantly lower in the Wistar/Tw strain at 4 months of age, suggesting the dysfunction of glomeruli of the Wistar/Tw males in this period. Serum total protein concentration, blood urea nitrogen value, serum albumin concentration were not different between the strains. Endocrinology 1091 EN 62 CHANGES OF ANDROGEN SECRETION AND HYPER- TROPHY OF LEYDIG CELLS AFTER HEMICASTRATION IN MALE RATS. T.Furuya, K.Miyoshi and M.Hokano. Dept. of Anatomy, Tokyo Medical College, Tokyo. The hormonal and testicular histological effects of hemicastration were examined using rodent models. Wistar strain rats were hemicastrated at 5, 10, 15, 20, 30 and 40 days of age, and autopsied 30 days after Surgery. Hypertrophy decreased as the age at hemicastration approached 15 days and did not occur in rats operated on 20 days of age or older. Serum gonadotropins, plasma testosterone and DHT were measured by RIA. A significant increase in FSH level was noted in the rats hemicastrated at 5, 10, 20 and 40 days when compared to the normal rats, but no difference in LH level was detected. A higher concentration of plasma testosterone was detected in 50 day-old control rats than in hemicastrated rats. In contrast, 70 day-old hemicast- rated rats showed plasma testosterone levels higher than in control rats. Plasma DHT in 60 and 70 day-old hemicastrated rats was higher than in control rats. Leydig cells in 70 day-old hemicastrated rats showed more active morphological features than in control rats. The size of Leydig cells, nuclei and mitochondria were larger and sER were more prominent than those in intact rats. Hemicastration at post- pubertal age increases Leydig cell size and androgen production. EN 63 MORPHOMETRIC ANALYSIS OF DIMORPHISM IN MOUSE PELVIS New edkahash Ty. WZ00O1.) Insti; Fac of .Sei:; Univ. of Tokyo, Tokyo. SEXUAL Sexual dimorphism of the pelvis of the mouse wWasS analysed morphometrically in normal male and female. In adult mice, relative length of pubis was larger in the female, While relative length of ischium and pubic symphysis was larger in the male. Caudal border of ischium was convex in the male, while it was straight or slightly concave in the female. Study of ontogeny of the dimorphism showed that relative length of pubis and shape of caudal border of ischium were different as early as 1 week after birth between male and female. Difference in relative length of pubic symphysis was already apparent at 3 weeks. Relative length of ischium was different at 8 weeks. These observations revealed that the sexual dimorphism appears earlier than ever described. Comparison of adult male, female, and androgen-insensitive mutant male (Tfm/Y) indicated that relatively long pubis and short pubic symphysis were characteristic of female, and that relatively long ischium and convex shape of caudal border of ischium were Characteristic of male. This suggests that the — former features are estrogen-dependent and the latter ones are androgen-dependent. EN 64 CHANGE IN BOMBYXIN CONTENT IN THE BRAIN OF Bombyx mori. A.Mizoguchil, H.Ishizakil, H.Nagasawa2 and A.Suzuki2. BLOW ENSE Sj Face of Sei, Nagoya Univ., Nagoya, 2Dept. of Agr. chem., The Univ. of Tokyo, Tokyo. Bombyxin from Bombyx mori, previously called 4K-PTTH, exhibits the prothoracico- tropic hormone(PTTH) activity on the pupal prothoracic glands(PG) of Samia cynthia ricini, but does not on the Bombyx PG. For the purpose of elucidating the physiologic- al role of this peptide in Bombyx, we examined the change in bombyxin content in the brain during Bombyx development. The ‘PTTH' activity in Bombyx brain extracts on Samia PG was high(4 units/brain) in the animals immediately after final larval ecdysis(on day-0 of fifth instar, Vo), gradually decreased in the course of larval development (V3,V¢), and was undetectable in the newly ecdysed pupae(Pg) and pharate adults (PA) (< 0.5 units/brain). Immunoblot- ting study of the brain extracts using bombyxin antibodies also revealed the quan- titative change of bombyxin compatible with the change in the biologic activity. Immunohistochemistry of the Bombyx brain demonstrated that bombyxin which accumulat- ed in the 4 pairs of large medial neuro- secretory cells was most abundant in the Vg brain among the developmental stages tested (Vo,V3,V6,P9,PA). The dramatic change of bombyxin content in the brain suggests that this peptide is involved inthe regulation of the development of Bombyx. EN 65 DEVELOPMENTAL CHANGE OF THE CEPHALIC NEURO- SECRETORY CELL ACTIVITY OF BOMBYX MORI AS REVEALED BY IMMUNOHISTOCHEMISTRY USING MONOCLONAL ANTIBODIES RAISED AGAINST PTTH. C.Suzukil, H.Ishizakil, H.Kataoka? and A. Suzuki2. 1lBiol. Inse., Lacs Of Sci. Nagoya Univ., Nagoya, 2Dept. of Agr. Chem., Fac. Of AGE <1,, Univ. Of Tokyo,. Tokyo. Mouse spleen cells were cultured for 4 days with an extensively purified Bombyx PTTH(22K-PTTH) and fused with NS-1l to produce monoclonal antibodies. Two clones (L101 & L104) produced IgMs which immuno- stained the brain neurosecretory cells in the medial and lateral regions and the peripheral nerve fibers of corpora allata, the neurohaemal organ for PTTH release. The immunohistochemical results showed that the amount of immunoreactive material disap- peared at around the head critical periods for PTTH necessary to larval moulting and larval-pupal commitment. Attempts so far made to demonstrate the immunobinding of these antibodies with PTTH activity turned out unsuccessful. Furthermore, immunoblot analyses with L101 after SDS-PAGE of Bombyx brain extracts revealed a single band of 23 kD which disappeared when a heated extract was tested; PTTH is heat-stable. These facts suggset that these antibodies were not directed to PTTH but to other brain peptides which resemble it closely in their purification behavior and are synthesized and released in close association with PRE 1092 Endocrinology EN 66 CHANGE IN HEMOLYMPH JUVENILE HORMONE TITER IN THE SILKWORM, Bombyx mori. S.Niimi, S.Sakurai and T.Ohtaki Department of Biology, Faculty of Science, Kanazawa University. Kanazawa 920. Juvenile hormone (JH), released from corpora allata, participates with ecdy- steroids in hormonal control of larval molting and metamorophosis in insects. A Simple, micro assey system of JH may be indispensable for the study of the role of JH in insect post-embryonic development. We prepared antiserum against JH-I, a major JH of Bombyx mori. Relative reactivity JH-I, ii, LEE and JH=1 acid was ]00, 20.4, 3.4 and ]00%, respectively. By RIA uSing this antiserum, change in JH titer in hemolymph was determined from early 4th throug the 5th instar. The titer in the 4th instar was relatively high in early half of the instar and then declined to one-fouth. The titer increased again simultaneously with the molting to the 5th instar but the maximum level was half of that in early 4th instar. It then decreased to low level and increased again after the onset of spinning. During those two large peaks, amall but significant peak was found in late feeding stage. This peak in female occurred one day earlier than that in male. Change in JH titer was expressed in a summation of JH-I and JH-I acid since the antiserum we used showed the same cross- reactivity to JH-I and acid. We are making an effort to measure the each amoutt. EN 6/7 CHRACTERIZATION OF ECDYSTEROID BINDING PROTEIN (Ecd-BP) IN WING IMAGINAL DISCS OF BOMBYX MORI. Y.Ishikawa, S.Sakurai and T.Ohtaki Department of Biology, Faculty of Science, Kanazawa University, Kanazawa 920 Ecdysteroid binding protein(Ecd-BP) in the wing imaginal discs of Bombyx mori was studied using 3H-ponasterone A (PNA) with high specific activity. Equilibrium Kd of the BP in cytoplasmic and nuclear fractions were 4.1 and 4.3 nM, respectively. Numbers of binding site (NBS) were 49.2 fmole/mg cytosol protein and 341.5 fmole/mg nuclear protein. According eo) Sechreelnerecl aineilySats, B7o3 a? 3625 OH iclae total binding capacities was found in nuclear fraction. Addition of Mgttincreased the binding capacity in cytoplasmic fract- ion, but addition of Cat+tdecrased it. Effects of three reagents on the binding capacity were examined. SH blocking reage- nts, iodoacetic acid and N-ethylmaleimide, inhibited the binding between PNA and Ecd- BP. Arachidonic acid, that exhibited dose- response inhibition on androgen receptor binding capacity in rat glandula prostati- ca, increased Ecd-BP binding capacity. Purification of the binding protein is under the progress using ligand affinity chromatography. EN 68 ECDYSTEROID CONJUGATES IN MATURE LARVAE OF THE FLESH FLY, Sarcophaga peregrina. K.Sakurai, S.Sakurai and T.Ohtaki Department of Biology, Faculty of Science, Kanazawa University. Kanazawa 920. In mature larvae of the flesh fly, Sarcophsga peregrina, ecdysone is rapidly metabolized into polar metabolites (free ecdysteroids and OA and OB), which could be separated on TLC. Very polar metabolity, OA, liberated free ecdysteroid by treatment with snail enzyme. IS-RP-HPLC analysis indicated that the very polar metabolites, OA, are consisted with two major compounds, whose side chain may be modified according to RIA date using two types of antiserum. Hydrolysis of these conjugated ecdysteroids by treatment with phosphatase revealed that one of them was phoaphate ester and the other was not. When the less polar metabolite was ana- lysed on RP-HPLC, these was no UV absorp- tion in the fraction with high radioac- tivity. UV spectrum of this fraction showed the maximum absorption at 2]2 nm whereas no absorption at 243 nm which is characteris-— tic to conjugated enone of ecdysteroids. Thus OB appeared to be a fragment of side chain, becouse OB fraction exhibited a high radioactivity which was originally located in the side chain. EN 69 3-DEHYDROECDYSONE: A MAJOR ECDYSTEROID PRODUCED BY PROTHORACIC GLAND OF THE TOBACCO HORNWORM, MANDUCA SEXTA. Sis Sakurai‘, Sip Kiriishi-, Okevlys Warren-, D.B. Rountree? and L.I. Gilbert2 IDept. of Biol., Fac. of Sci., Kanazawa Univ. Kanazawa 920 and 2Dept. of Biol., Univ. of North Carolina, N.C. 27514, U.S.A It is generally accepted that prothoracic glands produce ecdysone. When protein fraction of hemolymph was added to the in vitro incubation of larval or pupal pro- thoracic glands of Manduca sexta, ecdyste- roid content of the medium was increased by 8-fold. Comparative increase was observed when only the medium precondi- tioned with prothoracic glands was added with the protein fraction. A combination of analytical techniques (NMR, CD, MS) demonstrated that the major ecdysteroid released from the glands is a mixture of 2-dehydroecdysone and 3-dehydroecdysone (1:2), which is promptly converted into ecdysone if incubated with the hemolymph protein fraction. Comparative study showed that the major ecdysteroid secreted by ring gland of Sarcophaga peregrina and prothoracic gland of Bombyx mori is ecdysone whereas the glands of Papalio xuthus, P. protenor, P. machaon and Mamestra brassicae released dehydroecdysones aS a major ecdysteroid. Endocrinology 1093 EN 70 IN VITRO BIOSYNTHESIS OF JUVENILE HORMONE III BY THE CORPORA ALLATA OF THE COCKROACH, DIPLOPTERA PUNCTATA, DURING LARVAL LIFE. S.Kikukawa’ and S.S.Tobe Toyama Univ., Toyama and Dept. of Zool., Univ. of Toronto, Toronto. The rates of Juvenile Hormone (JH III) release by the corpora allata (CA) were determined in female larvae (I to IV instars) of the viviparous cockroach, Diploptera punctata, using an in vitro radiochemical assay. Higher rates were observed in the latter half of larval stadia I-III, whereas JH release was undetectable in the latter half of the final (IV) stadium. These findings suggest that the 'critical period’ for JH sensitivity may not be correlated with the period of increased JH synthesis. Thus, a reexamination of the ‘critical period’ was necessitated, using allatectomy and denervation of the CA. These data provide insights into the endocrinological programming of larval- larval molts and metamorphosis. EN 71 ISOLATION AND CHARACTERIZATION OF ECDYSTER- OID CONJUGATES FROM OVARIES OF BOMBYX MORI 2 crahetedy -Fujimoto-, io. Eintemcee and Netkekawae. »~Dept- (of Biology, Fac. of Sci. , Nagoya Univ., Nagoya and Dept. of Chem., Hac Or Ser, Tokyo inst. .of Technol ., Tokyo. In ovaries of Bombyx mori, ecdysteroids accumulate in free as well as conjugated forms. Major components of the free forms have been identified as follows: ecdysone, 20-hydroxyecdysone, 2-deoxyecdysone, 2- deoxy-20-hydroxyecdysone, 2,22-dideoxy-20- hydroxyecdysone and bombycosterol. Isolat- ion of the conjugates was achieved by the combination of liquid chromatography on Sephadex G15, silicic acid, and Sephadex LH-20 with high performance liquid chromato graphy. Six ecdysteroid conjugates have sofar been isolated. Upon incubation with snail juice or alkaline phosphatase from calf intestine, they yielded free ecdyster- oOids of the structures above mentioned, indicating that they are phosphate esters. This supposition was supported by the results of fast atom bombardment mass spec- trometry. Analysis by use of proton magnet- ic resonance revealed that 4 of them are 22-phosphate and 2 are 3-phosphate. Further analysis is now in progress. College of Liberal Arts, EN 72 EXTRACTION AND PARTIAL PURIFICATION OF SUMMER-MORPH-PRODUCING HORMONE IN THE ASIAN COMMA BUTTERFLY, POLYGONIA C-AUREUM ie K. Endo, T. Masaki and K. Kumagai*. BNVEeOn = Bb tOL habe Blo insta BACs lot Sci., Yamaguchi Univ, Yamaguchi. * Biol. Inst., Fac. of lLiberal-Arts, Yamaguchi Univ., Yamaguchi. In the Asian comma butterfly, Polygonia C-aureum, a physiological system under- lying the photoperiodic control of sea- sonal morphs was shown to involve a factor producing summer morphs (SMPH). The Poly- gonia SMPH as well as a Bombyx factor showing SMPH-activity could be extracted with 23. NaCl, but unsuccessful with acetone or 80% ethanol. Doses of the SMPH and 4K-PTTH (bombixin) were evaluated by Polygonia and Papilio pupal assays, res- pectively. Bioassay for SMPH was made on the basis of the color of the wings. The SMPHs were thought to be peptide hormones since it could be precipitated by ammonium sulfate and were inactivated by hydrolizing the extracts with trypsin. Molecular sizes of Polygonia and Bombyx SMPHS were estimated to be about 4,500, which were judged as being almost the same size as bombixin (4K-PTTH: M.W. 4,400) on the basis of the gel-filtration pattern through Sephadex G-50. But, based on the chromatographs of reversed-phased HPLC, the factors showing SMPH- and 4K-PTTH- activities were judged as being different. EN 73 IN VITRO SYNTHESIS OF VITELLOGENIN BY FAT BODY AND OVARY OF ARMADILLIDIUM VULGARE. S. Suzuki*, K. Yamasaki** & Y. Katakura***. *Biol. Lab. Kanagawa Pref. Col., Yokohama. **Dept. Biol. Tokyo Metropol. Univ., Tokyo. ***Dept. Biol. Keio Univ., Yokohama. Vitellogenin (Vg) synthesis was investi- gated in vitro tissue culture at each molting stage during vitellogenesis. Synthesized Vg was analyzed by polyacryl- amide gel electrophoresis. The fat body synthesized four forms of Vg, mainly Vg-1-2, and the rate of Vg synthesis was correlated with the molting cycle: low levels at stages A-C, E and a maximal level at stage D. Each form of Vg appears to be synthesized respectively in the fat body. The ovary also synthesized a slight amount of Vgs at stages D and E of the molting cycle. The lower forms (Vg-3- 4) were synthesized, not the higher (Vg-1- 2) in the ovary. The fat body from Y-organ ectomized females maintained very low synthetic level of Vgs. By the injection of 20-hydroxyecdyson, a slight induction of Vg synthesis was observed in vitro fat body culture. These results indicate that in A. vulgare most of the Vg may be synthesized by the fat body at stage D of the molting cycle, although vitellogenin synthesis could not be induced by the molting hormone. 1094 Endocrinology EN 74 ON THE MORPHOLOGICAL COLOR CHANGE AND NEUROSECRETORY SYSTEM IN THE MANTIS, TENODEA ARIDIFOLIA KeiliwaGantilmanGda Ya Ootas sb lOle lnisite mace of Sci., Shizuoka Univ., Shizuoka. Like other insects, mantises display two types of morphological color change green and dark body coloration (BC). The BC changes in relation to environ- mental factors such as temperature, pho- toperiod and humidity. Usually, the pigment modifications are associated with developmental stages. The present experiment was undertaken to investi- gate the fine structural changes of the brain-corpora cardiaca (CC )-corpora allata (CA) system during different phases of the developmental stages at various experimental conditions. During first instar, the larvae display the dark BC. In the proceeding instar, the BC of larvae exhibit a responsible reactions. Environmental factors such as constant lightness, green background and wet humidity cause green BC. On the other hand, constant darkness, brown background and dry humidity cause dark BC. On the basis of electron microscopic studies of the brain-CC-CA system, considerable difference is demonsit¢ra vec suite mcr. One of the remarkable feature of the CC in the dark BC animal is the presence of numer- ous electron-opaque granules. EN 75 ANNUAL CHANGES IN BIDDER'S ORGAN IN BUFO JAPONICUS FORMOSUS: HISTOLOGICAL OBSERVATION. Yoshiyuki Moriguchi and Hisaaki Iwasawa. Biol. Insite... Naagatal Unuives, eNasicatzal. Annual changes in germ cells and follicle cells in Bidder's organ were studied histologically. Secondary oogonia and leptotene»-zygotene-epachytene-stage oocytes markedly increased in number in June. In July, however, the development of oocytes was remarkably retarded, and remained in this condition through the autumn months, and no yolk accumulation was observed in hibernating toads, though yolk deposition began in the summer months in ovarian oocytes. Numerous degenerated oocytes were seen in March. It seems, therefore, that most oocytes in Bidder's organ degenerate during the hibernating period. The fine structure of oocyte- follicle complex in Bidder's organ was quite similar to that in the oocytes in the previtellogenic stage in the ovary. In Bidder's organ, however, a considerable amount of interstitium was seen, and the nuclei of the follicle cells were more ellipsoidal in shape, especially in August-September specimens, than those in the ovary. Well-developed oocyte- follicular cell interdigitations were seen through the year, and no remarkable change was recognized in this structure, EN 76 ULTRASTRUCTURAL STUDY ON RESPONSIVENESS OF SPERM TRANSPORT ROUTE AT HCG-INDUCED SPERMIATION IN RANA NIGROMACULATA. Tohru Kobayashi and Hisaaki Iwasawa. Biol. Inst., Niigata Univ., Niigata. the" responsiveness Vo sthessspieran transport route was examined electron- microscopically 0 and 20 min. and 2 and 4 days after hCG injection (low dose; 3.3, high dose; 15 IU/g B.W.) in adult summer frogs. In the control frogs, the cells of efferent duct-linked seminiferous tubules with the nephron were characterized by filament-rich cytoplasm in all regions of the intratestis, ductuli efferense, and intrakidney. After the~treatment, a remarkable development of ER and Golgi complex was seen in these cells, and the swelling of cytoplasm was also observed irrespective of the passage of spermatozoa. In the cells of the renal tubules, no noticeable changes were seen, even though spermatozoa passed through the tubules. The epithelial cells of the ampullar portion of the Wolffian duct released PAS-positive granules after the treatment, and the release of numerous granules was recognized 4 days after the high dose-treatment. The passage of spermatozoa in each route was recognized at the all fixative times in the high dose-treated group, but was not recognized in the route beyond the intratesticular efferent duct in the low dose-ones. EN 77 CIRCANNUAL CHANGES IN PLASMA CONCENTRATION OF GLUCOSE IN TOADS, BUFO JAPONICUS. K.Kubokawa and S.Ishii. Dept.Biol., Waseda Univ., Tokyo. Studying circannual changes of plasma corticosterone and aldosterone levels, Jolivet-Jaudet et al.(Ref.1,2) suggested a possibility that aldosterone is the main glucocorticoid and corticosterone is the main mineralocorticoid in the toad, Bufo japonicus. In order to examine this pos- sibility, we studied the monthly change of the plasma glucose level with the same materials which were used in the previous corticoid studies. Among plasma levels of corticoid and weights of the liver and fat body, only aldosterone showed a high and significant positive correlation with glucose. The peaks of glucose and aldos- terone were preceded by a sharp peak of the fat body and followed by a rapid increase of the ovarian weight which is due to -the yolk accumulation. This sequence of events supports the idea that aldosterone stimu- lates supply of glucose to the plasma from the fat body. In breeding period, we measured the plasma concentration of glucose. The glucose level increased tremendously when toads came into water. It have shown that PRL, T4, LH and FSH also increased. These results indicate that when toads breed in the pond, a number of regulatory mechanisms becomes active simul- taneously. Ref. 1. Jolivet-Jaudet,G. et al.(1984)Zool.Sci.1,317: 2.Jolivet-Jaudet, G.et al.(1984)Gen.Comp.Endcrinol.53,163. Endocrinology 1095 EN 78 EVOLUTION VERTEBRATES Susumu Ishii, Department of Biology, Waseda University, Nishi-Waseda 1-6-1, Tokyo 160 OF GONADOTROPIN MOLECULES IN We have limited information to discuss the molecular evolution of gonadotropins, as the complete or even partial amino acid sequence of gonadotropin has been reported only in mammals and fishes. The author Galeutaccosthe Sum of the diftterence of the CoOmbeninwmoOnseach) amine acid «cesidue for all amino acids except Cys and Trp between two hormones, and used the sum as a quanti- tative index to show the difference of the two hormones. The index between homologous FSH and LH was least in amphibians and largest in mammals. This indicates that the difference of FSH and LH molecules is larger in phylogenically higher vertebrat- es. When LHs of various vertebrates were compared with Bufo LH, the index was also larger when higher vertebrates were com- pared. When FSH was similarly compared, the indice or differences did not show any tendency. Detailed comparisons of various LHs with Bufo LH revealed that the content Ciel RrOnWaSeLarper. imnihuegher vertebrates, while the contents of Lys and Asp (or Asn) Were less wm lauleicer wemuclowmences> Wdese results show that the evolution of the LH molecule has a certain direction, while UIdGOEReGAes HSH molecule: as “rellative ly, BANdOM une sODservied. change of ithe! 9 EH motecule maybe. the first example ‘of the molecular orthogenesis in peptide hormones. EN 79 EFFECT OF PROLACTIN AND TESTOSTERONE ON THE NEWT MAUTHNER NEURON. Y. Suzuki and S. Kikuyama* Map abo Dept labl. Arts, Asia Unibre, Musashino-shi, Tokyo and *Dept. Biol., Sch. Edu., Waseda Univ., Shinjiku-ku, Tokyo. There is evidence that Mauthner neurons in the medulla oblongata play a roll in tail movement in some amphibians as well as in fishes. During the breeding season the male newt (Cynops pyrrhogaster) vibrates his tail vigorously to perform the court- ship behavior in front of the female. Morphological study revealed that the nu- clear volume of the Mauthner neuron both in male and female newts captured in the breeding season was larger than that in the newts in the non-breeding season and that in the breeding season the nuclear volume is much greater in the male than in the female, Implantation of testosterone (T) pellet to hypophysectomized newts increased the nuclear volume of the Mauthner neuron to some extent. Administration of prolac- tin (PRL) makedly increased the nuclear volume of the Mauthner neuron in the newts bearing T pellet. The present result, together with the previously obtained re- sults that treatment of the male newt with PRL and T elicits the courtship behavior and cloacal gland development, indicates that PRL is playing important rolls in reproduction in the male newt. EN 80 CHANGES IN PRL CELL BY THE ADDITION OF TRH OR DA IN VETRO- T: Shaman) Me Takahashi and H. Ooka!. DepityesOk wn Bolas) wang Cedhe “Guikeeaialbt; Tokyo Metropol. Inst. of Gerontol., Tokyo. Rat pituitary PRL cells are subdivided into three types : small secretory granule type (PE), mean granule type (PIL) “and large granules type (PIII). We observed morphological changes in female rat PRL cells in primary culture for 4 days by the SiClGlalifsLGi OLt WWidal Oye IY TG IL ING; im- munocytochemical technique. PRL cells which were cultured in the absence of TRH or DA had large granules, parallel-arrayed rough endoplasmic reticulum (RER) and denser Wolpe | 'Giow gat la:piplaicial tills (GA). a crear Ciera they were similar to PLU, WR (10 increased PRL levels in vitro.The eine granules changed into those of PII by the addition of TRH but GislnSig Uc@eliqnkeSoy «Che | fein “GElilis » Weiss. = iavone Simaleie GO Wlus Meany DWN Sells sinoweac highly active synthesis and secretion, and a few of them had RER which changed into vacuoles. These results suggest that the PRL cells stimulated by TRH contain imma- ture granules which resemble those of PII in Shape and size. DA (10 *M) decreased PRL Vevels in vitro. Decay of RER and GA, and increase of lysosome were observed in the cytoplasm. These ultrastructural features of PRL cells indicate inactive synthesis and release of PRL. EN 81 AGE-RELATED CHANGES IN PROLACTIN RELEASE BY LACTOTROPIC CELLS OF FEMALE RATS IN VITRO. M.Takahashi’, T.Shinkai* and H.Ooka? ‘Cell CUE sIhalG 5 HS DEOIESOI IAEILs, WolsyoO MiEnereojoo Ibe IMNSite Cie Carcome@lls pwWels7O> To study the possible mechanisms involved in hyperprolactinemia in aged rats, anterior pituitary cells from young(6 mo.) and old(22 mo.) female rats were CUCU COMUNne Ave EmO aro) Cxamimess thevsbasad: prolactin(PRL) release and responsibility to So Rewu ies and TRH. The basal PRL release of 10~- PRL cells from young and old rats wees 2355 Aime AileS mej pieSspecetwvoely, however, no significant difference was found between the two values(p>?0.05). eps ae concentration in the range from to 10 ~ M inhibited the PRL release of ele COLLIS iwicom Owe; WEES sli El COSC dependent manner. Similar inhibitory effect of dopamine was also observed for IEInNe) CEILS stigOm OilGl icklicSs 4 Comiciceuay Wows, Ehe PRL cells from old rats were more responsible to TRH than those from young rats with respect to the PRL release, that is, the PRL release of the cells from old rats was significantly stimulated by 10 M TRH, whereas the a from young rats was responsible to 10 M TRH. The present resumes isuggesin that the inereased susceptibility of the PRL cells to TRH in aged rats may be involved in age-related hyperprolactinemia in vivo. 1096 Endocrinology EN 82 INDUCTION OF METAMORPHOSIS IN THE LARVAL LAMPREY. III. THE EFFECT OF PINEALECTOMY. Shintaro Suzuki. Dept. of Comp. Endocri- nol., Inst. of Endocrinol., Gunma Univ., Maebashi. The Larval lampreys, Lampetra reissneri were pinealectomized by making a longitudi- nal incision. After one week these larvae were treated with potassium perchlorate. At 50 days after treatment metamorphosis was induced in the larger larvae. Complete- ly metamorphosed larvae were obtained at 100 days after treatment. In these larvae thyroid