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Sie ; 4 Bee: | re 5 eae i IOSHLINS S31NVYSIT_LIBRARIES SMITHSONIAN er ere ON! CNYINOSHAING | uw z ee Zz TY) = u a ” ce w a =| Bf: o = oc a oc aa ENS : ZZ 4 < a) < = “tae Ns VE S 2 S : = SE . a —, a 6 3 a ras a Ske : a 2a aa - 4THSONIAN INSTITUTION NOILNLILSNI” NVINOSHLINS ~S31NVUGIT LIBRARIES SMITHSONIAN 4 — Gm & a, scan rs ~ 0 S : 1, co Co 2 oe cs Os 8 CS 2 GMD = OL." eC » | \ = o bee i ’ 1 i | v y | i | s Zz 2 ZOOLOGICAL SCIENCE continuation of the ‘Zoological Magazine’ and the ‘Annotationes Zoologicae Japonenses’ VOLUME 1 1984 published by The Zoological Society of Japan tee : 7 nO ee ALS ie | | Jenna i = vd tyarhentelog, Py oh 7 be gino? Inpaolbes oat CONTENTS VOLUME 1 REVIEWS Kawamura, T.: Polyploidy in amphibians ... 1 Sakai, H., H. Murofushi and G. Matsumoto: High molecular weight proteins of nerve cells inducing cross-linking or bundling of cyto- Soe 22) DROS ts ee eee 16 Dan, K.: The cause and consequence of unequal cleavage in sea urchins Kitamura, A. and K. Hiwatashi: Cell contact and the activation of conjugation in Para- C22 eS ol 2 ee 161 Yamada, T.: Developmental control of cell COMERS 20 Cir) 2 0 ean: ee 835 Chang, M.C.: Experimental studies of mam- RAMEE AAUON . - cscs doco ode tev edecdiaes 349 Ishikawa, H.: Molecular aspects of intracel- lular symbiosis in the aphid mycetocyte ...... 509 Kobayashi, M. and T. Hoshi: Analysis of respiratory role of haemoglobin in Daphnia magna Hisada, M., M. Takahata and T. Nagayama: Local non-spiking interneurons in the arthro- pod motor control systems: Characteriza- tion and their functional significance ......... 681 ORIGINAL PAPERS Physiology Hiramoto, Y., M.S. Hamaguchi, Y. Nakano and Y. Shoji: Colcemid UV-microirradi- ation method for analyzing the role of microtubules in pronuclear migration and chromosome movement in sand-dollar eggs ocak BBE eee 5 en Oe 29 Gomi, S. and H. Sugi: Factors influencing the force-extension relation of the series elasticity in glycerinated rabbit psoas muscle fibers ... 35 Hisada, M., M. Takahata and T. Nagayama: Structure and output connection of local non-spiking interneurons in crayfish ......... 41 Tomioka, K. and T. Yamaguchi: Response modification of cricket sensory interneurons TE ETE Oe ee SO) ee 169 Kawasaki, M. and K. Aoki: A directionally selective binocular unit in the midbrain tegmentum of Tribolodon hakonensis Takasu, N. and M. Yoshida: Freeze-fracture and histofluorescence studies on photorecep- tive membranes of medusan ocelli Tomioka, K. and _ Y. Chiba: Effects of nymphal stage optic nerve severance or optic lobe removal on the circadian locomotor rhythm of the cricket, Gryllus bimaculatus ...375 Kobayashi, M.., M. Nakamura and_é£@=*T. Hasimoto: Innervation of the heart of a prosobranch mollusc, Rapana thomasiana ...383 Kawakami, H. and M. Kobayashi: Pharma- cological approach to the analysis of regula- tion of molluscan heart activity Hashimoto, H.: Frustule movement of fresh- water polyp, Microhydra sowerbii (Lankester) Itoh, T., F. Yokohari and Y. Tominaga: Two types of antennal hygro- and thermoreceptive sensilla of the cricket, Gryllus bimaculatus CDESG EEE) MT ee, Eh ok 533 Oshima, N. and R. Fujii: A precision photo- electric method for recording chromatophore FESHONSESHINWVILV ONE date Reet nt 545 Kasukawa, H. and R. Fujii: Potassium ions act to release transmitter from ‘“‘cholinergic’”’ sympathetic postganglionic fiber to the glass catfish melanophore Kikuchi, M. and K. Aoki: The structure of photoreceptor cells in the pineal organ of the Japanese common newt, Cynops pyrrhogaster ‘DYVENOSUSTCIART ttt othe ts en 701 Oshima, N., R. Fujii and 4H. Kasukawa: Simultaneous recording of motile responses of light-absorbing and reflecting chromato- PHOLES 17 VITO ae eI ele coke eee dds asia el Satou, M., T. Matsushima and _ K. Ueda: Neuronal pathways from the tectal “‘snap- ping-evoking area”’ to the tongue-muscle-con- trolling motoneurons in the Japanese toad: il Evidence of the intervention of excitatory interneurons (COMMUNICATION) ......... 829 Cell Biology Tamura, R., M. Takahashi and Y. Watanabe: Molecular mechanism of cell division in Tetrahymena thermophila. 1. Analysis of execution period of a division-arrest ts- mutant, Gecko wee he eee 50 Yasuda, T., R.Tamura and Y. Watanabe: Molecular mechanism of cell division in Tetrahymena _ thermophila. II. Ultrastru- ctural changes found in a division-arrest ts- Mutants. sence es Se eee 62 Hashimoto, M., E. Nishida and _ H. Sakai: Effects of betaine, taurine and isethionate on microtubule assembly Maruo, F. and M. Okada: Functions of a maternal gene are required for the synchro- nous nuclear division during early develop- ment of Drosophila melanogaster. Analyses On: a ts=mutant ice: Jes ee eee 405 Mitsuhashi, J.: Isolation of a continuous cell line from larval fat bodies of an arctiid moth, Spilarctia seriatopunctata (Insecta, Lepidop- teray Arctidae)ink 4: AeeeuRReee ate ae 415 Takayama, S. and K. Matsumoto: Nucleolus organizer region lateral asymmetry induced by BUdR incorporation in mouse chromo- Yamamoto, M.: Photoreceptor collaterals in the cuttlefish retina (COMMUNICATION) Saitoh, T. and H. Asai: Saturation density of Tetrahymena with or without agitation at various surface-to-volume ratios of culture test tubes (COMMUNICATION) Maruyama, K., K. Tsukagoshi and I. Mabuchi: Direct visualization of the effect of Mg?* on the elongation at the pointed ends of Limulus acrosomal actin bundles (COMMUNICA- TION) Biochemistry Yoshioka, S.. M. Masada, T. Yoshida, T. Mizokami, M. Akino and N. Matsuo: A- typical phenylketonuria due to biopterin deficiency: Diagnosis by assay of an enzyme involved in the synthesis of sepiapterin from dihydroneopterin triphosphate .................. 74 Kodama, T.: Further studies on the phys- iological role of hexose-6-phosphate dehy- drogenase: No positive correlation between hexose-6-phosphate dehydrogenase and en- zymes other than NADPH-cytochrome c reductase ..cccse.0)s os nether 205 Katoh, S. and T. Sueoka: Pteridine-metab- olizing enzymes in rat erythrocytes during development «032.0055. 6.05.40). 4lst ee DVS Izumi, S., K. Kiguchi and S. Tomino: Hor- monal regulation of biosynthesis of major plasma proteins in Bombyx mori ............... P23 Saito, Y. and H. Watanabe: Partial bio- chemical characterization of humoral factors involved in the nonfusion reaction of a botryllid ascidian, Botrylloides simodensis ...229 Sugita, H. and K. Sekiguchi: Lactate dehydrogenase of the horseshoe crabs and their hybrids Hamazaki, T., I. Iuchi and K. Yamagami: Chorion glycoprotein-like immunoreactivity in some tissues of adult female medaka (COMMUNICATION ).....(55..c0.2. See 148 Developmental Biology Morisawa, M., S. Morisawa and R. de Santis: Initiation of sperm motility in Ciona in- testinalis by calcium and cyclic AMP ......... 237 Osanai, K. and K.Kyozuka: Cross-fertili- zation between sea urchin eggs and mussel spermatozoa: Sperm entry without egg ActivatiONn o....isb.Geee et... s. ee eee 245 Yoshizaki, N. and Ch. Katagiri: Necessity of oviducal pars recta secretions for the forma- tion of the fertilization layer in Xenopus LQ@VES occas leOeleds sclie asa URe ee ee 255 Miyachi, Y.. M.Iwata, H.Sato and E. Nakano: Effect of fibronectin on cultured cells derived from isolated micromeres of the sea urchin, Hemicentrotus pulcherrimus...265 Ito, S. and K. Takamune: Early genesis of slow action potentials in co-cultured ecto- dermal cells at different ages ..................4. 273 Shirane, T.: Regulation of gonadal differ- entiation in frogs derived from UV-irradiated eggs Nishioka, M. and H. Ohtani: Hybridogenetic reproduction of allotriploids between Japa- nese and European pond frogs ................ 291 Sawai, T.: Surface movement in the first Gleavaze Stage Of NEWt E8ES ........ccsccccessees 427 Kinoshita, T. and K. Okazaki: Jn vitro study on morphogenesis of sea urchin larval spicule: Adhesiveness of cells ................. 433 Masuda, M. and H. Sato: Reversible resorp- tion of cilia and the centriole cycle in dividing Gemsoonsea urchinblastulac .2:22.......0..560008 445 Itow, T. and K. Sekiguchi: Acid mucopoly- saccharide in embryos of the horseshoe crab, Techypleus tridentatus (Chelicerata, Arthro- SITE aS Sane Se SRO a eke ss mene EM 463 Tanaka, K. and Y. Watanabe: Choanocyte differentiation and morphogenesis’. of choanocyte chambers in the fresh-water sponge, Ephydatia fluviatilis, after reversal of developmental arrest caused by hydroxyurea Hayashi, K.-I. and T. Hamano: The com- plete larval development of Caridina japonica De Man (Decapoda, Caridea, Atyidae) reared MN IT LORY) ois.) 5 EN eas. SEIS SWAll Iwasawa, H. and kK. Yamaguchi: Ultra- structural study of gonadal development in MIEN PARIS «4 812122... : BIO ALS. POE 591 Watanabe, Y.G.: Differential cell prolifer- ation and morphogenesis in the developing adenohypophysis of the fetal rat ............... 601 Yasumasu, I., A. Fujiwara, A. Hino and K. Asami: Effect of several redox dyes on the respiration of unfertilized eggs of sea MRMEEETILY 2, RE SEASEIRIDSS «Ss he PRM (P43) Kishida, Y. and E. Asai: Influence of nerve cord removal on pharynx induction by head piece transplanted in postpharyngeal body region of the planarian, Dugesia japonica IEEE 019. ROLES, SUI 6 oe 3 Nad Ries 3 Terada, M.: Comparison of zoeal develop- ment between the two carcinoplacid crabs, Carcinoplax longimana (De Haan) and Eucra- te crenata De Haan, reared in laboratory ...743 Haraguchi, T., Ch. Katagiri and H. Nagano: Changes in level of DNA polymerase a activi- ty during oogenesis of the toad, Bufo bufo japonicus (COMMUNICATION )............... 505 ill Hasegawa, Z. and E. Ohzu: Differentiation of hatching gland cells in the explants from anuran embryos (COMMUNICATION). ...673 Endocrinology Oguro, C., M. Fujimori and Y. Sasayama: Changes in the distribution of calcium of the frog, Rana nigromaculata, following ultimo- branchialectomy and calcitonin § admini- SUCTION LOE EE TR ORM ORs Sol ole 82 Urano, A.: A Golgi-electron microscopic study of anterior preoptic neurons in the bullixoetamatihent@ades.....uee. a eeeeett aoe te 89 Mainoya, J. R. and H. A. Bern: Influence of vasoactive intestinal peptide and urotensin II on the absorption of water and NaCl by the anterior intestine of the tilapia, Sarotherodon mossambicus Hirano, T.and S. Hasegawa: Effects of angio- tensins and other vasoactive substances on drinking in the eel, Auguilla japonica ......... 106 Watanabe, K. and E. Ohnishi: The mode of ecdysteroid accumulation in ovaries of Bombyx mori during the pupal and pharate ACUI PEHIOMUE A.A 248.08 Foes ilo ee ene 114 Takasugi, N. and T. Kato: Acceleration by vitamin A of the permanent proliferation of mouse vaginal epithelium induced by neo- natal treatment with progestin .................. 120 Jolivet-Jaudet, G., M. Inoue, K. Takada and S. Ishii: Circannual changes in corticos- terone plasma levels and binding of corti- costerone to plasma in Bufo japonicus formosus Chan, P. J. and W.R. Dukelow: Variations in squirrel monkey in vitro fertilization responses with seasonal and environmental conditions Moriya, T., C.R. Thomas and E. Frieden: Binding of triiodothyronine by nuclei of bullfrog tadpoeleieryuirocyte™. «... 20.05. ce eis 477 Ishii, T. and S. Kikuyama: Uptake of [°°S]- sulphate by Xenopus cartilage: The in- fluence of growth hormone and prolactin ...609 Takada, K. and S. Ishii: Purification of bullfrog gonadotropins: Presence of new subspecies of luteinizing hormone with high ISICICC EI MOMS Ee hse oy seeders ceelee stacecvedenve 617 iv Numata, H. and T. Hidaka: Termination of adult diapause by a juvenile hormone analogue in the bean bug, Riptortus clavatus Aaa SEGRE Oe he Shain ee Oe ee an pte WS Sasayama, Y., C.Oguro, R. Yui and A. Kambegawa: Immunohistochemical de- monstration of calcitonin in ultimobranchial glands of some lower vertebrates ............... 755 Takami, S., Y. Jokura and A. Urano: nuclear organization of the preoptic nucleus in the toads BuUsoyaponicusy. s.ieas- ee eee 759 Hutz, R.J., M. Ghosh and W.R. Dukelow: Steroid uptake and *H-uridine incorporation by early hamster and squirrel monkey (in vitro fertilized) embryos: Effects of ovulatory regimen Tachibana, H., T. Iguchi and N. Takasugi: Different perinatal periods of vitamin A administration for prevention of the oc- currence of permanent vaginal changes in mice treated neonatally with estrogen ......... Udy Oguro, C., H.Nogawa and _ Y. Sasayama: Occurrence of a _ physiologically active calcitonin in the brain of the bullfrog, Rana catesbeiana (COMMUNICATION) ........... 841 Behavior Biology and Ecology Yamanouchi, K., Y. Nakano, M. Fukuda and Y. Arai: Mesencephalic central gray as supraspinal neural substrates for lordosis reflex: Deprivation of serotonergic influ- ence by p-chlorophenylalanine ..................126 Yamamoto, A. and S. Ohba: Temperature preferences of eleven Drosophila species from Japan: The relationship between preferred temperature and some ecological character- istics in their natural habitats Yamamoto, A. and S. Ohba: Heat and cold resistances of sixteen Drosophila species from Japan in relation to their field ecology Watanabe, M., T. Kobayashi and H. Terami: Changes in eye color during aggressive interaction in the chocolate _ cichlid, Cichlasoma coryphaenoides .........ccccccceeeeeee 787 Genetics Sakaizumi, M.: Rigid isolation between the northern population and the southern population of the medaka, Oryzias latipes Taxonomy Aoki, J.: New and unrecorded oribatid mites from Amami-Ohshima Island, southwest JAP an ince ccsccauieslediecdiieledleeeeese TR REeEE RE aeEEee 132 Sawada, I.: Two new species of cestodes belonging to the genus Vampirolepis (Cyclo- phyllidea: Hymenolepididae) from cave bats Of Tatwan 5 2c8idc Js a ee 327 Hasegawa, H.: Skrjabinelazia machidai sp. n. (Nematoda: Seuratidae) from Gekko japoni- cus on Okinawa Island, Japan .................. 483 Kawakatsu, M. and R.W. Mitchell: Oahu- hawaiiana kazukolinda gen. et sp. nov. (Tur- bellaria, Tricladida, Maricola), a new freshwater planarian from Honolulu, Oahu Island, Hawaii, U.S. A. Iwamatsu, T., H.Uwa, A.Inden and K. Hirata: Experiments on interspecific hy- bridization between Oryzias latipes and Oryzias celebensis Matsui, M. and K. Miyazaki: AHynobius takedai (Amphibia, Urodela), a new species of salamander from Japan Ohnishi, S. and T. K. Watanabe: Systematics of the Drosophila montium species subgroup: A biochemical approach Shimazu, T. and S. Shimura: Paralepidapedon g. n. (Trematoda: Lepocreadiidae), with descriptions of metacercariae of Para- lepidapedon hoplognathi (Yamaguti, 1938) comb. n. and of two other species from sea UNCHINS fwd. len See 809 Sawada, I.: Cestodes of bats from Japan, with descriptions of new species of the genus Vampirolepis (Cestoda: Hymenolepididae) ...819 Hasegawa, H. and M.Otsuru: Pseudab- breviata yambarensis sp. n. (Nematoda: Physalopteridae) from the tree lizard, Japarula polygonata polygonata (Agamidae), on Okinawa Island, Japan (COMMUNI- CATION). (22a ORlo Rs Ae 677 Others Abstracts of papers presented at the Fifty-Fifth Annual Meeting of the Zoological Society 2 DS PES pants hates eS ee eee eee eee 854 Kobayashi, H.: President’s message ............ 852 Egami, N.: Editorial—‘‘Zoological Science’, the new official publication of the Zoological SUGISHFOl Wapall seca. wrecks veces oss eutereues ess i COSY TUE ESS i a i ec NO 845 IBOOKSKE VIE WISH teen acetic tces eoseceacanecuisee panies 1012 PNMINOUMCEMEMUS meee cscccuceuab aes seccsuocees iv, vil, 1014 wali Otti 1 rise ray by bi a \ | & = | viv Fike + : Ly te -. > be ’ nie : {a a 3) ra oo ., ‘ | aN d “f- - a : e ‘ = ; , i y ga! ils /_ me ae ct} 4 it : — “a * ég ; aiej- , OR me i f*« hand in ei te haan Be @ eT { 4 | : ' | " eae hata aE at ¢ = / ZOOLOGICAL SCIENCE \tinuation of the “Zoological Magazine” and the © ‘Annotationes modal [ha Japonenses— ZOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Nobuo Egami (Tokyo) Toshin-building, Hongo 2—27-2, Bunkyo-ku, Associate Editors: Tokyo 113, Japan. (03)-814—-5675 Susumu Ishii (Tokyo) Officers: Seiichiro Kawashima (Hiroshima) President: Hideshi Kobayashi (Funabashi) Koscak Maruyama (Chiba) Secretary: Makoto Asashima (Yokohama) Assistant Editors: Treasurer: Kiyoaki Kuwasawa (Tokyo) Takeo Machida (Hiroshima) Librarian: Ryoichi Arai (Tokyo) Kazuyoshi Tsutsui (Hiroshima) Editorial Board: Howard A. Bern (Berkeley, endocrinology) Aubrey Gorbman (Seattle, endocrinology) Robert B. Hill (Kingston, physiology) Yukio Hiramoto (Tokyo, cell physiology) Yukiaki Kuroda (Mishima, cell genetics) Hiromichi Morita (Fukuoka, sensory physiology) Tokindo S. Okada (Kyoto, developmental biology) Andreas Oksche (Giessen, neuroanatomy) Hidemi Sato (Nagoya, cell biology) Koichi Sekiguchi (Tsukuba, phylogeny) . Mayumi Yamada (Sapporo, systematic zoology) Ryuzo Yanagimachi (Honolulu, reproductive biology) 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, Associate Editor, Zoological Science, Zoological Institute, Faculty of Science, Hiroshima University, 1-1-89 Higashisenda-cho, 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, and in Asia between Pakistan and Korea, it is distributed by Business Center for Academic Societies Japan, 6-16-3 Hongo, Bunkyo-ku, Tokyo 113. Subscriptions outside Asia between Pakistan and Korea should be ordered from the sole agent, VNU Science Press BV. Park Voorn 4, 3454 JR De Meern, P. O. Box 2073, 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 volumes. 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 1984, 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 1: i (1984) i EDITORIAL———“Zoological Science”, the new official publication of the Zoological Society of Japan The Zoological Society of Japan is at the core of scientific zoology in Japan today, and owes its origin to the Tokyo Biology Society which was founded in 1878. Credited to a large extent with founding this Society was E.S. Morse, an American professor of natural history who was teaching biology at the University of Tokyo as a professor at the time. The Society was dubbed the Tokyo Zoological Society in 1885, and was essentially an informal discussion group until 1888 at which time Vol. 1, No. 1 of the ““‘Dobutsugakuzasshi”’ (‘The Zoological Magazine’’), the official society publication, appeared. Initially, the ““‘Dobutsugakuzasshi” contained articles in Japanese with the primary objective of educational edification. However, along with the development of zoology in Japan came the necessity for publishing papers in Western languages (English, German, French), thus leading to the ‘“‘Annotationes Zoologicae Japonenses” (“The Dobutsugaku Iho’’) an offshoot of the ‘“‘Dobutsugakuzasshi”’, which was first published in 1897. Thereafter, the format and contents of the ‘““Dobutsugakuzasshi’’ were changed in order to give increased emphasis mostly to original papers in Japanese language. This metamorphosis exemplifies the changing course of zoology in Japan at the time. Except for the World War II years these two journals have published regularly, with the ‘““Dobutsugakuzasshi’”’ distributed to all Society members and the “Annotationes Zoologicae Japonenses’”’ made available to special members on a subscription basis. These publications continue to this day to make important contributions to the development of zoology. At this point, special mention must be made of the tremendous support offered to the journals by all Society members, and particular homage is due our predecessors for their efforts in operating the Society and editing the journals. Membership in the Society increased markedly after 1970, and along with diversification in the fields of zoology and the remarkable advancements in modern biology came a fundamental change in thinking on the part of the members in the Zoological Society of Japan. A number of committees were convened in answer to a call for a re-examination of the nature of the official Society publications, resulting in the decision to combine the two journals with a view forwards creating a truly international publication. Accordingly, the journals the ““Dobutsugakuzasshi” and the “‘Annotationes Zoologicae Japonenses”’ (of which were published Vols. 1-92 and 1-56, respectively) were replaced by the “‘Zoological Science’, which was inaugurated as the official publication of the Zoological Society of Japan. We wish to express our deep appreciation for achieving this re-organization to Professors Katsuma Dan, Masutaro Kuwabara, and Hideshi Kobayashi, former and present Society Presidents, as well as the Council of the Zoological Society of Japan, the Re-organization Committee, and all the members of the Zoological Society of Japan. As the