follicles were formed from endo- style, as seen in spontaneous metamorphosis However,in the smaller larvae partial meta- morphosis was induced.Oral apertures, bran- chiopores, and eyes were at different meta- morphic stages, and the cell type 1 of the endostylar cells degenerated and no thyroid follicles were formed, even after pro- longed treatment. On the other hand, the larvae which were not treated with potassi- um perchlorate after pinealectomy did not metamorphose, and the plasma thyroxine lev- els were high, AG) alin Jalinie@aete dhemavyaee In the larvae treated with potassium per- chlorate after pinealectomy, the plasma thyroxine levels were very low. These results suggest that pineal complex is not involved directly in the metamorphosis of the larval lamprey. EN 83 A COMPARATIVE STUDY FOR IN VITRO RESPONSE OF THYROID GLANDS BY THYROID STIMULATING HORMONES IN MAMMALS AND AVES M. Sato, H. Sakai, S. Wakabayashi and T. Yoshida* Dept of Biol., Nihon Univ. School of Dentistry, Tokyo, and *Tsukuba Primate Center for Medical Science, N.1I.H., Tsukuba-gun. To establish thyroid stimulating hor- mone (TSH) bioassay, in vitro of thyroid glands by TSH were determined with Japanese quails, newly hatched cockerels, mice and monkeys. When quail thyroid glands were tissue-cultured in medium containing TSH preparation, the release of thyroxine (T4) from the glands showed significant increase after 2 hours. With mice and monkeys, it took more than 6 hours to reveal the same significant T4 increase, and the ratios of increase in both animals were similar. On the other hand, thyroid glands of any of the animals showed the same response using TSH from any of the animals. Seasonal changes in the response of avian thyroid glands were observed. Different response from mice could be ascribed to diurnal effects. These observations indicate that the responses of thyroid glands between mammals and aves’ differ, and that the thyroid glands used did not show high specificity to TSH from the animals. EN 34 CHANGES IN THYROID HORMONE CONCENTRATIONS IN DEVELOPING CHUM SALMON. Masatomo Tagawa and Tetsuya Hirano. Ocean Research Institute, Univ. Tokyo, Tokyo. In order to examine the role of thyroid hormones during early developmental stages of chum salmon, techniques were developed and validated for quantitative extraction of thyroid hormones from eggs, embryos and juveniles, and changes in the hormone concentrations in tissue and plasma were examined. Significant amounts of both thyroxine (T4) and triiodothyronine (T3) were found in unfertilized eggs, and the same levels were maintained until hatching. The embryonic thyroid follicles were still small in number and in size at hatching. During the course of yolk absorption, whole body concentrations of T4 and T3 decreased steadily, and then tended to increase tem- porarily toward the end of yolk absorption. Both T4 and T3 were detected in plasma during later stage of yolk absorption, and their concentrations increased gradually. T3 levels in both whole body and plasma were always lower than the T4 levels. Embryonic thyroid gradually developed during the yolk absorption, and looked mildly activated thereafter. These find- ings suggest an important role of maternal thyroid hormones during early development, followed by activation of the larval thyroid after the yolk absorption. EN 85 cDNA CLONING OF THE PORCINE THYROTROPIN B-SUBUNIT T.Hirai®, H.Takikawa’®, Y.Kato'. Dept. of ‘Protein Chemistry and of #Pharmaceutical Chemistry, Inst. of Endocrinol., Gunma Univ., Maebashi. A porcine anterior pituitary cDNA library was constructed in expression vector Agtll. Three clones for the precursor of the B-subunit of thyrotropin (pre-TSHB) were identified by hybridization with a synthetic nucleotide probe (85mer) and with a DNA fragment encoding human TSHB (Hayashizaki et al. 1985). The nucleotide sequence of these clones were determined. These clones cover a part of the signal sequence, the entire sequence of mature protein, and 3'-untranslated sequence. The nucleotide sequence showed that the mature protein of porcine TSHB consist of 118 amino acid residues. In comparison with the amino acid sequence of secretory form of porcine TSHB (Pierce et al. 1971)", there are four differences, and the extended carboxyl terminus of six amino acid residues. The nucleotide sequence of cording region have a homology of 90% for human, 93% for bovine, 84% for mouse, and 83% for rat. Northern analysis showed that the length of porcine pre-TSHB mRNA is about 500 bases with no heterogeneity. a) FEBS LETTERS 188, 394-400 b) Recent Prog. Horm. Res. 27, 165-212 Endocrinology, Morphology 1097 EN 86 PURIFICATION AND CHARACTERIZATION OF Y.Hanaoka and H.Hayashi emi om, Biol. , \Sch. of iduen) Waseda Univ., Tokyo. asic, OH Maclocrinoel., Gunma Univ., Maebashi Thyroid-stimulating hormone(TSH) was purified from the pituitary glands of bull- frogs, Rana catesbeiana, using ethanol precipitation, hydrophobic interaction and anion exchange chromatography, and affinity chromatography employing monoclonal anti- bodies against bullfrog LH and FSH®. This preparation was eluted from TSK G3000SW column as a single peak and its molecular weight was estimated to be about 32,000. LES INL@acitiviity, AS measvicecl Joy ela© i vitro release of Ty from the thyroid glands taken from hypophysectomized prometamorphic bullfrog tadpoles was about 2 times higher CIMAM CIMA Oi INO\VWalL ine MES els UES yee Ee concluded that this preparation is exactly TSH. Isoelectric focusing electrophoresis revealed that this TSH is of multiple isoelectric points, ranging from 4.5-5.0. BuUlliwvocq WSs was SepoeteeeSecl Whieo© icwWe subunits by reverse-phase HPLC after dissociation. One of these was bound to a monoclonal antibody against bullfrog FSH «a, and the other to a polyclonal antibody against human TSH 8, but not to monoclonal antibodies against bullfrog FSH and LH &. Bulltrog TSH B Ehus identified, Consisting of about 100 amino acid residues,seems to be of more acidic and hydrophilic nature than human one. EN 87 THE PRESENCE OF THYROID HORMONE BINDING PROTEINS IN PLASMA OF BULLFROG. K. Yamauchi, S. Koya, R. Horiuchi and H. Takikawa. Institute of Endocrinology, Gunma University, Maebashi. Using affinity labeling with N-bromoacety] (225) ]-L-triiodothyronine (T3), the presence of a distinct T3 binding protein (TS3BP) was demonstrated in the plasma of metamorphosing tadpoles and adult bullfrogs, respectively. The both proteins showed higher affinity for Wey eldeLia stOie Jo thyroxine. Tadpole type T3BP was found anoda] to albumin on polyacrylamide gel electrophoresis, and was 54 K dalton protein composing of four subunits. The molecular features resembled those of human thyroxine binding prealbumin. On the Other hand, adult type T3BP was found cathodal to albumin, and was 56 K dalton protein composing a single peptide. The replacement of tadpole type T3BP by adult type T3BP took place at stage XXIV. Since thyroid hormone levels in bullfrog blood increase during prometamorphosis and decrease at metamorphic climax, tadpole type T3BP might be important as thyroid hormone carrier. EN 88 EFFECTS OF PHOTOPERIOD AND TEMPERATURE ON PLASMA T, AND T, LEVELS IN THE DJUNGARIAN HAMSTER. 1 5 1 he MIEVSTOWlaY 5 IK 5 USiuESuEL™ einyel IU, Oalshoil— - oDept. of Biol., Nara Women's Univ., Nara, Dept. of Zool., Hiroshima Univ., Hiroshima. We investigated the effects of photo- period and temperature on plasma thyroid hormones and testosterone levels in the Djungarian hamster. In Experiment I, adult male hamsters were maintained in conditions of four combinations of photo- periods and temperatures (LD16:8-25°C; LD8:16-25°C; LD16:8-7°C; DBE AG=7PC)) . Miter Onaweeks,mEplasman l= ml andes tesito= sterone were measured “by ‘radioimmuno- assay. Plasma T, level was significantly higher at low temperature than at high temperature, regardless photoperiods. At high temperature, plasma T level was Significantly higher in long day than in short day. Plasma testosterone level was also higher in long day than in short day. In Experiment II, male hamsters were maintained in three conditions (long day, short day and short day with testosterone implantation) for 19 weeks. Plasma T level of testosterone implanted hamsters in short day was higher than that of short day control and similar to the value of long day control. 1A COMCIUSiLEM, i) Plasma T level was increased by low temperature. 2) Plasma T level was increased by long photoperiod, probably due to the high plasma testosterone level. MO 1 OVARIAN STRUCTURE AND OOGENESIS OF PYCNO- GONIDS K.Miyazaki and T.Makioka. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. The subphylum or class Pycnogonida is a small group of marine arthropods. Some problems remain concerning the phylogenetic relationships of pycnogonids among the arthropod groups. In the present study, we examined the ovarian structure and oogenesis in 10 species, including 4 families, of adult pycnogonids in order to compare them with those in the chelicerates and mandibulates. In most species, the tubular ovary was localized in the trunk and legs on the dorsal side of the gut. A cord-shaped germarium including the oogonia was present in the dorsal ovarian epithe- lium through almost the entire length of the ovary as in chelicerates. The growing oocytes occurred in the ovarian cavity as in mandibulates, and in most species, they were found only in the pedal ovarian branches. However, they were not arranged in a line in order of size as seen in mandibulate ovarioles. In Propallene longiceps, the tubular ovary was localized only in each leg, and the germarium was not found. No follicle cells occurred in any species as in horseshoe crabs. Thus, Ppycnogonids have both chelicerate-like and mandibulate-like characteristics in ovarian structure and oogenesis. 1098 Morphology MO 2 Ultrastructure of the tooth of sea urchin, Hemicentrotus pulcherrimus. O.Ochi and M.Watanabe. Dept.of Biol.,Fac. of Sci.,Ehime Univ.,Matsuyama. Morphological investigations on teeth of Hemicentrotus pulcherrimus isolated by maceration in antiformin or decalcified by ascorbic acid solution and energy disper- sive X-ray microanalyses on the teeth were performed. No great difference between the tooth and the other Echinoid one described by Markel, Chen, et al. is recognized. The teeth are divided into the plumula (growth region), the shaft and the chewing part. They are used up during chewing and continually renewed. The tooth skeleton is composed of two rows of tooth elements sticking alternately one within the other. Each element consists of the primary and secondary plates and the lamellae-needle- complex, and its carcareous deposits are formed intercellularly by syncytial odon- toblasts. Secondary calcareous deposits unite the tooth elements to form the com- plete tooth skeleton. The secodary deposit and the greater part of the tooth element are composed of almost CaCOz, but hardest part of the tooth, the stone part, has a high amount of MgCOz (about 2 per cent). MO 3 ABSENCE OF THE PINEAL IN THE ELECTRIC TELE- OST, GYMNARCHUS NILOTICUS. Ke Tstnekapy DepeEMioL Biol shumane Uniivaen, Matsue. Although there are several vertebrate genera that lack the pineal (e. g. Myxine, LOLPeCdO, = CEOCOGMIUST ReEEC.)y NOmeelLeOsts have been known to lack the pineal. How- ever, in the course of histological study of the brain of various teleosts, I found that the electric teleost, Gymnarchus nilo- tlcus; lacks they pineal. Enmehemepathalias mic roof, the saccus dorsalis is highly folded, but there is no pineal stalk here. In front of the saccus dorsalis, the para- physis extends rostrodorsally. The tip of the paraphysis is sacculated, but it does not contain the pineal end-vesicle. For comparison, I studied the epithala- mic region of several electric teleosts (e. g. Malapterurus, Electrophorus, Eigenman- nia, Sternarchella, Ste All these pos- sessed a well-developed pineal. According to the literature, blind or semi-blind te- leosts living in the cave or in the deep sea possess a well-developed pineal. The reason why only Gymnarchus niloti- cus lacks the pineal is unknown, but this is the first demonstration that there is a teleost without the pineal. MO 4 STUDIES ON THE KIDNEYS AND URINARY BLADDERS OF THREE ANTARCTIC TELEOSTS, Mizuho Ogawa, Dept. of Biology, Fac. of Liberal Arts and Science, Saitama Univ., Urawa, Saitama, 338, Japan. The structures of the kidneys and urin- ary bladders of the three Antarctic teleo- sts, Pagothenia borchgrevinki, Trematomus bernacchii and T. hansoni were compared, It was reconfirmed that their kidneys are aglomerular. It was found that their bladders are well developed in size. In the first two species, the bladder epithe- lium is composed of tall columnar cells continuous with the collecting tubule and which gradually transform to low columnar © cell near the cloaca. The epithelium of T. hansoni, however, is composed of simple cuboidal cells that cover the whole surfa- ce of the bladder. Although T. hansoni is a mid-water fish, P. borchgrevinki is ada- pted to life in and under the platelet ice and T. bernacchii dwells on or near the sea floor. These two fishes are exposed to different osmolalities during the free- zing and the thawing seasons. It is ther- efore necessary for them to produce dilute urine under hypotonic condition during the thawing season. This situation appears to be similar to that occurring the bladder of euryhaline marine fishes during their migrations into brackish environments. A functional difference in the urinary bladder between these two Antarctic teleo- sts and T. hansoni is suggested. MO 5 LIGHT AND ELECTRON MICROSCOPIC STUDIES ON THE LUNG OF HYNOBIUS NEBULOSUS TOKYOENSIS. T.Gomi, Y.Kikuchi, A.Kimura, Y.Ishikawa, T.Hashimoto and K.Kishi. Dept. of Anat., Sch. of Med., Toho Univ., Tokyo. gi ee EE ——e—eeEeeeeeeeee The lungs of the Hynobius nebulosus tokyoensis were observed by light and electron microscopy (SEM, TEM). In this species, the lungs were mainly divided into two groups, airway portion and respiratory portion. Smooth muscles were well developed in the airway portion. Elastic fibers and collagen fibers were more developed in the respiratory portion than in the airway portion. In the airway portion, ciliated cells and goblet cells were seen to exist. In the respiratory portion, epithelial cells were cuboidal or columnar in shape. In the cytoplasm of these cells, osmiophilic lamellated bodies were found to exist, and their secreting figures were observed. Moreover, these cells possessed the features of both Type I and Type II alveolar cells, demonstrating membranous cytoplasmic projections which extended from the lateral surface of the cell and covered the capillaries. The most characteristic feature was the presence of the secretory granules containing material of moderate electron density, which were found under the cell surface. These granules proved to be PAS and colloidal iron reaction positive. Sudan black B positive granules existed in the cytoplasm of the alveolar epithelial cell. Morphology 1099 MO 6 FIN RESORPTION IN TAIL OF THE AXOLOTL DURING METAMORPHOSIS. H.Takahama, T. Kinoshita, F. Sasaki and Ke Watanabe. Dept. of Biol.,, School of Dent. Med., Tsurumi Univ., Yokohama. Morphological changes of the dermis in the tail fin of an axolotl( Ambystoma mexicanum) which induced metamorphosis by T3 administration were observed by light and electron microscopy. The endocytotic capacity for FITC-dextran injected into the animals was compared between fibroblasts and macrophages. At the non-metamorphic stage the fibroblasts which undercoated basement lamella are spindle-shape and contain many smooth-surfaced vesicles near the cell membrane. Intermediate junctions are seen between the fibroblasts. The macrophages have higher endocytotic Capacity for FITC-dextran than the fibroblasts. At the metamorphic stage prominent degenerative changes occur only in the apical regions of the tail fin. In these region orthogonal arrangement of collagen fibers disappears in the basement lamella and the disordered collagen fibers accumulate. Many of the fibroblasts and macrophages are seen among the collagen fibers. Collagen-containing structures (phagosomes) are observed in the cytoplasm of the fibroblasts in those degenerative regions, where some fibroblasts and nerve tissue degenerate and the macrophages phagocytose them. However, there is no evidence that the macrophages directly phagocytose the degraded collagen fibers. MO 7 THE DIFFERENT LIFE CYCLES IN BIPALIUM SP (PERH. B,PENNSYLVANICUM BY R.OGREN)OF NON- FISSION TYPE SPECIES. N.Makino and Y.Shirasawa. Dept,of Biol., Tokyo Med,, Coll., Tokyo. Materials were first collected at Hino Shi in 1982, Authors made a paper open to the public about their characteristic stru- cture of copulatory organ in 1984, They were unknown species in our own and other lands, They have three longitudinal lines keep dark color in median line of the dor- sal side, showing dark green on the whole and have no line and lighty in the ventral side.In the breeding season,they have copu- lated at 100 to 200 mg within one year old, from the latter part of March to July in Tokyo.Their egg laying are very active,for they can lay egg 1 by 1 over 10 cocoons at last.After the copulation,they lay egg for some months, and after last egg laying, they die natural death within several months, But authors observed that materials were born in 1985, did the copulation only in April of the next year,and laid one cocoon only in July in 1987. Then,the weight of adults became over 1000 mg. Recently,author recei- ved a paper of new Bipalium from Dr.R.Ogren The new Bipalium;B.pennsylvanicum by R. Ogren seems to the same worm with our new material,and the body weight of B.penn.are less than 300 mg. Thus,the same species in adult have different body weight and differ ent aging,and about egg laying, there is nothing to choose between the two in differ ent adults, Interestingly,their cells are composed of 2n=15 by the air-drying method, MO 8 THE KARYOTYPES OF GENUS; BIPALIUM IN BOTH THE FISSION AND NON-FISSION TYPES. N.Seo, N.Makino and Y.Shirasawa. Dept. of Biol shokyo Medien. Coda ms LOkyols Genus; Bipalium have active regenerative ability, and have two characteristics; fission and non-fission type. The non-fis- sion type worms are usual species in Japan and propagate only bisexual reproduction and they have thick and short of external forms, for example, B. fuscatum, B. fusco- lineatum, B. hilgendorfi and B. pennsyl- vanicum(new species) etc. The fission type worms have been reared in our laboratory about three species; B. nobile, B. kewense and B. multilineatum and they have very long slender of external forms. These two types of Bipalium are different fromthe structure of the copulatory organs also. Authors have been studied the relation of regeneration and cell division in Bipalium. From this angle of vision, authores studied the analy- sis of karyotypes in Bipalium, both fission and non-fission types. Fission type species ; cosmopolitan worm, B. kewense in our mate- rial is 2n=18, though L.Winsor reported 2n= 16. B. multilineatum which do not bisexual reproduction in Tokyo is 2n=10. Giant Bipalium, B. nobile which does sexual and asexual reproduction is investigated both regular and translocated types in chromo- somes, namely 2n=10. Non-fission type species; B. pennsylvanicum is composed of 2n=15 and B. fuscatum is 2n=10. For the analysis of karyotypes, the air- drying method was used. MO 9 OBSERVATIONS ON THE REGENERATION OF THE SAGITTAL SMALL PIECES IN THE LAND PLA- NARIAN, BIPALIUM NOBILE. Y. Shirasawa and N. Makino. Dept. of Biol. Tokyo Med. Coll., Tokyo. Morphological and histochemical studies on the head-regeneration of the sagittal small pieces have been made in B. nobile. The head-frequency of the sagittal pieces showes a decrease of 50% under the trans- verse one. And the former took much more times for the head-regeneration than the latter. There was no difference in the head frequency between the pre- and the post pharyngeal region. In several hours after the cutting, most sagittal pieces curved toward the lateral cut surface and the rest formed a ring by the adhesion of the ante- rior and the posterior surface. Unlike the transverse pieces, almost all the sagittal pieces did not move actively. The lateral cut surface was covered by the stretched dorsal epidermis in about 24hrs. The histo- logical preparations stained by AF(Gomori's aldehyde fuchsin) demonstrated that also in the sagittal pieces, the AF-positive gran- ules released from the intestine appeared in the parenchyma at the stage of 3-5 days. And that in this stage, the anterior end of the ventral nerve cord approached to the anterior cut surface, and the nerve cells observed in this portion were slightly AF- positive. Electron microscopically, these cells had developed nucleoli, small dense bodies in the proximal of the nuclei, many free lobosomes and secondary lysosomes. 1100 Morphology MO 10 MO 12 FINE STRUCTURAL LOCALIZATION OF ACID PHOS- UNUSUAL VESICULAR STRUCTURE IN PHOTO- PHATASE ACTIVITY IN THE REGENERATING FRE- SENSITIVE CHROMATOPHORES. SH-WATER EARTHWORM, BRANCHIURA SOWERBYI. M. Obika. Dept. of Biol., Keio Univ., M. Shirasawa and N. Makino. Dept. of Bi- Yokohama. Oley WOOO MECls Collis; Wolves Dermal chromatophores of some verte- brates are photosensitive, responding to The morphology and AcPase activity were photic stimuli with rapid intracellular investigated electron microscopically in pigment translocation. Ultrastructural the fresh-water earthworm, Branchiura so- studies on melanophores, xanthophores' and werbyi and were compared among the early leucophores of Oryzias latipes and tail stages of the head-regeneration. On the fin melanophores of Xenopus’ laevis cut-surface of regenerating worms, the en- tadpoles have revealed that these photo- zyme was localized in the lysosomes of sensitive chromatophores possess’ charac- the propagating epidermal cells. In their teristic vesicles that bulge out from cell Golgi zones, the vacuoles and the reticu- surface. The size and number of vesicles lar parts were strongly and the cisternae are variable but they always contain mem- were slightly positive. But in intact wo- branous components such as small vesicles, rms, these activities were not conspicu- either spherical or tubular, or lamellated ous. In the intestinal epithelial cells, membranes. No other cytoplasmic organel- the dense bodies came from the Golgi vacu- les are observed in this area. Studies oles were strongly and the cisternae were on serial sections indicate that each slightly positive. Some of these, in rege- vesicle is connected to the cell body by nerating worms, showed the diffusion of a stalk that contains fuzzy, filamentous the lead phosphate and resulted in autoly- inclusions. Microtubules are only rarely sis. The intestinal granular cells had the encounterd in the stalks of Oryzias me- most remarkable activity in intact worms, lanophores. No significant difference in but the activity, in the large granules, the internal structure of the vesicles has rapidly declined in the regeneration. The been found between the dark- and light- nerve cells of the ventral ganglia had adapted chromatophores. From their distri- strong AcPase activity, unlike other cells bution and structural characteristics, mentioned above, in the well-developed Go- these unusual vesicular compartments found lga cCiternae. The Golligi vacuoles) jand) che in photosensitive chromatophores of the irregularly shaped dense bodies were also fish and the toad appear to be the likely positive. In regenerating worms, the acti- candidates of photoreceptors. vity of the cisternae was the most remark- able in the stage of 1-2 days. MO 11 MO 13 ULTRASTRUCTURAL INVESTIGATIONS OF MELANO- IMMUNOCYTOCHEMICAL AND ELECTRON MICROSCOPIC CYTE AND MACROPHAGE IN THE MOUSE HARDERIAN STUDIES ON THE GLIAL CELLS IN THE BRAINS OF GLAND. ELASMOBRANCHS. 1 K. Shirama and M.Hokano. Dept. of Anatomy, A.Chiba! and Y.Honma?. Dept. of Biol., Tokyo Medical College, Tokyo. Nippon Dental Univ., Niigata and “Sado Mar. Biol. Stat., Fac. of Sci., Niigata Univ., The presence of dendritic cells contain- Niigata. ing melanin granules have been demonstrated employing silver impregnation and with ele- Immunocytochemical and electron micro- ctron microscopy in the interstitial tissue scopic studies were conducted to elucidate of the mouse Harderian gland. Two types of the cytological features of glial cells in melanocytes, either with or without the the brains of elasmobranchs. For this various developmental stages of melanin purpose, three species of elasmobranchiate granules, were found in the gland. The cell fishes, Seyltorhinus torazame, Mustelus with developing granules was more dendritic manazo and Dasyatis akajet, collected from and contained a large number of cytoplasmic the coastal waters of Sado Island were organelles. The other cell was ellipsoidal used. : or slender in shape and contained few cyto- By means of the peroxidase anti- plasmic organelles and a large number of peroxidase method, S~-100 protein-like fully melanized granules, but no developing immunoreactivity was demonstrated in the granules. In general, the granules of the tanycytes and astrocytes including Harderian gland melanocyte resembled Bergmann's glia in the cerebellar cortex. granules from other organs(particularly the These cells with their endfeet formed a skin of the eyelids ). The general size distinct boundary membrane on the brain range of the granules was 0.2-0.9 pm. Each surface (membrana limttans gltae super- granule was enclosed by a membrane. fictalis) and the periphery of the blood The Harderian gland macrophages contained vessels (malaga pertvuaseularys) ya Byaaene fully pigmented melanin granules of various electron microscopic immunoperoxidase sizes. The granules were enclosed ly a method, S-100 protein-like antigen appeared membrane either singly or in groups. Some to be localized in the cytoplasmic matrix. of the melanin granules within the phago- Gilivallfalbrniallany, acidacs protein ke somes showed signs of degranulation,reveal- immunoreactivity was also detected in the ing the underlying matrix. tanycytes and astrocytes. Conventional electron microscopy demonstrated three types of glial cells, astrocytes, oligo- dendrocytes and microglia in these fishes. Morphology 1101 MO 14 ULTRASTRUCTURAL CHANGES IN THE RENAL GLOME- RULUS OF MALE AND FEMALE WISTAR/TW RATS. Win Win Yee, S. Takahashi and S. Kawashima. Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima. Changes in the renal glomerulus be- tween male and female Wistar/Tw rats were electron microscopically examined at l, 3, 6, 12 and 18 months of age.Common changes were observed after 3 months in male rats and 6 months old female rats in the glome- rular capillaries basement membrane (GBM) , epithelial cell foot processes (EpF) , and epithelial cell cytoplasm. Initial lesions detected in a few scattered area in 3-month -old male rats were the slight thickening of the GBM with some fusion of the EpF and the appearance of vesicles and vacuoles in the epithelial cells. Lesions became more extensive as age advanced, and at 18 months profound lesions were detected . In female rats the initial lesions were not apparent until 6 months of age . Every 12-month-old female rat showed focal changes of the GBM thickening with some fusion of the EpF and at 18 months focal changes became segmental ones . From 12 months there were’ striking sex differences in the development of renal lesions . Only aged male rats showed extensive thickening of the GBM with nodu- lar folds which intermingled with mesan- gial matrix , extensive fusion and _ then denudation of the EpF and degeneration of the epithelial cell cytoplasm. MO 15 METAMORPHOSIS OF LARVAL TRACHEAE AND FORMATION OF PUPAL ABDOMINAL TRACHEAE IN DROSOPHILA MELANOGASTER T.Matsuno.Kyoto Prefec.Univ.of Med.,Kyoto. Adult tracheal discs are present on the spiracular tracheae in the third instar larvae. After pupation the tracheae poste- rior to the fifth spiracles exclude air and do not function for gas exchange. Cells of discs proliferate, migrate along the tracheae and replace larval tracheal cells to form adult tracheae. Pupal abdominal tracheae (PAT) which function during the pupal stage arise from the fourth and fifth spiracular tracheae in the third instar larvae. Growing through cell division, PAT rudiments become elliptic lobes lying on the larval tracheae (trans- verse connectives). The lobes swell and bulbous buds are formed on the side being in contact with the larval tracheae. After pupation PAT rudiments stand up and tracheoles are discernible in some cells of buds. Tracheole cells elongate and PAT formation is completed. The branching pattern of PAT consists of ‘leaves! (tracheole cells) and 'branches'. ‘Leaves! crowd at the tips of 'branches'. ‘Branches! appear to correspond to buds. Bud formation seems to reqire contact between PAT rudiments and the larval tra- cheae. MO 16 REPAIR AND REGROWTH OF THE DETACHMENT SITE OF THE CORAL FUNGIA FUNGITES H.Yamashiro', and K.Yamazato2. lRadioiso- tope Lab. and 2Dep. of Biol. Fac. of Sci., Univ. of the Ryukyus, Okinawa. Structural changes of the detachment Sites in both disc and stalk after detachment were studied in the hermatypic solitary coral F. fungites. Truncated end of the remained stalk was covered rapidly with intracalicular soft tissues within a day or two. Then the stalk regenerated the complete polyp with mouth and tentacles (about 10 days later). Regenerated skeletal elements on the stalk were formed by direct growth of corresponding structures in the former stalk. Ina disc, the detachment plane was covered with the intracalicular soft tissues and then thin calcareous plates (about 10 pm thick) expanded horizontally from the lower ends of the septa or columellae. Before detachment, more or less in most specimens, interseptal spaces already had been narrowed, filled up or plugged up, partly or fully, with calcareous matter caused by the thickening of the septa, columellae and synapticulae. When the interseptal spaces become to be small and also the skeletal pulverization is insufficient to detach, the soft tissues below the detachment plane are suffocated, degenerate and sometimes result in the death of the stalk. MO 17 THE DIFFERENTIATION OF THE NAUPLIUS EYE IN CYPRIS LARVAE OF A BARNACLE, BALANUS AM- PHITRITE HAWAIIENSIS BROCH. M. Takenaka, T.Yamamoto and M.Yoshida. Ushimado Marine Lab., Okayama Uiniiavien; Okayama. The nauplius eye in cypris larvae is symmetrical in form and composed of two Pigment cells and fourteen visual cells. The Pigment cells form three concave depressions. Visua] cells are correspond- ingly divided into three groups, two lateral and one ventral components. At the metamorphosis into the adult stage, the three components of the nauplius eye are separated and differen- tiate into the adult ocelli independently. Each lateral component develops’ into the adult lateral ocelli, forming newly pig- ment cells and tapetum cells, while the ventral component develops into the adult median ocellus. It is concluded that the adult photoreceptors are originated from the ocellar components’ of the planktonic nauplii. 1102 Morphology MO 18 MONOAMINE CONTAINING CELLS IN THE TASTE BUDS OF THE MOUSE VALLATE PAPILLA. M.Kudoh. Dept. of Biol., Fukushima Med. College, Fukushima. The taste buds in the vallate papillae of the mice were observed by means of fluo- rescence histochemistry (aqueous glyoxylic acid method) and electron microscopy (gly- oxylic acid-Mg-KMn0O,method). The specific fluorescence appeared faintly in the taste bud of both untreated and nialamide-treated mouse, and was intensively enhanced by the administration of the monoamine precursors such as 5-HTP and L-DOPA. Electron micro- scopic observations of OsO4-fixed materials revealed that by the injection of the amine precursors a large number of small clear vesicles approximately 40-60 nm in diameter appeared throughout the cytoplasm of the type III cells which are supposed to be the gustatory cells ,and these vesicles aggre- gated especially around Golgi body and in the presynaptic regions of the nerve termi- nals. Electron microscopic histochemistry demonstrated that after injection of the amine precursors,numerous small dense vesi- cles, of which the size and the density of the content are variable, are found among the small clear vesicles in the cytoplasm of the type III cells. It is suggested from the present observations that the type III cells may be capable of taking up amines and storing them in the small clear vesi- cles. The stored amines might play a role in neurotransmission in taste transduction. NGwS MORPHOLOGICAL OBSERVATIONS ON NEUROSECRE- TORY CELLS OF A FRESHWATER OLIGOCHAETE, TUBIFEX HATTAIL. H. Jaana. Biol. Lab., Asahikawa College, Hokkaido Univ. of Education, Asahikawa. The distribution of neurosecretory cells and the morphological characteris- tics of them were studied by light and electron microscopes. Their cytological activity was also examined during an egg- laying period. Cells stained intensely with paraldehyde-fuchsin were observed in a subpharyngeal ganglion (Sub.G) and ven- tral segmental ganglia (Seg.G), while weakly-positive cells were detected in the COc@oiee il Ceinglaoim (Ceie5sE)), Swo.G aime! Seg.G. Electron microscopy revealed three types of granular cells in the Cer.G (type Lp UE eincl LEU CSIs), WHO Oeln@r CWvjoeS Oit granular cells (type IV and V cells) were intermingled with type II cells in the Sub.G and Seg.G. Among them only type II cells showed morphological changes during an egg-laying period. The number of gran- ules of type II cells decreased after oviposition, while well-developed rough ER and Golgi apparatus emerged in their cyto- plasm. Several hours later, swollen parts of axons accumulating granules came to be observed frequently. It was suggested that the release of granules of type II cells may trigger the oviposition, or the release after oviposition may activate epithelial cells of the clitellum which have taken part in a cocoon formation. MO 20 STRUCTURE OF SEMINIFEROUS TUBULES AND TESTICULAR VASCULAR PATTERNS IN AGING CHINESE HAMSTERS (CRICETULUS GRISEUS). H. Ninomi al. K. Yamazaki? , S. Kondo2 and 6 HOIMN1OING g lDept. of Labo. Animal Sci., Azabu Win a7 5 Sagamihara, ? Technical Sect., Mitsubishi- Kasei Inst. of Life Sci., Machida, Tokyo. The age-related changes of the histo- logical structures and vascular systems in the testes of the Chinese hamster(17-43 months) were examined both at the light and electron microscopic (TEM and _ SEM) levels. Scanning electron microscopy was used for the examination of testicular vasculatures as their acrylic resin casts. The senile changes noted were: depletion of spermiogenesis, edema of interstitial tissues, tubular sclerosis, degeneration and/or hyperplasia of Leydig cells, dilation of seminiferous tubules, arterial stasis, and embolism. Degenerative changes were also observed in the endo- thelial cells of intertubular and peritu- bular capillaries surrounding seminiferous tubules. The observation on the resin casts showed that those capillaries were torn-off and strangulated. The aetiology and pathogenesis of these changes in semi- niferous tubules are briefly discussed in relation to blood supply. MO 21 REMOVAL OF COLLAGEN BUNDLES IN MURINE UTERUS DURING POSTPARTUM INVOLUTON. K.Shimizu,T.Harada,M.Hokano. Dept.of Anat. Tokyo Med.Coll., Tokyo. The localization of collagenolytic activity within the mouse uterus ( IVCS strain was investigated during postpartum involution.The rate of collagenase activity was measured by analyzing of tissue levels of hydroxyproline from the day of parturition to the 20th postpartum day ( Woessner's method ). Collagenase activity was high during the first two postpartum days. Collagen distribution in tissues was analyzed by viewing birefringence induced by the picrosirius technique ( Sweat's method ). An attempt was made to interrelate the quantitative analysis with the histologic distribution of collagen during the first two postpartum days. Histologic and quantitative evidence indicated that ( 1 ) the collagenous compartments of the endometrium and myometrium differ in their response to the postpartum rise in collagenase activity. Collagen degradation occurs primarily in the endometrium, i.e. the myometrial collagen remains but much of the endometrial collagen is removed. ( 2 ) Endometrial collagen is degraded particulary in the immediate subluminal compartment. Morphology 1103 MO 22 MORPHOLOGICAL VARIATIONS IN THE Kt hk bt S)H BRAIN PRODUCED Bay, DOMESTICATION VYousaco, Dept. of Anat.) Yokohama City. Univ. Sch. of Med., Yokohama The external and internal features of the brains of the medaka, Oryzias latipes and its albino form, the "hi- medaka", were examined. On the dorsal aspect the proportion of the size of the telencephalon to the whole brain in greater in the hi-medaka than in the medaka. But the proportion of the size of the optic tectum is greater in the medaka than in the hi-medaka. The optic tectum of these killifish shows the same strata as in other bony fishes: stratum marginale (SM), st. opticum (SO), st. fibrosum et griseum sup. (SFGS), st. griseum centrale (SGC), st. album centrale (SAC), and st. periventriculare CSIEWA)R The SO and SFGS received retinotectal afferent fibers. The SO and SFGS together occupy about 32% of the total tectal thickness in the medaka, but only about 26% in the hi- medaka. Since the hi-medaka is an artificially produced strain, these morphological variations imply that the structure of the optic tectum has been affected by domestication. MO 23 ON THE MORPHOGENESIS OF FREE NEUROMAST OF FRESHWATER TELEOST, ORYZIAS LATIPES. S.Nagai, M.Otsuka and K.Ishii. Dept. of Biol., Dokkyo Univ. School of Med. Mibu. The developmental process of the free neuromast in embryo and larva of Oryzias latipes alb. was studied using an electronmicroscope and a scanning one. Distinction of hair cells and supporting cells, and interlocking formation by desmosomes and invagination were observed at 120 hours after fertilization. In addition, initial formation of cupula pore with stereociliae and kinocilium was ob- served at 125 hours. The number of hair cells increased according to the develop- ment of embryo: one cell at 120 hours and 2 cells at 125 hours after fertilization; 5 cells just aikter hatching; ‘11 cells for-2= day old and 15 cells for 7-day old larvae, respectively. The formation of afferent neurons was found at 132 hours after fer- tilization while that of efferent neurons was observed just after hatching. Secretory granules in supporting cells were observed from 120 hours after fertilization. MO 24 A GOLGI STUDY ON THE CUTANEOUS NERVES AND THEIR TERMINALS IN THE SALAMANDER, HYNOBTUS NEBULOSUS. N. Iwahori, Dept. of Anat., Fac. of Med., Nagasaki Univ., Nagasaki. SEE ee a i ete ee be a The morphology of the cutaneous nerves and their terminals was studied in the sal- amander using the rapid Golgi method. The cutaneous nerves are derived from the spi- nal and some cranial nerves and travelled superficially among muscles as a number of discrete bundles which contained numerous axons of diverse diameters. Upon arriving at the dermal-epidermal transitional region, the cutaneous nerves swung in a tangential direction and branched out into numerous collaterals to form a dense fiber network. Many fibers emerged from the fiber network and proceeded toward the surface, branching profusely to terminate mainly in the epi- dermis. The majority of the fibers termi- nated as free nerve endings; only a few ended with some terminal expansion. The free nerve endings were seen in the middle and deep epidermal areas forming a dense terminal plexus, while the expanded tip terminals were impregnated mainly in the middle epidermal region. The distribution area of the terminals derived from one cutaneous nerve fiber overlapped to a con- siderable extent with that from other nerve fibers. In addition to the above fibers, in the upper dermal regions, a few fine fibers were seen to travel toward the mucous glands to terminate in conjunction with glandular tissue. MO 25 MORPHOLOGICAL CHANGES OF ACHR-AGGREGATES DURING MUSCLE REGENERATION OF URODELE. T.Tabuchi and S.Inoue. Dep. of Comp. Endocrinol., Inst. of Endocrinol., Gunma Univ. Maebashi. Acetylcholine receptor (AChR) -aggregates of urodele limb muscles were observed during development and regeneration. The binding of tetramethyl rhodamine-labelled a-bungarotoxine (TMR-aBT) to AChR, and the histochemical reaction for acetylcholine esterase were observed in the same sample. The materials used were the upper arms of the larval Hynobius nigrescens and the musculus flexer digitorum communis of adult Cynops pyrrhogaster. During the larval period of Hynobius, AChR-aggregates appeared in spot and/or plate forms. They became linearly arranged dring metamorphosis and then acquired the mature shape of the neuromuscular junction (n=m=3). During the regeneration of lower arms of Cynops, the labelling intensity of the n-m-j of stump muscles decreased to some extent throughout almost all the phases of regeneration. In the early period of regeneration, spot and plate form AChR- aggregates became lost. After 3 post- amputation weeks, they reappeared not only in the original n-m-j location in the stump muscles but also near the cut ends of muscles. These spot and plate aggregates were considered to form the new n-m-j through a similar way as in development. 1104 Morphology MO 26 A HISTOLOGICAL STUDY ON THE NERVE INNERVA- TION IN THE HEART OF CLEMMYS JAPONICA. H. Ishihara. Biolog.Lab., Fac. of Sci.& Engineer., Aoyama Gakuin Univ., Tokyo. The morphology of nerve innervation and nerve endings in the heart of Clemmys ja- ponica was investigated according to the Silver impregnation method devised by the present author. In the adventitia, the nerve plexus was relatively well developed. The nerve bundles which separated from fun- dus plexus were distributed over the adven- titia. The nerve bundles were covered with neuroplasmamass containing numerous nuclei. They were observed to run a wavy course. After repeating ramification, the nerve bundles became thinner and thinner in structure and then radiation was seen running to all direction. The nerve bun- dles which ha@ entered myocardium, togeth- er with capillaries or by themselves di- verged from adventitial plexus. The nerve bundles penetrated the muscle fiber, after repeated branching was seen to form rich plexuses. In this plexus, nerve cells and triangular cells attaching to the nerve fiber were frequently observed. A fine nerve fiber which ran paralel or obliquely in the muscle layer was detected. Various nerve endings were very often found in the myocardium and adventitia. Further, the fine nerve fibers were observed to run parallel to the capillaries or surrounding them. In addition, the nerve fibers bear- ing nuclei here and there were found to run in serpentine course. NCa27 INNERVATION OF THE SHORT THUMB MUSCLES OF THE FRUITBAT Pteropus medtus Khin Maung Saing. Inst. of Biological Scien- ces, Univ. of Tsukuba , Ibaraki 305, Japan. The thumb of the fruitbat found in Burma is well developed in structure and function com- pared to insectivorous bats. As the forelimb morphology in bats plays an important role in locomotion, functional specializations con- cerning gross anatomy, innervation and lipid content of the short thumb muscles of the fruitbat were examined. Three muscles i.e. flexor pollicis brevis, adductor pollicis and abductor pollicis brevis were present. All muscles were innervated by metacarpal nerve I derived from a single nerve’ trunk composed of combined median and ulnar nerves. All muscles had diffuse type of motor end- plates. Single motor endplates on_ single muscle fibres were found predominantly. But occasionally a single axon supplied two motor endplates on different muscle fibres or two diferent axons ended in two motor endplates on a single muscle fibre. Complex and inter- mediates types of neuromuscular spindles, Pacinian corpuscles’ and SHeineclated Golgi tendon organs were found.All spindles’ had nuclear bag and nuclear chain types of intra- fusal muscle fibres. Three extrafusal muscle fibre types ie. red, white and intermediate based on lipid content were noted. Correla- ship of function to structures in the thumb of the fruitbat is discussed with comparison to other mammals including insectivorous bats and monkey. MO 28 THE ULTRASTRUCTURE OF SIF (SMALL INTENSELY FLUORESCENT) OR SGC (SMALL GRANULE-CONTAI- NING) CELLS AND NERVE TERMINALS’ IN THE CARDIAC GANGLIA OF THE SHREW MOLE, Urotrichus talpoides. S.Kikuchi DAoeEc@Or*e BiloOlls 5 Selnc@ir illosAirctesS aincl Seis , Iwate Med.Univ., Morioka. The cardiac ganglia in the atria and inter- atrial septum of the shrew mole contain clusters of small cells which show a strong glyoxylic acid-induced fluorescence of dopa- min. Ultrastructurally these cells are char- acterized by large nerve terminals and an abundance of dense-cored vesicles in the cy- toplasm. These SGC(SIF) cells are in glomus- like structures within the ganglia in close proximity to capillaries, of which endothe- lial cells. are fenestrated | in) the vayeaoll contact with the SGC cells and their proc- esses. Each SGC cell with a thick process is innervated by at least one large nerve terminal, together with several small ones. A large nerve terminal frequently makes synaptic connections with two or three SGC cells surrounding it. Three types of synap- tic connections, afferent, efferent and reciprocal, ane) y found) yom (the oCGycolskse These characteristics resemble those of the SGC celblis {iin ‘the scarotid) body fandiwaomene body rather than the other autonomic gan- glia or paraganglia, suggesting a chemo- receptive function of SGC cells aineehie cardiac ganglia. The chemoreceptors may be not confined to the arotkic) Tecionssbuemmonge widely distributed in the heat. MO 29 WHERE DO NEURONS RELEASE THEIR CHEMICAL MESSENGERS? —— TRANSMISSION AND SCANNING ELECTRON MICROSCOPIC STUDY. Y. ENDO*, Dept. of Anatomy, Yamanashi Med. Coll., Yamanashi. Generally, it is believed that neurons release their chemical messengers toward the effector cells through a synapse. I investigated exocytotic release of secretory granules from the neurons of the posterior pituitary, adrenal medulla and enteric nervous system of the rats stimulated with perfusion of high Kt Ringer solution. Tannic acid-glutaraldehyde- osmium tetroxide fixation was applied. Unexpectedly, as far as observed, exocytosis of large-cored vesicles occurred at non-synaptic sites where no structural specialization of membrane was found. Scanning electron microscopy of the enteric nervous system using a HCl- digestion method indicated that varicose neuronal processes were exposed elswhere to the surface of unmyelinated nerve fascicles. Exocytosis of large-cored vesicles occurred not only at these exposed area but also at the interspace between neuronal processes and Schwann cells. *Present adress: Dept. of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606. Morphology, Behavior Biology 1105 MO 30 OBSERVATIONS OF LIMB WOUNDS IN MICE OF WHICH LIMB WERE SURGICALLY AMPUTATED T,Nobunaga! and S.Inoue?. J/iInst. for Exper. Animals, Tohoku Univ., Sendai and 2Inst. of Endocrinol., Gunma Univ., Maebashi. PRISMS SST Tr As previously reported, the wound heal- ing of the amputated limbs in the fetus seemed to proceed faster than the ampu- tated limb of the newborn mouse. The sur- faces of the amputated limbs in utero at 6 post-operative days showed slight moundings. We further studied the amputated limbs which were harvested at 6 and 49 days after birth, the limbs having been ampu- tated in utero at 13 or 14 days after mating in proestrus (in utero operated + 6 day-or 49 day-limb series). The external appearance of the in utero operated + 6day limb series was not so different from the amputated limbs in utero at 6 post opera- tive days. However, the limbs at 49 days after birth (in utero operated + 49 day limb series) were cone shaped, showing large amount of fibrocartilage-like callus formation at the ends of the amputated bones. BB 1 LEARNING ABILITIES AND OTHER BEHAVIORAL CHARACTERISTICS OF EXCELLENT AND DULL LINES OF ddY MICE. N.Mishima, Y.Maeda, T.Mizuta, R.Iguchi. Dep. of Biology, Fac. of Pharm. ,Tokushima- Bunri Univ., Tokushima. The present experiment was aimed to study behavioral characteristics of 8th generations of "Excellent line" (Ex-8) and "Dull line"(Dull-8) which were obtained by selective brother-sister breedings of ddyY mice exhibiting "good"and"poor" retentions in 1-trial passive avoidance task,respec- tively. In the retention test of the task, Dull-8 displayed significantly lower avoidance than non-selective ddy mice (control), but there was no difference between Ex-8 and the control. On the other hand, Ex-8 established much more quickly active avoidance task in T-maze and two appetitive tasks; Skinner box and radial maze than Dull-8. Although the two lines were selected by results of retention test but not acquisition session, Ex-8 had Superior learning abilities and Dull-8 had inferior abilities than the control in the all tasks as far as we tested. On the contrary, there are no significant difference in general activities such as wheel running activity or open field activity. These results suggest that there exsists significant differences in some characters specific to learning ability, but not general activity between the two lines. BB 2 ONTOGENETIC CHANGES IN LEARNING AND MEMORY ABILITIES OF ddY MICE IN AVOIDANCE TASKS. H.Nakano., R.Iguchi and N.Mishima, Dep. of Biol.,Fac.of Pharm., Tokushima Bunri-Univ. Development of learning and memory aloiv thes Orme mdGyY aamlce werner msiticiecin ain various tasks as a function of age. Passive avoidance task was acquired with Similar numbers of trials to adult mice, on the day of eye opening. However, the MCC Cl omcaySmolad) couldmnotileansmlactt ve avoidance tasks such as 1-way and 2-way iomaze tasks. shearnangs or "the two tasks became possible between 16-19 days and 18- 22 days, respectively. On the other hand, long term memory was scarecely established by 15 days old mice, but rapid improvement of the memory retention appeared between 17-19 days. These results demonstrate that avoudanece Kearning: Fand "tts menony are suddenly developed between eye opening and weaning. On the contrary, open field activity or wheeling activity, at weaning was about 50% of adult levels. Catechol- amine levels of brain were measured at various ageS since our previous data suggested possible involvement of dopami- nergic system in learning and memory mechanitom pe Ormadulit mice. s At. 20mdays DA was only 52 % and NE was 50% of each adult levels.These results show that emergence of learning and memory abilities are not dependent upon maturation of CA system or general activity. BB 3 FEMALE MICE PREFER ODORS OF AGGRESSIVE MALES. S.Hayashi. Dept.Biol.,Fac.Educ.,Kagoshima Univ. Kagoshima. A dominant male develops preputial glands and attacks subordinate ones cus- tomarily even in a population where a dominance relationship has been estab- lished. In this experiment, advantages of these aggressions and functions of prepu- tial glands were investigated . Preputial glands of a male mouse increased when the animal could smell of strange male odors and increased mark- edly when it could display aggressions. The development of the glands did not continue without males to fight with or attack. When 11 aggressive males were isolated for 24 days, the weight of their preputial glands decreased from 46.5 mg to 32.2 mg, on the average. On the other hand, female mice pre- ferred a box which had contained an aggressive male over that contained a submissive one .-in 5-min test. These female preferences for a dominant male disappeared after resection of the prepu- tial glands. Effects of preputialectomy were more noticeable in aggressive males than in submissive males. Aggressiveness of a male mouse and some adequate rivals seem to be essential to maintain developed preputial glands and sexual attractivity. 1106 Behavior Biology BB 4 HORMONAL FACTORS INVOLVED IN COUNTER- MARKING BEHAVIOR IN MICE. M.Daumae and T.Kimura. Dept. of Biol., Coll, oO Actes ancl S@ig, UmlWo OF WOsvo, Tokyo. Counter-marking in mice was inves- tigated by analyzing urine deposition pattern on filter paper previously marked with other male and female mice urine. Intact males deposited larger numbers of urine spots and more amount of urine on the area with female urine than on the area with male urine, but castrated males marked both area equally. Ovariectomized and androgenized females deposited larger number of urine spots than intact female, but they, as well as intact females, showed no marking preference towards pre- viously marked area with male or female urine. Neonatally androgenized females deposited larger numbers of urine spots in total than intact females, and marked more amount of urine on male urine area than on female urine area. Ovariectomy erase the marking preference for male urine in these neonatally androgenized females. Although further experiments are needed, these results suggest that (1)male-type urina- tion pattern (large numbers of small spots) is established by neonatal andro- gen, (2)preference for counter-marking on female urine area is induced by later androgen only in mice showing male-type pattern, and (3)estrogen possibly enhances counter-marking on male urine area. BB 5 ANALYSIS OF FEMALE-ATTRACTING ACTIVITY OF MALE MOUSE URINARY FACTORS AND PREPUTIAL GLAND SECRETION. ISG INalin@nnslyel" ,- 40. Kimura! and kK. Mori?. Dane5 Ce Nols, Collis OH Maes aiacl Sex, alate 2Dept. Qe Nene, Chino, PAGS NGieo yp Winsiyo Ox Tokyo, Tokyo. Two androgen-dependent compounds (dehy- dro-exo-brevicomin[I] and 2-sec-butyl dihy- drothiazole[II]) in male mouse urine are known to attract females when mixed with castrated male urine, but stereospecificity of these compounds has not been studied. As both enantiomers of [I] were synthesized by one of us(Mori), We compared the effects of each when mixed with castrated male urine plus [II](racemate). Although the study is still in preliminary stage, results so far obtained strongly suggest that the coexis- tence of both enantiomers is necessary for female-attraction. This is rather excep- tional and interesting case, since most of other pheromones show one-sided stereospe- cificity. We reported in the last Meeting that preputial gland secretion mixed in male urine is another essential factor for fe- male-attraction. To confirm this, we mixed [I] and [II](both racemate) with castrated or castrated-preputialectomized male urine. When tested with both mixtures, female mice significantly preferred the former. This indicates that factor(s) in preputial gland secretion which is probably androgen-inde- pendent is indispensable for attracting females. BB 6 IMPAIRMENT AND RECOVERY OF OLFACTORY BEHAVIOR FOLLOWING LESION OF THE ACCESSORY OLFACTORY BULB IN RAT. M.Ichikawa. Dept. Anat. & Embryol., Tokyo Metropol. Inst. for Neurosci., Tokyo. Wie) halve relpio ritieidiit halt listymiapierEe IGOCISC/EiNabAC elon OOCwWies iain wei mecia I amygdaloid nucleus(MAN) following denervation of fibers Erom accessory olfactory bulb (AOB)!