success of this re-organization hinges on the contents of the journal, we are inexorably dependent upon the criticism and support not only of the Zoological Society of Japan, but of scientists everywhere concerned with zoology and biology. January, 1984 Nosuo EGAMI Editor-in-Chief ha f 2 | teas fad ; sith $77) oe ae Hoi Sic =F 7 ‘ ; eas + :. i wm Tes et ri bik Tie aa 7 cA i teh Oe Wy de : See) Sit Fy po ital Watters ic ae 1 4 | sah Gata ZOOLOGICAL SCIENCE 1: 1-15 (1984) © 1984 Zoological Society of Japan REVIEW Polyploidy in Amphibians TOSHIJIRO KAWAMURA Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Hiroshima 730, Japan CONTENTS Introduction PRONE FAl DOL DIOIY. «0.50. ccssececaccucaesacnccesces’ A. Polyploid species 1. Leptodactylidae, 4. Bufonidae, 5. Ranidae, B. Spontaneous polyploids C. Accidental polyploids Il. Artificially induced polyploidy PM eeTMITOUTIDIOIOS <2. <. sccckcccssceiccccsccsceacvecses B. Allotriploids ee) PAUOLEETADIOIGS oie. 5scccdecoedscecceccsevceees D. Allotetraploids E. Pentaploids, hexaploids and heptaploids ce DIGS OT bee eta ti a aE Oe) eae a References INTRODUCTION About half a century ago when the present author first attempted to study polyploidy in amphibians, it was the general belief that any polyploid animal in vertebrates cannot survive even if they are produced. In such an atmosphere, the only exception was the existence of 18 triploid tadpoles discovered by Hertwig and Hertwig [1] in the control of hybridization experiments between female Rana esculenta and male Bufo viridis. It was rather strange that nobody reex- amined the problem of polyploidy in amphibians for nearly twenty years thereafter. Vertebrates were in an extreme contrast to flowering plants in absence of polyploids. It is well-known that most of the flowering plants are various kinds of polyploids. The present author Received August 6, 1983 2. Hylidae, 3. PSOE H EEE HEHEHE HEHE HEHEHE HEE EEEEE HEHEHE EEE HEHEHE EEE EEE HH EEEEEH EHH EEEEE EEE EEE See eee eee reese eeesseeee POORER E HEHEHE HHH EEE EEE HEHEHE HEHEHE HEHEHE EEE HEHEHE EE EEEE HEHEHE HEEEEEHEEE a SPREE HEHEHE EEE HEHEHE HEE HE EEE EEE EEE EEE EEE EEE EEE HEHEHE EE EH EE EEE HEE EE EEE HEHE EEE EHEE EHH EEEEES POPPE HR E EEE EEH HEHEHE HEHEHE EHH HHH HEHEHE HEHEHE EES TOP RHHHHHEHHEHEHHHEHEEEEHEEEE HEHEHE EEE HEHEHE EH EEEHEHH EEE EEE EES SRR H HERR HHH EEE EEE HEHE EEE EE EEEE EEE EEE EE EE HEHEHE EEE HEHE EEEEE HEHEHE EEEEEE HEHE EEE EEE EHEHEE HEHEHE H HEHEHE EEEES Pipidae, 6. Ambystomidae was one of the investigators interested in obtaining polyploids in amphibians and in comparing them with those in flowering plants. It seemed to be a most worthy work to the present author at least to produce various kinds of polyploids comparable to those of the vegetable kingdom by artificial means in place of the evolutional force that worked during the geological age. Since the time about 20 years ago, many poly- ploid species or populations belonging to six families have been discovered in various areas of the world. These natural polyploids as well as artificially induced polyploids will be reviewed here. I. NATURAL POLYPLOIDY A. Polyploid species 1. Leptodactylidae The existence of amphibians with a high number 2 T. KAWAMURA of chromosomes in South America was briefly reported by Saez and Brum [2]. According to them, Odontophrynus americanus belonging to Leptodactylidae was 42 in diploid number, while Ceratophrys ornata belonging to the same family was 88, 92, 98 or 108 in diploid number. These chromosome numbers were remarkably higher than those of three other species which were 2n=22. Becak ef al. [3, 4], Bogart [5], Becak et al. [6] and Becak [7] confirmed that the chro- mosome number of Odontophrynus americanus distributed in Brazil and Argentina is 2n=—44. As the chromosomes of Odontophrynus cultripes distributed in Brazil [6, 7] and of O. occidentalis distributed in Argentina [7, 8] are 22 in diploid number, O. americanus is considered to be a tetraploid species. In fact, Becak et al. [9], Barrio and de Rubel [10] and Becak and Becak [11] discovered an Odontophrynus americanus popu- lation having 22 chromosomes in Argentina and Brazil. The tetraploid and diploid populations are not overlapped in distribution. These inves- tigators insisted that the tetraploid Odontophrynus americanus have originated by autopolyploidy from the diploid O. americanus, although they are morphologically indistinguishable. Becak et al. [12] obtained triploid hybrids between tetraploid female Odontophrynus amer- icanus and diploid male O. cultripes. These hybrids were viable and apparently did not show any abnormality until seven months after hatching at least. Becak and Becak [13] assumed that natural triploids were phylogenetically an inter- mediary step to higher degrees of ploidy. In fact, Becak et al. [9] obtained artificial triploids from intraspecific matings between diploid and tetraploid specimens of Odontophrynus amer- icanus. Then, Becak and Becak [11] insisted that the tetraploid Odontophrynus americanus could have originated by autotriploidy from the diploid O. americanus and is already isolated from the latter to such an extent that both may be considered as different species, as no natural triploid hybrids have been found up to date. The exact chromosome number 104 of Cera- tophrys ornata was determined by Bogart [5] and Barrio and de Chieri [14]. Barrio and de Chieri described that there are two populations of Ceratophrys ornata, one having 26 chromosomes and the other having 104 chromosomes. They are distributed in the northern and the east-central area of Argentina, respectively. These two popu- lations are evidently cryptic species and one is octoploid as compared with the other. Becak et al. [3, 6] and Becak [7] reported that another Brazilian horned frog Ceratophrys dorsata has also 104 chromosomes, while Colombian C. calcarata similar to C. ornata in appearance is N= 2H | Barrio and de Chieri [15] examined the chromo- somes of several Pleurodema species distributed in South America. P. bibronii (=P. darwinii) from Uruguay and P. kriegi from the Central Argentina are 44 in chromosome number, while P. bufonina and P. thaul from the southern Argentina, P. cinerea, P. guayapae and P. tucumana from the northern Argentina, and P. nebulosa from the Central Argentina are 2n=22. Thus the two species having 44 chromosomes are tetraploid as compared with the others. 2. Hylidae Johnson [16] reported that the two call-types, fast and slow, of Hyla versicolor distributed in eastern North America are isolated by genetic incompatibility. Thereafter, he [17] confirmed by hybridization experiments that the chance of gene exchange between the two call-types is reduced due to high hybrid sterility between them and inviability of the F, backcrosses. Based on these findings, he considered that the two call-types of Hyla versicolor may actually be two species. However, Johnson [18] divided the Hyla versicolor complex into two subspecies, Hyla y. versicolor and H. vy. chrysoscelis. While the former is of slow call-type and mostly found in the northern area, the latter is of fast call-type and abundant in the southern area. Ralin [19] described the two subspecies as two discrete species, Hyla versicolor and Hyla chrysoscelis, and disclosed their ecological differences. While Hyla chrysoscelis tends to call from trees and bushes, Hyla versicolor usually calls from the ground. Wasserman [20] described that Hyla versicolor is a tetraploid species having 48 chromosomes and an allied Hyla andersonii is a diploid species ——_ Polyploidy in Amphibians 3 having 24 chromosomes. The fact that Ayla chrysoscelis is also a diploid species like Ayla andersonii was reported by Bogart and Wasserman [21] together with the confirmation that Ayla versicolor is a tetraploid species. They postulated that the tetraploidy could independently arise in the diploid species through an intermediate triploid stage. Ralin [22] obtained hybrids be- tween tetraploid female Hy/a versicolor and diploid male Ayla chrysoscelis. These hybrids are tri- ploid and poor in viability. Maxson ef al. [23] stated on the basis of their immunological researches that the eastern and western populations of Ayla chrysoscelis have been genetically isolated for about 4 million years and that tetraploid Hyla versicolor are allotetraploids produced recently from hybrid- ization between the eastern and western Ayla chrysoscelis. Ralin and Rogers [24] and Ralin and Selander [25] made comparisons between the two populations of Hyla chrysoscelis as well as between Hyla chrysoscelis and Hyla versicolor in 13 external morphological measurements and in the electrophoretic patterns of enzymes encoded by 12 structural gene loci. The results of these comparisons showed that Hyla versicolor evolved from Hyla chrysoscelis after the latter had dif- ferentiated into the two populations. These investigators assumed that Hyla versicolor arose either through allopolyploidy or by autopoly- ploidy in a population of Hyla chrysoscelis which is genetically intermediate between the eastern and western populations of Hyla chrysoscelis. Wiley [26] examined the G-banded karyotypes of 12 Hyla species collected from six states of the USA and confirmed that the populations of Hyla chrysoscelis have probably been involved in the origin of the tetraploid species Hy/a versicolor. Danzmann and Bogart [27] examined the gene dosage effects on MDH isozyme expression in triploid offspring obtained from reciprocal matings between Ayla chrysoscelis and Hyla versicolor, and confirmed that progeny phenotypes expected from parental genotypes are observed in all the interspecific cross combinations per- formed. The finding that Phyllomedusa_ burmeisteri collected from Sado Paulo, Brazil is a tetraploid species was reported by Becak ef al. [8]. This treefrog possesses 52 chromosomes in mitotic figures. Thereafter, Batistic et al. [28] discovered a diploid population (2n=26) of Phyllomedusa burmeisteri from state of Rio de Janeiro, Brazil, while a population of P. burmeisteri from state of Sao Paulo, Brazil is tetraploid. They assumed that the tetraploid population recently evolved from the diploid population by autopolyploidy, because tetraploid frogs are very similar to diploid frogs in appearance as well as in chromosome morphology and reveal quadrivalents in meiosis. Barrio [29] examined the chromosomes and mating calls of four Argentine Phyllomedusa species and confirmed that P. burmeisteri is a tetraploid species and P. sauvagii, P. pailona and P. hypochondrialis are diploid species. He stated that acoustic divergence among them could be an effective prenuptial isolating mechanism in an overlapping area. 3. Pipidae Tymowska and Fischberg [30] reported on the chromosomes of 10 species and two sub- species of the genus Xenopus distributed widely in Africa. While X. /. laevis, X. I. petersi, X. l. victorianus, X. (laevis) borealis, xX. (laevis) gilli, X. muelleri, X. clivii and X. farseri, are 2n=36 in chromosome number, X. (/aevis) bunyoniensis and X. ruwenzoriensis possess 72 (4n) and 108 (6n) chromosomes, respectively. A single Xenopus tropicalis from Cote dIvoire is 2n=20 in chromosome number. In sperma- togenetic metaphase I of Xenopus bunyoniensis having 72 chromosomes, only 36 bivalents and no multivalents are observed. The 108 chromo- somes of Xenopus ruwenzoriensis can be classified into characteristic groups of six chromosomes. The first spermatocytes usually show 54 bivalents at the meiotic metaphase, although one or two big multivalents are also found in some of them. The hexaploid and tetraploid species of Xenopus from western Uganda were repeatedly described by Fischberg and Kobel [31], although YX. (/aevis) bunyoniensis with tetraploid 72 chromosomes was treated as Xenopus species nova. The latter was given the name Xenopus wittei n. sp. by Tinsley et al. [32]. 4 T. KAWAMURA Tymowska ef al. [33] reported that Xenopus vestitus distributed in eastern Central Africa was a tetraploid species possessing 72 chromo- According to Tinsley [34], this species is commonly sympatric with Xenopus laevis victorianus. Tymowska et al. observed the for- mation of 36 bivalents at the first meiotic meta- phase, and stated that this tetraploid species represents either a case of ancient autotetraploidy or, more likely, one of allotetraploidy of more modern origin. Kobel ef al. [35] discovered a new tetraploid species having 72 chromosomes in western Cameroons. They named this species Xenopus amieti. According to them, what was known hitherto as Xenopus fraseri includes three distinct species which differ in chromosome num- ber, that is, X. fraseri (2n=36), X. amieti (4n=72) and X. ruwenzoriensis (6n=108). Fischberg and Kobel [31] stated that Xenopus ruwenzoriensis strikingly resembles X. fraseri, although both species slightly differ in external structures as well as in color and pattern. Distribution of Xenopus fraseri, X. amieti and X. ruwenzoriensis was shown by Kobel et al. [35]. Xenopus fraseri is widely distributed in Central Africa from the western end districts to Uganda, while X. amieti and X. ruwenzoriensis are found in West Cameroon and western Uganda, respectively. Vigny [36] analyzed the mating calls of 12 species and subspecies of the genus Xenopus including X. ruwenzoriensis (6n=108), X. wittei (4n=72) and X. vestitus (4n=72). It was found that the mating calls remarkably differ from one another and there are no definite characteristics of polyploid species in both call rate and trill rate. somes. 4. Bufonidae Mazik ef al. [37] reported that there are two forms of Bufo viridis which differ in distribution and chromosome number in Kirghizia, USSR. One form is diploid (2n=22) which lives in the low land, while the other is tetraploid (4n=44) which lives in the mountains. They stated that the tetraploid form appears to have been produced by polyploid- ization in the course of evolution. Pisanetz [38] reported that Bufo danatensis distributed in Turk- men, USSR is tetraploid (4n=40). In Ethiopia, diploid and tetraploid Bufo species were dis- covered by Bogart and Tandy [39]. These two are 20 and 40 in chromosome number, respec- tively, and are similar to Bufo kerinyagae in morphology. 5. Ranidae Bogart and Tandy [39] discovered two species consisting of diploid and tetraploid populations in African Ranidae. One of them is Pyxicephalus delalandii, in which the populations collected from the central part of South Africa are diploid having 26 chromosomes, while those collected from the southeastern part of South Africa are tetraploid having 52 chromosomes. The other species is Dicroglossus occipitalis. The populations col- lected from Central Africa are 2n=26, while those collected from West Africa are 4n=S2. 6. Ambystomidae The existence of almost all-female hybrids in the Ambystoma jeffersonianum complex distributed in Michigan and Indiana, USA was described by Clanton [40] and Minton [41], respectively. Thereafter, Uzzell [42, 43] elucidated that the Ambystoma jeffersonianum complex occurring wide- ly in North America is constructed of two diploid (2n=28) bisexual species and two all-female triploid (3n=42) parthenogenetic species. The. diploid species are A. jeffersonianum and A. laterale, while the triploid species are A. platineum and A. tremblayi. Ambystoma jeffersonianum are distinguished from A. laterale by larger body size, broader snout and lighter or less heavily spotted coloration. While Ambystoma jeffersonianum and A. laterale are generally distributed in the southern and northern areas, respectively, the ranges of these two species overlap in the eastern area. The two triploid species occur adjacent to each other and in areas where the diploid species are close together. Especially, Ambystoma tremblayi and A. platineum are found in association with A. laterale and A. jeffersonianum, respectively. Al- though the females of the two triploid species are intermediate between those of the two diploid species in body size, snout width and color, A. platineum and A. tremblayi are more similar in these respects to A. jeffersonianum and A. laterale, respectively. On the basis of distributional and Polyploidy in Amphibians 5) morphological data, Uzzell assumed that the triploid populations arose by hybridization of A. laterale and A. jeffersonianum, and that the di- chotomy of the triploid species may reflect dis- crimination by the males of diploid species. Macgregor and Uzzell [44] observed that the oocytes of triploid females contain hexaploid (6n= 84) lampbrush chromosomes which are joined into ‘pairs by chiasmata and form 42 bivalents. They considered that meiosis in triploid females is pre- ceded by a premeiotic mitosis without cytokinesis (endomitosis) and the resulting sister chromosomes form pseudo-bivalents. Sperm from diploid males stimulates development of the triploid eggs without contributing chromosomes to the triploid nucleus. Uzzell and Goldblatt [45] analyzed the serum proteins of the four species of the Ambystoma jeffersonianum complex by _polyacrylamide-gel electrophoresis. From the results of electro- phoretic analysis, together with morphological and geographical observations, they considered that the triploid species are reciprocal allotri- ploids, that is, A. tremblayi has two sets of laterale chromosomes and one set of /jeffersonianum chromosomes, whereas A. platineum has two sets of jeffersonianum chromosomes and one set of laterale chromosomes. The origin of the triploid species was assumed by Sessions [46] on the basis of analysis of C-band heterochromatin and cold-induced secondary con- strictions in the chromosomes of diploid and triploid species. According to him, the chromo- some constitutions of the triploid species suggest that the putative ancestral diploid female hybrids were produced from a mating between female Ambystoma jeffersonianum and male A. Jaterale, and that both kinds of allotriploids, A. platineum and A. tremblayi, were produced from unreduced eggs (WZ) of these diploid female hybrids by fertilization with sperm of male A. jeffersonianum and A. Jaterale, respectively. He also stated that Ambystoma platineum may produce a high percentage of diploid offspring with karyotypes identical to A. jeffersonianum, and that tetraploids are occasionally produced from A. tremblayi, although diploids have not been detected among their offspring. Concerning the ecological relationship between diploid and triploid species, Uzzell [47] found that males of the diploid species do not court the triploid females as extensively as they court the conspecific diploid females, and stated that the strong differential mating by the males of the complex may control the success of the triploids in expanding or maintaining their range. From some experiments performed in field enclosures, Wilbur [48] confirmed that the larvae of bisexual, diploid Ambystoma laterale and those of all- female, gynogenetic triploid A. tremblayi have different ecological requirements but still interact significantly. Downs [49] reported that diploid and triploid unisexuals of the genus Ambystoma are dis- tributed on Middle Bass and North Bass Islands in Lake Erie, North America. Bisexual species are absent on North Bass Island, while diploid bisexual Ambystoma texanum is present on Middle Bass Island. The electrophoretic patterns of serum proteins as well as morphological characters indicate that the diploid unisexuals possess one laterale genome and one texanum genome, while the triploid unisexuals have one /aterale genome and two texanum genomes. The triploids on the Bass Islands are considered to have been produced from the allodiploids by backcrossing with A. texanum. The apparent absence of bisexual species on North Bass Island argues for a parthe- nogenetic mode of reproduction, while on Middle Bass Island where A. texanum occurs with the unisexuals, the latter may have a gynogenetic or hybridogenetic relationship with males of the dip- loid bisexual. As a possible example of parthe- nogenesis, Uzzell [50] also stated that the populations of Ambystoma platineum and A. tremblayi exist without their normal bisexual hosts in Central Indiana, although these females are perhaps gynogenetic by the aid of some other bisexual species of Ambystoma that do occur with them. B. Spontaneous polyploids The first polyploid amphibians were discovered by Hertwig and Hertwig [1] in Rana esculenta (2n=26). When they made a crossing experiment between a female Rana esculenta collected from Germany and a male Bufo viridis, all the eggs 6 T. KAWAMURA became diploids in place of haploids. On the other hand, 18 control tadpoles produced by mating with a male Rana esculenta