*s '~. However, functional significance of synaptic reorganization following the denervation of afferent fibers has been unknown. To clarify the relationship between synaptic reorganization and functional recovery, as a first step, we studied the change of olfactory behavior following denervation of the AOB fibers. It is known that male rat shows the preference in odor of female rat. In the present situdy, thus; thie preference of male rat in female odor was examined before and after a lesion of the AOB. The preference decreased to 10 % of intact after the AOB lesion ( 6 - 10 days atten the Lesion): Thereat ten, the preference of AOB lesioned rat increased gradually and showed about 80 % of intact at 2 month survival time. The present study indicates that time course of recovery of odor preference after lesion of the AOB is similar to the time course of recovery of the synaptic density in MAN following a lesion of the AOB“. 1. Ichikawa, M. Zool. Sci., 3 (1986) 982. 2. Ichikawa, M. Brain Res., 420 (1987) 243 3. Ichikawa, M. Brain Res., 420 (1987) 253 BB 7 COLOUR LEARNING IN THE BLOWFLY, LUCILIA CUPRINA. T.Fukushi Dept. of Biol., Miyagi Coll. of Educ., Sendai. The compound eyes of flies provide a variety of spectral types of receptor that could be used in colour vision. By means of learning experiment, colour vision has been proven in Musca (Fukushi, 1976), Drosophila (Menne and Spatz, 1977) and Lucilia (Fukushi, 1985). What use does a fly make of its colour learning capacity under natural circumstances where objects with many hues of colour exist? The Australian sheep blowflies Lucilia Cuprina were trained to a colour by presenting a droplet of the sugar solution on a patch with various colours. Then, the flies were tested on visits to colours in the arena where four kinds of colour patches were arranged so that the fly encountered each colour in the same probability in random walks. The flies visited preferably the colour to which they had trained. The effect of training was most remarkable in blue and yellow. Green was discrimanated from blue but confused with yellow. The red-trained fly behaved in the test situation as black-trained one, suggesting that the fly is hardly discernible red. Behavior Biology 1107 BB 8 PHOTIC ENVIRONMENT, BIOLUMINESCENCE AND VISION.OF A SQUID, Watasenia scigtillans. Y.Kito ,,M,Seidou , M.Michinomae™ and A. Jokuyama , Dept of Biol., Osaka Univ. and Dept. of Biol., Kounan Univ., Kobe. Mechanism of bioluminescence of this squid has been clarified (1,2) and the light is thought as emitted from some fluo- rescent substance, excited by the lucifer- ase system. This paper suggests that bio- luminescence of the squid is not corres- ponded with blue monochromatic light of the deep sea environment but with a particular trichromatic vision recently found (3). Half bandwidth of light from large photo- phores oF the fourth arm was broad and 4600 cm (Em max:475 nm) as detected by spectrograph. Fluorescence from the small photophore of mantle were determined with microspectrofluorometry. Bandwidth and Em max were varied according to characteristic shape and color of the photophore. Then, fluorescent substance was extracted and Partially purified by HPLC. Two substances (Em max:460 and 490 nm) with MW<20K were found. Thus, various fluorescences from photophores are explained as emitted from mixtures of a certain proportion of the two fluorescent substances. The light may fac- ilitate mutual communication among species. 1. T.Goto in Marine natural products, Acad. Press, 1980. Peon, Proc. NAS.,82,(1985) 4629 3. S.Matsu et al., J.Gen.Physiol. in press BB 9 LARVAL RELEASE RHYTHM IN THE GRAPSID CRAB SESARMA PICTUM. M. Saigusa. College of Liberal Arts and Sciences, Okayama Univ., Okayama. Sesarma pictum is a terrestrial crab which inhabits banks at the seashore. Fe- males incubate eggs in early summer, and after a few weeks they release zoea-larvae at the water's edge. This study examined the daily timing of larval release in the laboratory. For this purpose ovigerous females were collected from the field at Kasaoka, Okayama Pref., and maintained under arti- ficial 24-h light-dark cycles. When they were exposed to a light regime whose phase was much the same as in nature, i.e. light-off at 20:00 and light- on at 5:00, the daily timing of larval release roughly coincided with the times of high water in the field. The correlation between the release timing and tidal cycles was recognized over 2-3 weeks. The phase of this free-running rhythm was clearly bi- modal, not unimodal. Both phases of this circa-tidal rhythm gradually delayed under a 24-h LD in which light-off was set at 1:00. However, it could not be confirmed whether the release timing was actually related to the onset of darkness, because of poor number of experi- ments. BB 10 THE LOCOMOTOR ACTIVITY IN THE HAGFISH, EPTATRETUS BURGERI, UNDER THE VARIOUS LENGTH OF LIGHT-DARK CYCLES. S.Qokal and H.Kabasawa2. latomi College, Tokyo, ?Keikyu Aburatsubo Marine Park Aquarium, Miure. the locomotor activity in the hagfish, kotatretus burgeri,was recorded by means of an infra-red light photocell systen. The animal showed nocturnal activity in 12L:12D and had clear circadian rhythm in constent darkness. ~The animals were exposed to various light-dark cycles greater and less than 24 hours which were gradually changed with 20 minutes interval successively for 7 months. The animals showed stable entrainment in 2441 hours. Entrainment to greater and less than 2441 hours produced @ gradual decrease in activity time, and an entrance of activity to light time. The activity crossed over the light time, then settled in the adjacent dark time. The animals showed similar "beats" through the experiments. The maximum range of locomotor activity cycles was about 24243 hours. These results are explained in correlation with the effect of light pulse on the length of circadian rhythm in a previous work. BB 11 LOCOMOTOR RHYTHM OF THE MUTANT CRICKET, GRYLLUS BIMACULATUS. K. Hamada, H. Ito and I. Nakatani. Dept. Of Biolks. hac. 5.01 SCL), svanagatasUniivicr, Yamagata. The mutant cricket with white compound eyes was segregated from normal _ black eyed one in our laboratory. Melanin granules do not exist in the iris and the retinular pigment cells of the white eyed cricket. The records of locomotor activi- ties of the crickets were done under the condition of two illuminating intensities (aboult > 2b0 6 pix © ‘and G00, © tlocc)r: jin.» ahi ght period and with (or without of | dark Shelter gin eae plastresbox, ine swhieh ja Cricket wis confined, jjrespectively.. Uhese records were continued from the stage of last instar nymph to two weeks after of the imaginal molt under 12-h light and 12-h dark photoperiodical condition at 24° C. Nocturnal rhythms in the wild cricket and diurnal rhythms in the mutant cricket were can not observed through the stage of larva to imago, while several mutant crickets showed nocturnal rhythms through larval to imaginal stage when they reared with shelter. The difference of locomotor rhythm due to illumination intensity was not remarkable. These results may be show that the compound eye of mutant’ cricket to illumination slightly sensitive’ than the wild cricket. 1108 EC a PLEVALENCE OF INTESTINAL HELMINTHS OF THE FROG( RANA BREVIPODA PORESA AND RANA JAPONI- CA) Y. Sasaki and N. Makino. Dept. of Biology, Tokyiow Medi (Cole LOO Prevalence of enteric helminths were parasitic on the frog(Rana brevipoda porosa and Rana japonica) were studied during a 3 month period (Sep. from Jul.) This investi- gation is a part of studies that are going on one year. Jaabizieyy abigoyofs} {(( Ievalel lois )) joeie month were captured at Inzai cho, Inba gun, Chiba Prefecture. Sex ratios of 150 frogs were approximately equal (48%6:52%9). Frogs (Rana j.) were collected small in number at the same place, and they were also examined in) Ondem sO, COmpanch= watch Ranawbr Per nae cestodes; Ophiotaenia sp. were observed at a low rate in the small intestine of Rana b.p..The nematodes;Oswaldocruzia sp. showed a high prevalence in the stomach and the duodenum of Rana j.. This nematodes were observed that they were laying eggs in September.The mature and immature trematod- es; Diplodiscus amphichrus j. were observed in the small intestine of Rana _b.p.and Rana Sloe The nematodes ; Cosmoserca japonica displayed a very high prevalence in the rec- tum of both frogs. There were many females. And we always observed eggs and larvae for 3 months. Females laid eggs in the physi- ological salt solution and larvae hatched out in no time. Interestingly, some frogs were parasitic on larvae only in the rectum. Above-mentioned effects were no significant difference between males and females of host. CZ THE ECOLOGICAL STUDY OF INLAND ANT-LION T.Ohhashi,Biological Institute.Hyogo Univercity of Teacher Education,Hyogo. Larvae of Hagenomyia micans MacLachian mainly constructed the pit under an over- hanging precipice along a path and in case of Myrmeleon formicarius Limme under the floor of a temple and a shrine.Both species inhabited frequently in the same place.It seemed that there was no differ- ence in habitat between the two species. It was observed that both species pass- ed the first winter in either the first or the second instar and second winter in the third(last)instar.It seemed to take three years from the egg to the adult in some cases.The reproductive activity of H.micans was a little later than that of M,formicarius. These ant-lion larvaes mainly captured ants.Third instar larvae of M. formicari- us living in larger-sized pits captured lager-sized and various prey. Ecology EC 3 SEXUAL REPRODUCTION IN FOUR SYMPATRIC SPECIES OF THE CORAL GENUS MONTIPORA FROM OKINAWA. fObie® ie T. Yeemin and K. Yamazato. Dept. of Biology, Univ. of the Ryukyus, Okinawa. The mode and timing of sexual reproduction, mating pattern and embryogenesis were studied in four species of coral genus Montipora, namely, M. aequituberculata, M. peltiformis, M. digitata and M. sp.1, on the reefs of Sesoko Island, Okinawa. All species had an annual gametogenic cycle and were simultaneous hermaphrodites, bearing male and female gonads in the same polyps. Three species released gametes on a few nights following the full moon in June and the other, M. peltiformis in July, August and September. Feggs (bearing zooxanthellae) and sperm were packaged in a single cluster in each polyp before spawning. Egg diameters ranged from 312 um (M. digitata) to 416 um (M. sp.1). Fertilization and development were external. Cross-fertilization was the major mating pattern. Only M. sp.1 was capable of self- fertilization. Embryogenesis and larval development were observed. Developmental patterns and rates were similar in all the four species. First cleavage occurred at approximately 2 hours after spawning. The majority of larvae had settled within 4-7 days in aggregates on small tiles and limestone substrate under laboratory conditions. EC 4 REPLODUCTIVE ECOLOGY OF A SCLERACTINIAN CORAL GALAXEA FASCICULARIS LINNAEUS (GAMATEGENESIS AND FERTILIZATION). M. Minei and K. Yamazato. Dept. of Biology, Univ. of the Ryukyus, Okinawa. Galaxea fascicularis has two types : Soft and Hard. In 1987, the spawning of Soft type was observed on June 17-19 in the freshly collected colonies and on June 18 and July 17 in the field, and that of Hard type on July 16-18 and August 15-17 in the freshly collected colonies. Main spawn- ing season of Soft type was June while that Hard type was August. All spawning occured about 7 days after full moon. G. fascicularis have been reported as herma- phroditic coral. However, our studies showed that G. fascicularis had female colony that contain eggs only in the mesentery and hermaphroditic colony that contain both eggs and testes in the mesentery. The ratio of female and hermaphroditic colonies are about 3 : 2. Male colony was not found. On both types almost all eggs from female colony could be fertilized and develop. However, eggs from hermaphroditic colony did not develop by self or cross fertilization. Thus, eggs from herma- phoroditic colony had no function. EG 5 DISTRIBUTION OF THE DEEP SEA SHRIMP,EUALUS BIUNGUIS IN THE JAPAN SEA. H.Ito. Nansei Reg.Fish.Res.Lab.,Hiroshima. Eualus biungquis is genarally known as a deep sea shrimp in the Northern Pacific Ocean. An ecological survey was carried out on the shrimp from 1969 to 1971 in the cen- tral areas of the Japan Sea. The results obtained are as follows: 1) The animal inhabits the coastal waters of Hokkaido,Honshu,and Peter the Great Bay. It is also distributed in offshore reefs and bank areas such as Yamato-tai,Kita- Yamato-tai,Shin-Oki-tai,and Hakusan-se. The southern extremity is located around 36°06' N and this may be considered the southern most distribution of the species in the northern hemisphere. 2) The shrimp is distributed in the strata from a depth 208m down to 1250m,but most frequentlly dwells within the depth range of 600m and 1000m. 3) Judging from the carapace length compo- sition of males and females,there is no evidence of sexual habitat segregation in the strata of the water depths EC 6 HISTOCOMPATIBILITY GRAFTING BIOASSAY IN A CORAL PORITES ( SYNARAEA ) RUS K. Miyara and K. Yamazato. Dept. of Biol. , Univ. of the Ryukyus, Okinawa. The screlactinian coral Porites ( Synaraea ) rus is living over approximately 1 km on the reef of south-eastern coast of Sesoko Is. , Okinawa. They were dioecious and made of six color-morphs. Twenty seven colonies containing either one or both sexes of five color-morph from this reef were used for histocompatibility grafting bioassay during six months. All iso-graft pairs ( N = 27, for control ) fused ( p<0.001 ), while allo-graft pairs ( N = 224 ) showed fusion reaction only in the case of the same color-molph with the same sex ( N = 65, p< 0.005 ) with few exception. Moreover the colony size ( in a diameter ) that could be expected to have been derived from fragments ( N = 87, mean diameter = 8.8 cm, measured within two weeks after the typhoon No. 12 ) tended to increase in number of colonies within each population. However, the number of small size colony that were expected to have been recruited from planulae did not increase. In conclusion, P. (_S. ) rus in Sesoko Is. occur in clonal populations characterized by each color-morph, which were derived from fragmentation. Ecology 1109 EC::7 MECHANISM OF DIEL ACTIVITY OF CONTRACTION AND EXPANSION OF A FAVIID CORAL, GONIASTREA ASPERA. Y.Nakano and K.Yamazato. Dept. of Biol., Univ. of Ryukyus, Okinawa. We studied the adaptive strategy and the me- chanisms of diel activity of contraction and ex- pansion of the polyps of a faviid coral, Goniastrea aspera. This coral occupies the upper limit of coral distribution, that is, the infralittoral subzone, and is found abundant on the reef near Sesoko Marine Science Center. The polyps of this coral contract during the day and expand during the night in the field. Our study revealed the following in the la- boratry: 1) They remained expanded under continuous light and contracted under continuous darkness. 2) When the light-dark periods shortened (L:D= 6:6, 3:3), the contraction-expansion periods be- came shorter accordingly. 3) Under the red or green light, they expanded. These results suggest that the contraction and expansion behavior of the polyps were directly con- trolled by light condition. However, the behavior was indirectly by the energy supply of zooxanthellae The behavior is closely connected with photo- synthesis. EG © REGRESSION OF THE VAEVULA CEREBE OF PERCIDA. K. UCHIHASI SEKIHAN LABORATOTY, AKASHI. As for the habitats of the species of Percida, I classified them into five, i.e., the surface layer, the ridge area, the middle layer, the bottom layer and the deep sea bottom, from each of which I got the sample. Next I made median longitudinal sections ( 15-20A) of valvula cerebelli, stained them with Nissl’s method and examined them. As the result of the survey, I found out that the shapes of valvula cerebelli of every one of the forms that have differenti- ated from primitive Thunnus alalunga to Allolepis. pollandi, are going on with regression. This fact shows the adaptive radiation of Percida, and I also presume that this is because there may have been the diversification of the oceans in- eluding the enlargement of the floors of the oceans during the Cretaceous periqd. Now, while Clupeida, which came into being during the Jurassic period, shows the progresive differentiation of its valvula cerebelli, Percida which appeared during the Cretaceous period shows exces~ sive regression. 1110 Taxonomy and Systematics ts i KARYOLOGICAL AND TAXONOMIC STUDIES OF THE DUGESIA SPECIES IN SOUTHEAST ASIA. XI. CHROMOSOMES OF DUGESIA JAPONICA JAPONICA AND DUGESIA SP. FROM TAIWAN. S.Tamural, I.Okil, M.Kawakatsu2, M,Takai%, K,-Y.lue4, H.Hori5, A.Muto6, and S.Osawaé. 1jsaka Pref.Inst.Publ.Health, Osaka, ?Biol. Lab., Fuji Women's Coll., Sapporo, © Biol. Lab., Saga Med.Coll., Saga, #Natn.Taiwan Normal Univ., Taipei, Taiwan, 5Gen-Iken, Hiroshima Univ., Hiroshima, and SLab.Molec. Gens, Depu Biol. Paciisci. j Nagoya Univ), Nagoya. The animals of Dugesia species from ad- ditional 5 localities of Taiwan were ex- amined cytologically. The 8 karyotypes of animals of Dugesia japonica japonica were found from 4 local- ities (Uulai, Hsiaoi, Liukuei, and Chihpén Hot Spring). They are as follows: 2x=16, 3x=24, (3x=-1)4+1LB=24 (3x-1)+1LB+SB=24+SB, (3x-1)4+2LB=25, 3x & (3x-1)4+7LB=24 & 24, 3x +1LB & (3x-1)+2LB=25 & 25, and (4x-1)+2LB & (3x-1-1)+3LB=25 & 25. The mixokaryotypes were found in animals from Uulai, Hsiaoi and Chihpén localities. The chromosome numbers (including no. of SB chromosomes) of Dugesia sp. from Lankhstiyistand ware las) FoMlowseN Se 19) k&e2d i 25, IS) by 2a & 25% 2, siacl 19 & 24 ee 2h & 25 & 26. The karyotypes are unidentified yet. Judging from the shape of each chro- mosome of Dugesia sp. of Lanhsti Island, the species is different from D. japonica and Dugesia sp. (species of Taiwan) re- ported in 1979 (Kawakatsu et al.). WS 2 KARYOLOGICAL AND TAXONOMIC STUDIES OF THE DUGESIA SPECIES IN SOUTHEAST ASIA, XII. A SYNTHETIC REPORT ON THE TAXONOMY OF FRESHWATER PLANARIANS FROM TAIWAN. M.Kawakatsul, I.0ki2, S.Tamura?, M.Takai?, K.-Y.Lue4, H.Horid, A.Muto6, and S.Osawaé. Ipiolstab., fuga Womens (Coli; \Sapporo, 26saka Pref.Inst.Publ.Health, Lab., Saga Med.Coll., Saga, 4 Natn. Taiwan Normal Univ., Taipei, Taiwan, 5Gen-Iken, Hiroshima Univ., Hiroshima, and ®Lab.Molec. Gen., Dept. Bole, Pactser.5) Nagoya Univ, Nagoya. For the past 20 years, the freshwater planarian fauna of Taiwan and the karyo- logy of this animal group have become very clear. Dugesia japonica japonica Ichikawa et Kawakatsu, 1964. This species is commonly found both in the lowland and alpine re- gions of Taiwan. Its chromosome number is essentially n=8 and 2x=16. Some other homo- and mixokaryotypes were also found. D. j. ryukyuensis Kawakatsu, 1976, is not distributed in Taiwan. Dugesia sp. (species of Taiwan). This species 1S only recorded from the swampy places in the vicinity of Taipei. Its karyotype includes a pair of subtelocen- tric chromosomes (2x=16). There is a high possibility that the species is identical with D. austroasiatica Kawakatsu, 1985. Dugesia sp. (species of Lanhsti Island). Although its karyotypes are unidentified WU, wilds SOSeLES sley Cimilyy aceybwacl alsa Ieualasl Island now. Sakale Bios IS, 8 HYBRIDIZATION EXPERIMENTS IN CYPRINIDA (II). CROSS BETWEEN CARASSIUS CARASSIUS AND CTENOPHARYNGODON IDELLUS. M.Kasama and H.Kobayasi. Dept. of Biol., Jap. Wom- en's Univ., Tokyo. Intergeneric cross was performed with Carassius carassius and Ctenopharyngodon idellus . About 41 % of eggs produced from this cross was developed into embryos. Almost all of embryos hatched, but many of hatched larvae suffered from edamatous syn- drome, bend body and circulatory failture. Finally 56 hybrids could be reared until the adult form. Forty-one of them were analyzed morphologically and karyologically at the age of about one and a half years, and the others were subsequently reared in the laboratory. Based upon 17 characters selected on the basis of marked and consistent differences between paren- tal species, the hybrid index was within the range considered intermediate. But only 4 of 17 charac-— ters were intermediate, and 10 were closer to Cc. Carassius. The phenotype of hybrid was more simi- lar to C.carassius than intermediate. The chromosome number of all hybrids was 124. These chromosomes were distinguished into 28 meta— centrics, 50 submetacentrics and 46 acrocentrics, which was karyotype incorporating two genomes of Cc. Carassius and one of C.idellus. These data confirm that the hybrids are rather similar to C.carassius than intermediate. And it seems that the trend of resembalance to C.carassius in almost all characters results from the un- balanced reception of inheritance from parental species. TS 4 MARINE HYDROIDS FROM OKINAWA ISLANDS. M. Yamada. Biol. Lab., Oshamambe Branch, Science University of Tokyo, Oshamambe, Hokkaido. The marine hydroid fauna of Okinawa Islands was studied chiefly in the vicinity of Sesoko Marine Science Center of the Univ. of the Ryukyus in 1985-86. Four athecate and nine thecate species were able to be identified. In addition to them over ten Species were found, but these were not identifiable owing to their lacking of gonophores. Most of the species identied are the species already known from the Japanese waters but two ones are newly added to the Japanese fauna. Among the species identified the biogeographically interesting ones are as follows: Asyncoryne ryniensis Warren which is ever known from South Africa and the Seychelles is first recorded from the Pacific; Myrionema amboi- nense Pictet known from the Philippines and the Indo-Pacific is also first recorded from Japan. The finding of Eugymnanthea inqui- lina is the southernmost record from Japan. It is indicated that the marine hydroid species in Okinawa Islands are mostly temp- erate or subtropical ones and no boreal species were found. Taxonomy and Systematics BLT TS 5 URBAN TARDIGRADES IN HOKURIKU AREA. K.Utsugi. Dept. of Biol.,Tokyo Women's Medical College, Tokyo. For studying the urban tardigrades, mosses or lichens were collected at 105 stations from 11 cities in the Hokuriku area (Tsuruga, Fukui,Kanazawa,Nanao,Takaoka, Toyama, Uotsu,Itoigawa,Joetsu,Kashiwazaki and Niigata) during a period from 1984 to I SXSV7 he The) presence of tandigrnades were micKo- scopically examined in the precipitates after macerating the mosses or lichens for a few hours. Then most of the specimens were treated with heat and mounted in Gum- chloral for phase microscope observation. Some of them were fixed in 5 % formalin after heat treatment and prepared for SEM observation. isespeetes were found at 53 Stations iin 11 cities. 6 species including Macrobiotus harmsworthi, M.hufelandi, M.intermedius, M.occidentalis, Milnesium tardigradum and Echiniscus japonicus were commonly found in many stations,however,the other 13 spe- Cies including M.richtersi, Hypsibius sp. H.dujardini, Isohypsibius schaudinni, Diphascon scoticus, Echiniscus baius*, Eve lapponicus, E.rugospinosus*, E.testudo gquadmriibiss; Pseudehiniscus suillus, Pp. facettalis, P.ramazzotti £.facettalis and Cornechiniscus lobatus were rarely found in urban areas. Among these tardigrades, three species indicated by "*" were reported for the HUTESE TiS Win Geyorywos Ts. 6 BIOCHEMICAL EVIDENCE FOR THE GENETIC DIS- TINCTION BETWEEN Ciona intestinalis AND Ciona savignyi. IN5 IG), Bee Kobayashi2 and K. Numachi2. \Dept. of Biology, Nagoya Univ., Aichi and Otsuchi Marine Research Center, Ocean Res. Institute, Univ. of Tokyo, Iwate. Ciona intestinaris and C. savignyi are distinguished from each other by several minor but distinct feature (Hoshino and Nishikawa, '85). An electrophoretic in- vestigation was designed to obtain further information on the taxonomic and genetic relationship between these two morphologi- cally similar species. Mature specimens of both species were collected at Onagawa bay. Ovary and intestine were mixed and homoge- nized, then these supernatant were subjected to horizontal electrophoresis in 12.5% starch gel (6mm thick). Gels were holizon- tally sliced into 1mm thick pieces and stained in the required fashion. MDH, AK and EST-2 showed a single (monomorphic) bands and all of their mobility were different between species. In ICDH, single- and double-banded phenotypes which corresponded homo and heterozygous state were detected. These bands showed same mobility between species. Polymorphic patterns were also obtained in PHI, EST-2 and catalase and the mobility of their bands was different between two species. Theallele frequencies of seven loci were calculated from each 32 individual samples. Based on these data, the genetic distance (Nei,'72) between Ciona intestinaris and C. savignyi was computed at 0.869. TS 7 GENETIC POLYMORPHISM DEDUCED FROM RESTRIC- TION PATTERNS OF DNA IN THE GENUS, CIONA K. Owada and M. Hoshi. Biol. Lab., Fac. of SiGe LOKVO LAS E LO WEOMNAE Ma 7 WONESVOe Genetic polymorphism was studied in the genus Ciona; C. intestinalis from OsSuchay, Yamada, Misaki, Nagoya, Toyama and Maizuru and C. savignyi from Asamushi, Yamada and Misaki. Nuclear DNA extracted from indi- viduals was digested with Bam HI. The deg- radation products were electrophoretically analyzed by ethidium bromide (EtBr) stain- ing and by Southern hybridization. A 6.6kb mouse rDNA fragment including 18S and 28S DNAs and the non-transcribed spacer region (NTS) were used as a probe. Polymorphic patterns were observed in EtBr-stained gel. The visible bands sug- gesting highly repetitious sequence were Clceececl Aer Ac8p Go, Sosy VoO, a AaOueuac! 