were all tri- ploids. Although there was no doubt that the eggs of the female were diploid, the origin of these diploid eggs was unknown. Dalcq [51] found two triploid embryos of Rana esculenta each in 22 embryos of an experimental series of androgenesis and in 16 control embryos. There was also no doubt that these triploid embryos developed from diploid eggs by fertilization with normal haploid spermatozoa. Wickbom [52] reported that four of eight Rana esculenta collected on Bornhorm Island near the exit of the Baltic Sea were triploids and the other four were dip- loids. It was strange that triploids were found in Rana esculenta alone, although chromosomes were examined in many anuran species collected from Europe. The problem of triploidy in Rana esculenta was most earnestly examined by Gtnther [53]. Of 24 frogs collected from Berlin, Magdeburg and Dresden, East Germany, seven were triploids, including five males and two females. Thereafter, he [54] examined the chromosomes of Rana escu- lenta collected from 19 places in the northern and eastern parts of East Germany. The results showed that 39.6% of all the frogs were triploids and, especially, more than 80% of the frogs col- lected from each of two places were triploids. While the sex ratio of diploids was about 1:2 (99SS and 18622), that of triploids was nearly 1:1 (12886 and 1362-2). Since the time when Berger [55] insisted that Rana esculenta are hybrids between Rana lessonae (2n=26) and Rana ridibunda (2n=26), this con- ception has generally been recognized. Thus, triploid Rana esculenta are considered to be allotriploids. Autotriploid Rana lessonae and R. ridibunda have not yet been found in the field. Uzzell and Berger [56], Giinther [57], Berger and Roguski [58], and Giinther ef al. [59] recognized that the size of erythrocytes is an easily determined trait in distinguishing between diploidy and triploidy in the Rana esculenta complex. According to Uzzell and Berger [56], Uzzell et al. [60], Giinther and Hahnel [61], Giinther er al. [59] and Giinther [62], there are two classes of triploid Rana esculenta; those with two genomes from R. lessonae and one from R. ridibunda (LLR), and those with two genomes from R. ridibunda and one from R. lessonae (RRL). In the popu- lations consisting of Rana esculenta alone or R. esculenta and R. lessonae, there was a preponder- ance of frogs with two lessonae and one ridibunda genome (LLR), while in the polulation consisting of R. esculenta and R. ridibunda, those with two ridibunda and one lessonae genome (LRR) dom- inated. Ebendal et al. [63] and Ebendal and Uzzell [64] reported a high frequency of triploid individuals in some Swedish populations of the Rana esculenta complex. According to them, the triploids in one population had two Jessonae genomes and one ridibunda genome, while those in the other populations most likely carried one lessonae genome and two ridibunda genomes, although no Rana ridibunda have been collected hitherto in Sweden. Uzzell et al. [60], Berger and Uzzell [65], Berger and Roguski [58] and Berger e¢ al. [66] found that diploid Rana esculenta usually lay three kinds of eggs, large, medium and small. Most of the large eggs at least become LLR and RRL allo- triploids by fertilization with sperm of Rana lessonae and Rana ridibunda, respectively. Uzzell et al. [60] assumed that the large ova derive from tetraploid first oocytes which contain two genomes from each of the parental species. From these tetraploid oocytes, diploid ova with one Jessonae genome and one ridibunda genome are produced by two meiotic divisions after normal synapsis. According to Berger and Roguski [58], about 90% and 10% of the large eggs obtained from seven field-caught female Rana esculenta became triploids and diploids, respectively, while small and medium eggs became diploids. On the other hand, Berger et al. [66] described that small and large eggs of a female LR hybrid obtained from a cross between a female Rana lessonae and a male R. ridibunda, became diploids and triploids, respectively, by fertilization with sperm of male R. lessonae ot R. esculenta (LR). Gunther et al. [59] and Gunther [62] were confident that the presence of two genomes from one or the other parental species in allotriploids permits synapsis to occur more readily and Polyploidy in Amphibians 7 results in producing haploid gametes, although the mechanism eliminating the remaining genome is unknown. The results of experiments per- formed by these investigators clearly showed that the LLR triploids collected from East Germany produce haploid gametes with a /essonae genome, while the RRL triploids collected from Poland produce haploid gametes with a ridibunda genome. C. Accidental polyploids Polyploids seem to occur in almost all am- phibians, although they are usually very rare. Fankhauser [67] detected two triploid embryos of Triturus helveticus (2n=24) in the control of experiments of merogony. This was the first discovery of polyploids in urodeles. There- after, Fankhauser and Kaylor [68] discovered two triploid embryos of Notophthalmus viridescens (2n=22) in the control of androgenetic experi- ments. Then, Fankhauser [69] examined the chromosomes of 100 larvae of this species by the tail-tip method and discovered that four of them were triploids. As these triploids completed metamorphosis, he observed their morphological characters in detail. Fankhauser [70] also re- ported that 13 triploids and two tetraploids were included in 134 larvae of Eurycea_bislineata (2n=28). The frequencies of accidentally oc- curring haploids, polyploids (triploids, tetraploids and pentaploids) and chromosome mosaics found by Fankhauser and his collaborators in four species of urodeles were listed by Fankhauser [71]. It was found that the frequencies of poly- ploids distinctly differ from species to species. For example, 44 (1.8%), one and seven (0.28%) of 2448 larvae were triploids, a tetraploid and pentaploids, respectively, in Notophthalmus viri- descens, four (1%) and one of 398 larvae were triploids and a pentaploid in Cynops p. pyrrho- gaster (2n=24) and 28 (5.1%) and two of 546 larvae were triploids and tetraploids in Eurycea bislineata, while there were only one triploid, one tetraploid and two pentaploids among more than 3,000 larvae of Ambystoma mexicanum (2n=28). A mature triploid male of Triturus vulgaris (2n=24) was collected by B66k [72] from the field. While he examined the mitotic and mei- otic chromosomes in the testes, the somatic characters of this triploid was not observed, as the body parts other than the gonads were not preserved. Il. ARTIFICIALLY INDUCED POLYPLOIDY A. Autotriploids Triploidy is easily induced by exposing eggs to an extreme temperature shortly after insemination in order to suppress the extrusion of the second polar body. In urodeles, numerous triploids have been produced by this method in Notophthalmus viridescens by Fankhauser and Griffiths [73] and Fankhauser and Watson [74], in Cynops p. pyrrhogaster by Fankhauser et al. [75], Fankhauser [71] and Kawamura [76], in Taricha granulosa (2n=22) by Costello [77] and Costello and Holmquist [78], in Ambystoma mexicanum (axolotl) by Fankhauser and Humphrey [79], in Triturus vulgaris (2n=24) by BObk [80], in Triturus vulgaris, T. alpestris (2n=24), T. helveticus and T. cristatus (2n=24) by Fischberg [81~83] and in Pleurodeles waltl (2n=24) by Beetschen [84] and Ferrier and Jaylet [85]. Ferrier and Jaylet [85] also obtained viable triploids by subjecting eggs to hydrostatic pressure after insemination in Pleuro- deles waltl. Of these artificially produced triploids, Triturus alpestris [81, 83], Ambystoma mexicanum [86, 87], Cynops p. pyrrhogaster [88] and Pleuro- deles waltl [84] attained sexual maturity. Although spermatozoa were distinctly fewer in the testes of triploid males than those in the control diploid males, various aneuploid offspring were produced together with a few diploid ones from triploid males by mating with diploid females in Cynops p. pyrrhogaster [88] and Ambystoma mexicanum [89]. Humphrey and Fankhauser [86] reported that triploid female axolotls occasionally spawn as many as several hundred eggs. When such triploid females were mated with diploid males, the offspring were mostly hyperdiploids and non- viable, while a few viable tetraploids were found by chance in certain spawnings. Fankhauser and Humphrey [87] elucidated chromosome num- bers of 445 larvae, of which 377 (84.7%) were 29 (2n+1) to 41 (Gn—1), 48 (10.8 %)were tetra- 8 T. KAWAMURA ploid (56-L), three were pentaploids (70+) and two were heptaploids (98+), while no triploids were found. According to Gallien and Beetschen [90] and Beetschen [84], triploid males in Pleurodeles waltl produced no viable offspring by mating with diploid females. Most of the offspring were 25 (2n+1) to 32 (3n—4) in chromosome number, while the other were hypertriploids. Fischberg [81, 83] reported that mature triploid female Triturus alpestris were sterile, although their ovaries contained a few, considerably large oocytes distributed separately. A similar phe- nomenon was found by Kawamura [88] in mature triploid females of Cynops p. pyrrhogaster, al- though two triploid females mated with diploid males produced a few abnormal larvae. In anurans, Parmenter [91] obtained a single triploid tadpole by artificial parthenogenesis in Rana pipiens (2n=26). Although this tadpole could not live more than 17 days, it was the first triploid produced by artificial means. Kawamura [92] produced many triploids by the same method in Rana nigromaculata (2n=26) together with haploids, diploids, tetraploids, hexaploids and various chromosome mosaics. Some _parthe- nogenetic triploids produced by Kawamura [93] in Rana nigromaculata and R. japonica (2n=26) completed metamorphosis. Abundant triploids were produced by Kawamura [94, 95] in Rana nigromaculata by refrigerating eggs shortly after insemination in order to suppress extrusion of the second polar body. In the testes of mature triploid males, spermatozoa were much fewer than those of the control diploids. Mature triploid females had small and underdeveloped ovaries. No offspring were produced from tri- ploid females by mating with diploid males. Kawamura assumed that the cause of under- development of the ovaries in the triploid frogs could not be explained by other than abnormality of trophic conditions common to both sexes. This assumption was confirmed to be correct by Kawamura et al. [96]. The trophic conditions of frogs were remarkably increased in our laboratory by changing their food from mosquitoes and domestic flies to two-spotted crickets, Gryllus bimaculatus, since 1976. Autotriploid females in Rana nigromaculata and R. brevipoda (2n=26) produced a small number of diploids, triploids, tetraploids and aneuploids or others by mating with diploid males. Triploid Rana pipiens em- bryos were produced by Briggs [97] in a large number by heating eggs shortly after insemina- tion. According to Humphrey et al. [98], triploid tadpoles autopsied during metamorphosis usually appeared to be males, owing to sex reversal of genetic females. Triploid Rana japonica produced by Kawamura and Tokunaga [99] after cold treat- ment of eggs were similar to the above triploid R. pipiens in that the froglets were all males. Kawamura [100] reported that triploid male Rana nigromaculata produced embryos and _ tadpoles which were intermediate between diploid and triploid in chromosome number by mating with diploid females. Dasgupta [101] obtained a high percentage of triploid embryos by subjecting eggs to hydrostatic pressure after insemination in Rana pipiens. Nishioka [102] has described the reproductive capacity of triploid male Rana nigromaculata and R. brevipoda. The behavior of the three homologous chromosomes of each triplet in the first meiotic metaphase of triploid male Rana nigromaculata and R. brevipoda was analyzed in detail by Okumoto [103]. Ueda [104] obtained mature triploid males and females in Bombina orientalis (2n=24) by cold treatment of eggs after insemination. There was nearly an equal number of males and females. While triploid females had very small, abnormal ovaries, triploid males produced a few abnormal embryos and tadpoles by mating with normal diploid females. Nishioka and Ueda [105] pro- duced numerous mature male and female triploids in Hyla arborea japonica (2n=24) by refrigerating eggs after insemination. B. Allotriploids In urodeles, Kawamura [106] obtained allo- triploids from Cynops p. pyrrhogaster eggs by refrigeration shortly after inseminating with sperm of C. p. ensicauda (2n=24). Fischberg [107] also produced allotriploids from Triturus helveticus eggs fertilized with sperm of 7. alpestris by the same method. Kawamura [108] reared four reciprocal allotriploids between Cynops p. Polyploidy in Amphibians 9 pyrrhogaster and C. p. ensicauda for several years. All of them were females with ill-developed Ovaries and were intermediate in external charac- ters as a whole between diploid hybrids and one of the parental species. In anurans, the first allotriploids were produced by Kawamura [106] from Rana japonica eggs fertilized with sperm of R. ornativentris (2n=24) by the refrigeration method. Kawamura [109] reported that 19 allotriploids between these two species were all males like the diploid hybrids and triploid Rana japonica. The testes of mature allotriploids contained bivalent and _ univalent chromosomes which were arranged somewhat regularly in the equatorial plate of the first meiotic metaphase, while no normal spermatozoa were produced. Becak and Becak [13] obtained allotriploids (3n=33) from females of a tetraploid species (4n=44), Odontophrynus americanus, by mating with males of a diploid species (2n=22), O. cultripes. In male allotriploids, spermatocytes mostly showed a high frequency of bivalents and univalents and a few trivalents in the first meiotic metaphase. The gametes were aneuploids in the range of 12~21 chromosomes or 23~32 chromo- somes, besides euploids with 11, 22 or 33 chromo- somes. Nishioka [102} produced four kinds of allotriploids consisting of different combinations of cytoplasm and genomes between Rana nigro- maculata and R. brevipoda. Although they were generally abnormal in spermatogenesis, two kinds of allotriploids were slightly fertile and produced many abnormal embryos. In reciprocal triploid hybrids between Rana nigromaculata and R. brevipoda, Nishioka [110] has observed that two chromosomes of each triplet in metaphase plates are usually equal to each other and differ from the remaining in size. Miiller [111] dis- covered triploids in the offspring of five of 24 cross combinations among six species and two subspecies of Xenopus. These triploids must be allotriploids, as they are produced from unreduced diploid eggs of one species by fertilization with haploid spermatozoa of another species. Ohtani [112] observed that the lampbrush chromosomes in most of the oocytes of a female allotriploid between a female Rana brevipoda and a male R. nigromaculata consisted of a mixutre of one to five triploids, 8~12 bivalents and 8~12 univalents. Okumoto [103] found that the mean number of trivalents per cell was 1.58 in male allotriploids between female Rana nigromaculata and male Rana brevipoda and 0.94 in the reciprocal allotriploids. In these allotriploids, the two homologous chromosomes derived from _ the mother seemed to be prone to conjugate quickly with each other owing to their affinity. Kawamura et al. [96] reported that allotriploid females, (N)NNB, obtained from eggs of a diploid female Rana nigromaculata, (N)NN, fertilized with sperm of a diploid male R. brevipoda, (B)BB, produced a small number of diploids, triploids, allotetraploids and aneuploids or others by mating with diploid male R. nigromaculata or R. brevipoda. C. Autotetraploids In urodeles, Kawamura and Sanada [113] found a single tetraploid among many larvae raised from cold-treated fertilized eggs of Cynops p. pyrrhogaster. This tetraploid could not attain completion of metamorphosis. Fischberg [82] obtained four tetraploids besides 184 triploids among 553 larvae raised from _ cold-treated fertilized eggs of Triturus alpestris. One of these tetraploids completed metamorphosis and died without attaining the age of 10 months. Humphrey and Fankhauser [86] and Fankhauser and Humphrey [87] reported that in axolotls about 11% of the larvae produced from triploid females by mating with diploid males were tetraploids. Some tetraploids of mixed axolotl and Ambystoma tigrinum (2n=28) ancestry reached the size typical for diploids. A considerable number of tetraploid females became sexually mature and produced triploid or near-triploid offspring by mating with diploid males. Unlike the tetraploid females, tetraploid males were subnormal in size and vigor, and completely sterile. Thereafter, Humphrey and Fankhauser [114] reported again that tetraploid axolotls occurred with a frequency of 3 to 4% in the spawnings of triploid females mated with diploid males, probably as a result of failure of the meioses in the egg. The spawnings of tetraploid females averaged 225 eggs, while those of triploid and diploid females laid 300 and 10 T. KAWAMURA over 650 eggs on the average, respectively. Sanada [115] refrigerated eggs of Cynops p. pyrrhogaster immediately before the first cleavage and obtained 10 tetraploids, of which nine meta- morphosed at the same rate as the controls. Kawamura and Utsunomiya [116] produced tetra- ploids in Cynops p. pyrrhogaster and C. p. ensicauda by exposing eggs to supersonic waves when the first cleavage furrow began to appear. Fischberg [117] obtained high percentages of autotetraploids in Triturus vulgaris and T. hel- veticus by heat-shock treatment of eggs usually at the 2-cell stage. Romanovsky and Spicarova [118] reported that 28 larvae raised from Triturus vulgaris eggs heat-shocked by Fischberg’s method were all tetraploids. Beetschen [119, 120] pro- duced tetraploid larvae in Pleurodeles waltl from matings between triploid females and diploid males. Eleven of the tetraploid larvae became adults, and five mature female tetraploids laid 1365 fertilized eggs by mating with diploid males and produced 211 triploid adults. Jaylet [121] and Gaillard and Jaylet [122] also reported that all the larvae obtained by Fischberg’s method were tetraploids in Pleurodeles waltl. In anurans, the first four tetraploids were discovered by Kawamura [92] in 413 parthe- nogenetically developed individuals of Rana nigromaculata, although the oldest of them died at the age of 30 days in spite of its nearly normal appearance. The second tetraploid anuran was obtained by Kawamura and Moriwaki [123] in Rana limnocharis (2n=26) together with many triploids from cold-treated eggs. This frog began metamorphosing earlier than the controls, but was killed at the age of 151 days because of infection. It was a female whose ovaries were normal and contained many growing oocytes. Briggs and King [124, 125] and King and Briggs [126] obtained tetraploid embryos in comparatively high frequency by transplantation of embryonic nuclei into enucleated eggs in Rana pipiens. Fischberg et al. [127] also obtained tetraploid embryos by the same method in Xenopus laevis. Sambuichi [128] produced tetraploid embryos from both fertilized and unfertilized eggs transplanted with blastula nuclei in Rana japonica. Gurdon [129] reported that 11 tetraploid adults were obtained in Xenopus laevis by nuclear transplantation. Whereas both males and females of these tetra- ploids were almost identical to normal diploids in external morphology, they were very abnormal in gametogenesis and entirely sterile. Kawamura et al. [130, 131] reported the repro- ductive capacity of tetraploids obtained from fertilized eggs by heat-shock treatment or trans- plantation with a blastura nucleus in Rana japonica. Ten of 14 tetraploid larvae obtained by these two methods completed metamorphosis and all became males. Seven of them attained sexual maturity. While three tetraploids were abnormal in inner structure of the testes, the other four were nearly normal in this respect and produced a small number of triploid offspring by mating with diploid females. Kawamura and Nishioka [132] obtained 19 tetraploid tadpoles from fertilized eggs of Rana nigromaculata by heat-shock treat- ment. Six of them metamorphosed normally and became males. Three tetraploids attained sexual maturity. Two of these tetraploid males produced diploid, triploid and tetraploid offspring from mating with diploid females by refrigerating eggs shortly after insemination. It is noteworthy that autotetraploid males of Rana japonica and Rana nigromaculata are normal in growth and produce viable offspring by mating with diploid females. In this respect, they distinctly differ from those of Xenopus laevis [129], axolotls [114] and Pleurodeles waltl [119, 120]. Kawamura et al. [96] reported, as stated above, that a small number of tetraploids were produced from female triploids by mating with diploid males in Rana nigromaculata and R. brevipoda. Three metamorphosed tetraploid Rana nigromaculata were males. Nishioka and Ueda [105] obtained abundant tetraploids in Hyla arborea japonica from triploid females mated with diploid males, while no tetra- ploids were obtained from triploid males. When female and male tetraploids were mated, 85% of the offspring were tetraploids. On the other hand, when tetraploid females were mated with diploid males, about 80% of the offspring were triploids. In these first-generation offspring, com- paratively many hexaploids or pentaploids ap- peared besides tetraploids or triploids. While the sex ratio of femlaes and males in. the first- Polyploidy in Amphibians 11 generation offspring of female and male tetraploids was about 3:1, it was about 1:1 in the second- generation offspring. No differences were ob- served in external characters among diploids, triploids and tetraploids. D. Allotetraploids External characters and reproductive capacity of six kinds of allotetraploids between Rana nigro- maculata (N)NN and Rana brevipoda (B)BB, that is, (N)NNNB, (B)BNNN, (N)NNBB, (B)BBNN, (N)NBBB and (B)BBBN, were briefly reported by Nishioka [102]. The most interesting of these allotetraploids are two kinds of amphidiploids, (N)NNBB and (B)BBNN, as amphidiploids hold an important position in the vegetable kingdom. The first four artificial amphidiploids were obtained by Kawamura and Nishioka [133] between Rana nigromaculata and R. brevipoda by three different methods. One of them was raised from a fertilized Rana brevipoda egg trans- planted with a diploid blastula nucleus of R. nigromaculata. This individual grew into a male frog which attained sexual maturity. Kawamura and Nishioka [134] confirmed that this frog was completely fertile in contrast to diploid hybrids, and produced allotriploids by mating with females of the two species. Nishioka [135] obtained 14 amphidiploid frogs between two Japanese brown frog species, Rana japonica (2n=26) and R. ornativentris (2n=24). Four of them were produced by the nuclear transplantation method, and grew into two males and two females. The other ten were produced by the heat-shock method and grew into seven males and three females. Two of the male amphidiploids were partially fertile and produced a small number of allotriploids by mating with diploid female Rana japonica. Two of the three female amphidiploids had normal or almost normal ovaries. These findings are in remarkable contrast with that all the diploid hybrids are males and completely sterile. Kawamura and Nishioka [136] obtained numer- Ous metamorphosed amphidiploids from matings between diploid female Rana brevipoda and autotetraploid male R. nigromaculata by cold treatment of eggs. About 30% of these amphi- diploids were males. In the first-generation offspring of female and male amphidiploids, there was nearly an equal number of females and males. The females and males of these amphi- diploids seemed to be XXXX and XXXY in sex chromosome constitution, respectively. Two kinds of amphidiploids, (N)NNBB and (B)BBNN, were obtained by Kawamura et al. [96] from female (N)NNB allotriploids mated with male R. brevipoda, (B)BB and from female (B)BBN allotri- ploids mated with male R. nigromaculata, (N)NN, respectively. Two other kinds of allotetraploids, (N)NNNB and (B)BBBN, were also obtained from female (N)NNB allotriploids mated with male Rana nigromaculata and from female (B)BBN allotriploids mated with male R. _ brevipoda, respectively. When sex of these four kinds of allotetraploids was examined, males were re- markably preponderant over females in number. In total, 44 (86.3%) of 51 allotetraploids were males. Nishioka [137] reported that numerous amphidiploids are produced from triploid back- crosses of hybrids between Rana nigromaculata and R. plancyi chosenica (2n=26). When female (N)NNC triploid backcrosses were mated with male Rana plancyi chosenica, (C)CC, numerous (N)NNCC amphidiploids were obtained together with some triploids, diploids and aneuploids. About 77% of the amphidiploids were males. Nishioka and Okumoto [138] examined the developmental capacity and sex ratio of the first-, second- and third-generation offspring of a female and two male (N)NNCC amphidiploids obtain: d from Rana nigromaculata and R. plancyi It was found that the developmental capacity of the offspring of amphidiploids re- markably increases and the sex ratio of amphi- diploids becomes nearer to 1:1 from male pre- ponderance with the advance of generation. The amphidiploids were intermediate between Rana nigromaculata and R. plancyi chosenica in external characters as a whole. chosenica. E. Pentaploids, hexaploids and heptaploids According to Fankhauser [71], ten spontaneous pentaploid larvae including seven Notophthalmus viridescens, one Cynops p. pyrrhogaster and two axolotls were discovered during the several 12 T. KAWAMURA years before 1944. The morphological and behavioral characters of one of the pentaploid larvae of Notophthalmus viridescens were ob- served by -Fankhauser [139]. Besides, three pentaploid larvae and one triploid-pentaploid and one diploid-pentaploid mosaic larvae were pro- duced by cold treatment of inseminated eggs in axolotls [79]. Of a total of eight pentaploid larvae of Notophthalmus viridescens including one produced experimentally, three were raised to metamorphosis. However, they were greatly reduced in growth and remained longer in the larval stage than the controls. While two of them underwent the first molt about three or five weeks later than the average diploids, the remainder died without having molted. The single pentaploid Cynops p. pyrrhogaster was also greatly reduced in growth rate and metamorphosed almost eight weeks later than the diploid controls. All these pentaploid Notophthalmus_ viridescens and Cynops p. pyrrhogaster showed S-shaped curvature of body axis and coiling or pronounced curvature of the tail. Fankhauser and Humphrey [87] discovered three pentaploids and two heptaploids among 445 larvae produced from matings between triploid females and diploid males in axolotls. There- after, Humphrey and Fankhauser [114] noticed that pentaploids are most commonly the offspring of diploid parents in axolotls. They constituted over 0.14% of the total progeny. Fankhauser and Humphrey [140] reported again that 190 (0.17%) of 109445 larvae produced from matings between diploid females and males in axolotls were penta- ploids. A single hexaploid was found among these abundant larvae. It was evident that the pentaploids were produced from a tetraploid egg with complete suppression of meiosis by fer- tilization with a normal haploid spermatozoon. All the pentaploid of which the sex was determined were females having WWZZZ in sex chromosome constitution. Humphrey and Fankhauser [114] stated that pentaploids were frequently malformed and non-viable, but many survived to ages of several months. They tended to develop cur- vature and to become retarded in growth, and usually died before attaining the age of two years. The ovaries of older pentaploids contained very few or no mature ova. | Sanada [141] found a single pentaploid among larvae raised from cold-treated fertilized eggs of Cynops p. pyrrhogaster. This pentaploid larva was comparatively larger than the diploid controls and showed a slight curvature of body axis. It began metamorphosis a week later than the diploid controls. Subtelny [142] briefly reported that artificial insemination of eggs of a female Rana pipiens induced to ovulate by the routine pro- cedure of pituitary injection yielded larvae of which 12° were pentaploids, 859% were diploids, and 3% were haploids or mosaics. Cytological observations indicated that the pentaploid larvae resulted from a failure of emission of the first and second polar bodies in the fertilized eggs. Bogart [143] described that there are many cross com- binations of Bufo species which have been found to produce only triploids or pentaploids, or diploids and pentaploids, besides those which produce dip- loid or diploid and triploid offspring. According to him, triploid and pentaploid hybrids result from diploid and tetraploid eggs produced by female Bufo species in a small percentage by fertilization with sperm of males of foreign species. Nishioka and Okumoto [138] obtained six hexaploid and 11 diploid-hexaploid mosaic larvae from matings between a female and two male (N)NNCC amphidiploids produced from Rana nigromaculata and R. plancyi chosenica, -while eight pentaploid and two _ diploid-pentaploid mosaic larvae were produced from a mating between the above female amphidiploid and a diploid male Rana nigromaculata or R. plancyi chosenica. f/ 58 59 60 61 62 63 64 65 66 67 68 69 70 71 WZ 1B 74 75 76 ad) 78 79 80 81 82 83 84 85 86 87 88 89 Gunther, R. (1977) Biol. Zbl., 96: 457-466. 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B, Div. 1, 13: 151-154. Subtelny, S. (1966) Amer. Zool., 6: 357. Bogart, J. P. (1972) In Evolution in the Genus Bufo. Ed. by W. F. Blair, Univ. of Texas Press, Austin and London, pp. 171-195. Nishioka, M. and Ohtani, H. (1983) La Kro- mosomo, II—29: 898. Acad. Sci. ZOOLOGICAL SCIENCE 1: 16-28 (1984) © 1984 Zoological Society of Japan REVIEW High Molecular Weight Proteins of Nerve Cells Inducing Cross-Linking or Bundling of Cytoskeletal Proteins HIKOICHI SAKAI, HIROMU MUROFUSHI and GEN Matsumoto! Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, and ‘Optoelectronics Section, Electrotechnical Laboratory, Niihari-gun, Ibaraki 305, Japan INTRODUCTION Microtubules, microfilaments and neurofilaments are three major filamentous elements constructing cytoskeletons in a variety of nerve cells. Recent studies on cytoskeletal networks of nerve cells have visualized pronounced complex structures con- sisting of neurofilaments, microtubules and cross- linking thin filaments [1, 2]. These cytoskeletal networks may play various roles in the maintenance of the cell shape, in the axonal transport and in the axonal growth. For nerve cells in constructing these cytoske- letons, mutual association of these filaments seems to be established by the:aid of cross-linkers rather than entanglement of the filaments themselves. In many animal tissues, various cross-linking proteins have been identified so far in bundling of homo- logous filaments such as actin [3] or microtubules [4-6] in vitro. Moreover, some proteins have been known as cross-linkers of heterogous fila- ments, linking microtubules and microfilaments [7]. In addition, microtubules are known to be capable of forming networks with neurofilaments provided that microtubules are assembled in the presence of neurofilaments [8]. It was recently proved that the 200 K subunit of the neurofilament triplet which attaches to the filament core made of the 70K subunits stimulates polymerization of tubulin, thereby causing cross-linking of neuro- Received July 13, 1983 filaments [9]. The plasmalemmal undercoat originally termed by Ishikawa [10] is a site of biological importance for membrane functions at which some high mo- lecular weight proteins are localized. The layer lining the axolemma is also composed mostly of cytoskeletal networks by which functions of the neuronal axon will be maintained. This article intends to review three species of high molecular weight proteins, microtubule- associated proteins (MAPs), fodrin, and 260K protein found in nerve cells with characteristics giving rise to cross-linking or bundling of cyto- skeletal filaments and to discuss possible biological implications. HIGH MOLECULAR WEIGHT MICROTUBULE-ASSOCIATED PROTEINS Microtubule-associated protein 1 and 2 (MAP-1 and MAP-2) have originally been isolated from brain tissues through purification of microtubule proteins by cycles of temperature-dependent as- sembly and disassembly. MAP-2 is a heat-stable protein, thus capable of being separated from MAP-1 and tubulin by heat treatment [11]. Many works have been focused on the assembly- promoting characteristics of MAP-2 after its purification [11, 12]. MAP-1 also has recently been shown to have ability to initiate microtubule assembly [13]. The high molecular weight MAPs have been High Molecular Weight Proteins 17 believed to associate periodically with microtubules [11, 14] with the one end of smaller domain (35 K daltons) attaching to the surface lattice leaving most of the filamentous strand projecting from the microtubules [15]. The fine structure of the MAP- 2 molecule was recently visualized by using the platinum-replication technique [16]. It extends outward from the microtubule wall with a length ranging from 80 to 90nm. It is reasonable to suppose that the long filament allows microtubule to associate with some other intracellular structures like actin filaments, neurofilaments or various membraneous compartments. The association of microtubules with actin filaments was originally detected by Griffith and Pollard using a falling ball viscometer [7], who demonstrated that MAPs favor their interaction causing the formation of networks. In vitro experiments clearly show that the cross-linking of actin filaments is caused by purified MAP-2 and modulated by phosphorylation of MAP2 as demonstrated by the low shear viscometry [17, 18]. Figure 1 shows that unphosphorylated MAP-2 causes a large increase in low shear viscosity of pre-formed actin filaments at a 1: 1 molar ratio of MAP-2 to actin. In contrast, phosphorylated MAP-2 fails to cross-link actin filaments. Many works were undertaken to understand cellular functions of MAPs besides the well estab- lished property of MAPs in initiating microtubule assembly. However, we do not have clear evi- dence that MAPs play roles in other functions than cross-linking actin filaments. Only an evidence we have is that MAP-2 binds to pituitary secretory granule membrane [19] or links insulin-containing secretory granules [20] or clathrin coated vesicles [21] to microtubules in vitro in a cAMP-dependent fashion, suggesting that MAPs favor microtubule- mediated transport of cytoplasmic vesicles. Recent results indicate that MAP-2 is associated only with microtubules of neuronal dendrites being absent from microtubules present in axons [22, 23]. By immunohistochemical staining, MAPs are shown to be closely associated with the growing tips of dendrites before the appearance of micro- tubules [24]. MAPs are also present as patches beneath the surface membrane of nerve cell body before the emergence of dendrites. These results 150 100 50 0) 200 400 MAP 2 (ig/ml) Fic. 1. Formation of actin filament network by MAP-2 and its inhibition by phosphorylation of MAP-2. Pre-formed actin filaments (0.15 mg/ml) was mixed with either unphosphorylated (@) or phosphorylated (0) MAP-2 (U-MAP2 and P- MAP2, respectively) and viscosity was measured 10 min after the mixture was drawn up into a falling ball viscometer (From Nishida et a/. [17]). suggest that MAP-2 acts as a framework for building up microtubule cytoskeletons rather than working directly in nerve functions. However, the possibility cannot be excluded that MAP-2 which is localized at the plasmalemmal undercoat of the dendrites and nerve cell body are involved in the membrane excitation. FODRIN Recently more than several groups succeeded in isolating a spectrin-like protein from mammalian brains. A pair of high molecular weight poly- peptides which are axonally transported had been detected in rabbit retinal cells [25]. Levine and Willard [26] succeeded in partial purification of these proteins and prepared antibody against them. Immunohistochemical studies revealed that the antigens were highly concentrated in the cortical cytoplasm of neurons. They designated this 18 H. SAKAI ef al. protein as ‘“‘fodrin”’ after Greek term, fodros which means lining in English. The same protein was found by another groups in the course of studying calmodulin-binding proteins in the particulate fraction of mammalian brains. Davies and Klee [27] termed this protein ‘‘CBP-I’’ (calmodulin- binding protein I), and a name, “‘calspectin” (calmodulin-binding spectrin-like protein) was pre- sented by Kakiuchi et al. [28, 29]. Another approach to the finding of this protein was made by Shimo-oka and Watanabe [30]. They found a doublet of high molecular weight proteins in brain which co-precipitated with actomyosin and stim- ulated actomyosin ATPase activity. They called this protein “BABP” (brain actomyosin-binding protein). As stated here, there is a discrepancy in the terminology of this protein. Moreover, a name, “brain spectrin’ was presented to this protein [31-33]. In the present review, we use the term “‘fodrin”’ because it is most widely used now. Fodrin can be purified from the particulate fraction of mammalian brains, from which it is solubilized with high concentration of KCI [26, 27, Fic. 2. Electron micrograph of fodrin mol * ecules rotary shadowed with tantalum/tungsten. 34, 35] or at low ionic strength and 37°C [29, 31, 32]. The latter method is based on the procedure for the purification of spectrin from erythrocyte ghost. Fodrin can be purified by means of gel filtrations and chromatographies. Some groups used affinity chromatography using immobilized calmodulin [27, 29]. Electrophoretic analysis revealed that purified fodrin consisted of two high molecular weight polypeptides with apparent molecular weights of 240,000 and 235,000 in an equal molar ratio [26, 27, 29, 31, 34]. This heterodimeric nature resembles those of spectrin and TW 260/240 protein present in the terminal web of the small intestinal brush border [36]. The former consists of two polypeptides having molecular weights of 240,000 (a-spectrin) and 220,000 (6-spectrin), and the latter has two subunits with molecular weights of 260,000 and 240,000 [36]. Glenny ef al. [37] showed that the MW 240,000 components of the above three different protein fractions resemble each other not only in terms of the apparent molec- ular weight in SDS-polyacrylamide gels but also in immunological cross-reactivity. That is, antibody om, x" ad Fodrin seems to be composed of two strands which are tightly associated along its length. Magnification x 120,000. (From Glenney ef al. [34]. Reproduced with permission of The American Society of Biological Chemists, Inc.). High Molecular Weight Proteins 19 against each of the MW 240,000 subunits of the three protein fractions cross-reacted with each of the other two, although the cross-reactivity between fodrin and TW 260/240 was stronger than those between spectrin and fodrin or TW 260/240. On the other hand, immunological homology was not detected between the other subunits. They also showed that one dimensional peptide mapping patterns of the MW 240,000 components of the three proteins resembled each other, those of the other subunits being dissimilar to each other [37]. As mentioned before, fodrin is capable of binding calmodulin in a calcium-dependent manner. The same was shown for spectrin [38] and TW 260/240 [37]. The calmodulin-binding activity for each subunits of fodrin, TW 260/240 and spectrin was examined by ***I-calmodulin overlay after electro- phoretic separation of the subunits in the presence of sodium dodecylsulfate [37]. It was revealed that the MW 240,000 subunits of the three proteins bound calmodulin. Glenny ef al. [36] suggested that fodrin, spectrin and TW 260/240 belong to the Same group of proteins, consisting of two species of polypeptides, one of which possesses common characteristics and the other does natures different from each other according to the cells or tissues they derive from. Electron microscopic studies using the rotary shadowing technique [31, 34, 36, 39] or negative staining [35] revealed that the fodrin molecule shows a shape of long flexible double strand having a contour length of about 200 nm (Fig. 2). This value is smaller than that of TW 260/240 (264 nm [36]) and slightly longer than that of spectrin tet- ramer (194 nm). It was also shown that fodrin is in the form of tetramer having a contour length of 200 nm under lower ionic strength and, at higher salt concentration it changes to dimer of about 100 nm in length suggesting that head to head binding of double-stranded dimer occurs in the former condition [39]. One of the most important characteristics of fodrin is its ability of binding to actin filaments. Fodrin is co-precipitated with actin