17.0kb. The bands of 2.8, 6.5 and 9.0kb comprised rRNA gene. The repetitious bands were observed in most of C. savignyi but iin Mess} elon “SO Ce Cy salaciesonsul sus, The rDNA 18S-28S probe detected five INeVNGlss Cue Bosse Oso, 7s, Os5. Ave 2 Olid. These patterns showed variability of the NTS within rDNA repeating units. The 7.4kb band was found at Misaki and 8.5kb band at Asamushi in C. savignyi. This diversity suggests a heterogeneity in the sequence and/or length of NTS. Restriction patterns are remarkably dif- ferent among the populations from differ- ent localities, which suggests a regional isolation of Ciona reproduction. IS @ BIOCHEMICAL STUDY ON THE TAXONOMIC RELA- TIONSHIP OF THE TWO MORPHOLOGICALLY VERY SIMILAR SEA-URCHINS, ECHINOSTREPHUS ACICULATUS AND E, MOLARIS, Nc Matsuoka and jl, Suzuki... Dept. of Biol. BAC tO es Caney aliihnnO Sakis Und vee bico'sakey. The genus rchinostrephus of the family tchinometridae (Echinoida: Echinoidea) comprises only two living sea-urchin species, They are E,aciculatus A,Agassiz and E,molaris(Blainville). These two species are very similar morphologically and therefore have often been confused with each other, The taxonomic relation- ship of these two morphologically very Similar sea-urchin species was examined by means of gel electrophoresis of 18 different enzyme systems, The results demonstrated that the two species shared common allozymes at 25 of the 26 genetic loci studied here and that the Nei’s genetic identity value(I=0.963) obtained between the two species was comparable to those already reported between conspecific local populations of many other animals, Their genetic similarity verified by the present electrophoretic study and their morphological and ecological similarity strongly suggest that ELE, aciculatus and E, molaris may belong to one and the same species, 1112 Taxonomy and Systematics Teg APPLICATION OF A TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS METHOD TO SYSTEMATIC STUDY OF LAND SNAILS IN SUBGENUS LUCHUPHAEDUSA. J.Miyazaki, R.Ueshima, and T.Hirabayashi. imeiesg Iwao >- S@Gitse, Windy, Ov Wsukwloa, Ibaraki. Thie) Halduilal tom) spi Olcle/sis manna: aiteine phylogeny of land snails in subgenus Luchuphaedusa are intriguing subjects, since its distribution in the southwestern Japan is peculiar and is not interrupted by biogeographical boundaries suggested by other examples, and the phylogenetic relationships of members in this group are not clear. We compared by 2D PAGE the large form of Tyrannophaedusa (Luchuphaedusa) ophidoon with 8 members of this subgenus and 2 species of different subgenera of the same genus, and obtained the similarity ranging from 0.989 to 0.667. The similarity between the lbange fEonrm and 2. (Decolliphaedusa) bilabrata was lowest, while two species of the other subgenus, Nesiophaedusa, were very similar to the large form. The difference was very small between two specimens of the large form. The small form of T. (L.) ophidoon was significantly different from the large form. We estimated the relationship of the large and small forms to be closely related species or distantly differentiated intraspecific populations by comparing our result with available data obtained previously. iS 10 A NEW SPECIES OF GENUS JULIA FROM OKINAWA ISLAND S.Mizofuchil and T.Yamasu2 IDept.Biol., Coll. Sei., Univ. Ryukyus, ‘Div.Gen.Educ., Univ. Ryukyus, Okinawa. Several hundred living specimens of genus Julia were collected at Sunabe, Okinawa in 1986 and 1987. We found about 20 specimens’ showing some different characteristics from other Known species of the same genus. General morphology of these specimens is similar to that of J. mishimaensis except for some remarks. The specimens are light greenish yellow both on the body and the shell. Several red spots are scattered near the top of each valve. Each rhinophore provide with double brown bands at the mid portion. exclusively and not on C. ambigua as the other four species of the same genus did. Spawning habits of the adults are closely similar to those of J. mishimaensis. Shape of radular teeth are different from those of other species. The tooth is wider in width and thinner in thickness. Comb-like sculpture on the lateral sides of the teeth is faint than those of the other species. Considering morphological features and habits of the Specimens, it seems to be reasonable that the specimen is a new species in genus Julia. TS lal A NEW SPECIES OF MARINE INTERSTITIAL OSTRACODA OF THE GENUS PSAMMOCYTHERE FROM KUSHIRO, HOKKAIDO. So ielalieuhwels IealOils Lalo, Musiaaliee Colleaas Hokkaido Univ. of Educ., Kushiro. A new species of the genus Psammo- cythere (Ostracoda) was collected from a sandy shore at Kushiro, Hokkaido. This is the fourth record of the genus from the world. The new species resembles Ps. santacruzensis Gottwald, 1983 from Gala- pagos in appendage morphology, but the structure of carapace is clearly differ- ent from each other. The family psammocytheridae which con- tains only the genus psammocythere is considered as a sister group of all the other cytheracean groups, because the former has four-segmented endopodite of the legs (except leg 1 in male), while the latter has three-segmented endopodite (Gottwald, 1983). The primitive character (four-segmented endopodite) has been pre- served in interstitial environment. Judging from the worldwide distribution of the genus Psammocythere and the fossil record of the superfamily Cytheracea to which the genus belongs, it is considered that the genus would colonize in marine interstitial habitat during the lower Mesozoic at the latest. IS 12 ON THE ANAL OPERCULUM OF HARPACTICOID COPEPODS. Y. Kikuchi. Itako Hydrobiol. Stat., Fac. OL Scie, ehbarwaki Umino) lvalko—macinnan For several years, I have examined on morphology of anal operculum of freshwater and terrestrial harpacticoid copepods using SEM and phase contrast microscope. The anal opercula are various in shapes: semi-circular, triangular, transverse narrow plate and comb. Some of the anal Opercula being semi-circular attached to one to five spines on its hind margin, for instance, as Epactophanes richardi. Elaphoidella grandidieri, semi-terrestrial species, have comb-sShaped anal operculum of muscular membrane. The anal opercula of several species of genus Canthocamptus were transverse narrow plate, triangular and semi-circular. Consequently they were able to be distin- guished in each species by the morpholo- gical differences of anal operculum. Functions of anal operculum are a little known. It seems that anal operculum shields harpacticoid anus from harm or danger. Anal opercula of terrestrial harpacticoid cope- pods trended to elongate by comparison with freshwater ones. It has been suggested, or implied that anal operculum is advantageous for preventing evapolation from anus in low humid conditions as leaf litters of LOMOSU Taxonomy and Systematics 1113 Ts 35 THE PREZOEA STAGE IN DECAPOD CRUSTACEANS R. Quintana and K. Konishi. Zool. Enst<, Fac. of Sci., Hokkaido Univ., Sapporo 060. Some basic problems on prezoea larva are reviewed. A discussion on misunderstand- ings due to its nature and very short duration (normally few minutes) is made. Recent evidences have proved that the prezoea occurs as a normal stage in the ontogenic development of a wide variety of decapod crustaceans. Transverse sections of the prezoea stage of Pagurus brachiomastus (Thallwitz) were made to determine the fine structure of the prezoeal cuticle and the exoskeleton of the first zoea, by using transmission electron microscopy. The thin cuticle covering the newly hatched prezoea is 15- 17 nm thick, which is approximately 24 times thinner than the zoeal cuticle. High resolution revealed a trilaminar structure of the prezoeal cuticle, which is composed of an outer layer, approxi- mately 4.4 nm thick, a mid lighter layer, 3.5 nm thick, and an inner layer, 7.7 nm Ehaiek: During the prezoeal stage, the inner zoeal exoskeleton consists of an epi- cuticle and exocuticle, measuring appro- ximately 0.36 um thick. At this stage, there were no indications of the formation of endocuticle. TS 14 IDENTIFICATION OF BIFURCATE PARAOCULAR PROCESS AND POSTOCULAR FILAMENTARY TUFT OF "CYPRIS Y" (CRUSTACEA ) T. Ité?, M. Takenaka?. ‘Seto Mar. Biol. Lab., Kyoto Univ., Shirahama, Wakayama, ?Ushimado Mar. Lab., Okayama Univ., Ushimado, Okayama. Ultrastructures of the bifurcate paraocular process and the postocular filamentary tuft in "cypris y" were examined using TEM. The bifurcate paraocular process is accompanied proximally with a mass of complex lamellar structures that are apical branches of cilia arising from what appear to be nerve cells, and each cilium contains 9+0 microtubules. It is concluded that the bifurcate paraocular process is an external portion of the organ of Bellonci, which is well known in cirri- pedes and some other crustaceans. There is little doubt that the bifurcate, frontal filaments known in nectiopodans are also an external portion of the same organ. The postocular filamentary tuft is continuous with two kinds of compactly aggre- gated, prominent cells, in which one is charac- terized by the possession of dense mitochondria, the other is characterized by granular inclusions. The filaments rise from the latter kind of cell. This organ, although it is not connected with any duct, is similar in ultrastructure to the cement glands known in cirripede and rhizocephalan cyprids. | DISCOVERY OF A NEW SPECIES OF THE PHYLUM LORICIFERA FROM THE IZU-OGASAWARA TRENCH Y.Shirayama! and R.M.Kristensen*. ! Ocean Res. Inst., Univ. of Tokyo, Tokyo, “Inst. Cell Biol. Anatomy, Zool. Inst., Univ. Copenhagen, Copenhagen @, Denmark A new species of the most recently described phylum, Loricifera Kristensen, 1983, was discovered from the red clay collected at ST. 9 of a cruise of RV Hakuh6 Maru, ORI, UT. The station was situated close to the axis of the Izu-Ogasawara Trench at a depth of 8260 m. The present species was found to belong to the genus Pliciloricus (Pliciloricidae) and is the most closely related to P. profundus Higgins and kristensen, 1986 in the presence of single pair of P-flosculi, the type of mouth cone and clavoscalids, and the claw-shaped spinoscalids. Unique to the present species is its bulb-like shape of the lorica, the mucous coat of the larval lorica and the 30 sculp- tured plates in the adult thorax. This is the first record of loriciferans from the Indo-Western Pacific area. In addition, the habitat of the present species is completely different from the other congeneric species. Though the former was dis- covered from very fine sediment of the hadal depth, all de- scribed species so far have been collected exclusively froin sublittoral coarse sands off the southeastern coast of the USA. Moreover, in contrast to the interstitial nature of the other species, the present species probably burrows within the sediment. These findings suggest that loriciferans have great ability to adapt to various kinds of habitat and the phylum is distributed ubiquitously throughout marine environments. TS 16 THE STRUCTURE OF THE DIGITS IN ORZELISCUS TARDIGRADA AND ITS TAXONOMIC SIGNIFICANCE H.NODA (SETO MAR.BIOL.LAB.,KYOTO UNIVERSITY SHIRAHAMA, WAKAYAMA. ) The detailed structure of the digit of the genus Orzeliscus is described. Digit is composed of four parts; a stump-shaped proximal portion of the stalk rising directly from the tibia, a round distal portion of the stalk, a ventral club-shaped swelling, and a dorsal rod forming itself a middorsal ridge of the swelling. The swelling is somewhat depressed dorsoventrally and is fringed with a narrow membrane. Some previously unknown similarities in legs are recognized between Orzeliscus, which was formerly belonged to Batillipedidae but which currently belongs to Halechiniscidae, and Batillipes (Batillipedidae). "E,toe disc", "D,brace”, "C,distal stalk enlargement" and "A,distal portion of stalk" in Batillipes in the sense of McKirdy(1975) correspond to the ventral swelling, the dorsal rod, the distal portion of the stalk and the proximal portion of the stalk in Orzeliscus, respectively. Based upon this observation, the taxonomic problems between the two families are discussed. 1114 Taxonomy and Systematics Say SYSTEMATIC POSITION OF THE FAMILY MARINONISCIDAE (CRUSTACEA: ISOPODA) N.Nunomura. Toyama Science Museum, Toyama ee eee A new family Marinoniscidae was created for the specimens collected in the tidal zone, Amakusa, Kyushu and later the second species was collected in the supertidal zone of Okinoerabu Island, Ryukyu Islands. Judging from absence of pseudotracheae and having a single genital papilla, Marinoniscidae should be included in the group, Crinocheta-Oniscoidea. Among this group, Marinoniscidae resembles the families Scyphacidae, Halophilosciidae and Olibrinidae both in many morphological features and halophil habitat. Marinonisc- idae has some primitive features such as long 6-7 segmented flagellum of antennae, presence of 12 teeth on the outer lobe of maxillule and presence of distinct inci- sions on palp of maxilliped, otherwise, Marinoniscidae has also some advanced features such as a Single tuft of molar process of mandibles and presence of noduli lateralis. From the results compared above, Marinoniscidae and other above-mentioned families considered to have been derived independently from some SwOC|< O12 WaljfoyllOCaiSwey ali MOw SOQ jweOSSianw wWEVES ¢ US dis ON SOME ISOPOD CRUSTACEANS FROM THE JAPAN TRENCH. S. sGamoOnm Dept sor eB iolis,) onach yormeEcwcry, Yokohama Natl. Univ., Yokohama. Four new deep-sea asellote isopods were taken from far off Kinkazan by the marine biological research of the Japan Trench and its vicinities, which was carried out by R/V Hakuho-Maru of the Ocean Res. Inst., Univ. of Tokyo, during) the ecxuise KH-81-4 (6 Jul.= 4 Aug., 1981). The speci- mens were collected by beam-trawl and box- corer. I) Sp Macnostyiiisitsp.) y(A) ie eaciesema Keys e/a mm long, collected from 6386-6450 m deep. Body compact, elliptical in dorsal view, more than 3 times as long as its width. IVS) ENOSShe5 (7) We Sq (eon 2 Teele Boz mm, from 6380 m. Body compact, elliptical, nearly 2 1/2 times as long as its width. Eye absent. These species differ from the other members of the genus by their ellip- tical shape of body. 3) Janirella sp., a female, 2.6 mm, from 6380 m. Body ellipti- cal, with sparse spines. Each body segment has 1-3 lateral projections with 2-3 api- cal spines. Eye absent. This species is allied to di, seuberculatagbanste in) 9163), from 5350-7817 m. 4) Janirallata sp., a female, 10.9 mm, from 1890-1900 m. Body flattened, oblong. Each body segment has 2 lateral projections with serrated borders and 1-2 long apical setae. Eye present. WouNs Goeeles CKesouloles Wo Seyeiesicel Bilie= SeSiin,g LVGSI, seco WAIL im Ses ALPHEID SHRIMPS (CRUSTACEA, DECAPODA) FROM THE PACIFIC COAST OF N.- AND S.-AMERICAS IN THE COLLECTION OF THE ZOOLOGISCHES MUSEUM, UNIVERSITAT HAMBURG, W. GERMANY. Y. Miya. Fac. of Liberal Arts, Nagasaki Univ., Nagasaki. In the collection of the Zoologisches Museum, Universitut Hamburg there are found 12 species of Alpheidae collected from the Pacific coast of N.- and S.-Amer- icas. They were taken from various local- ities from San Pedro, California southward to Tocopila, N. Chile in date from the 1890s to the 1910s by captains of merchant ships for the Museum Godeffroy. Then they were placed under the control of the ZMUH. During the World War II the museum was de- stroyed, but the specimens were stored away in a safe place. The species are listed as follows: Alpheus clamator, A. cf. chilensis, A. cristulifrons, A. cylindricus, A. formosus, A. lottini; Synalpheus charon, S. fritz= muelleri, S. spinifrons, S townsendi brevi- spinis, Betaeus emarginatus, B. truncatus. Among them A. ai tetatiteone: and A. formosus are newly recorded from the Pacific coast and should be enumerated as the amphi- American species. Morphological charac- teristics of S. €. brevispinis, wilenmers represented by a young male specimen (6.8 mm in body length) from Iquique, Chile, are closely compared with 3 other subspe- cies. is 20 GENETIC AND ENVIRONMENTAL EFFECTS ON THE VARIATIONS IN REPRODUCTIVE CHARACTERISTICS IN HYNOBIUS NIGRESCENS Hisashi Takahashi and Hisaaki Iwasawa. Biol. Inst., Niigata Univ., Niigata. In 6 populations of H. nigrescens living at various altitudes, and with various spawning dates, the relationships of clutch size, egg size and total egg volume to female body size were studied. Body size significantly differed among populations, being larger in earlier breeding populations. There waS a nega- tive correlation between body size and total egg volume in all the populations examined. On the other hand, as for the relationship of clutch size and egg size to body size, the populations were divided into 2 groups; the body size correlated with clutch size in one group, and corre- lated with egg size in the other one. Adjusted means of clutch size, egg size and total egg volume, which mean the aver- ages except for the effect of difference in body size, significantly differed among populations. It is conceivable that the interpopulational variations in the adjusted means of clutch size and egg size are caused by the difference in the allocation of ooplasm among populations. These phenomena may be due to genetic factors. It seems that the difference in the’ adjusted means of total egg volume among populations is affected by environ- mental factors, such as breeding periods. Taxonomy and Systematics 1115 Tsg2t MORPHOLOGICAL FEATURES OF HYNOBIUS SP. (URODELA) OF CENTRAL JAPAN. H. Nambu. Toyama Science Museum, Toyama. Morphological features of Hynobius sp. collected from the mountain areas of Japan Sea side of Central Japan were studied comparing with species belonging to "nebulosus group", H.lichenatus, H. tokyoensis, H.nebulosus and H.takedai. The present species is characterized by unique skull feature, unique shape of vomer teeth series, less number of teeth and lack in the 5th toe of hind limb. Nasal, prefrontal, frontal and vomer of this species are not developed, but pari- etal is well developed. Skull length of this species is apparently smaller than that of H.lichenatus. In vomer teeth series of this species, outer branch is short and principal branch is narrow in comparison with that of the other species compared. Number of vomer, upper jaw and mandible teeth are smaller than that of the other species compared. The 5th toe of hind limb is almost lacking, although in other species, the 5th toe is developed. Number of trunk vertebrae of this species, H.lichenatus, H.takedai and H.nebulosus from Tottori are 17. However, that of H. tokyoensis and H.nebulosus from Okayama and Nagasaki are 18. From the results obtained, it is concluded that the present species is apparently discernible from the Hynobius species hitherto described and is new to Science. ts 22 CHARACTERISTICS OF ADAPTATION TO TORRETIAL ENVIRONMENT FOUND IN BUFO TORRENTICOLA LARVAE. Hisaaki Iwasawa and Kenichi Saito. Biol. Inst., Niigata Univ., Niigata. HEC Pol. seOrLnenticola s(Bot.) and of B. japonicus formosus (B.j.f.) were reared at 8, 18°C and room temperature, and the larval morphology of the two species was compared. iim WOUmMG Io 15 Merwe, ielne external gills were remarkably under- developed. In the swimming larvae, the body was rather depressed, and well- developed musculature and poor fin were seen in the tail. The tail was rela- Eaveby sShoneEer in B. &. The oral part was strikingly well-developed in B. t.; the area of this part was ca. 8 mm’ in full- grown larvae (St. 41), and this value was Seceeumess Larger, than in B...j. £... The labial processes were well-developed and the average number was 139 in the larvae Ghse-w40),, and, was)8.4, in, Be. jis, Lf. im .the Same stage. The neuromasts were well developed in B. t. Metamorphic changes in the oral part occurred more suddenly in B. t. At 18°C, B. t. larvae metamorphosed on the 52nd day after fertilization on average, and B. j. £. ones on the 45th day. The adaptation to torrential environment in B.t. larvae was seen most clearly in the sucker-like development of the oral part. Tse23 PHYLOGENY OF RANA LIMNOCHARIS AND TWO ALLIED SPECIES. M.Nishioka and M.Sumida. Lab. for Amphibian Biol.,Fac.of Sci.,Hiroshima Univ.,Hiroshima Phylogenetic relations were biochemically examined among five populations, the Oka- yama, Hiroshima, Okinawa, Iriomote and Tai- wan, Of Rana limnocharis, two populations, the Philippine and Tai, of Rana cancrivora and the New Guinea population of Platyman- tis papuensis. Nineteen kinds of enzymes extracted from skeletal muscles and livers and two kinds of blood proteins of 64 frogs in total were analyzed by the method of starch-gel electrophoresis. It was found that there were 31 loci controlling these enzymes and blood proteins. At each locus, there were 2.6 alleles, 3.8 on the average, which revealed 2.8 phenotypes, 4.4 on the average. The mean proportion of hetero- zygous loci per individual, the mean pro- portion of polymorphic loci per population and the mean number of alleles per locus in the eight populations of the three species were 5.8%, 18.4% and 1.18, respectively. A dendrogram generated from Nei's genetic distances (D) by the UPGMA method showed that the Okayama, Hiroshima and Okinawa populations resemble one another very closely (D=0.018~7 0.053) and considerably distinct from the Taiwan and Iriomote popu- atvons! (D=05170 . O2321) sank: faimnochars= There is a high degree of dissimilarity be- tween R. limnocharia and R. cancrivora (D= 1.43011.747) and remarkably greater genetic dissimilarity between these two species and Py papuensas” (D=3.217 3.269). TS 24 ISOZYMIC DIFFERENTIATION AND SPECIATION IN RANA NARINA FROM THE RYUKYU ARCHIPELAGO AND TAIWAN M. Matsui, Biol. Lab.Yoshida Coll., Kyoto Univ., Kyoto. Isozyme variation was studied in 96 individuals of Rana narina from throughout the species range (Amami-Oshima, Toku-no- shima, Okinawajima, Ishigakijima, Iriomote- jima, and Taiwan). Starch gel electrophore- Sis was done to resolve 11 enzyme systems encoded by 15 presumptive loci. The calcu- lated Nei's and Rogers' genetic distances indicated the presence of extreme genetic divergences among island populations. Al- though two allopatric populations from Iri- omotejima were genetically almost identical (Nei's D= 0.03), two syntopic populations from Ishigakijima showed a clear genetic division from each other (D= 0.44). The result well agreed to the morphological evidence. The large-sized type was genetic- ally very similar to two populations from Iriomotejima (D= 0.08), but the small-sized stood in the isolated position from other populations. The distribution pattern of several electromorphs (e.g., Ldh-B-a,b,d) indicated the occurrence in the past of multiple invasions by this frog complex in- to the Ryukyu Archipelago. Constructed UPGMA phenogram indicated the presence of five distinct lineages (Amamit+ Toku, Okina- wa, Ishigaki-small, Ishigaki-large+ Iriomo- te, Taiwan), and suggested the possibility of further taxonomic divisions in this spe- cilles’. 1116 Taxonomy and Systematics S125 BIOCHEMICAL VARIATION IN CYNOPS PYRRHO- GASTER FROM WESTERN DONE TN - Hayashi and M. Matsui“. Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto and “Biol. Lab., Yoshida Coll., Kyoto Univ., Kyoto. Using starch gel electrophoresis, we ana- lysed 15 loci of 12 enzymes in 40 popula- tions of C. pyrrhogaster collected from Kinki, Chugoku, Shikoku and Kyushu Dis- tricts. The UPGMA phenogram based on Nei's genetic distance (D) showed the following: (1)Three poplations from southern Kyushu are genetically differen- tiated from the other populations (mean D value = 0.145). (2)The latter group is divided into two subgroups with the mean D value being 0.050. One subgroup con- sists of populations from western Chugoku, Shikoku and middle and northern Kyushu, and the other from eastern Chugoku and Kinki. C. pyrrhogaster has been morphologically divided into six local races, of wich three (Hiroshima, Sasayama and Intermediate races) occur in western Japan. The analysis of allozymic variation revealed the following: (1)The division between Sasayama and Hiroshioma races are supported. (2)The division be- tween Sasayama and Intermediate races is not supported. (3)Hiroshima race is not homogeneous but contains two distinct groups. TS Zo MICROHABITAT OF TWO SYMPATRIC EUBRANCHIID NUDIBRANCHS, £. RUSTYUS AND £. OLIVACEUS (AEOLIDACEA) FROM CAPE ARAGO NEAR COOS BAY, OREGON. Y.J.Hiranol, J.H.R.Goddard? and Y.M.Hirano% l3Mukayrshamay Mar. Biol. Lab. Bac. Of SCL. Hiroshima Univ. 20regon Inst. Mar. Biol., Una; (Of Oregon), Uie cA. pasiniesrapiler asia ie Se We Mee EOS a ote ae fe a ee Eee La Eubranehus rustyus (Marcus, 1961) and E. olitvaceus (O'Donoghue, 1922) were found together from a hydroid, Plumularta sp. growing on the seaweed, Cystoceira sp. in Cape Arago. EF. rustyus always occurred on the part of Plumularia stalk and E£, oltvaceus always around the base of Plumu- larta. Although other species of hydroids, such as Calycella sp. and Halecitum sp. were found around Plumularta stolon, observation of nematocysts in the cerata apparently showed that £. rustyus eats Plumularia hydranth and £., olivaceus eats Plumularia hydrorhiza. Obelia spp., which had been reported as major food resources of E£, oltvaceus, was not found nearby. Difference of microhabitat between £, rustyus and FE, oltvaceus is considered to be caused by partition of feeding parts on the same hydroid. It is interesting as contrasted with the relationship between two Eubranehus species (E. mtsaktensts and E. hortt) in Japan which occur together and eat completely different species of hydroids (Hirano & Hirano, 1985). Se27 TWO SYNGENS OF EUPLOTES HARPA (CILZOPHORA), BISIEVVAINOCO, 4OOILSA UMSies, WAGs CO Sei.) Hiroshima Univ., Hiroshima. Using the encysting euryhaline ciliate Euplotes harpa, the way of sexual repro- aiticienvony number of mating types, and kinds of breeding systems have been in- vestigated. In this species, two sibling species, syngen 1 and 2, were found, which have separate breeding systems from one another. Stocks of syngen 1 were collected from the Seto Inland Sea and the Sea of Japan, and undergo both inter- and intra- clonal conjugations as the way of sexual reproduction. Viability of exconjugants from interclonal conjugations varied from cross to cross. When crossing two differ- ent stocks from areas being relatively far QYOeiee, Walalosl dalicy Oir exconjugants was extremely low. But when crossing two stocks from the same area, viability was always high. On the other hand, viability of exconjugants from intraclonal conjuga- tions was always more than 75%. This syn- gen has a multiple mating type system with seven mating types. Stocks of syngen 2 were collected from Ariake Bay and Beppu Bay, and undergo two kinds of conjugation the same as syngen 1 do. Viability of exconjugants from interclonal conjugation as well as those from intraclonal conjuga- tion was constantly high. This syngen has a multiple mating type system with six mating types. There is no morphological difference between syngen 1 and 2, but no conjugations occur between them. Ts: 28 AUTOGAMOUS STOCK IN SYNGEN 1 OF _BUPLOTES WOODRUFFI (CILIOPHORA). We iMOSalea MOC, Iinleies, MACs Ol SCLo; Hiroshima Univ., Hiroshima All of the stocks studied belong to syngen 1 that is a brackish-water dweller, which are capable of undergoing conjuga- tion, but not autogamy. One of 29 stocks that were recently collected and isolated cago Joneciel+* -channells. The jelly coat also contains a third factor, presumably an oligopeptide(s), that is not obligatory for the induction of the acrosome reaction but facilitates it by increasing the intracellular pH of sperm. Upon encountering the jelly coat, the spermatozoa respond to a co-operation of these three components of the jelly coat and eventually undergo the acrosome reaction in a few seconds. ile SECOND CENTURY: NEW CHALLENGES AND’ NEW STRATEGIES FOR STUDYING EGG ACTIVATION. D. Epel and R. Swezey. Hopkins Marine Station, Department of Biological Sciences, Stanford Universiby. Pacitic Grove, CA 93950 USA. The visualization of fertilization with the microscope preceded by just a few years the founding of the Misaki Marine Laboratory. This lecture focusses on a new technique for study- ing egg activation, examining enzyme activities in electrically-permeabilized eggs and embryos of sea urchins. Glucose-6-phosphate dehydrogenase (which was first studied here at Misaki) exhibits high and equal activity in homogenates prepared from either eggs or embryos and there is no change in activity at fertilization. However, unfertilized, permeabilized eggs show highly repressed activity, equivalent to only 1-2% of the available enzyme. When permeabilized after fertilization, however, the activity of this enzyme increases 8-15 fold. Thus, a post- fertilization change in activity is only seen in permeabilized cells. This same behaviour is also seen for five other enzymes we have assayed, suggesting that a general step-up in activity takes place which is only apparent in the permeabilized cell. Our experimental analysis indicates that this activation occurs in vivo and is controlled by both Ca** and pH. Clearly, understanding the nature of this change will provide new insights into the fertilization process. This study is one example of new types of approaches which will become increasingly important for this 2nd century of study of egg activation - staying as close as possible to the in vivo situation. Another approach will come from the development of optical "reporter" molecules which can assess directly the status of intracellular enzymes etc. A third approach, is microinjection of enzymes and antibodies to assess function in vivo. Fertilization is one of the best understood developmental programs - a major challenge for the next century will be understanding how the responding elements are set up during oogenesis to be unfolded at fertilization. 1124 Perspectives in Marine Biology FERTILIZATION IN MARINE INVERTEBRATES Christian Sardet. Biologie Cellulaire Marine CNRS, Station Marine, F-06230 Villefranche sur mer Our recent research deals with 3 questions: -Is sperm attracted to the egg? -Does it enter in a particular region of the egg? -What events does it trigger in the egg? In some chosen species, these questions can be answered with great clarity, and it has been a major contribution of Marine Stations to provide a place to explore the diversity of species, discover and develop appropriate biological systems. In Siphonophores (planktonic cnidarians), the egg secretes an extracellular material (the cupule) that attracts the spermatozoids in a specie specific manner. The attractant (a small acidic protein) modifies the trajectories of spermatozoids in such a way that they remain trapped near the source of attractant (collaboration with D. Carré, M.P. Cosson and J. Cosson). If in most species sperm enter anywhere, in many species, the spermatozoid enters in the animal hemisphere, near the maturation pole (insects, cnidarians, fish, amphi- bians,...). The ascidian egg where sperm is said to enter at the vegetal pole appeared one of the rare exception. Our recent studies on denuded ascidian eggs (Phallusia mam- millata) indicate however that sperm enter preferentially in the animal hemisphere. Using time lapse video recording and an Imaging Photon Detector to detect light emis- sion from aequorin loaded eggs (collaboration with L.F. Jaffe and J.E. Speksnijder), we have analyzed the reaction of the ascidian egg to the entering sperm. A large loca- lized increase in intracellular calcium (=3 uM) propagates (=7 ym/sec) across the egg. As it reaches the animal pole, it is followed by a wave of cortical contraction that propagates from the animal to the vegetal pole in 2 minutes (=2 um/sec), moving simul- taneously subcortical organelles and extracellular markers to the vegetal pole. Perio- dic oscillations in the level of intracellular calcium and cytoplasmic contraction- relaxation of the egg precede second polar body formation. Subcortical organelles that have accumulated at the vegetal pole finally move toward the sperm aster and the futur posterior pole of the embryo. THE INDUCTION OF MOTILITY IN SPERMATOZOA BY IONS AND CYCLIC AMP. M. Morisawa. Ocean Research Institute, University of Tokyo, 1-15-1, Minamidai Nakano-ku, Tokyo, 164 Japan, After leaving from the testis, the spermatozoa pass into sperm duct or epididymis. From there they are released into the place in which fertilization occurs. Throughout this travel, the spermatozoa are suspended into fluid environments differing properties: The chemical and physical nature of fluids in vas deferens and spawning ground progressively or drastically changes, In the course of trip, function of spermatozoa are also changing: They acquire the potential for motility during passage through the sperm duct or epididymis and then they initiate motility at spawning or at ejaculation, and it has been implicated that composition of the surrounding fluid may result the induction of sperm function. A number of factors which seem to be responsible for the acquisition or initiation of sperm motility have been proposed since these phenomena were treated by Tournade or Gray in early twenty century, and they are still poorly understood. These problems are recently reinvestigating by the author and colleagyes using salmonid fish spermatozoa and it was suggested that change of HCO, and H concentrations in the sperm duct is the external factors for the acquisition of sperm motility and gradual increase in intracellular cAMP which occurs during transit of sperm in the sperm duct is involved in the expression of the acquired potential for motility. We further demonstrated ’a cascade process of the initiation of sperm motility in salmonid fishes: Seminal K at the concentration of approximately 35-85 mM suppresses the motility of sperm even though they have already acquired the potential for motility at the posterior end of the sperm duct and decrease in environmental K at spawning in river fresh water causes instantaneous and drastic increase in intracellular cAMP and then phosphorylation of axonemal protein with a molecular weight of 1,5000(15K protein) by newly synthesized cAMP triggers the final step of the initiation cascade of sperm motility. Studies based on these evidence further suggested that similar process occurs and regulates sperm motility in marine invertebrates such as sea urchins, tunicates and mammals. Perspectives in Marine Biology 1125 THE MOTILE MECHANISM OF THE SEA URCHIN SPERM: ANALYSES WITH REACTIVATED ‘MODEL SYSTEMS: Keiichi TAKAHASHI. Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo. Echinoderm spermatozoa have been shown to be very useful in the study of flagellar motility and its control. We have used demembranated sea urchin sperm flagella to determine the mechanical properties of ATP-driven microtubule sliding that powers the flagellar motion and to elucidate the mechanism by which the sliding is controlled to generate patterns of flagellar beat. Some of our recent results are reviewed. To study the dynamic properties of microtubule sliding, particularly the force- velocity relation, glass microneedles that have previously been calibrated for their compliances are attached to the demembranated flagellum. The positions of the microneedles are monitored by projecting their microscopic images onto a photodiode- array position sensor. Microtubule sliding is initiated by perfusing a reactivating solution containing ATP and elastase and the force and velocity of the sliding be- tween the microtubules attached to the needles are determined by analyzing the deflection of the needles caused by the sliding force. A piezoelectric device is used to change the distance between the microneedles so that abrupt length steps can be applied to the flagellum during the force generation. This made it possible to carry out a "slack test" for more reliable determination of the mechanical parameters of sliding than has previously been possible. The results indicate that the shape of the force-velocity curve differs substantially from that of the familiar hyperbolic force-velocity curve of skeletal muscle and is independent of the ATP concentration over a wide range. VIDEO MICROSCOPY OF MITOSIS. Shinya Inoue, Marine Biological Laboratory, Woods Hole, MA 02543, U.S.A. The application of video technology has provided enhanced image contrast, resolution, and recording speed in microscopy, as well as making possible noise reduction, thin optical sectioning, 3—-D reconstruction, and discrimination and quantitation of image features (for summary, see Inoue, 1986, Video Microscopy, Plenum Press, NY). With video microscopy, time-lapse analyses of cell division and mitosis can be carried out at high optical resolution using well-corrected optics and aided by the excellent image quality and versatile playback capabilities of the optical memory disk recorder (OMDR; laser disk recorder/player). I will demonstrate, in Haemanthus endosperm cells that Drs. A.S. and J. Mole-Bajer and I recorded in rectified Plan Apo polarization optics: (1) the behavior of chromosome arms in anaphase; (2) very thin serial optical sections showing the dichroism of microtubules decorated via anti-tubulin to 5 nm colloidal gold spheres. Finally, I will present stereo-paired images reconstructed, using a novel computer program, from sequential optical sections stored in the OMDR (Inoue, S. and T.D. Inoue, 1986, Ann. N.Y. Acad. Sci. 483: 392-404). These images show surprising distributions of microtubules including: splaying and extensive mixing of kinetochoric and polar microtubules towards the spindle poles; some very long, curved microtubules criss—crossing the others which run more or less parallel to the spindle axis; and distal ends of polar microtubules wrapping tightly around the metaphase and anaphase chromosome arms, forming a loosely woven basket of microtubule helixes. These arrangements of microtubules were confirmed in the living cell, but their functional roles are yet to be understood (Inoué, S., A.S. Bajer, and J. Molé—Ba jer, 1985, in M. DeBrabander and J. DeMey, eds., Microtubules and Microtubule Inhibitors, Elsevier, Amsterdam, pp. 269-276). 1126 Perspectives in Marine Biology MICROMANIPULATION OF DIVIDING ECHINODERM EGGS. Yukio Hiramoto. University of the Air, 2-11 Wakaba, Chiba 260, Japan. The location and nature of the motive force for chromosome movement and spindle elongation during anaphase were analyzed in sea urchin eggs by micromanipulation and Colcemid-UV method in which Colcemid could be applied to a restricted region of the cell. Chromosome behavior and spindle elongation during mitosis were observed by differential interference microscopy and birefringence indicating the state of microtubules at various regions of the cell were measured by polarization microscopy, while various micro-operations were made to the mitotic apparatus by micromanipulation and the Colcemid-UV method. By micromanipulation experiments, it was found that poleward chromosome movement occurred in the spindle after removing the aster(s), the region of the spindle near its polar end, or protoplasm at the interzonal region of the spindle by sucking it out with a micropipette, or severing the spindle in two parts at the equatorial plane or at the region between the equatorial plane and the polar end. The Colcemid-UV method revealed that chromosome movement occurs only in birefringent region of the spindle, irrespective of the presence or absence of birefringence in other regions of the same spindle. It was concluded that chromosome movement during anaphase is driven by microtubule-dependent engine(s) located near chromosomes. The spindle elongation during anaphas was inhibited by removing asters attached to the ends of the spindle by micromanipulation or destructing them by Colcemid-UV method. A hypothesis was presented that the spindle elongation is caused by forces generated at the surface of astral microtubules acting in the direction away from each centrosome attached to the end of the spindle. MODULATION OF SPINDLE acelin Ly IN DIVIDING SEA URCHIN EGGS. H. sSatomvl. J... toh Ohanags amdiae Sato2. lsugashima Marine Biol. Lab., Nagoya Unive, Lobasy Mies olviand 2Shoin Women's Univ., Biol. Lab., Nada-ku, Kobe 657. Well preserved isolated spindles from the first cleavage of dividng sea urchin eges revealed that3,000 to 5,000 orderly arranged microtubules as the major cytoskele- tal components. Considering the sensitive nature of the spindle birefringence, it is highly probable that the spindle assembly is regulated by the labile association of pooled tubulin and the kinetic centers such as the kinetochores and MTOC's in centro- somes. In order to examine molecular basis of spindle assembly, mitotic arresters and Chlortetracycline were used as the molecular probes. When fertilized sea urchin eggs of Hemicentrotus pulcherrimus, Pseudocentrotus depressus or Clypeaster japonicus were exposed to dilute solutions of alkylresorcinols, i. e.,)°T=1, its derivatives, Ansamycin, Cullvularin or Macbecin I; spindles were tend to arrested in metaphase and also turned into barrel-shape. Anaphase chromosome move-— ment can be initiated but with very slow pace. Efficacy of these drugs is comparable with detergent and can be interpreted as freezing the dynamic state of equilibrated molecular association of tubulin along the microtubules. Bundles of straight microtu- bules with an even length composed barrel-shaped spindles, and we thought it might re- flects the stabilized state of tubulin polymers caused by the local increase of mole- cular hydrophobicity. We confirmed this unusual spindle shape also due to the disc- oidal redistribution of MTOC's in centrosomes. Chlortetracycline was employed as a fluorescent chelate probe to determine the distribution of membrane-associated calcium in dividng sea urchin eggs. Following the change of the fluorescent pattern durint mitosis, we found that the CTC-fluorescence co- incided with that of ER-like membrane which were revealed in electron micrographs. Tetracaine, which was known to displace membrane-bound calciun, reduced fluorescence and spindle birefringence. Results obtained suggested that intracellular free calcium ions were sequestered in the membrane system associated with the mitotic apparatus during mitosis. (Supported by Grants-in-Aids for Scientific Research; 59380026, 60480020 and 61304008. ) Perspectives in Marine Biology 1127 THE CENTROSOME: ORGAN OF CELL ARCHITECTURE D. Mazia Hopkins Marine Station of Stanford University Paetene Grove, CA 93950, U.S.A. A century ago, the centrosome was recognized by Boveri as a major permanent organ of the cell. At the present time, the centrosome commands renewed attention and it is prominent in the present symposium. (Contributions of Schatten, Sakai, Sato and Mazia). The cell cycle includes a centrosome cycle: a consistent cycle of changes of the shapes of centrosomes. Compact centrosomes flatten, spread and divide, thus forming the poles for the next mitotic cycle. Since the centrosomes generate microtubules, the shape of the mitotic apparatus (e.g. pointed poles or flat poles) will depend on the stage of the centrosome cycle relative to the stage of the chromosome cycle. The general facts of the centrosome cycle have now been confirmed in many kinds of cells including types of cells (e.g. in higher plants) in which the very existence of centrosomes has been denied previously. Currently, immunofluorescence studies with anticentrosome antibodies are especially revealing. It is proposed that the centrosome is the architectural organ of the cell; the conformations of centrosomes generate cell morphologies that are based on microtubules. A model of the structure of the centrosome present it as a very long flexible linear element, carrying large numbers of sites that determine the origins and directions of individual microtubules. There is new evidence for the model, but specific molecular characteristic of centrosomes are still unknown. Reterence:) | Mazia, D. 1987. Int. Rev. Cytol., 100:49-92. CENTROSOMAL INHERITANCE AND MOTILITY DURING FERTILIZATION. G. Schatten and H. Schatten Integrated Microscopy Resource for Biomedical Research, University of Wisconsin, 1117 West Johnson Street, Madison, WI 53706 U.S.A. The organization of the cytoskeleton and the regulation of motility have been investigated during fertilization in sea urchins and in mice. Microfilaments inter- acting with non-erythrocyte spectrin or fodrin are found at the regions of sperm-egg fusion in sea urchins, but not in mice. Latrunculin, a new microfilament inhibitor, interferes with sperm incorporation in sea urchins but not in mice, reinforcing the suggesting that these two system accomplish fertilization in different manners. In sea urchins, an actin-spectrin gel interacts with the plasma membrane at the site of sperm incorporation whereas spermhead incorporation in the mouse occurs without any apparent microfilament involvement; the formation of the second polar body requires microfilament activity. Centrosomes are traced in sea urchins with a human autoimmune antibody and a mouse monoclonal antibody generated against Drosophila intermediate filament protein, which reacts with a 68 Kd antigen in sea urchins. The sperm contributes this structure which spreads and duplicates as shown by Boveri at the turn of the century. Surprisingly the mouse sperm does not bind centrosomal antibodies, and instead it appears that this structure is maternally inherited in this mammal. Parthenogenesis experiments support this unexpected conclusion. Centrioles are not found in the unfertilized mouse oocyte, as studied with serial thick sections and high voltage electron microscopy. Though the sperm contributes a centriole-like structure at fertilization, it does not seem to be involved in the formation of the first mitotic spindles. Instead mitoses occur in the absence of centrioles. Remarkably centrioles are not observed until the second trimester of fetal development. These studies demonstrate that, contrary to expectations, fertilization in this mammal raises several significant questions involving the fundamentals of cell organization and motility. 1128 Perspectives in Marine Biology ASTER FORMATION IN VITRO INDUCED BY 51K PROTEIN, A COMPONENT OF THE CENTROSOME. K. Ohta, M. Toriyama, S. Endo and H. Sakai. Dept. of Biophys. and Biochem., Fac. of SCios UMW OF MOMWO>5. IOKWo, The pericentriolar material of sea urchin egg consists of the clusters of granules with a diameter of ~90 nm originally found by Endo in 1979. After isolation and homogenization of the mitotic apparatus (MAs) at low temperature, granules in the suspension formed many small asters when mixed with tubulin. The protein fraction responsible for this aster-forming activity was solubilized and partially purified by phosphocellulose column chromatography. This fraction which contained more than several components with a major protein having a molecular weight of 51,000 and an isoelectric point of 9.8 (51K protein fraction) was reconstituted into granules by dialysis and initiated astral microtubules in the presence of tubulin with the plus end distal to the center of the asters. The antibody was raised in mouse against the 51K protein and affinity-purified. The antibody stained only the center of asters reconstructed from both the granules in the homogenized MAs (centrosomal fragments) and those reconstituted from the 51K protein fraction. The antibody also stained the centrosome as well as the peripheral region of the centrospheres and the half spindles. Therefore, the 51K protein is a component of the centrosome and it is involved in the formation of the clusters of granular materi- al (Endo, 1979) in the centrosome and plays a role in the initiation of astral and spindle microtubules at mitosis. SEA URCHIN ARYLSULFATASE AND ITS EXPRESSION DURING DEVELOPMENT. Hl. Shimadarelins Ga OimeZool mMihac.) OSes Umi. IC OKyor mokyor Arylsulfatase (ARSase) of sea urchin embryos which begins to increase at the hatched blastula stage is an extracellular protein which becomes localized in the cell sur- face of the embryo during gastrulation (Rapraeger and Epel, 1981). However, littie is known about its role in embryogenesis and the mechanism controlling its expression during sea urchin development. We have recently purified the ARSase from the sea urchin embryo. The Enzyme consists of ten identical 63KD-subunits, and it occupies as much as 0.5% of the total proteins of pluteus larvae. The activity of purified ARSase was markedly inhibited by 20 mM sulfate ions. It showed the maximal activity between pH 5 and 6, and showed little activity around pH 8 (the pH value of sea water). Thus, the enzyme seems to be inact- ive in an extracellular environment. Moreover, the purified ARSase did not hydrolyze any sulfated polysaccharides extracted from the sea urchin embryo. These results, together with its extracellular localization, suggest the ARSase of the sea urchin embryo functions as one of the cell-surface structural proteins rather than functions as an enzyme. By use of the inhibitors of transcription and translation, we found that the low lev- el of ARSase observed in the blastula is dependent on the translation of maternal mRNA, and the increase in the ARSase activity after this stage is due to transcrip- tion of the genomic ARSase gene. In order to further confirm this conclusion, the Northern analysis of the embryonic mRNAs were performed using the ARSase cDNA as the probe. The ARsase cDNA was cloned in our laboratory by use of the chemically synthe- sized DNA fragment corresponding to the amino acid sequence of a part of the purified ARSase polypeptide. The result of the Northern analysis agreed well with the results obtained by use of the inhibitors of transcription and translation. It was found that transcription of the ARSase gene starts at the pre-hatched blastula stage, and conti- nues thereafter, while a detectable amount of the ARSase mRNA is stored in the unfer- tilized eggs. The size of the ARSase mRNA was estimated to be about 2.9 kb. Perspectives in Marine Biology 1129 MOLECULAR CLONING AND DEVELOPMENTAL EXPRESSION OF HOMEO BOX CONTAINTING GENES IN SEA URCHIN EMBRYOS. Tom Humphreys, University of Hawaii, Pacific Biomedical Research Center, 41 Ahui Street, Honolulu, Hawaii 96813, USA. Hybridization of Drosophila Homeo Box DNA seqeunces to Southern transfers of sea urchin genomic DNA indicated the presence of 5-10 homeo boxes. Screening of a sea urchin genomic recombinant DNA library, with Drosophila probes from both Antenna- pedia-like and engrailed homeo boxes has so far yielded six unique homeo boxes _ confirmed by nucleotide sequencing. Four of these homeo box sequences are Antennapedia-like with 66-72% nucleotide sequence homology to the Antennapedia gene homeo box. They encode amino acid homeo domains that show 70-88% homology with the Drosophila Antennapedia gene homeo box domain. One of the sequences isolated, though definitely a homeo box sequence, shows no special homology to any other homeo box which has been sequenced. The amino acid sequence encoded by this homeo box shows 42-47% homology to all of the approximately 30 known homeo domain sequences. The one engrailed-like homeo box in the sea urchin genome was isolated and sequenced. Its encoded amino acid sequence was 70% homologous to the Drosophila engrailed homeo domain and 77% homologous to the mouse engrailed homeo domain. The expression of these homeo box sequences in sea urchin embryo was examined by isolating RNA from various stages of the embryo and preparing Northern transfers of this RNA to react to the various sea urchin gene probes. These experiments detected one homeo box gene which was expressed very actively during the embryonic stages. This sea urchin homeo box was most homologous to the Antennapedia homeo box with 88% homology in the encoded amino acid sequences. Transcripts of 6.9 kb and 7.5 kb could first be detected in blastula stage. They increase several fold by gastrula stage and then decreased significantly as development preceded to pluteus stage. Insitu- hybridization showed that these transcripts appeared in the dorsal (aboral) ectoderm of the embryo. MOLECULAR ANALYSIS OF THE CHICKEN GENE CONTAINING THE HOMEO BOX A. Kuroiwa and E. Yokoyama. Division of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, 2-6 Musasidai, Buchu-Caty,.lokyo',, Japan In the course of Drosophila embryonic development, segmentation genes and homeotic genes control a formation of metameric structure of body segment and a determination of the identity of each segment, respective- ive easnioOmeoeLTe genes and Some silegmentat ton. genes , so fan cloned, contain "homeo box" sequence in the 3' exon. The homeo box is translated into a certain part of a protein and such a region act as a DNA binding demain Gb Ene proteim, “that regulate a expression of other genes. The homeo box is found not only in invertebrate but also in vertebrate gemon. Genes carrying homeo box in vertebrates are expected to have dastiEbaretunctkaon asiainy Deosophila - In order to analyze a function of the homeo box genes in chicken embryonic development, I have isolated 15 different genomic clones containing a Antp-type homeo box. As in the case of Drosophila, (i.e. ANT-C and BX-C), chicken homeo bex genes make several clusters in the genom and homeo domain locate near the end of open reading frame. At leaset 3 homeo box gene cluters are found in the chicken genom. Choxl cluser has 5 homeo box genes in 52Kb DNA in same transcriptional orie- Ntation (CHoxl. 0-1.4). ClusterII has 3 homeo box genes in 57Kb DNA (a, 2o-7) 2 o—) ) mand claSteneiiin has! Sehomeoe box) genés* in 35Kb DNA (x; 226),Y:)- Since the homeo box sequence and gene arrangement of the CHoxI cluster are almost identical as the gene in HoxI cluster of mouse, CHoxI is a chicken homologue of HoxI. All these genes encode a characteristic mRNA(s), experessing in embryonic development and adult central nervous system. Byun stun hybasrdizatron dialysis, CHoxl.4 as found to express in entire spinalcord and certain region of somites of 4 days embryo. 