filaments by centrifugation [26, 27, 30]. Low shear viscometry (Fig. 3) revealed that fodrin cross-links actin filaments to make a gel [31, 32, 34]. The cross- linking of F-actin by fodrin has been confirmed by cP get 100 50 O 10 20 30 40 50 FODRIN (g/m!) Fic. 3. Formation of actin filament network by fod- rin. Actin solution (0.1 mg/ml) was mixed with varying concentration of fodrin and viscosity was measured by a falling ball viscometer (Ishikawa and Murofushi, unpublished). electron microscopic studies [34, 35, 39]. This actin-binding and _ cross-linking activities are shared by spectrin and TW 260/240 [36]. It is also shown that fodrin promotes actin polymerization in vitro [40]. Although binding of fodrin to F- actin has well been studied, interaction of this protein with another cytoskeletal components has not yet been fully investigated. The only one exception is the studies on the interaction of fodrin with microtubules in vitro [6]. It was initially observed that fodrin causes unusual increase in turbidity of a tubulin solution under the condition that supports microtubule assembly (Fig. 4). Soon, it was found that fodrin co-sediments with microtubules assembled from phosphocellulose- purified tubulin. The dark-field light microscopic and electron microscopic studies revealed that fodrin bundled microtubules (Figs. 5 and 6, re- spectively). The binding of fodrin to microtubules was inhibited by the addition of microtubule- 20 H. SAKAI et al. O 0 10 20 30 40 INCUBATION TIME Fic. 4. Enhancement by fodrin of turbidity increase (min) of tubulin solution. Varying amount of fodrin was added to purified tubulin solution and change in turbidity was monitored at 350 nm. Fodrin con- centration: Omg/ml (1), 0.1 mg/ml (2) and 0.3 mg/ml (3). Hatched area represents cold treat- ment to depolymerize microtubules (From Ishika- wa et al. [6]). associated proteins (MAPs), suggesting that fodrin and MAPs shear the same binding site with micro- tubules. It has been reported that fodrin is phospho- rylated by protein kinases [41]. Two species of protein kinases that phosphorylate fodrin were partially purified from the membrane fraction of brain, one of which was Ca’*-dependent and the other Ca’*- and calmodulin-dependent for activity [42]. Along with the result that fodrin molecule itslef is capable of binding calmodulin, the calmodulin-dependent phosphorylation of fodrin suggests that some regulatory system involving calmodulin works for the function of fodrin. As stated before, fodrin can be purified from the membrane fraction of brain. Immunohistochem- ical studies reveal that anti-fodrin antibody stains cortical cytoplasm of the entire neuron [26]. Fodrin occupies a similar location in Schwann cells. Moreover, fodrin antigens are also localized in the peripheral cytoplasm of a wide variety of cells in various non-neural tissues including skeletal muscle, uterus and intestinal epithelium. Anti- fodrin antibody stains cortical cytoplasm of many species of culture cells, for instance, 3T3 cells, fibroblast [26], and lymphocyte [43]. These data do not necessarily indicate that fodrin molecule itself is widespread in many species of cells. In- stead, it may be reasonable to consider that many species of proteins shearing the same immunolo- gical determinants as for fodrin exist and function in many types of cells. What is the function of fodrin in the neural cells? This question has not yet been answered well. Sub-membraneous localization of fodrin and its ability of binding actin filaments strongly suggest that like spectrin, fodrin is a component of mem- brane skeleton functioning as a lining framework of the plasma membrane. In the case of erythrocyte a model of membrane skeleton is widely accepted, that is a meshwork structure consisting of spectrin tetramer, actin oligomer and band 4.1 is anchored via ankyrin to band 3 which is integrated in the lipid bilayer [44, 45]. It may be considered that fodrin, actin oligomer and un- known proteins make a sub-membraneous network in the neural cells. It is also possible that fodrin cross-links actin filaments present in the cortical region of cytoplasm as does TW 260/240 in the terminal web of small intestinal brush border [46]. Along with its ability to cross-link actin filaments, fodrin was shown to bind to microtubules in vitro [6]. Two possibilities on the ways fodrin interacts with microtubules can be considered. One is that microtubules or tubulin oligomers, like actin oligomer in the erythrocyte membrane skeleton, are involved in the skeletal structure underneath the plasma membrane of neural cells. another possibility is that fodrin present in the sub-membraneous structure stretches inward the cytoplasm and interacts with cytoplasmic micro- tubules. In this case, fodrin may act as a mediator of signals between the membrane skeleton and internal cytoskeletons. It has been reported that fodrin is one of the major components of postsynaptic densities [47]. Actin, tubulin and calmodulin are also included in this structure. Although the function of this sub- High Molecular Weight Proteins 21 membraneous structure is not clear, a close inter- connection is postulated between post-synaptic densities and acetylcholine receptors which are integrated to the lipid bilayer. On the other hand, specialized sub-membraneous _ structure which resembles that of erythrocyte membrane skeleton is detected in the node of Ranvier [9]. It is also possible to consider that fodrin is involved in the sub-membraneous network of this specialized region of the axon. In fact, the node of Ranvier is Stained with anti-fodrin antibody with fairly high intensity [26]. Sub-membraneous structures in neurons may function as a support of receptors and channel proteins which exist in a form of cluster in the special region of the plasma membrane. It may also be possible that the sub-membraneous architectures, presumably containing fodrin, are directly involved in the machinery of nerve excita- tion. 260 K PROTEIN This protein was initially isolated from the squid giant axon, axoplasm of which was extruded out by the roller method. The empty axon is cut longi- tudinally and extracted with a solution of high ionic strength [48]. SDS-polyacrylamide gel electro- phoresis of the extract reveals that a protein having a molecuiar weight of 260,000 is a major component comprising 36% of the total proteins present in the extract. Since the extruded axoplasm contains little of this protein, the 260 K protein is considered to be localized at the axolemmal undercoat. A layer of several microns from the axolemma intact after extrusion of most of the axoplasm. The protein was originally purified by using high performance liquid chromatography [49]. Later, since it was shown that the protein band was PAS-positive, it was further purified by using Con A-Sepharose affinity chromatography [4]. Extraction of the fragmented empty axons with a solution of low ionic strength is incapable of solubilizing the 260 K protein. The treatment with Triton X-100 also fails to extract the 260 K protein. remains Fic. 5. microtubule suspension (1.5 mg/ml) was observed by a dark-field light microscope. (0.3 mg/ml) causes microtubules to form thick bundles. Light micrographs of microtubules assembled in the absence (A) or presence (B) of fodrin. The See that fodrin Bar, 10 um (From Ishikawa et al. [6]). 22 H. SAKAI ef al. Fic. 6. Electron micrographs of microtubules assembled in the absence (A) or presence (B) of fodrin. 100 nm. (From Ishikawa eft al. [6]). Only a solution of high ionic strength is capable of extracting this protein. Furthermore, internal perfusion of the axon with the extraction medium A350 1.6 14 1.2 1.0 08 0.6 04 0.2 O ‘@) 5 10 15 20 = 25 (min) INCUBATION TIME Bar, of the 260 K protein results in release of this protein into the perfusate (unpublished data). These results also seem to indicate that, at least, a part of the 260K protein localizes at the axolemmal undercoat possibly lining or associating with the axolemma. A pronounced characteristic of this protein is that it induces remarkable increase in turbidity when it is added to MAPs-free tubulin solution under the assembly condition [4]. The final levels of the turbidity increase with increasing the amount of the 260K protein (Fig. 7). The turbidity of pre-formed microtubules is also increased by the addition of the 260K _ protein. Evidence is Fic. 7. Pronounced enhancement of increase in turbi- dity of tubulin solution by 260K protein. Varying amount of purified 260K protein was added to purified tubulin solution and change in turbidity was monitored at 350nm. The concentration of 260K protein: 0 mg/ml (1), 0.02 mg/ml (2), 0.05 mg/ml (3), 0.1 mg/ml (4), 0.2 mg/ml (5) and 0.35 mg/ml (6). Figures in the right edge represent the levels of turbidity after cooling to 0°C (From Murofushi e¢ al. [4]). High Molecular Weight Proteins 23 presented that the 260 K protein binds to micro- tubules assembled from purified tubulin. The dark-field light microscopy and electron micros- copy (Fig. 8, A and B, respectively) reveal that most of microtubules are tied into thick bundles which resemble those induced by fodrin (Figs. 5 and 6). Most recently, the rotary shadow technique revealed that the 260 K protein has a shape of rod with a knob at one end showing a contour length of about 100 nm (Fig. 9) [50]. The 260K _ protein resembles the dimer of fodrin in their electron microscopic images after rotary shadowing. Both the proteins show the contour length almost identical to each other. It is emphasized that it is only this protein that the biological function is clearly demonstrated among several high molecular weight proteins in nerve cells. Electrophysiological and biochemical studies of squid giant axons prove that micro- tubules which are fully occupied with tyrosine at the N-terminus of a-tubulin and the axonal 260K protein are inevitably required for the maintenance i = Fic. 8. of bundled microtubules (1.5 mg/ml) with 260K protein (0.2 mg/ml) are shown. SA). is seen. Bar, 100 nm (cf. Fig. 6A). B. Electron micrograph of bundled microtubules. (From Murofushi et al. [4]). of the membrane excitability. This conclusion comes from a series of studies proving the fol- lowing. (i) Numerous microtubules are found just underneath the axolemma running parallel to the longitudinal axis of the axon as observed by electron microscopy. The closer to the axolemma, the more the number of microtubules, about 90 microtubules per ym’ of the cross section being found just underneath the axolemma [51, 52]. (ii) The membrane excitability of the giant axon is quickly lost by perfusing the axon with colchicine, podophyllotoxin or Ca ions which destroy micro- tubules underlying the axolemma [49, 53, 54]. (iii) Even when the membrane excitability of the axon is deteriorated by microtubule poisons such as Ca ions or colchicine, the axon does restore the exci- tability upon being perfused with the 260 K protein and the reaction system that supports tyrosination of a-tubulin in the medium favorable for micro- tubule assembly [49, 55]. In such experiments, tubulin must be tyrosinated by tubulin-tyrosine ligase [56] because the excitability is never restored Dark-field light micrograph Bar, 10 wm (cf. Fig. Inclusion of 260K protein within the bundles 24 H. SAKAI et al. if the enzyme is omitted. It is also proved that the 260 K protein is indispensable for steady mainte- nance of the recovered excitability. Furthermore, no restoration occurs when microtubule assembly is totally suppressed in the axon by the lack of GBP [S255 i: Such a pronounced restoration of the axonal excitability was initially detected by monitoring the action potential, the resting potential and the threshold as parameters of the membrane exci- tability. Figure 10 shows that the action potential almost disappeared by perfusing the axon with a Ca**-containing medium. Such axons never re- store the excitability even if Ca’** be removed, while the resting potential is restored considerably in the standard perfusion medium. However, upon perfusing the axon with a solution of micro- tubule assembly system containing microtubule proteins, tubulin-tyrosine ligase, tyrosine, ATP, Mg and K ions, GIP, cAMP and the 260K protein, the axon shows a substantial restoration of the action and resting potentials and the threshold after a lag period which is necessary for assembly of the tyrosinated tubulin at a tem- perature being not optimal for assembly in such an electrophysiological experiment [49]. It is also stressed that the resting potential is kept restoring beyond the original level. Cyclic AMP favors promotion of the restoration, possibly by cAMP- dependent phosphorylation of some components in the axon because perfusion of the axon with phosphatases quickly destroys the excitability [52]. The voltage-clamp method further allows to confirm that the purified tubulin-tyrosine ligase [56] and the purified 260K protein are indis- pensable for the restoration of the sodium conductance as shown in Figure 11 [57]. In this experiment, the membrane excitability was partly Fic. 9. Electron micrograph of the 260K protein molecules rotary shadowed with platinum. Some molecules are shown in the form of a ring. Magnification x 120,000. Tsukita et al. [50]). (From High Molecular Weight Proteins 25 2 3 | Il om’ J fa) Potentials, mV = < a) £ ge Al _ oOo O OL_O O 4 3 6 7 8 IV = action potential O O O ees 8 - threshold _—__—_—_——_e— resting potential 100 150 Time , min Fic. 10. Destruction of the membrane excitability by Ca?* and its restoration by microtubule as- sembly system accompanying tubulin-tyrosinating enzyme system and the 260K protein. Solution I, standard perfusion medium (360mM KF—40 mM K-phosphate buffer—4 % (v/v) glycerol-0.01 mg/ ml protease VII, pH 7.2). Solution II, 360 mM K-glutamate-40 mM K-HEPES, pH 7.2. Solution Ill, Solution II plus 0.2mM CaCl,-1mM MgCl). Solution IV, Solution I plus 0.1 mg/ml 260 K protein—1 mg/ml bovine brain microtubule proteins—3 units/ml tubulin-tyrosine ligase- 0.5 mM ATP-30 uM tyrosin-0.5 mM MgCl,-3 »M cAMP-0.1mM GTP. For detail, see Matsu- moto et al. [49]. deteriorated by perfusing the axon with colchicine, followed by the addition of the restoration medium containing purified 260 K protein. It is clear that the sodium conductance is kept restoring after the perfusion medium is replaced with the restoration medium. The addition of the 260K protein is again inevitable for steady restoration of the excitability. These results allow one to suppose that the 260 K protein which is lining the axolemma and possibly attaching to the channel protein with one end cross-links microtubules running parallel to the longitudinal axis of the axon with the other end, thereby framing the axolemmal undercoat com- posed of microtubule array. In other words, the reconstitution or repair of the functional ax- olemmal undercoat is capable of being accom- plished only by tyrosinated microtubules and the 260 K protein. We have described cross-linking or bundling property of MAPs, fodrin and the 260 K protein. It is now becoming clear that fodrin and the 260 K protein have characteristics resembling each other in the molecular shape and size, in bundling microtubules and in the distribution in the cortical cytoplasm of neuron or axon. Although fodrin has not been investigated as for its action on electrophysiological properties of axons possibly because of the technical problem for mammalian 26 H. SAKAI et al. Colchicine SIS Porcine brain C3S 18.5mM Tyrosine, ligase, 260K | = £ NO Membrane current [mA/cm2] ro) iM =2 = iio 20.3°C =A 1 1 Imsec (pp eee eee O 20 40 60 80 100 120 (B) Time (min) Ona 9k Gt [mMS/cmé?] Vey 100,40; 4 Colchicine SIS Porcine brain CaS “RANA a 18.5mM | | Tyrosine,ligase,260K ~30,70 ee ee eS Vwa “|_60/50 le VR ba . | e “| fo) | ® i! ONa, 50;20; 24's Bene OP! io °. Lath & seee* x se x | ° tee | x ely al , ey 0 o% : ge Ix X Pol of of oF! wee O 20° 40 GO 80 100). 120 Time [min] Fic. 11. Decrease in Na and K conductance by colchicine and its restoration by microtubule assembly system accompanying tubulin-tyrosinating enzyme system and the 260K protein. Restoration medium contains 0.1 mg/ml 260K protein, 1 mg/ml porcine brain microtubule protein, 3 units/ml tubulin-tyrosine ligase, 60 »M tyrosine, 1 mM ATP, 0.5mM GTP, 3mM MgCl, 355mM KF, 25mM K-HEPES at pH 7.3. The axon is voltage-clamped at a membrane potential of 20 mV after pre-conditioned with a pulse of -100mV for 30 msec. The peak Na conductance (gy,), steady K conductance (gx), leak conductance (g,), resting potential (Vg) and reversal potential (Vy.) are plotted as a function of the perfusion time (From Matsumoto ef al. [57]). nerves, the 260 K protein is now clear to be an insucha way that the 260 K protein functions, both important component for the activation of the the proteins might have the same ancestor in a sodium channel in the axolemma. If fodrin is sense of molecular evolution. shown to be involved in the membrane excitation 10 11 12 13 14 15 16 17 18 19 20 21 High Molecular Weight Proteins 27 REFERENCES Tsukita, S., Usukura, J., Tsukita, S. and Ishikawa, H. (1982) Neurosci., 7: 2135-2149. Hirokawa, N. (1982) J. Cell Biol., 94: 129-142. Craig, S. W. and Pollard, T. D. (1982) TIBS, 7: 88-92. Murofushi, H., Minami, T., Matsumoto, G. and Sakai, H. (1983) J. 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UIl- trastr. Res., 80: 374-382. Nishida, E., Kotani, S., Kuwaki, T. and Sakai, H. (1982) In “Biological Functions of Microtubules and Related Structures”, Ed. by H. Sakai, H. Mohri and G. G. Borisy, Academic Press, Tokyo, pp. 285-295. Pollard, T. D., Selden, S.C. and Griffith, L. M. (1982) In “Biological Functions of Microtubules and Related Structures”, Ed. by H. Sakai, H. Mohri and G. G. Borisy, Academic Press, Tokyo, pp. 311-319. Sherline, P., Lee, Y.C. and Jacobs, L.S. (1977) J. Cell Biol., 72: 380-389. Suprenant, K. A. and Dentler, W.L. (1982) J. Cell Biol., 93: 164-174. Sattilaro, R. F. and Dentler, W.L. (1982) In “Biological Functions of Microtubules and Re- lated Structures’. Ed. by H. Sakai, H. Mohri and G. G. Borisy, Academic Press, Tokyo, pp. 297- 309. 22 23 24 25 26 27 28 29 30 39 47 48 Black, M. M. and Lasek, R. J. (1980) J. Cell Biol., 86: 616-623. Matus, A., Bernhardt, R. and MHugh-Jones, T. (1981) Proc. Natl. Acad. Sci. USA., 78: 3010-3014. Berrhardt, R. and Matus, A. 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ZOOLOGICAL SCIENCE 1: 29-34 (1984) Colcemid UV-Microirradiation Method for Analyzing the Role of Microtubules in Pronuclear Migration and Chromosome Movement in Sand-Dollar Eggs YUKIO HIRAMOTO!>*, MryAKo S. HAMAGUCHI’, YOSHITARO NAKANO! and YOKo SHOJI! 1Biological Laboratory, Tokyo Institute of Technology, O-Okayama, Meguro-ku, Tokyo 152, and *Department of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan ABSTRACT — A method is described in which a definite area in a cell immersed in a medium con- taining Colcemid is irradiated with ultraviolet rays of 365 nm wavelength which eliminate the Colcemid effect on microtubule formation. In eggs of the sand-dollar, Clypeaster japonicus fertilized in sea water containing 5 ~M Colcemid, a sperm aster was formed in the area irradiated with UV rays, provided that the area contained the sperm centriole. When the irradiated area was displaced after the formation of a sperm aster in the locally irradiated eggs in Colcemid sea water, the aster moved to the newly irradiated area forming astral rays in the newly irradiated area and destructing astral rays in the shaded area. When the area of the mitotic spindle was irradiated with UV rays in eggs immersed in sea water containing 10 »~M Colcemid during prometaphase to metaphase, a birefringent spindle was formed within the irradiated area and anaphase chromosome movement occurred in the spindle. The spindle elongation during anaphase depended on the width of the irradiated area: The spindle did not elongate when the width was more or less the same as the length of the reformed spindle, © 1984 Zoological Society of Japan whereas the spindle elongated when the area covered a sufficient extent outside the spindle. INTRODUCTION It is a well-known fact that microtubule struc- tures in the cell such as the spindle and asters are destructed by application of colchicine or Colcemid (N-methyl-N-desacethylcolchicine). Borisy and Taylor [1] reported that colchicine inhibits polymerization of tubulin into micro- tubules by binding with tubulin molecules and thus decreasing their concentration. Aronson and Inoué [2] showed that irradiation of cells with ultraviolet rays of about 365nm_ wavelength isolated from rays emerged from a high pressure mercury arc caused a large decrease in absorbance at 355nm and thus eliminated the effects of colchicine and Colcemid which inhibit tubulin polymerization. It is expected that the effects of colchicine and Accepted September 13, 1983 Received July 18, 1983 Colcemid can be eliminated within a definite area in the cell if this area is locally irradiated with 365nm ultraviolet rays with an appropriate intensity. If so, it may be possible to form or destruct microtubule structures in a limited area in the cell, and this method may be useful in analyzing the role of microtubules in living cells. In the present paper, we report this method together with some experimental results for analyz- ing the formation of sperm aster, the pronuclear migration, and the chromosome movement during mitosis in fertilized sand-dollar eggs. MATERIALS AND METHODS In the present study, we constructed an apparatus with which a definite area in the cel! can be irra- diated with ultraviolet rays of 365 nm wavelength during observation by differential interference microscopy or polarization microscopy (cf. Fig. 1). Cells (C) were placed on the stage of a microscope 30 Y. HirAMOTO et al. Fic. 1. A: analyzer. C: cell. units. DM: dichroic mirror. 365 nm UV-rays. and M,: mirrors. N, polarizer. Ca: camera. and N,: Nomarski camera for polarization microscopic observation. W: window for limitting the irradiated area. recorder. the microscope are not shown in this figure. that can be used either as a differential interference microscope or as a polarization microscope by simply replacing prisms in the optical path (cf. ref. 3, the prototype of Nikon ‘‘Nomarski-Recti- fier microscope’). A dichroic mirror (DM) in Nikon fluorescence microscope was set at the base of the microscope so as to transmit visible rays for illumination emitted from the built-in light source (L;, a high-pressure mercury arc, HBO 100 W, OSRAM) and to reflect UV rays from a Co: compensator. F,: filters for transmitting visible rays. L,: light source for observation. prisms. VC,: video-camera for differential interference microscopic observation. Experimental setup for locally irradiating the cell with UV rays. CU, and CU,: video-camera control F,: filters for transmitting L,: light source for the UV-irradiation. M,;: Ob: objective lens. Oc: ocular lens. P: VC,: video- VM, and VM: video-monitors. VIR: video-tape The Condenser lens and trinocular tube of high-pressure mercury arc (L2, USH-100, Ushio Electric Co. Tokyo). Rays of about 365 nm wavelength (rays of 365.015nm, 365.483 nm, 366.288 nm and 366.328 nm emitted by the mer- cury arc) were isolated by filters (F., UV-D36A, UV-35 and IRA-25S, Toshiba Glass Co.) in the optical path. For determining the area of irra- diation, a window (W) was placed at the conjugate point of the position of the cell on the stage of the microscope in reference to the condenser lens. Colcemid UV-Microirradiation Method 31 The size, the shape and the position of the area to be irradiated were adjusted by changing those of the window. The cell was usually observed and recorded with a video system—a video camera (VC,, CTC 2600, Ikegami Tsushinki Co.), a video tape recorder (VTR, VITR-1100SH, Sanyo Electric Co. or NV 8030, Matsushita Electric Co.) and a video monitor (VM;) after an appropriate magnification with the differential interference microscopic optics. Bire- fringence of the cell was observed directly with eyes or after recording with a SIT camera (VC:, C 1000-12, Hamamatsu TV Co.). Because the video cameras were sensitive to UV rays, the size, the shape and the position of the irradiated area were known by the video system. The dose of UV irradiation was determined to be 310° erg/wm’ with a photocell (S780-5BQ, Hamamatsu TV Co.) placed on the microscope stage in the same condition as that in the experi- ment. As biological materials, eggs of the sand-dollar, Clypeaster japonicus were used. The procedure for analyzing the formation of sperm aster and pronuclear movement was as follows. Five minutes later, eggs were inseminated, then were put on a glass slide on the stage of the microscope for observation together with pieces of polyester film of about 100 ~m thickness, and were covered with a coverslip. Shortly after the penetration of the sperm head into the egg, irradiation with ultraviolet rays of about 365 nm wavelength (UV rays) in a circular area of 50 ~m diameter in the egg was started, and was continued later on. In some experiments, the irradiated area was dis- placed from one part to the other part of the egg. Effects of Colcemid sea water and the UV irra- diation on the formation of sperm aster and the movements of sperm and egg pronuclei were observed with a differential interference micro- scope connected with video system as mentioned above. The procedure for analyzing the formation of the spindle, the chromosome movement and the spindle elongation during mitosis was as follows. Eggs were deprived of the fertilization membranes and hyaline layers by treating the eggs with 1 M urea solution (pH 8.2 by NaHCO;) for 1 or 2 minutes shortly after insemination and then put into Ca-free sea water. During prophase to pro- metaphase of the first division, eggs were put into normal sea water and then put on a polylysine- coated glass slide together with pieces of polyester film of 60 ~m thickness, and a cover slip was put over them. By compression of eggs, most of the mitotic apparatuses were oriented in parallel with the planes of the glass slide and the cover-slip. During mid-metaphase, the medium was replaced by sea water containing 10 ~M Colcemid. Irra- diation of 365 nm UV ray was started at metaphase over the area of the mitotic apparatus. Details of the procedure for each experiment will be described in “‘Results’’. RESULTS Inhibition of the sperm aster formation and the pronuclear migration by Colcemid As reported by previous authors [4, 5], the for- mation of the sperm aster was inhibited in eggs immersed in sea water containing 5 ~M Colcemid, though both the penetration of sperm into the egg and the elevation of fertilization membrane oc- curred. After the penetration, the sperm head was at a standstill and changed into sperm pro- nucleus there. The egg pronucleus stayed at its original position. No fibrous structure such as astral rays was formed in the egg. Formation of the sperm aster in the Colcemiad- treated egg by irradiation with ultraviolet rays When a circular area of 50 wm diameter con- taining the sperm head was irradiated with UV rays, astral rays were formed radially from the posterior region of the sperm head where a cen- triole is to be located (cf. arrowhead in Fig. 2a). The astral rays exterded until their tips reached the periphery of the irradiated area. Formation of astral rays was never observed unless the irra- diated area contained the sperm head, irrespective of whether the area contained the egg pronucleus or not. When the irradiated area contained both the sperm head and the egg pronucleus, the egg pro- nucleus started migration toward the center of the 32 Y. HiRAMOTO ef al. xe Fic. 2. Formation of a sperm aster by locally irradiating a sand-dollar egg in Colcemid-sea water UV rays. the start of irradiation. centriole are indicated by arrowheads. 26°C. aster when astral rays extending from the center of the aster reached it. After approaching the astral center, the egg pronucleus finally fused with the sperm pronucleus which had changed from the sperm head. The migration of the egg pro- nucleus never occurred when it was located outside the irradiated area, and consequently, no astral rays reached it (cf. Fig. 2b). Migration of the sperm aster by displacing the UV-irradiated area in the Colcemid-treated egg After complete formation of a sperm aster by irradiation with the UV rays at a limited area in a Colcemid-treated egg, the irradiated area was displaced to other part of the egg. In this case, astral rays extended into new irradiated area while the rays left in the area where the UV-rays had been cut off gradually faded away. The center of the aster slowly moved toward the new irra- diated area, so that the whole aster finally dis- a: 1’20’’ after the start of irrandiation (5’30’’ after insemination). The irradiated area is indicated by a circle. x 800. b: 13’50’’ after Positions of the sperm placed to this area. The displacement of the aster occurred in any directions: toward the center of the egg, away from the center or in parallel with the cell surface, depending on the direction of the displacement of the irradiated area. Formation of birefringent structures by UV irradiation in eggs during mitosis The mitotic spindle and astral rays of the amphiaster have positive birefringence with re- spect to their axes mainly due to microtubules [6, 7]. Similarly to Sluder’s observation [8], the birefringence completely disappeared and the spindle shortened within a few minutes when the eggs in prophase to metaphase of the first division were put into sea water containing 10 uM Colcemid in Clypeaster eggs. Anaphase chromo- some movement never occurred in those eggs without spindle birefringence. If the whole eggs were irradiated with the UV-rays, the birefringence Colcemid UV-Microirradiation Method 33 - Fic. 3. irradiated with UV rays at limited areas of the spindle. 2 Time (min) Chromosome movement and spindle elongation of sand-dollar eggs in Colcemid sea water The irradiation of UV rays was started after complete disappearance of spindle birefringence by application of sea water containg 10 uM Colcemid during metaphase. fringence. spindle elongation are on the right. 25 C: recovered, the spindle elongated, and meanwhile, chromosomes moved toward the poles. When the Colcemid-treated egg was irradiated with the UV rays in a limited area of the mitotic apparatus, the recovery of birefringence and sub- sequent chromosome movement were practically restricted in the irradiated area (cf. ref. 4 and 9). When the area of the whole mitotic apparatus including “‘extra-polar” regions of the Colcemid- treated egg in metaphase was irradiated as shown in Figure 3a, spindle elongation accompanied by poleward migration of chromosomes occurred after the recovery of birefringence of the spindle and asters (cf. Fig. 3a, right). However, when the irradiated area did not contain the “extra-polar” regions as shown in Figure 3b, no spindle elonga- tion occurred, while the chromosomes migrated Chromosome movement started after the recovery of spindle bire- The irradiated areas are indicated on the left, and the chromosome movement and the poleward after the recovery of spindle bire- fringence (cf. Fig. 3b, right). DISCUSSION The result of the UV-irradiation in a limited area in Colcemid-treated egg shortly after insemi- nation in the present study indicates that the sperm aster is formed in the irradiated area when the area contains sperm centriole and that the astral rays extend over the entire irradiated area but not outside the area. The experiment in which irra- diated area was moved after the formation of sperm aster by local UV-irradiation of Colcemid- treated egg shows that astral rays are reversibly formed and destructed in a limited area by switch- ing on and off the UV-irradiation. Spindle 34 Y. HIRAMOTO et al. elongation did not occur across the boundary of the irraidiated area, while chromosome movement occurs in the spindle formed within the irradiated area (cf. Fig. 3). These facts, together with the result of previous report [9], show that astral rays of the sperm aster and the amphiaster and mitotic spindle fibers can be reversibly formed and de- structed by switching on and off the UV-irradiation at the area expected from the image of the window set in the optical path of the UV-irradiation in the present experimental set-up. It has been understood that colchicine and its derivative Colcemid inhibit formation of micro- tubules by binding with tubulin [1] and that these compounds are changed by the UV-irradiation into compounds which do not bind with tubulin ““Lumicolcemid” (cf. ref. 2). Therefore, it is considered that the concentration of Colcemid decreases at the irradiated area and consequently, microtubules are formed by polymerization of tubulin molecules freed from Colcemid. It seems that Colcemid molecules diffuse into the irradiated area passing through the boundary of the irra- diated area and tubulin molecules diffused out of the irradiated area. The coincidence between the UV irradiated area and the area in which microtubule structures were formed may indicate that the area of normal tubulin concentration in the cell practically coincided with the irradiated area in the present experimental condition. The distribution of tubulin concentration around the irradiated area may be determined by various factors, e.g. the rate of Colcemid-tubulin binding, the diffusion constant of Colcemid, and the rate of destruction of Colcemid by the UV-rays de- pending on their intensity. The distribution of Colcemid concentration in and around the irradiated area may be determined by microphotometry during the irradiation, be- cause Colcemid has an absorption spectrum definitely different from that of Lumicolcemid, the substance changed from Colcemid by the UV-irradiation [2, 10]. Details of the pronuclear migration upon fertilization and chromosome movement during mitosis will be reported else- where. ACKNOWLEDGMENT This work was supported by Grants-in-Aid for Sci- entic Research from the Ministry of Education, Science and Culture, Japan awarded to Y. H. REFERENCES 1 Borisy, G. G. and Taylor, E. W. (1967) The mech- anism of action of colchicine binding of colchicine -*H to cellular protein. J. Cell Biol., 34: 525-533. 2 Aronson, J. and Inoué, S. (1970) Reversal by light of the action of N-methyl N-desacethyl colchicine on mitosis. J. Cell Biol., 45: 470-477. 3 Hamaguchi, M.S. and Hiramoto, Y. (1980) Fertilization process in the heart-urchin, Clype- aster japonicus observed with a differential inter- ference microscope. Develop., Growth and Differ., 22: 517-530. 4 Aronson, J. F. (1979) A Motile system acting on the cell nuleus. In “‘Motility in Cell Function’’. Ed. by F. A. Pepe, J. W. Sanger, V. T. Nachmias, Academic Press. 5 Schatten, G. and Schatten, H. (1981) Effects of motility inhibitors during sea urchin fertilization. Microfilament inhibitors prevent sperm incorpora- tion and restructuring of fertilized egg cortex, whereas microtubule inhbitors prevent pronuclear migration. Exp. Cell Res., 135: 311-330. 6 Sato, H., Ellis, G. W. and Inoué, S. (1975) Micro- tubular origin of mitotic spindle form birefrin- gence. Demonstration of the applicability of Wiener’s equation. J. Cell Biol., 67: 501-517. 7 Hiramoto, Y., Hamaguchi, Y., Shoji, Y., Schroeder, T. E., Shimoda, S. and Nakamura, S. (1981) Quantitative studies on the polarization optical properties of living cells. II. The role of microtubules in birefringence of the spindle of the sea urchin egg. J. Cell Biol., 89: 121-130. 8 Sluder, G. (1976) Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs. J. Cell Biol., 70: 75-85. 9 Hiramoto, Y. and Shdji, Y. (1982) Location of the motive force for chromosome movement in sand-dollar eggs. In “Biological Functions of Microtubules and Related Structures’. Ed. by H. Sakai, H. Mohri and G.G. Borisy, Academic Press, New York. 10 Wilson, L. and Friedkin, M. (1966) The Bio- chemical events of mitosis. I. Synthesis and prop- erties of colchicine labeled with tritium in its acethyl moiety. Biochemistry, 2: 2463-2468. ZOOLOGICAL SCIENCE 1: 35-40 (1984) Factors Influencing the Force-Extension Relation of the Series Elasticity in Glycerinated Rabbit Psoas Muscle Fibers SETSUKO GomI and HARuo SUGI Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173, Japan ABSTRACT — 1. The force-extension relation of the series elasticity (SE) in glycerinated rabbit psoas muscle fibers activated with Ca was examined over a wide range of stretch and release velocities. High-speed cinematography indicated no appreciable compliance localized at both ends of the preparation. 2. The force-extension relation of the SE became steeper as the velocity of applied fiber length changes increased. The minimum amount of quick release required to drop the Ca-activated force to zero decreased to some extent when the release velocity was increased from 5-6 to 30-40 L,/sec. 3. The glycerinated fiber preparations were found to develop forces up to five times the maximum isometric force (P,) in response to large stretches, in contrast with the intact fiber preparations in which the force development by stretch is known to exhibit an upper limit around two times Po. 4. The decay of force after the completion of a stretch consisted of three exponential components with half decay times of about 0.25, 1 and 25 msec. The first component was less pronounced as © 1984 Zoological Society of Japan the stretch velocity was decreased, and virtually absent with slow stretches. 5. The change in ionic strength of the surrounding medium had no appreciable effect on the force-extension curves. 6. These results are discussed in connection with the properties of the cross-bridges and other mechanical components within the muscle fibers. INTRODUCTION It is well known that a muscle contains an elastic component in series with the contractile compo- nent [1]. Jewell and Wilkie [2] showed that a part of the elastic component is distributed along the length of muscle fibers, while the rest of it is located at tendinous fiber ends. In the contraction model of Huxley and Simmons [3], the series elasticity (SE) along the fiber has been assumed as being due to the elasticity of the cross-bridges, which are the projections from the thick filaments, and are believed to generate force and motion by their cyclic reaction with the sites on the thin filaments. On this basis, the force-extension rela- tion of the SE has been studied on intact, skinned and glycerinated fibers to give information about the cross-bridge properties [4, 5, 6]. In spite of the extensive studies on the SE in muscle fibers, Accepted August 17, 1983 Received May 17, 1983 no systematic experiments have been made on how the velocity of stretch and release influence the force-extension relation of the SE. The present experiments were undertaken to study the effect of stretch and release velocities on the force-extension relation of the SE in glyceri- nated rabbit psoas muscle fibers activated with externally applied Ca ions. MATERIALS AND METHODS Strips of rabbit psoas muscle (2-4 mm in width and 3-4 cm in length) were tied to glass rods, kept in a solution containing 50% glycerol, 2mM EGTA and 10 mM histidine (pH 6.8) at 0°C for 12-14 hr, and then transferred to a fresh solution of the same composition for storage at —20°C until use (2-4 months) [7]. Small bundles con- sisting of three to five muscle fibers (about 0.5 cm at slack length, Lo) were excised from the glyc- erinated muscle under a dissecting microscope, and mounted horizontally in a glass experimental 36 S. GoMI AND H. Suct A B-1 He-Ne laser chamber (3 ml) filled with a relaxing solution containing 65mM KCl, 5mM MgCl, 1mM EGTA, 5mM ATP and 10mM histidine (pH 6.8). As shown in Figure 1A, both ends of the glycerinated fiber bundle preparation were glued with Aron alpha A adhesive (Sankyo) between the arm of a servo-motor (GP-100, General Scanning) and a force transducer (AE 802, Aksjeselskapet) having a compliance of 2 wm/g and a resonance frequency of 5kHz. The length changes of the preparation were sensed by a differential trans- former incorporated in the servo-motor. The preparation was made to contract maximally with a contraction solution, which was prepared by adding CaCl, to the relaxing solution to result in pCa 5.5, taking the apparent stability constant of Ca-EGTA complex to be 1.95 x10°/M [8]. The sarcomere length of the preparation was measured with He-Ne laser light. All experiments were made at room temperature (20—23°C). The preparation was first kept isometric at the slack length ZL, with sarcomere lengths of 2.0-2.2 ym, and when the maximum isometric force (Po) was developed in the contraction solution, the force-extension relation of the SE was studied by applying constant-velocity stretches and releases of various amounts and recording the resulting force changes as a function of time together with the fiber length changes on a cathode-ray oscil- loscope (565, Tektronix) (Fig. 1B-1). Thus, the force-extension curve of the SE was obtained by plotting the force (P ) immediately after the stretch or release against the amount of fiber length change (4L) expressed in percentage of Lo (Fig. 1B-2). In some cases, the force-extension curve of the SE were