1130 Perspectives in Marine Biology A MOLECULAR ANALYSIS OF HETEROCHRONY IN THE EVOLUTION OF DIRECT DEVELOPMENT IN SEA URCHINS. Rudolf A. Raff Department of Biology, Indiana University Bloomington, Indiana 47405, U.S.A. If we are to understand:the evolution of animal morphology we must define the mechanisms by which evolutionary changes occur in embryonic development. An excellent experimental system to study such processes is provided by direct developing sea urchins closely related to those with a typical mode of development. A significant number of sea urchin species have eliminated the typical pluteus larva, and instead produce large yolkly eggs that rapidly and directly develop into juvenile sea urchins. Development in such forms is radically modified in several ways. The most evident changes result from heterochronies in which larval features are lost and development of adult features is accelerated. We are investigating the development of an Australian direct developing sea urchin, Heliocidaris erythrogramma. This species is sufficiently closely related to Strongylocentrotus purpuratus that cDNA and monoclonal antibody probes to primary mesenchyme cells of S. purpuratus cross react readily. We have been able to establish the homology of mesenchyme and other cell lineages between the two species, and have begun to investigate the heterochronies in two aspects of cell behavior. The first is in changes in cell cleavage dynamics. The second is in timing changes in cell lineage-restricted gene expression. The use of molecular probes to mesenchyme cells demonstrates that the predicted heterochronies are demonstrable at the cellular and molecular levels. A large number of primary mesenchyme cells are present by the completion of gastrulation, and they initiate an adult pattern of gene expression. STUDIES OF MESENCHYME IN SEA URCHIN EMBRYOS Robert D. Burke, Allan W. Gibson, and Colin R. Tamboline. University of Victoria, Victoria, B.C. V8W 2Y2 Canada A distinctive aspect of sea urchin development is the mesenchyme. These cells, which migrate into the blastocoel from the vegetal plate and the tip of the archenteron, are associated with the principal morphogenetic events in the formation of the pluteus larva. Classically two subsets are recognized; primary and secondary mesenchyme. Studies we have undertaken using monoclonal antibodies have enabled us to document the ontogeny of morphologically indistinguishable subsets of mesenchyme, such as pigment cells. The antibody, Sp-l recognizes a 110 kd glycoprotein characteristic of presumptive pigment cells. Using this antibody the release of these cells from the vegetal plate, their migration through the blastocoel and insertion in the epidermis has been documented. Mesenchyme is known to give rise to skeletogenic cells, pigment cells and a population of cells that span the blastocoel. Our studies indicate that a number of components of the extracellular matrix are associated with the skeletogenic and blastocoelar cells. The antibody Sp-12 is specific to several peripheral membrane components of all the classes of mesenchyme. A glycoprotein recognized by this antibody is a major component of the extracellular matrix of adult body wall and is present in the embryo from the mesenchyme blastula stage. Sp-14 is an antibody that binds to a fibrillar component of the ECM of early embryos. This material is present in the blastocoel and associated with the primary mesenchyme. Primary mesenchyme cells and mesenchyme from dissociated embryos appear to produce both Sp-12 and Sp-14 positive material in vitro. We hypothesize that an important role of the mesenchyme in the sea urchin embryo is to produce or modify the extracellular matrix of the blastocoel. Perspectives in Marine Biology 1131 ROLE OF A SITE IN FIBRONECTIN MOLECULE IN MIGRATION OF PRIMARY MESENCHYME CELL IN SEA URCHIN EMBRYOS. H. Katow, Biology Laboratory, Rikkyo University, Toshima-ku, Tokyo 171. The cell migration is an elemental process along with the cell division in the morphogenesis in multicellular animals including sea urchin embryos. In the most sea urchin embryos the primary mesenchyme is a group of cells to be formed primarily in the blastulae as a mass of free cells, and they possess a property of migration. The migration is held until the formation of cell aggregates prior to the spicule differentiation. During the migration the cells have an extracellular glyco- protein, fibronectin, on their surface. Anti-fibronectin antibodies inhibit the cell migration in vivo. In vitro the cells are able to bind with exogenous fibronectin, and their migration responds to the amount of the molecule in culture medium. In proper amount, 40pg/ml in Pseudocentrotus depressus, fibronectin increases not only the number of cells which were promoted the migration but also the distance of the migration performed by each of these cells. The synthetic peptides that contain a particular amino acid sequence, Gly-Arg-Gly- Asp-Ser, which is known to represent the cell binding domain of fibronectin also inhibit the binding of the molecule to the cell surface as well as the fibronectin-promoted migration of the cells in vitro. The synthetic peptides can be injected into a large number of blastulae simultaneously using a micro-mass injection technique, and they inhibit the cell migration in the blastocoel. The inhibition of cell migration by these synthetic peptides is erased by addition of extra amount of fibronectin to the culture medium. The inhibition, therefore, is regarded as of a competitive. All these results indicate that the amino acid sequence in cell binding domain of fibronectin molecule is the very site at which the molecule binds to the primary mesenchyme cells, and that the site is utilized in the cell migration in the blastocoel. THE HAGFISH AS A RESOURCE IN THE STUDY OF EVOLUTIONARY ENDOCRINOLOGY Aubrey Gorbman, University of Washington, Seattle, Washington 98195, U.S.A. The hagfish Eptatretus burgeri has figured prominently in the history of the Misaki Marine Biological Laboratory, particularly since Prof. Bashford Dean of Columbia University came in 1900 to study development of Eptatretus. It was one of the principal research materials of Prof. Hideshi Kobayashi during his directorship of the laboratory, and later. The hagfish at Misaki has two broadly Significant attributes that have attracted researchers from all over the world. First, it is the most primitive vertebrate available for study today. Hence, it is the best source of information to the zoologist from which to extrapolate and judge the evolution of any vertebrate system. The second cyclostome group, the lampreys, are much more like other lower vertebrate taxa, especially primitive cartilaginous fishes. A second feature of the Japanese Eptatretus that attracts study is the seasonality of its breeding cycle; it is probably the only seasonally breeding hagfish species, an indication that it may be less degenerate than other hagfish species, and therefore more representative of its proto- vertebrate primitive ancestor. Several examples of hagfish study, one of them completed at the Misaki laboratory, will be presented briefly. One deals with the brain-pituitary regulatory relationship. A second deals with development of the head and pituitary gland. Profound differences in these and other features between cyclostomes and higher vertebrates, and between hagfishes and lampreys are contributing to phyletie reasoning in two ways: 1) they provide criteria against which the full extent of evolution of vertebrate systems may be judged, and 2) they indicate a diphyletic origin of the two cyclostome groups, now included in a Single class. 1132 Perspectives in Marine Biology REPRODUCTIVE BIOLOGY IN HAGFISH A MARINE BIOLOGICAL ENIGMA. B. Fernholm Research Department, Swedish Museum of Natural History S-104 05 Stockholm, Sweden During the last one hundred years no other marine chordate has been the subject of so much research into its reproductive biology with so little result as hagfish. I will try to analyze the reasons for this and point out potentially fruitful approaches. 1. Hagfish are exclusively marine animals living fairly deep, at least during spawning time. Therefore direct observation in the sea is difficult. 2. Hagfish dig into soft bottoms thereby making direct observation difficult. 3. Most of the studied species of hagfish do not seem to have a well difined spawning time. 4, The investigated species seem to have particular spawning places which are difficult (E@ isla 4 5. In several species of hagfish it is particularly difficult to find males, especially males with mature sperm cells. Hagfish of different species occur in all oceans and many attempt to find their eggs and embryos have been made, mainly in Scandinavia, England, California, and Japan. One hundred years ago the first efforts to obtain ripe eggs from females were made in Scandinavia, keeping females with large eggs in wooden cases in the sea for 6 months. No eggs were laid. I have used plastic and metal cases in Japan and Scandinavia in a Similar way. Many seemingly normal eggs, all unfertilized, were deposited. The most successful attempts to secure embryos have been those in California using long lines and hooks. Since that method is less efficient than baited traps for catching large numbers of hagfich it has not been in much use recently. The method depends on the hooked hagfish sliming and wriggling and thereby catching nearby deposited eggs in its slime and was recommended already around the turn of the century as the best method to obtain eggs and embryos. I plan to use the long line method in Sweden, in addition to improved cases having muddy bottoms. VOLUME CONTROL PROCESSES IN ANIMAL CELLS R.GILLES Laboratory of animal physiology, University of Liége, 22, quai Van Beneden, B-4020 Liége, Belgique This brief overview will compare the physiological responses of cells from homeosmotic and euryhaline poecilosmotic species to osmotic chal- lenges.Both cell types are perfectly able to regulate their volume fol- lowing an osmotically induced swelling. However, mammalian cells cannot survive in diluted media while cells of euryhaline poecilosmotic species do. This shows that adaptation and survival of cells to changes in the osmolality of their environmental medium cannot be related solely to their possibility of volume regulation. In fact, their possibility to cope with the disrupting effects changes in intracellular concentrations o£ inorganic dons can have. on the: Structure and) activity of (di Beerene macromolecules must also be taken into consideration. In this respect, the amino-compounds usually found in rather large amounts in cells of Marine euryhaline poecilosmotic species could have an important part to play. They indeed seem to have a "Stabilizing" effect on protein struc-— tune that can oppose” the “disrupting! ertectes of Unorganic TONS Hun ede, their use as major osmolytes in the cells of euryhaline species allows a control of the intracellular level of inorganic monovalent ions other- wise used as major osmotic effectors. The cells can thus avoid the important disrupting effects of changes in these ions concentration. The mechanisms discussed can be considered as clues to survival and settlement of species in coastal ecotones with fluctuating salinities. Perspectives in Marine Biology 1133 OSMOREGULATORY ROLES OF PROLACTIN AND GROWTH HORMONE IN TELEOSTS. Tetsuya Hirano. Ocean Research Institute, University of Tokyo, Tokyo. It has been well established that prolactin (PRL) plays an important role in maintaining hydromineral balance of euryhaline teleosts in fresh water, whereas cortisol has been known as a sSeawater-adapting hormone. An improvement in seawater adaptability has also been shown when some salmonids are treated with mammalian growth hormone (GH). The opposite effects of PRL and GH are intriguing considering the close similarity between their primary structures. Recently, PRL and GH have been isolated and characterized from chum salmon pituitaries. Chum PRL was about 100 times more potent than ovine PRL in causing plasma Na retention in hypophysectomized killifish. When injected into seawater-adapted juvenile rainbow trout, PRL produced a dose dependent increase in plasma Na, and chum PRL was 2-10 times more active than Ovine PRL. On the other hand, when juvenile rainbow trout were transferred from fresh water to seawater after 3 injections of chum salmon GH, plasma Na levels were Significantly lower then the saline-injected control. In addition, only a single injection of chum GH was sufficient to reduce plasma Na levels of chum fry transfer- red from fresh water to seawater. Changes in plasma levels of PRL and GH during transfer of chum salmon and rainbow trout to different salinities were followed using recently established homologous radioimmunoassays. When immature chum salmon were transferred from seawater to fresh water, PRL levels increased, whereas GH levels fell. Furthermore, when transferred from fresh water to seawater, they exhibited a significant increase in plasma GH without any change in PRL levels. The importance of GH and cortisol in seawater adaptation was also assessed in mature chum salmon. Mature fish, especially females after completion of ovulation, are difficult to maintain in seawater, mainly due to a gradual increase in plasma ion concentrations. When plasma levels of various hormones were examined, an increase in plasma GH and cortisol was observed in parallel with the increase in plasma ions. Whether GH acts directly on osmoregulatory surfaces or indirectly through cortisol remains to be studied. SYMBIOSIS BETWEEN ANOMALOPID (FLASHLIGHT) FISHES AND LUMINOUS BACTERIA. Margo G. Haygood! and Daniel H. Cohn2. 1Scripps Institution of Oceanography, La Jolla, CA 92093, USA and 2University of Washington, Seattle, WA 98195, USA. _ Fishes from a number of families have light organs that contain extracellular symbiotic luminous bacteria. In most cases the bacteria have been cultured and identified, but in the anoma- lopids (flashlight fish) the symbionts have proved impossible to culture thus far. In the flashlight fishes, the bacteroids are contained in two conspicuous suborbital light organs. DNA extracted from the light organs of the Caribbean flashlight fish, Kryptophanaron alfredi, was hybridized with a probe derived from the luciferase genes of Vibrio harveyi, a free-living luminous bacterium. The probe showed stronger homology to the luciferase genes of the symbiont than to those of Vibrio fischeri, a readily cultured luminous symbiont. This result suggested a relatively close evolutionary relationship between the luciferase genes of the symbiont and V. harveyi, and was consistent with previously published kinetic analyses of the luciferases from these organisms. A clone bank was constructed with DNA from the K. alfredi light organ and the luciferase genes were cloned. The nucleotide sequence of luxA was determined. Comparison with the published V. harveyi luxA sequence showed that the two sequences were 75% homologous at the nucleotide sequence level and 83% homologous at the amino acid sequence level. Codon usage in the symbiont gene differed from the V. harveyi gene and exhibited a marked bias. This result raises the possibility that the symbiont may have acquired the luciferase gene by lateral gene transfer. Comparison of luciferase genes from other anomalopid species may provide an independent test of the proposed phylogeny of the fish species. In addition, examination of nucleotide sequence divergence of the bacterial genes within the context of evolution of the fish hosts may provide a rare opportunity to estimate nucleotide substitution rates in a relatively recently evolved bacterial gene family. 1134 Perspectives in Marine Biology MECHANICAL AND THERMAL SIGNS OF EXCITATION PROCESSES IN THE NERVOUS SYSTEM. I. Tasaki National Institute of Mental Health, Bethesda, MD 20892, U.S.A. By using synthetic piezoelectric and pyroelectric materials, sensitive divices were constructed for detection of mechanical and thermal changes in the nervous system. To record rapid mechanical changes in excitable cells and tissues, piezo- electric banders (purchased from Gulton Industries) were employed in conjunction with an operational amplifier (AD515, OPA111 or 128). It was found possible to demonstrate rapid swelling during excitation in squid giant axons, crab nerves, lobster and frog retinae, frog spinal cord preparations etc. These findings strongly suggest that movement of water is involved in excitation processes. In the squid giant axon, the peak of swelling was found to coincide with the peak of the action potential recorded intracellularly from the site of mechanical recording. It is suggested that the excitation process is associated with an increase in the water content of the excitable membrane sites. To detect heat production associated with excitation processes, thermal detectors were constructed by using multi-layers of polyvinylidene fluoride (manufactured by Kureha Chemical Industry). Using these detectors, it was found possible to measure a rapid rise in temperature of about 1/100,000 degree. Rapid heat production was detected during excitation of crab nerves, vagal nerves of the guinea pig, frog retinae and spinal cords, etc. It is probable that the observed heat production during excitation of nerve fibers is associated with exchange of bound calcium for univalent cations. In stimulation of the dark-adapted frog photoreceptor cells by a brief light pulse, the energy released by the cells in the form of heat was found to be more than one million times as large as the energy of the light pulse absorbed by the photopigment. In isolated spinal cord preparations of the bullfrog, it was shown that the observed heat production is associated with synaptic transmission of nerve impulses. SQUID : ANIMALS INDISPENSABLE FOR EXPERIMENTATION Gen Matsumoto. Electrotechnical Laboratory, Tsukuba Science City, Ibaraki 305. The important problems in neuro-physiology have been successfully approached through the study of animals with particularly large neurons that are amenable to experimentation. The squid Loltgo, Doryteuthts and Septoteuthts have large nerve cells suitable for studying molecular events of synaptic communication between the cells and neural transmission along the axon. Molecular organizations of the axonal cytoskeletons underlying the plasma membranes of squid giant neurons have been well analyzed, biochemically and morphologically. It has been found that the axolemma is specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons. Intensive studies on the subaxolemmal cytoskeletons will lead to better understanding of their physiological roles in membrane excitation. Axoplasm from the giant axon can be dissociated, allowing bidirectional organelle movements to be visualized along individual cytoplasmic filaments with video-enhanced differential interference contrast microscopy. The axoplasm deserves as a typical system for elucidating molecular basis of motile force generation. Difficulties in squid experiments result from regional and seasonal limitations on the availability of natural squid stocks. These limitations are now partially eliminated by successfully maintaining the squid jn an aquarium in the laboratory. Perspectives in Marine Biology 1135 NEURAL TYPE DIFFERENTIATION AND Na CHANNELS IN THE CLEAVAGE-ARRESTED AND ISOLATED BLASTOMERES OF THE ASCIDIAN EMBRYO. K. Takahashi, Y. Okamura and H. Okado. Dept. of Neurobiology, Inst. of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo. As previously reported, when the cell-cleavage of the Halocynthia embryo of the protochordate is arrested at the early developmental stages, such as 1- to 8-cell, and the arrested embryo is cultured further until the normal hatching time, the membrane of a large and multinuclear blastomere expresses one of four types of excitability, such as neural, epidermal, muscular and non-excitable types. The differentiation of excitability will be different depending upon the cytoplasmic factors in the blastomere and the cell-cell interaction with neighboring blastomeres. In the present study, the cell autonomous differentiation was analysed using blastomeres from the cleavage-arrested 4-cell embryo of Halocynthia roretzi. All four blastomeres in the 4-cell embryo evoked a long-lasting Ca action potential, suggesting epidermal type differentiation in terms of the excitability, but the other cell type may appear when each blastomere was cultured separately. The 4-cell embryo was dechorionated by pronase, and the cleavage-arrested blastomeres were isolated and identified by the vital staining patterns of mitochondria. The isolated blastomere was continuously cultured at 10°C in Jamarine artificial sea water containing extra 20 mM CaCl. Voltage-clamp measurements were performed more than twice for each blastomere. Changes in Na, Ca delayed K and anomalous K channels were examined during development and compared between anterior A3 and posterior B3 blastomeres. At the final stage of the development all blastomeres were penetrated again in order to determine differentiation types in terms of the existence of various ionic channels. Both anterior A3 and posterior B3 showed an increase in egg type Na channels with a shift of the critical level to hyperpolarized direction after 20 hours development. After 50 hours the increased Na channels in A3 which includes presumptive neural region were altered into the differentiated type with the depolarized critical level, while B3 blastomere showed an increase in differentiated Ca or differentiated Ca plus delayed K channels. INTERSPECIFIC CHIMAERAS : A TOOL TO STUDY THE DEVELOPMENT OF THE NERVOUS SYSTEM Nicole M. Le Douarin, Institut d'Embryologie du CNRS et du Collége de France, 49bis, Avenue de la Belle-Gabrielle 94736 NOGENT-sur-MARNE CEDEX (FRANCE) In the Vertebrate embryo, the peripheral nervous system (PNS) arises mainly from a pluripotent structure, the neural crest. A fate map of the neural crest, constructed by using the quail-chick marker system, showed that it is regionalized into areas from which only a definite set of phenotypes originate. However, if the position of the neural crest cells is changed by heterotopic transplantations before the onset of their emmigration, it appears that virtually all the cell types forming the PNS ganglia can arise from any region of the neural crest, provided it is transplanted in the appropriate position in the embryo. This reveals the critical role played by the microenvironment experienced by the neural crest cells during their differentiation (1) and raises two questions : l1- what are the tissue factors selecting among the neural crest cell potentialities ; 2 - what is the state of committment of the neural crest cells during migration, and when they reach the site of gangliogenesis. Experiments carried out both in vivo and in vitro revealed that the precursors of sensory and autonomic neurons have different requirements for survival and differentiation. The role of the primordium of the central nervous system in the development of sensory neurons has been demonstrated along with the effect of BDNF on the survival of the early sensory precursors (2, 3). A clonal analysis of neural crest cell developmental potentialities is in progress and has already shown that during the process of migration the crest cells are highly heterogenous with respect to their state of committment (4). 1136 Perspectives in Marine Biology ADVANCES IN MARINE BIOLOGY OF CHINA. Ruiyu Liu (J.Y. Liu) Institute of Oceanology, Academia Sinica, Qingdao, China Great progress has been made in marine biology of China since the founding of the People's Republic. Based on the materials obtained through a series of comprehensive oceanographic and fishery resources surveys over entire China seas, hundreds of papers and tens of monographs have been published on the fauna and flora of Chinese seas, and hundreds of new taxa described. Based on these data, the "Fauna Sinica" and "Flora Phycologica Marina Sinica” are now in compilation. Im the ecological Studies, ‘the characteristics of community structure and seasonal changes of quantitative distribution of plankton and benthos (as well as nekton and micro-organisms) were analysed and described; the biology and ecology of some of the important species have been studied in detail; delineation of marine faunal and floral regions in China seas have also been made; and the prediction of natural resources of important fishes and shrimps, necessary for marine fishery production and management, has been successively practiced. Sea ranch- ing experiments by releasing juvenile shrimps and fishes into the sea for the enhance- ment of living resources (shrimp and fish stocks) have been successfully carried out in Jiaozhou Bay and many other bays and coastal areas in Huanghai and Bohai Sea, and the East and South China Sea, the production of shrimps in these areas is therefore increased in recent several years. Experimental ecology, physiology and biochemistry studies on marine organisms have been conducted and great strides made. New findings in the studies on the nucleus- cytoplasm interrelationship during the early developmental stages of amphioxus, ascidians and fishes by nucleo-transplantation technique revealed that the segregation of the cytoplasm was gradually established during organogenesis and embryogenesis, but not during the fertilization after the entrance of the spermatozoid. Scientists led by the late Prof. T.C. Tung and his student Prof. S.C. Wu believed and have experiment-— ally proved with their cytochemical studies that the cytoplasm played an important role in cell differentiation and in the transfer of genetic information. In their comparative photosynthetic study of the marine algae, Prof. C.K. Tseng and his student have put forth new proposal concerning three independent lines of evolution leading to the Rhodocyanophytae, the Chromophytae and the Chlorophytae. In the study of neuro- physiology, marine biologists found the giant nerve fiber of the ventral nerve cord (with a diameter of 150-250 um) of the shrimp, Penaeus orientalis, is characterized by a very high conductivity of 80-200 m/sec, much higher than that of higher vertebrates. Phototaxis measurements of the pelagic fishes, Decapterus maruadsi, Pneumatophorus japonicus, etc. have been made and the results are useful and helpful for the improvement of the perse seine fishing technique. Based on the findings obtained in the experimental ecology studies of induced maturation, spawning, larval breeding, nutrition and growth of juvenile and young fishes, shrimps, molluscs and seaweeds, effective methods for the mass production of fries of fishes and shrimps, spats of mussels, scallops, clams abalone and pearl oysters, juveniles of jelly fish and sea cucumber, and sporelings of kelp, purple laver and other species of sea weeds have been popularized in mariculture production. Production of shrimp, mollusc and seaweed culture increased drastically in recent years; annual production of cultivated shrimps in China reached 80,000 tons in 1986. The international academic exchanges and cooperations in the fields of marine biology developed fast and are strengthened recently. In the past 37 years, marine biologists of China have contributed much in the development of marine fisheries production, as well as the progress of marine biological sciences, and shall certainly contribute much more in the future to the socialist contruction and mordenization of our country. Perspectives in Marine Biology 1137 PERSPECTIVES IN MARINE BIOLOGY. J.D. Ebert, Carnegie Institution of Washington and Chesapeake Bay Institute, The Johns Hopkins University, Baltimore, Maryland. It is a signal honor to celebrate with you the one-hundredth anniversary of the founding of the Misaki Marine Biological Station. Your invitation to speak about the future of marine biology was welcome, for the revolutionary thrust of science and technology today has the potential of eclipsing, even within the next decade, all of the advances wrought in the preceding century. I take as my theme our opportunity--indeed our imperative-- aS marine biologists to work in the true service of mankind, to respond to real human needs, to battle man's ancient enemies of hunger, disease and ignorance--in short to truly enhance the quality of human lives through basic research. 