directly displayed on the oscillo- scope by applying large constant-velocity fiber length changes and recording the resulting force changes as a function of fiber length (Fig. 1C). The velocity of fiber length changes ranged from 4 to 40 L,/sec. Since the velocity of unloaded shortening of the preparation (V.) is 2-2.5 L,/sec (Gomi & Sugi, unpublished), the slowest velocity of fiber length changes was about two times of Vo. To perform the experiments within the range of fiber lengths where the resting force was negligible, the maximum amount of stretch was limited to Bf so) Sree stretch eS ~Telease stretch 9 release Length AL/Lo Fic. 1. Methods of studying the force-extension rela- tion of the SE in glycerinated rabbit psoas muscle fibers. A: Diagram of experimental setup. The fiber bundle preparation (P) was mounted horizon- tally in an experimental chamber (C) between the servo-motor (S) and the force transducer (F). The sarcomere length was measured by optical diffrac- tion with He-Ne laser light. Solutions enter into the chamber at J and flow past the preparation to the exit O as indicated by arrows. B: Diagram showing the experimental procedure to obtain the force-extension curve by applying releases and stretches (4L) of various amounts to the prepara- tion and recording the resulting force changes. The force (P) immediately after release or stretch was expressed as a fraction or multiple of Po, and is plotted against the amount of fiber length change expressed relative to Lp. C: Diagram showing the force-extension curve on the oscilloscope by apply- ing large length changes to the preparation and displaying the resulting force changes as a function of fiber length. After each fiber length change, the preparation was made to relax completely in the relaxing solu- tion for more than 5 min before it was again activated in the contraction solution. The ex- periments were terminated when the value of Po fell by more than 20% of the initial value (2-2.5 kg/cm’) after repeated application of the contrac- tion solution. The oscillographic records were photographed with a 35 mm camera, and enlarge- ment was used for analysis. Series Elasticity in Muscle Fibers 37 RESULTS Absence of appreciable end compliance at both ends of the glycerinated fiber bundle preparation Since the glycerinated fiber bundle preparation used in the present study was glued to the servo- motor and the force transducer at both ends, it seemed desirable to examine whether the prepa- ration was damaged over an appreciable distance from both ends to produce an appreciable com- pliance localized at both ends; if this is actually the case, the response of the SE in the preparation would be greatly masked by this end compliance. To make this point clear, two kinds of preliminary experiment were made with several preparations. First, the preparation with a number of carbon markers on it was continuously observed under a microscope during the course of force development in response to the contraction solution. No ap- preciable localized extension of the end segments was Observed during isometric force development. Secondly, the length changes of the end segments were recorded with a high-speed cinecamera (Hicam, Redlake) at 1,000 frames/sec during the application of stretches (up to 5% of Zo and up to 40 L,/sec). Again, there was no appreciable localized extension at the end segments during the fiber length changes. These results indicate that there is no appreciable end segment compliance which may mask the response of the SE along the fiber length. Effect of velocity of fiber length changes on the force- extension curves Figure 2 is a typical example showing the dependence of the force-extension relation of the SE in glycerinated rabbit psoas muscle fibers on the velocity of applied fiber length changes. The force-extension curves were sigmoidal in shape, and their slope became steeper as the velocity of fiber length changes was increased; for a given amount of release or stretch, the resulting devel- opment or drop in force was more marked as the velocity of stretch or release was increased, though the effect was hardly appreciable for small length changes (within+0.25% of L.) (Fig. 2A). The minimum amount of release required to drop the maximum Ca-activated isometric force 5.0 A 35 Lo/sec 4.5 = P/Po 11 Lo/sec 7 40 ©) ee 0 3.5 (e) s ~ 3.0 od Z Ce) 5.5 Lo/sec \X 25 (@) C) ‘\ 2.0 4415 1.0 B 1.0 0.5 0.5 +8 & LS _ ») 6 4 2 0 2 stretch release ALY ton) Fic. 2. Force-extension curves with three different velocities of fiber length changes. The velocities are indicated alongside each curve (Fig.2A). The curves were obtained by the procedure illustrated in Fig. 1B. The force-extension relation obtained with releases is also shown on an expanded time scale at the lower right of the figure (Fig. 2B). to zero decreased from 1.5-2% to 1.0-1.5% of Lo when the release velocity was increased from 4—6 to 30-40 length/sec in ten preparations examined (Fig. 2B). An interesting feature concerning the SE in the glycerinated fibers was that, with increas- ing amount of stretch, the force in the preparation continued to rise at least up to five times Po, in contrast with the intact vertebrate muscle fibers in which the force development in response to applied stretches is known to exhibit an upper limit around two times P, [9, 10, 11]. 38 S. GOMI AND H. SuGI Effect of stretch velocity on the time course of force decay after stretch Although it is well known that, after the com- pletion of an applied stretch, the force in muscle fibers starts to decay exponentially with time [12], the effect of stretch velocity on the time course of the force decay after stretch has not yet been studied. To obtain some insight into the under- A B | 50ym Fic. 3. Length and force changes of the preparation during and after stretches (5% of L,) at three dif- ferent velocities (40 L,/sec in A, 8 L,/sec in B and 4 L,/sec in C). The upper trace shows the fiber length changes, while the middle and the lower traces show the force changes and the zero force level respectively. Records A, B and C were ob- tained from the same preparation. T 3rd component \ ee 2nd component Last \ component \ 10 5 Time (msec) 1st component 5 Time (msec ) lying mechanism of force development by stretch, the effect of stretch velocity on the early part of the force decay after the completion of stretch was examined. Figure 3 shows typical records show- ing the force changes in response to stretches (5% of Lo) at three different velocities. It can be seen that, the greater the stretch velocity, the force decay after stretch takes place more rapidly. In Figure 4, the force decay for the first 10 msec after the completion of stretch is plotted on a logarith- micscale. In all the fifteen preparations examined, the early part of the force decay was resolved into three exponential components with half decay times of about 0.25, 1.0 and 25 msec. The first component was most prominent with stretches above 30 L,/sec (Fig. 4A), but less prominent with slower stretches (Fig. 4B), and virtually disap- peared with stretches below 5 L,/sec (Fig. 4C). Effect of ionic strength on the force-extension curves Since it has been reported that the maximum isometric force and the unloaded shortening ve- locity in skinned vertebrate muscle fibers change markedly depending on the ionic strength of the bathing solution [13], the effect of ionic strength on the force-extension relation of the SE was studied on five preparations with various velocities of fiber length changes. Attention was focused on a part of the force-extension curves with small C 3rd component 3rd component 2nd component 2nd component 10 5 10 Time (msec ) Fic. 4. Analysis of early part of force decay after stretch into three exponential components with half decay times of about 0.25, 1 and 25 msec respectively. Open circles: Force decay after stretches at three different velocities (40 L,/sec in A, 8 L,/sec in B and 4 L,/sec in C). Filled circles: Force decay minus the third component. Triangles: Force decay minus the second and the third com- ponents. Forces are expressed as fractions of the force (J) at the beginning of force decay, and plotted on a logarithmic scale against time. Note that the first component is virtually absent in C. Series Elasticity in Muscle Fibers 39 1 stretch release aL/Lo (%) Fic. 5. Force-extension curves with three different KCI concentrations in the surrounding medium. The KCl concentration is indicated alongside each curve. The curves were obtained by the procedure illustrated in Fig. 1C. The velocity of fiber length changes was 40 L,/sec. fiber length changes, since the cross-bridge prop- erties are generally studied with small length perturbations. As shown in Figure 5, the force- extension curves did not change markedly when the KCl concentration of the bathing solution was increased from 50 to 150 mM. DISCUSSION Possible structural origin of the series elasticity in vertebrate muscle fibers The present experiments have shown the de- pendence of the force-extension curves of the SE in glycerinated fibers on the velocity of applied fiber length changes (Fig. 2). This result, together with the sigmoidal shape of the force-extension curves, may be taken to indicate that the SE in vertebrate muscle fibers does not arise from an undamped elastic structure, but is divided into some structural components with visco-elastic properties. The force-extension relation for small fiber length changes (0.25% of Lo) was almost linear, and did not change appreciably by changing the velocity of fiber length changes. With larger fiber length changes, especially with stretches, the curves deviated from each other depending on the velocity of fiber length changes. By stretching glycerinated rabbit psoas muscle fibers in rigor and observing the resulting extension of sarcomere structures, Suzuki and Sugi [14] have recently shown that the bare region of the thick filament and the whole thin filament have an appreciable elasticity, while the Z-band structure was very rigid. This implies that, in each sar- comere, three structures responsible for the SE may be connected in series, i.e. (1) the cross-bridges at the zone of overlap between the thick and thin filaments, (2) the thin filaments at the non-overlap zone and (3) the bare region of the thick filaments. It would be of interest to examine whether the three exponential components of the force decay after stretch (Fig. 4) result from stress relaxation oc- curring in the above three sarcomere structures after the completion of stretch. Due to the increase in the slope of the force- extension curves the minimum amount of release required to drop the Ca-activated force to zero decreased to some extent with increasing release velocity (Fig. 2B). In tetanized vertebrate muscle fibers, this value is also reduced as the release velocity is increased [3, 15]. Though Huxley and his coworkers attributed this result to the quick force recovery already occurring during the time of release, recent experiments of Sugi and Kobayashi [16] indicate that, if the fibers are re- leased too rapidly at one end, the mechanical impulse propagates along the fiber length without causing the accompanying sarcomere length changes to produce force drop at the other fiber end connected to the force transducer. The ve- locity of this mechanical impulse along the fiber increases with increasing velocity of applied length changes [17] to result in steeper force drop for a given amount of release. The same kind of explanation would apply to the present results. 40 S. GoMI AND H. SuGI Comparison of the force-extension curves of glyc- erinated fibers with those of intact fibers An interesting feature brought about in the present study is that, in glycerinated fibers, the force rises up to five times P> in response to large stretches (Fig. 2B). It is generally believed that the cross-bridges can not remain attached to the thin filaments when they are stretched over a large distance, so that detachment or slippage of the cross-bridges may take place during large stretches. In addition, it has been shown that tetanized vertebrate muscle fibers are lengthened rapidly under a load of more than about two times P» [18], indicating that the cross-bridges can not sustain forces above two times Py. In accordance with this, the force development in response to stretch in tetanized fibers exhibits an upper limit around two times P, [9, 10, 11]. One possible explanation for the ability of the glycerinated fibers to develop large forces in response to large stretches may be that the turnover rate of the cross-bridges is much Slower in glycerinated fibers than in intact fibers, as indicated by the slow rate of ATP splitting in glycerinated fibers [19]. If the cross-bridges detach with a slower rate from the thin filaments during contraction, they would be stretched over a longer distance to produce more force until the time of detachment. This implies that the force- developing capacity of muscle fibers is to some extent dependent on the turnover rate of the cross- bridges. In this connection, it is of interest that the force development of tetanized vertebrate muscle fibers for a given amount of stretch is more marked with decreasing temperature [10]. REFERENCES 1 Hill, A. V. (1938) The heat of shortening and the dynamic constants of muscle. Proc. Roy. Soc., B 126: 136-195. 2 Jewell, B. R. and Wilkie, D. R. (1958) An analysis of the mechanical components in frog’s sartorius muscle. J. Physiol., 143: 515-540. 3 Huxley, A. F. and Simmons, R. M. (1971) Pro- posed mechanism of force generation in striated muscle. Nature, 233: 533-538. 4 Julian, F.J. and Sollins, M. R. (1975) Variation of muscle stiffness with force at increasing speeds of shortening. J. Gen. Physiol., 66: 287-302. 5 Tsuchiya, T., Giith, K., Kuhn, H. J. and Riegg, 10 11 13 14 IS) 16 7 18 19 J. C. (1982) Decrease in stiffness during shortening in calcium activated skinned muscle fibers. Pflugers Arch. Ges. Physiol., 392: 322-326. Heinl, P., Kuhn, H.J. and Riegg, J.C. (1974) Tension responses to quick length changes of glycerinated skeletal muscle fibres from the frog and tortoise. J. Physiol., 237: 243-258. Tanaka, H., Tanaka, M. and Sugi, H. (1979) The effect of sarcomere length and stretching on the rate of ATP splitting in glycerinated rabbit psoas muscle fibers. J. Biochem., 86: 1587-1593. Portzehl, H., Caldwell, P.C. and Rtegg, J. C. (1964) The dependence of contraction and relaxa- tion of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. Biophys. Acta, 79: 581-591. Joyce, G. C., Rack, P. M. H. and Westbury, D. R. (1969) The mechanical properties of cat soleus muscle during controlled lengthening and shorten- ing movements. J. Physiol., 204: 461-474. Sugi, H. (1972) Tension changes during and after stretch in frog muscle fibres. J. Physiol., 225: 237- D3: Edman, K. A. P., Elzinga, G. and Noble, M. I. M. (1978) Enhancement of mechanical performance by stretch during tetanic contractions of vertebrate skeletal muscle fibres. J. Physiol., 281: 139-155. Abbott, B. C. and Lowy, J. (1957) Stress relaxation in muscle. Proc. Roy. Soc., B 146: 280-288. Thames, M.D., Teichholz, L.E. and Podolsky, R. J. (1974) Ionic strength and the contraction kinetics of skinned muscle fibers. J. Gen. Physiol., 63: 509-530. Suzuki, S. and Sugi, H. (1983) Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers. J. Gen. Physiol., 81: 531-546. Ford, L. E., Huxley, A. F. and Simmons, R. M. (1977) Tension responses to sudden length changes in stimulated frog muscle fibres near slack length. J. Physiol., 269: 441-515. Sugi, H. and Kobayashi, T. (1983) Sarcomere length and force changes in single tetanized frog muscle fibers following quick changes in fiber length. In “‘Contractile Mechanisms in Muscle’. Ed. by G. H. Pollack and H. Sugi, Plenum Press, New York, in press. Truong, X. T. (1974) Viscoelastic wave propagation and rheologic properties of skeletal muscle. Am. J. Physiol., 226: 256-264. Katz, B. (1939) The relation between force and speed in muscular contraction. J. Physiol., 96: 45-64. Arata, T., Mukohata, Y. and Tonomura, Y. (1978) Acceleration of the ATPase activity of glycerol- treated muscle fibers by repeated stretch-release cycles. J. Biochem., 84: 751-761. ZOOLOGICAL SCIENCE 1: 41-49 (1984) Structure and Output Connection of Local Non-Spiking Interneurons in Crayfish MITUHIKO HISADA, MASAKAZU TAKAHATA and TosHIKI NAGAYAMA Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan ABSTRACT — The structure of local non-spiking interneurons (LNSNs) in the terminal abdominal ganglion of the crayfish Procambarus clarkii Girard and their function in controlling the uropod motoneuron activity were investigated by intracellular staining and current injection using Lucifer dye filled microelectrodes. The LNSNs found were classified into bilateral and unilateral types. The unilateral type LNSNs were further divided into antero-lateral (AL) and postero-lateral (PL) type by their somata location. They were all monopolar, only a single process emerging from the soma. The AL type extended branches both antero-medially and postero-laterally while the PL type toward the anterior from the soma. Their branches extended both ventrally and dorsally. The branching density was distinctively higher than that of other neurons. Somata of all types of LNSNs were significantly smaller than those of intergangionic interneurons and fast motoneurons. The difference in the structure of LNSNs did not necessarily reflect that in the motor control reve- aled by the current injection test. One AL type LNSWN excited a synergistic set of uropod moto- neurons when depolarized, whereas another AL type LNSN of the similar structure excited also an antagonistic set of motoneurons in addition to the synergistic ones when depolarized. The possibility that the different parts of an LNSN functions as separate input-output units producing © 1984 Zoological Society of Japan this apparent disparity in the observed results was discussed. INTRODUCTION Interneurons which function without spikes have been recently reported in the central nervous system of crayfish [1-3]. These interneurons can modulate and control the motoneuron activity directly by their membrane potential change. The mode of motoneuron control by these non-spiking interneurons is diverse: some of them affect the synergistic set of motoneurons while others, the antagonistic ones (Nagayama et al/., in preparation). Morphologically, these non-spiking interneurons have no explicit axon structure, extending their branches only within one ganglion. This fact indicates that these anaxonic non-spiking inter- neurons can be characterized as the intraganglionic interneurons which are exclusively devoted to the local processing of neural signals as contrasted with the interganglionic interneurons which trans- Accepted August 5, 1983 Received June 23, 1983 mit signals in form of spikes to and from various parts of the central nervous system. The local non-spiking interneuron (LNSN) is thus a novel type of central nervous element both physiologically and morphologically, distinct from other types of neurons so far known. Similar interneurons of both the non-spiking nature and the anaxonal structure have also been reported in insect [4,5]. The transmissio 1 mechanism between the interneuron and the postsynaptic motoneuron has been studied in locust [6]. Their function as the active component in the integrative action of the central nervous system, however, is little understood in any species. We examined in this study the structure of LNSWNs in the terminal abdominal ganglion of the crayfish Procambarus clarkii Girard and its cor- relation with their mode of motoneuron control using the intracellular current injection and staining techniques. The result revealed that the LNSNs in the ganglion could be classified into three major groups according to their gross 42 M. HIsADA ef al. morphology. No significant correlation was ob- served, however, between the structure of inter- neurons and their mode of motoneuron control: similar interneurons with regard to their mor- phology had _ different effects on uropod motoneuron activity. On the basis of results obtained, we present a hypothesis that the motor effect of an LNSN may not be fixed but varied, if necessary, depending on the peripheral and central input to that interneuron. MATERIALS AND METHODS Animals and preparations Adult crayfish Procambarus clarkii Girard were obtained commercially (Sankyo Labo Service, Tokyo) and kept in laboratory tanks before use. Both sexes were used without any significant differ- ence in the obtained results. The abdomen was