'We cannot escape from the past'', wrote Rene Dubos in his paean to the symbiosis of humankind and the Earth, "but neither can we avoid inventing the future". But what future will we invent? How will we shape the world's marine DLological laboratories as they enter their second centuries? Often the question is put: '"'How effectively can we harness the special power of biotechnology, that is derived from genetic engineering and immunology, to better understand the develop- ment and function of marine organisms?"’ The answer is found in the program of our international seminar. However, our challenge goes beyond the thrust of today's technology and its applications to the often unspoken question, "Is there a continuing justification for marine laboratories?' Today's biotechnology permits us to focus, ever more rapidly, on ourselves, even to contemplate sequencing the human genome. Is there still a pivotal role for fundamental research in such fields as biodiversity and marine biomedicine, or must we focus our efforts on the preservation of the marine environment and marine resources through coastal mariculture and other economically attractive technologies? I will address these questions, which are critical to the future of marine biology. CLOSING REMARKS. S. Kinoshita. Dept. of Biol. Saitama Medical School, Saitama. On behalf of the organizing comittee of this symposium I would like to thank al] of you, speakers, chairpersons, and all participants, for the success of this very active meeting. These three days were very repleted days filled with stimulative and exciting presentations and discussions. The memory of this symposium will be the best monument for the centenial aniversary of the Misaki Marine Biological Station. Our Misaki Marine Biological Station is now making her first step to the second century. On the day of her new start, we are very happy to know that so many friends are together with us in solidarity. And at last, but never at least, we are grateful for the Japan Society for the Promotion ofScience and the Fujihara Foundation of Science, by whose support this symposium was able to be held in such a splendid success. Thank you and meet you again soon. ZOOLOGICAL SCIENCE 4: 1138 (1987) [Book Review| © 1987 Zoological Society of Japan New Horizons in Sperm Cell Research HipEO Monrl!, editor. Japan Scientific Societies Press, Tokyo, and Gordon and Breach Science Publishers, New York. 1987, 546 pp. This clearly organized book is based on papers presented at the Fifth International Symposium on Spermatology, held at Fujiyoshida, Japan, in 1986. Forty one invited and selected contributed papers are arranged according to the seven main topics considered at the meeting: 1. Spermatogenesis, Maturation, and Capacitation; 2. Evaluation and Control of Fertility; 3. Sperm Metabolism and Motility; 4. Sperm-egg Interactions; 5. Evolution- ary Aspects of Sperm; 6. Male Contraception; 7. X- and Y-bearing Sperm. Papers gathered under the last two headings represent the most accessible account of current trends devoted to analyzing the control of fertility in mammals including human. Four papers parti- cularly give us a comperehensive bird-view of the extent to which methods to develop repeatable means of separating male- from _ female- determining spermatozoa have been accom- plished. One of the more intriguing parts of the volume is the demonstration that certain products of post-meiotic gene expression are elaborated by developing germ cells in mice. Questions concern- ing the function of the products expressed by the haplotype gene remain open, but hopefully the elucidation of the significance of this gene express- ion should be done coupled with the in vitro culture of testis germinal explants that has now been successfully performed in amphibian system. Mammalian spermatozoa enjoy greater focus among the investigations gathered under the first four headings. However, the papers addressed to these topics confirm that the comparative approach to the functional aspects of spermatozoa is highly rewarding. Such investigations include the elucidation of the molecular cascade which leads to the events such as motility and the membrane fusion of the acrosome reaction, first discovered in the spermatozoa of lower vertebrates and echinoderms and now known to be shared among many mammals as well. In this respect, what is diverse may be the environmental condi- tions provided by the male and female reproduc- tive tracts, so that the efforts to elucidate the molecular basis for postgonadal maturation of sperm in diverse animal groups are warranted. A study on the biochemical characterization of iso- lated plasma membrane of sea urchin sperm also illustrates that invertebrates can still offer a para- digm for studies of gamete interactions. Papers collected under the fifth heading repre- sent the comparative and phylogenetic approaches that were particularly emphasized during the Symposium. These include the comparative ex- amination of sperm ultrastructure for elucidating the taxonomical problems at specific levels, in several invertebrates including molluscs and anne- lids. Another example of the comparative approach may be seen in the analysis of the production of polymorphic spermatozoa, notably found in oligochaete annelids. The occurrence of such dimorphism might be relevant to the more conceptual questions as to why grossly excessive numbers of sperm are produced by males, if indeed spermatozoa exist only to fertilize, as discussed in one paper in this section. These issues, together with the most fundamental ques- tions raised by C. R. Austin under the title “Are Sperms Really Necessary?”, certainly inspire the readers of Zoological Science who have a deep concern for the broad aspects of sexual reproduc- tion, including its genetic basis. CHIAKI KATAGIRI Hokkaido University Sapporo, Japan. ZOOLOGICAL SCIENCE 4: 1139 (1987) [Book Review] © 1987 Zoological Society of Japan Emotions—Neuronal and Chemical Control YUTAKA OomuRaA, editor. Japan Scientific Societies Press, Tokyo and S. Kager AG, Basel. 1986, 446 pp. This book is a collection of 38 rather independ- ent papers introducing mainly recent studies on the neural and chemical control of behaviors and physiological activities which compose the basis of our understanding of EMOTION. The papers are grouped into four chapters with the following titles: 1) Chemosensory Control of Feeding and Drinking, 2) Sensation-Energy Balance, 3) Neuroendocrine Affected Behavior and 4) Psy- cophysiological Correlates of Emotion. Many of the authors, as well as the editor, of this book are authorities of various fields of neurophysiology or psycophysiology, and studies presented in most of the papers are of the high level. The editor seems to attempt to elucidate mechanisms existing behind “EMOTION” by in- tegrating recent accomplishments in the field of studies on neural and neurochemical control mechanisms of feeding and drinking, since 20 papers of the first two chapters dealt with this problem. I do not know whether the editor thought his attempt successful or not, since he did not give his own conclusion in this book. Accord- ingly, each reader should conclude by him- or herself. However, this book still keeps its scien- tifically high quality and is useful to obtain current information on the control mechanism of food intake and related physiological functions. Neuroendocrine mechanisms are discussed in three papers of the third chapter and one paper of the last chapter. Some of them are too remote to emotion, although they can be highly evaluated. Two papers on the aggressive behavior also com- prises an interesting part of this book. The article most faithful to the title of the book is the paper by E. T. Rolls, entitled “A theory of emotion, and its application to understanding the neural basis of emotion”. In this article, the author aims to clarify the neural bases of emotion and to provide a theoretical basis to understand the complex neural system which is involved in emo- tion. He first discussed the function of emotion, and presented the function of the amygdala and orbitofrontal cortex which are indispensable to understand the neural bases of emotion. This important and interesting article occupies only 20 pages out of 446 pages. The editor could have provided more space to this article. In the last chapter, in addition to the paper by Rolls, there are a few papers which are more directly related to emotion. In conclusion, I felt that emotion is still far behind our scientific understanding, although neurophysiologists and psycophysiologists have achieved a great advance in these years. Many readers of this book might be inspired to challenge this hard target to attack. Susumu IsHII Waseda University Tokyo, Japan. ZOOLOGICAL SCIENCE 4: 1140 (1987) [Book Review| © 1987 Zoological Society of Japan Indo-Pacific Fish Biology TERUYA UYENO, RyoricH! ARAI, ToRU TANIUCHI AND KElicHI MATSUURA, Editors. The Ichthyological Society of Japan, Tokyo. distributed by Business Center for Academic Societies Japan, Tokyo. 1986, 985 pp. This is a comprehensive volume published as the Proceedings of the Second International Confer- ence on Indo-Pacific Fishes held at the Tokyo National Museum, Ueno Park, Tokyo from July 29 to August 3, 1985 under the sponsorship of the National Science Museum. It is honored by a foreward by His Imperial Highness Crown Prince Akihito as the Honorary President of the Conference. Almost all the representative Japanese ichthyologists of today and many authorities concerned from abroad comprising 24 countries assembled at the Confer- ence. Since most of the appropriate reviewers of this book are contributors, I took the liberty of substituting for them. This book is compiled in three major parts in phylogenetic order, Agnatha, Chondrichthyes and Osteichthyes. At the begin- ning is placed a brief statement on the history of Japanese ichthyology. Recent advances in ichthyology, as in many other branches of zoology, were due to the application of modern biological methodology. These include transmission and scanning electron microscopies, biochemical tech- niques, and electrophysiological approaches, all of which have served as powerful exploratory tools as described in this book. Part I Agnatha consists of eight papers selected from the symposium, the largest ever to be organized on the biology of cyclostomes. Part II Chondrichthyes composed of thirty papers deals with paleontology, systematics and zoophysiology, reproduction, growth, and behavior on elasmo- branchs and chimaeroids. Part III Osteichthyes constitutes naturally the largest part, being the most frequent in any water, most important natural resourses for food and most frequently used research materials among the fish classes. Fifty-five papers are devoted to zoophysiology, systematics, biology of larvae, reproduction, ecol- ogy and evolutionary genetics. Since the Devonian period, the age of fishes, stretching back less than half-a-billion years, most major branches of living fish classes were found as records preserved in rocks. The change from water to terrestrial life was first initiated in the Devonian by the early amphibians and completed by the reptiles in the late Paleozoic era. Later, from their reptilian ancestors, the warm-blooded birds and mammals were developed. Since good- bye fins and gills and welcome limbs and lung, these land dwellers have adapted themselves to almost every conceivable mode of life on land. In contrast, fishes have enjoyed inhabiting waters ever since those days. The modification and diversification of the basic design and function are so great in fishes as can be seen in this book that the fish world is comparable in taxonomic size to the rest of the vertebrates. However, it may be said that fishes have, nevertheless, maintained fundamentally the same basic design. Therefore, for a firm understanding of any vertebrate, know- ledge on fish is considered essential. Undoubtedly, this volume will become to be one of the dog-eared reference books for not only fish biologists but also those studying terrestrial verte- brates. The editors and the Ichthyological Society of Japan are to be commended for their contribu- tion to its publication. SEIICHIRO KAWASHIMA Editor, Zool. Sci. Hiroshima University Hiroshima, Japan. ANNOUNCEMENT 1141 The editors express their gratitude to the following reviewers, who evaluated papers for ZOOLOGICAL SCIENCE from September 1, 1986 to August 31, 1987. Without their assist- ance the journal could not function. Aketa, K. Akino, M. Amemiya, S. Ando, K. Aoki, J. Arai, R. Arai, Y. Asahina, S. Asami, K. Ashida, M. Baba, K. Berm, H. A: Chiba. Y. Egami, N. Eguchi, E. Eguchi, G. Endo, K. Fujii, R. Fujimoto, M. Funakoshi, K. Gorbman, A. Gotoh, T. Hamaguchi, Y. ata, 1. Hashimoto, H. Hayashi, K.-I. Hayashi, S. Hidaka, T. Hihara, F. Hiramoto, Y. Hirano, H. Hirano, T. Hisada, M. Hiwatashi, K. Hoshino, K. Ichikawa, M. Ichikawa, T. Ide, H. Iga, T. Tjiri, K. Imai, H. Imamura, T. Inoue, S. Inoué, S. Ishihara, K. Ishii, S. Ishizaki, H. Ito: Iuchi, I. Iwamatsu, T. Iwasawa, H. Kamishima, Y. Katagiri, Ch. Katakura, Y. Kato, K.-I. Kawahara, A. Kawamoto, K. Kawanabe, H. Kikuchi, T. Kikuyama, S. Kinoshita, S. Kobayashi, M. Kobayashi, Y. Kohno, S.-I. Koji, T. Kondo, H. Kondo, N. Konishi, K. Koshida, Y. Kimura, T. Kuramoto, M. Kuroda, Y. Kurokawa, H. Kuwasawa, K. Mabuchi, I. Machida, M. Maruyama, K. Matsui, M. Matsumoto, A. Matsumura, S. Matsuoka, N. Matsushima, O. Miki-Noumura, T. Mohri, H. Mori, T. Morino, H. Morita, H. Moriya, T. Muneoka, Y. Murakami, A. Nagahama, Y. Nagata, S. Naito, N. Naitoh, Y. Naka, K.-I. Nakamura, K. Nakane, T. Nakasone, Y. Nakauchi, M. Nishizuka, M. Nomaguchi, T. Noumura, T. Ochi, O. Ogawa, M. Oguro, C. Ohnishi, E. Ohoka, T. Ohta, Y. Ohtaki, T. 1142 Oide, H. Oishi, T. Oka, Y. Okada, M. Okada, T. S. Okajima, A. Ooka, H. Ooka, S. Oota, Y. Osanai, K. Osanai, M. Quintana, R. Saito, T. Sakai, H. Sakai, M. Sakaizumi, M. Sakurai, S. Sasaji, H. Sasaki, M. Sasayama, Y. Sato, H. Sato, T. Satoh, N. Satomi, D. Satou, M. Sawada, I. Shibuya, T. Shigei, M. Shigenaka, Y. Shima, A. Shimada, H. Shimazu, T. Shiokawa, K. Shokita, S. Sugi, H. Sugiyama, K. Suhama, Y. Suzuki, M. Suzuki, N. Suzuki, S. Suzuki, T. Tachi, C. Taguchi, Y. Takahashi, K. Takahashi, S. Takasugi, N. Takata, K. Takeda, H. Takeda, M. Takei, Y. Takeuchi, S. Terakado, K. Tomioka, K. Tsuchiya, T. Tsuneki, K. Tsutsui, K. Uchibori, M. Uchida, T. Uchikawa, K. Ueda, K. Uehara, T. UVéno, S.-I. Urano, A. Wada, M. Washio, H. Watanabe, K. Watanabe, M. Watanabe, T. K. Watanabe, Y. Yagi, S. Yamada, K. Yamada, M. Yamada, T. Yamagami, K. Yamaguchi, T. Yamamoto, K. Yamamoto, M. Yamamoto, Y. Yamanouchi, K. Yamasaki, T. Yamazato, K. Yanagisawa, T. Yasugi, S. Yoneda, M. Yoshida, M. Yoshizato, K. 1143 ANNOUNCEMENTS THE 59TH ANNUAL MEETING OF THE ZOOLOGICAL SOCIETY OF JAPAN The 59th Annual Meeting of the Zoological Society of Japan will be held at the Sapporo Medical College from October 8 to 10, 1988. Further information and application forms will be sent to the domestic members in the April issue of ‘Biological Science News’ (No. 197). The deadline for application is July 15, 1988. For application from foreign countries, please contact: Professor MITUHIKO HISADA Organizing Committee of the 59th Annual Meeting of the Zoological Society of Japan c/o Zoological Institute, Faculty of Science, Hokkaido University, Kita 10-jyo Nishi 8-chome, Kita-ku, Sapporo 060, Japan. Phone: 011-716-2111, Ext. 2749. ZOOLOGICAL SCIENCE AWARD Annual awards for the best original papers have been established through the donation of Narishige Scientific Instrument Laboratory, Tokyo. The sum of about 500,000 yen will be awarded annually at the Annual Meeting of the Zoological Society of Japan to a few papers published in ZOOLOGICAL SCIENCE during the preceding calendar year. Every original papers published in this journal will automatically be candiates for the award. The aim of the award is to encourage contributions to this journal. Selection Committee for the award will be organized every year. ZOOLOGICAL SCIENCE AWARD 1987 was given to the following three papers. Hiroshi Nojiri, Moriyuki Sato and Akihisa Urano: Increase in the vasopressin mRNA levels in the magnocellular neurosecretory neurons of water-deprived rats: in situ hybridization study with the use of synthetic oligonucleotide probe. Vol. 3, No. 2, pp. 345-350, 1986. Ryohei Kanzaki and Tatsuaki Shibuya: Identification of the deuterocerebral neurons re- sponding to the sexual pheromone in the male silkworm moth brain. Vol. 3, No. 3, pp. 409-418, 1986. Masakane Yamashita: Jn vitro maturation of the brittle-star Amphipholis kochii oocytes induced by cyclic AMP. Vol. 3, No. 3, pp. 467-477, 1986. 1144 AUTHOR INDEX A AGex Fak iol ccecs doe eee 1038 ADORE fc. 5 hi Oe eee 1055 ADOEMY 6.85 eS PE Cee 1017 Albes IM ae ret tence eae 1033 AIDES SII NA coc e ee ener meee 839, 1052 Adachi, Si \ intuit ae en ee eee 1085 Adachi. Bis beocsaieh inn. Seer mee ene a5 Ahmiad,Re Ais ite ob ox hae onion 93 AGING NE... as ae Ne ae soe ch 970 AidacK etic ati nk ioe eee eee 1084 Atigaks), 4. ..occs canoe ecm anne tak eee 1061 AvarainyMis 2 84 oe See x ok eee 523, 1080 Abas Sei au UO aitan Saronic eee a ee ga 889 ikasak aks. icine sts ace 739, 1024, 1050 AKC CaicdIKRE: is Mair sre teeset ecient 1028, 1029, 1030 Alki @ SiMe. doh ches ote ee eee 1009, 1013 Alkciyamla Dy. ccewcsccytoece prin oes euciee eee 990 Amakawas ls tape ca. Beer ces cer eee 965, 966 ANMAMAUNKK ioe. satis ahlstidawa awe Mase e eee 1025 AMOR OR IE ci. 5 ieee Me om crea 1070, 1071 Amemiiyat Sine youu acre ear 1049, 1058 Andersen... Ca ctsé taaccante cb astustecoet eae 123 ANGORA, hosts PUP PUNE aioli aves nee cance aes 1084 ANGOGMBS cpsh : inacd ewe eokes mete 37, 45, 960 NO KiGgE: okie cv ah 'arqanata poke et acudtoan oe eee 1054 AOA Keio nt Yo By 543, 967, 969, 970, 1061 NOK Resins 5.5 aesiays on hts logohcncd eetceslreeee he orinn 621 POM O TA co assoc ao a ret eee ar 1035 ENC} (0 ae) Ree RE APR TUM TORR AL Ge A ERR 1087 Aotsukas Tr ene eee ete eee ere 1022 Atalicdnat. £55 Sse. a eteevtaain. att os we opent 1000 AratnKesitccied asvx is case era ta ek 1043 Araineyil 220 Pee cee 197, 1078, 1079, 1082 Atakis Yorsauace londemswanp an (Maes. cpae 1034 ATHENA E6882 el eel cee eeu 813 PN SAC ALIN sc. aisle eect ecete, Saas ee eae 1001 Asainknad. tetssad. acto kibett.. aaaiebwed: 1040 PSA Ay) ie cise acs Sa ea a 929 NS alkuas Se KR Ui kote UE ora ea daae teers 839, 1052 PRS ELITE IRS 5 apts cls anaes Ws 1024 PSAO SR einer sk ue tect mare ce eme eons chars 1066 AsashimiagM icc sa 285, 411, 1065, 1068 Ashida, Mii cso. Aare es ee 1026, 1027 Ashida J9P.{ hii 4. 2208. 22S... eee 1003 Awano, Ma: . 4. < eee 1025, 1043 LJ EDD SO 1045, 1047, 1053 LM EDT 2. Ss 3 OO me 993, 994 _ZERION4L, IN, 5 es rrr 1093 WOE PIE DSS Oe Se 1051, 1065 COSTE bo9b000 == aoe nme © GET, er 1088 WOME ITAMENR AMO Gor ooo. 5 2545.5 Pe 81 1148 J UE G Cpe RRP re he eer RE GUrer chs 3 bi, 1102 Nacoos-Worena- eine ee eee eee PsA 1062 PACGMUEMONG, V- -scr.: racer eretenseateratetotetehe cele pane os 986 ames Bis 13.4 «casei coo oto pea eee 93 TOOM s Oc Rhine eke 1003 TOK UTA. Gc sacscnatatocectiecnbnts cae He De 675 K abasawadis Ae ‘eso iceaieneene ects hee 1107 Keath Os cess 9b. ncvncesnah assceeneere torctenokct none k 1 eA 627 ais Kee vag hin errs enteritis 2th eRe 1000 Kapitan) syaen.cWecut tae: et ctar mere o ad 889 Wea piubtiag plea red hcxscetininciss cy mera eensratennne neti ckks 649, 1028 I aIPIW aT Aol Cc Bfoe joarchensrire eueremasecncrovenceuetete eee wet 1023 | CVap thin) Gee emer ieee Reem res ues er Ie 4 oe 1039 CATING Eps lPM eh ete a be wale ge Ncw enlace eet ae 1065 aM GUI, Yes cis. iccr er Yoter near aeetancnetenotatonate 1003, 1004 CA TTNNUTTIN AAs Tins> asthe» wnat cine tee miase nap ace eo eTS 1007 IR Aris Ave AB eck yk ect shins ean cna Rcacnceso st done CO 93 J EVCOTS N00 Guan eee An ener enna n y 960, 1068 J REM As Ge 8 See een een ne err en Tee 6 1039, 1047 IST GOTH Tes wana hasten es cae ssi ee 28 dete ok hcmapa cect RO 978 IAT IM Ot OF HD se tin x excuses te hea kc arcechsasnarsetoue. eee 1116 I XevORN EIN) Cepeda eee y AES ee Ree e Rn dis Sic 1081 Iara ASAD co pesecs ei soeinctonnansantae Rieotctchanc atone Ae 523 asainavelVE: 8a ares cerca ee: 1110 Kashiwa eis AG oercecnoitnai cern oir meyee: 1067 Kashiwagitvee nc fe echoeah olka rohecrahtacb ana 3 1025 Kashiwamata, S. ......-. fai AEN pe Se SE 1054 Kasugas vlc. 96 evcnulirnaclaus dite sooweron ean 1061 Keasucas Nii io cory a pat ea A en alee eee 1078 Kasukawa, He .3ceccue. eee AEE Me 243, 982 asuyas Bes sek nec cba vaen seen sco ss. eee 693 CAS UY APO 40 8s etraiiois arenes east OE 998 IAS UNA 0:42 9t5y.d ir arse oraitr aad enicen re nce 969 Katagiri AG snvdcesnaccsclda conan yea wane 1011 NatagininC@h: i) 2853. see. 1052, 1053, 1067, 1138 eaten NO hat OR sic. ciclyei ha canes ane OS IRACASITIGN 5 os hy cna naed and cuncinahdioeaae O15 Aaa kunrar a Reecnis cine bed eneana ane ae 81 Natal unas Vin 4995025 c8 a wren wen ee 1093 IC Atal O Keay Fv 20 3h sea oh cal g MN Av ut vintce uc ay ee 1091 KReataokanwM 5 Of n25 0% 0 sinein aoe cole nina Se 1045 Katayamnias Hie. .xar cua sassoans dotson tose alt 981 Keatayanas Ds kiss viva ata nando oe ROO 968 | GOW o ORE At on RENE Dem NaMAE EE NS sg 1042 | CT (ond Hel oS Creme reners eneretnaes eee J. 997, 1071 IalOl Sees curtail da ee ea 187 Kato Y gs sales eee ee eee 969 IAT OSOY, eee. eek ee ie SI ee 1035, 1044 Nate S 6. Abd eee eee 1096 Katoh, Se + .0.4.hce snes arctersidetns ere 1033 Keaiwamuttia,,. Koa icctsesearctneiccrsrse otc eee 1041 aw,aninuira:, M1 |. «...2.:.:-ate-cestiates ker oteten ope 1019 IRA WAMU. 1 Si -snsessphsseseseettnekitinol one oe ee 979 Iawasakdl, HOU}. o.ocs:csc.srancuncinoe nna 1010 Kawashima, Se viens. 855, 867, 1080, 1081, 1082, 1083, 1101, 1140 Iearwaslnina,, Tx s.0005.+.::+stehs1.c0 tector et e 1059 ICAU CM TAL 8, «ce onc.cese ncncnorcnetl ee eo 1086, 1090 IAW AZOE 2D, vpesciecsrsrspdraraseenessranae eee 1090 | arrange Se REO E Se co: 1054 GMS, RG seh nthspsd saver onsrstononddonnidenno ee 763 Ae ea res MA Mie onsca fades syanchonetcdetoncruncet net cee 223 Khins/Maung Saing. 222-3734 eee 1104 ilaran. 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BRARI ES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLI | = = | Oe = ae = ae be = z = eB = S % 9 a oN os 9 ob AD = Z, E Nor 2 F 2 = ie : a hie 3 = 2 ES SMITHSONIAN _INSTITUTION ee INOS ES _LIBRARIES SMITHSONI 2 G2 : : = Yi e tig hes “4 — \ =~ 7 lz} < YP fv < Cc % Y fy eae z Sc WR ¢ te. ae ie oa = WS = td a 2 iy z hie z SNI NVINOSHLINS S3I4NVUGIT LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLI Saruvugin Satuvuatt INSTITUTION INSTITUTION INSTITUTION SMITHSONIAN INSTITUTION NOILALILSNI NVINOSHLINS S S3IYVHEIT LIBRARI NVINOSHIINS S3INVUEIT_ LIBRARIES ES. e SJIUVEGLI_LIBRARIES |= a ae fe = Ed ae = Z AYN 4 / Sx = = z a a Soe ZY Bw: oD . | © a OO %2 a8 O % ae _= = ‘i = “yy = = % se S = ee i. = = = . bis) eee Ww ae vy. = 77) : SNI NVINOSHLINS S31IYVYUYSIT LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI |Z 2 7 ” = a : = a = Yip 3 a = S cc = a ey, 5 om 5 ro) = S. QO *Gi Oo e ro) Zz Sea od z bul z= a = fe ES_ SMITHSONIAN INSTITUTION | NOILNLILSNI_NVINOSHLIWS _S31YVYalT LIBRARIES_ SMITHSON: Oo az wees oO os La oO pans ra) ay =a w = wo . = o = ty ae = x0 e 0 IS 2 Bs) = “LY 2 <> = eh Se AS Ss > ra Y fl, By Si 0) — Ps) EN ad a ras jy v0 - : = 2 WY = 2 ES © wn ee: wn a Wy n an wo = w = w hay = Ww Zz SNI_NVINOSHLINS S3IYVUYGIT_ LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLI if J 4 ae ad pes > ———— 2 ee os ¥S17 LIBRARIES SMITHSONIAN INSTITUTION NOJLNLILSNI NVINOSHLINS S31NYVYNEIT LIB! a e = iS Boe cobs z ~ : E 3 : : = wv pm 0] a = = = all rm > pin - = re = ri do } m = = ; m = m el m ” = w = un ey ee wn TION NOILNLILSNI_ NVINOSHLINS $3 luvud Tt IBRARIES SMITHSONIAN INSTITUTION NOI = ‘3 = a eae Se Se = = = = ‘Zz NS S z Se ae | 1@) els 1@) . Kt . Ale oO r of 5! g g ZW & ZN 8G Z E z = Ys 2 = N 2] S a s FA . B = 2 LIBRARIES SMITHSONIAN INSTITUTION NOILOLILSNI NVINOSHLINS S3IuVuRgIT LIB: ~_ uW? = Ww = w > & 2 & ~ Za uw ra 5 = = < = re : = a = : S e S Ps) oO — fee) ~ a os 7 Z j x i S$ a = er S$ TION | NOILALILSNI_NVINOSHLINS ~°3 iyVug ees LIBRARI ES_ SMITHSONIAN INSTITUTION o NOIL fe) aa eel rs) oss oO x me ) = wo . = mae o = > SS > = > 2 = me SS = ee = Fe: = eS = QV 5 a he - D ; — _ Ww Ee : > e > ~ > a = * Zz rm Zz = = JTION NOILOLILSN!I NVINOSHLINS S3IYVYURIT LIBRARIES SMITHSONIAN INSTITUTION NOld 0) =z uw = oe: uw z Oe Ww “a = =< = ‘s = < ne = ; 4 z = A : er ea. 8 z Ri : EQS FY bis a S a 2 B 3 Cc. 4 LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLINS S3Iyvuait LIB! ao Ww as w” — Ww = A a ro a 7) uw a oti wn ul n — = -j a — c x : e 2 . rs) = Z i) se 5 an O - J z oa = sf Zz ADERY NVINOSHLINS S3I¥vygit INSTITUTION NOIL & . = z = = g > SI o 2D ad = _ ~ = a 5 = a an cs - . i m zm m ah z Ww — Ww —_ — ce 2685 Mee a ee 3 9088 01261 neeeer Sa etn uy en en eres meecenten per he Peewee moe NEES arene ait ay aay peneaeneape Nee Be ae Day nia ode MeL ige nota ed ey Wem Aes gemyanguetly Ng Ryn emureesonpe: Peer ren erie t nrer ns le ct) Lyte he Ney og re See art me Ia pinay nnn eens anger te Pete gate Ae ee ay Haypitag eons et Toa Nida en Wo smiere tae ke hee LNAI HOES eed eyentey er PeeEe ‘ ‘ sueeiyeis ae erat worneae fam C8 PRE R SY etn gyre SNE nce TY oneenpe aegatoon Male yg avd a8 SM symp 08) pee en aarear yeas yAuaeae Go Rp anew run eases ee eet ee nave dette Wa A A be ay AN pes St pia oe A ON TNE ONS op ene eee Looe Lye PN AREER Pe ee core aap pier an, aN Mappa tags AURA re HN eer arin Lr) Ne aaa GUNN Ee WU wren we SER mV Nm Nn eye err pea rape ee aa re et Sr ed ee Mig at Oe Ue eprint veer Yeu ey OE Ma yer Ning ne A Pe Na DERE DEN RSE DNL EAA ES cree EME VOR SUTIS EAP Ned aL ef aue ere reese ee ene ce Dn eas Oe Kg eA Nn agin pS ERE ngeen eget ite ere eR SND MUNN ENT ene rte aya regeatiaung a ea yh ype gre hye Were eke RMN PS RANE I ERLE SS Sewer enee wee Beg YAS LYS AFA Oe eR HN OW ee Nag ee tyre UY Hag PO ery oe wy Brey etn Snrmeanrh SIN ELOY A SND Tee bee] MRM ARE MRS: Wee eae Aha Erne Ve reer Dene on ea a og ee a va oH = he ARE age 109 RRND wae rey eaters wae te hE Len eras weeny ore ARUN! 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