isolated and pinned ventral side up in .van Harreveld [7] solution. Details of the dissection procedure have been described previously [8]. Extracellular recordings Two extracellular oil-hook electrodes [9] were placed respectively on the closer and the opener motoneuron axons to monitor the effect of current injection into LNSNs on their activity. Spike activity of the closer motoneurons was recorded from the root 2 motor bundle just proximal to its bifurcation both to the reductor and to the adductor exopodite muscle. The nerve to the reductor exopodite contained at least 3 motor axons which were usually discharging spikes tonically [10]. The nerve to the adductor exopodite con- tained two large motor axons which were usually silent [8]. Spike activity of the opener moto- neurons was recorded from the root 3 motor bundle just distal to its bifurcation to the ventral rotator muscle, which contained those motor axons in- nervating the abductor exopodite lateral, the abd. ex. ventral and the abd. ex. dorsal. Intracellular recordings and current injection LNSWNs in the terminal abdominal ganglion were penetrated by glass microelectrodes filled with 5% Lucifer yellow CH [11] in 0.1 M lithium chloride solution. The electrode resistance was 100-200 M2. The right half of the ganglion was probed from the ventral side. We judged the penetrated cell to be the LNSN when the cell satisfied the criteria which have been described previously [2]. The cell was confirmed to be an LNSN by injecting the Lucifer dye into it after the electrophysiological study (see below). Current was injected into the penetrated cell through the recording electrode by a bridge circuit. Since the electrode resistance became higher than 200 M2 during the probing of the ganglion, it was sometimes difficult to record the membrane potential of LNSN and to keep the bridge completely balanced while the current was being injected. Intracellular stainings Lucifer yellow was injected iontophoretically into the LNSN by applying hyperpolarizing current pulses of 1 to 5 nA with 0.5 second duration at 1 Hz for about 10 minutes. But often the hyperpolari- zing current injection to check the motor effect of the penetrated cell was found to produce the cell staining. Therefore in some preparations, more than one neuron were stained after checking several neurons, although there was a difference in the fluorescence intensity of individual neurons. After the dye was injected, the ganglion was immediately dissected from the abdomen, mounted without further fixation and photographed under a fluo- rescent microscope. In some cases, the ganglion was then fixed in 10° formalin in phosphate buffer for 15 minutes, dehydrated in an alcohol series and cleared in methyl salicylate and photographed again. The morphology of the LNSN was ob- tained by tracing the photographs. In some preparations, the LNSN was stained with Co (NO;)2. The procedure is described elsewhere [2]. RESULTS Morphology of local non-spiking interneurons (LNSNs) Intracellular staining of LNSNs revealed that they could be classified into either the bilateral or the unilateral type. The bilateral type extended branches on both the ipsilateral and the con- Local Non-Spiking Neurons in Crayfish 43 tralateral side to that of soma location (Fig. 1A). Typically, the branches on both sides were con- nected with a process of about 10-20 “m in diameter but no distinct axon structure was ob- served. Branches extended to both the ventral and the dorsal half of the ganglion. Somata of this type of interneuron were usually located in the ventral ridge of the centro-medial portion of the ganglion (Fig. 1D). We have stained 6 of this type of LNSN. But since these LNSNs_ extended profuse branches over the whole ganglion, which were too extensive to infer their output con- nection by the present form of current injection test A C-9 (see Discussion), we had to omit them from the present analysis of the correlation between the structure and the output mode. The latter, unilateral type LNSNs extended their branches only within the ipsilateral half of the ganglion to their somata. They could be further classified into the AL (N=20) and the PL (N=12) types. Somata of the PL type LNSNs were located in the ventral ridge of the postero- lateral portion of the ganglion (Fig. 1B). The soma was connected to the thickened part of the neurite with a fine process. They have no distinct axon structure, extending their branches toward B Fic. 1. Local non-spiking interneurons (LNSNs) in the terminal abdominal ganglion of crayfish. A: A bilateral type LNSN. The cell was penetrated at the branch contralateral to the soma. Cobalt stain without intensification. C: An AL type LNSN. Lucifer stain. cation of LNSN somata in the ganglion viewed from the dorsal side. The electrode always probed the right half of the @ indicates somata of unilateral type and © indicates those of Three of the bilateral type LNSNs (shown with *) were penetrated at projected onto the standardized ganglion. ganglion from the ventral side. bilateral type LNSNs. the contralateral branch to the soma. B: A PL type LNSN. Cobalt stain without intensification. Calibration bar: 100 ym. All are dorsal view. D: Lo- Somata of 38 LNSNs were 44 M. HisaDA et al. Fic. 2. Photographs of a whole mount of the terminal abdominal ganglion in which an AL type local non- spiking interneuron (LNSN) was stained with Lucifer yellow CH. The ganglion was viewed from the dorsal side. focalized. Calibration: 50 ym. the anterior. The branching frequency was almost the same in any neuritic branch of the unilateral type LNSN. Therefore, we could not get any idea about the regional difference within an LNSN regarding its integrative function from _ the structural aspect, such as seen in the conventional motoneurons and interganglionic interneurons. The AL type LNSNs had their somata in the ventral ridge of the antero-lateral portion of the ganglion (Fig. 1C), had no axonic structure, and extended branches both antero-laterally and postero-laterally when viewed from above (Fig. 2). Characteristic to all these three types of LNSNs are the following points: 1) Size of somata was significantly smaller than that of fast motoneurons and ascending interneurons [2]. 2) Only a single process emerged from thesoma. 3) Their branches extended both ventrally and dorsally as contrasted with, for example, those of motoneurons which extended only in the dorsal half of the ganglion. 4) Their branching density was higher than that of other neurons in the ganglion. Motor effects of LNSNs revealed by current in- jection A variety of motor effects were observed with the intracellular current injection into unilateral type A: The dorsal branches are in the focal plane. B: The soma and the ventral branches are LNSNs. Details of the physiological analysis on their motor connections are given elsewhere (Nagayama eft al., in preparation). Here we briefly summarize their mode of motor control. The LNSNs were divided into two groups according to their motor effect on uropod moto- neurons. One group of LNSNs controlled the synergistic set of motoneurons. Some of them increased the spike discharge rate of these motoneurons when depolarizing current was injected. Others de- creased it with the membrane potential change in the same direction (Fig. 3A). These LNSNs had no significant effect on the motoneurons which were antagonistic to the affected motoneurons. They usually exerted bidirectional effect on the motoneuron activity: thus hyperpolarization of these LNSNs has opposite effect on the moto- neurons. Another group of LNSNs controlled the antag- Onistic set of motoneurons. Some of them affected these motoneurons in co-activation. When they were depolarized, activity of both the Opener and closer motoneurons was effectively inhibited (Fig. 3B) or excited. Hyperpolarization of these LNSNs had the reverse effect on the activity of both motoneurons but in some cases had Local Non-Spiking Neurons in Crayfish 45 B ive Op . Cl LN Gamnoles- 06 Op rant | CUHHH | LN Fic. 3. Effect of intracellular current — LNSNs on the uropod motoneuron activity. A: An LNSN (LN, third trace) which decreased the spike discharge rate of the synergistic motoneurons innervating the opener muscles (Op, upper trace) when the depolarizing current was injected (bottom trace). No significant effect was observed in the closer motoneuron activity (Cl, second trace). B: An LNSN which decreased the spike activity of both the opener and the closer motoneurons when depolarized. C: An LNSN which decreased the opener motoneuron activity and at the same time increased the closer motoneuron activity when depolarized. Calibration: A (0.1 V,0.1 sec, 5nA); B (SOmV, 0.2 sec, SnA); C (20 mV, 0.1 sec, 5 nA). no significant effect. Other LNSNs of this group affected the motoneurons in a reciprocal way. When some of these LNSNs were depolarized, the activity of closer motoneurons was increased and, at the same time, that of opener motoneurons was decreased (Fig. 3C). Hyperpolarization of these LNSNs usually had the reverse effect. Depolari- zation of other LNSNs caused a decrease in the closer activity and an increase in the opener activity while hyperpolarization usually caused, as other LNSNs, the reverse change in the respective motoneuron activity. A question here is what the basis of this diversity in the mode of motoneuron control by LNSWNs is. Does the difference in the control mode of LNSNs really reflect that in the structure of individual LNSNs? LNSN structure and the motor effect The following results were all obtained by penetrating the LNSNs at their neuritic branches and not at the somata. Of 6 LNSNs which decreased the activity of synergistic closer moto- neurons when depolarized and increased it when hyperpolarized, two were shown to be of the PL type (Fig. 4A left) and four were of the AL type (Fig. 4A right). All 3 LNSNs which had the reverse effect on closer motoneurons were of the AL type LNSN (Fig. 4B). Thus in the former case, the LNSNs which were defined as the same in function were found to be heterogeneous in their structure. Although the latter group was found to be homogeneous, it is very likely from the following fact that the group will prove to be heterogeneous when more LNSNs are examined. Of all 13 LNSNs which affected the antagonistic set of motoneurons in co-activation, 12 LNSNs decreased both the opener and closer motoneuron activities when depolarized and the remaining 1 LNSN increased them when depolarized. This LNSN was of the AL type. Five of the former group were of the PL type (Fig. 4C left) while other 7 were of the AL type (Fig. 4C right). Of other 10 LNSNs which affected the antagonistic motoneurons in the reciprocal way, two decreased the closer activity and at the same time increased the opener activity when they were depolarized. They were both the AL type LNSN (Fig. 5A). 46 M. HIsaDA ef al. Ls) L-29 C L-46 Fic. 4. Structure of LNSNs with their motor effect examined. A: Two LNSNs from two animals which decreased the activity of closer motoneurons when depolarized. B: Two LNSNs which increased the closer motoneuron activity when depolarized. C: Two LNSNs which decreased the activity of both the opener and closer motoneurons when depolarized. D: Two LNSNs which increased the closer motoneuron activity and decreased the opener motoneuron activity when depolarized. In A, C and D, the PL type LNSNs are drawn with the outline of the ganglion to show their position in it while the AL type LNSNs are drawn with higher magnification to indicate their fine morphology. Calibration: 100 um. The remaining 8 increased the closer and decreased the opener activity when depolarized. Five of them were of the PL type (Fig. 4D left) while other 3 were of the AL type (Fig. 4D right). The results indicate that a certain mode of motoneuron activation is not necessarily shown by those LNSNs of similar structure. It was frequently observed, on the other hand, that the LNSWNs of similar structure showed different mode of motor control (Fig. 5). The LNSN in Figure 5A decreased both the closer and opener motoneuron activities when depolarized while that in Figure 5B decreased the closer activity and, at the same time, increased the opener activity when depolarized. From the morphological viewpoint only, these two LNSNs would be considered to be identical. Reichert et al. [3] have also reported in their recent paper that unilateral type LNSNs could not be distinguished solely on the basis of their structure. They distinguished these LNSNs according to the effect which these neurons exerted on uropod efferents when depolarizing current was injected to them. It seems to be a general conclusion, there- fore, that there is no significant correlation between the LNSN structure and the motor effect revealed by intracellular current injection. DISCUSSION The structural types of LNSNs in the terminal ganglion shown in this study (Fig. 1) are not exhaustive. Reichert et al. [3] have reported several other structures of LNSNs in the same ganglion. We have also observed structures other than those ever described. Since we limited the electrode penetration to certain regions of the ganglion, there must have been a sampling bias. But this would not invalidate the results obtained in this study. In the following sections, we will first discuss the output connection of LNSNs and Local Non-Spiking Neurons in Crayfish 47 A B FP oh i, a § E—2p Sir Fic. 5. Structure of two LNSNs which had different motor effect. A: An LNSN which decreased the activity of both the closer and opener motoneu- rons when depolarized. B: An LNSN which decreased the closer motoneuron activity but increased the opener motoneuron activity when depolarized. Calibration: 100 um. These LNSNs were stained in different animals. then present a hypothesis on the functional fractionation of individual LNSNs. Two types of LNSN structure The unilateral type LNSNs were classified into either the AL or the PL type according to the location of their somata in the ganglion and the branching pattern of main processes (Fig. 1). In each class, LNSNs showed slight variations in the branching pattern of fine processes and their density, soma size and the location in the ganglion (Fig. 4A—D right). But it did not seem heuristic in any respect to classify further the LNSNs since 1) technically, the site of injection and the amount of Lucifer dye differed with each experiment and 2) following a general rule in arthropod neurons, even the explicitly identified neurons are known to differ in detailed structure from animal to animal [12]. Although the gross structure of these two types of LNSNs was different, the areas in which they extended branches seemed to overlap largely with each other (Figs. 1, 4). Nickel and cobalt back- filling of the opener and closer motoneurons in other preparations showed that their dendritic branches extended chiefly in the overlapping area of both types of LNSNs [see also 8]. Thus the neuroanatomical examination indicated that it would not be surprising if the two types of LNSNs had the similar output connections with uropod motoneurons. Electrophysiological analysis actually showed that there was no significant difference between these two types in the motor effect observed with the current injection test (see below). The possibility still remains, however, that the difference in the gross LNSWN structure may reflect that in the input organization. Output connection of LNSNs in the terminal ganglion Since the LNSNs do not generate a spike which usually travels without much decrement, we should be careful in interpreting the result of the intracellular current injection test using a single electrode. The voltage change evoked at the penetration site by the injected current would spread electrotonically over a limited length of branches with gradual decrement which is deter- mined by the cable properties of the LNSN membrane. Rall [13] calculated the voltage decrement in an LNSN of the locust metathoracic ganglion and showed that a voltage change at one site of the interneuron could be reduced to less than 5% of the original value when it spread to another site of about 300 m in distance. It has been shown that the transmission between the LNSN and the postsynaptic motoneuron is mediated chemically [5, 14, Nagayama ef al., in preparation]. The motoneuron spike activity was modulated in proportion to the amount of the current injected into the LNSN [2]. This indicates that the amount of transmitter released from the output synapse of the LNSN to the motoneuron is determined by the extent of potential change in the presynaptic membrane as in the neuro-muscular junction [15]. Which of the motoneurons are modulated upon the current injection test is mostly dependent upon the electrotonic distance between the injection site and the output synapses to individual motoneurons. 48 M. HISADA et al. Zettler and Jarvilehto [16] showed that the graded potential change spread actively over the non-spiking interneuron in the insect visual system. The crayfish LNSNs may also bear the active patch of membrane. In any case, the motor effect of LNSNs revealed by the intracellular current injection seems to be crucially determined by the site of electrode penetration, not truly reflecting the overall configuration of synaptic connection with motoneurons. The co-activation of antagonistic motoneurons (Fig. 3) could there- fore be non-physiological: injected current might drive a set of output synapses which in normal condition may never be activated simultaneously as being the parts of separate input-output units. Because the branching density of LNSNs was far higher than that of ascending interneurons and motoneurons in the ganglion, we could not pinpoint the site of electrode penetration by the method devised to this date [8]. To examine the above- mentioned possibility, it would be necessary to develop a new double-staining technique which can indicate both the electrode penetration site and the neuron structure. A novel mode of neuron function We have shown elsewhere (Nagayama et al., in preparation) that the spikeless control of moto- neuron activity by LNSNs is their normal mode of operation. Furthermore, we have found that the conventional spiking neurons can also operate without generating spikes [8]. The control of synaptic output not by the all-or-none spike potential but by the graded change in the membrane potential suggests a novel mode of neuron oper- ation which is different from that in the spiking neurons. As discussed above, the potential change produced by the current injection spreads over the LNSWN branches with a gradual decrement which depends upon their cable properties. This will also be the case with the membrane potential change due to the action of input synapses on the LNSN. Thus the electrotonic distance of indi- vidual output synapses from the site of input Synapses determines which output synapse or Synapses are activated. This argument raises a theoretical possibility that the synaptic potential generated at an end of the LNSN branch can not, or can at most very weakly, activate output synapses at another end [13, see above]. We therefore hypothesize that different parts of an LNSN function quite independently with each other as separate input-output units. Dendritic branches of the adductor exopodite motoneuron which innervates one of the closer muscles of the uropod has actually been shown to operate without spikes as the output pathway to another moto- neuron, separate from the axon of its main output pathway [8]. Since as many as 100 synaptic contacts are reported to be involved in the func- tional connection of two neurons [17] in the stomatogastric ganglion of lobster the interaction occurring among the input synapses of the LNSN from different neurons would be far more com- plicated than our present surmise. Detailed knowledge on the distribution of output and input synapses on the LNSN branches is prerequisite in testing the hypothesis. ; ACKNOWLEDGMENTS We thank Dr. W. W. Stewart for providing a sample of Lucifer yellow CH. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture to M.H. (56440006) and to M.T. (57740384). REFERENCES 1 Heitler, W.J. and Pearson, K.G. (1980) Non- spiking interaction and local interneurones in the central pattern generator of the crayfish swimmeret system. Brain Res., 187: 206-211. 3 2 Takahata,M., Nagayama,T. and Hisada, M. (1981) Physiological and morphological charac- terization of anaxonic non-spiking interneurons in the crayfish motor control system. Brain Res., 226: 309-314. 3 Reichert, H., Plummer, M.R., Hagiwara, G., Roth, R. L. and Wine, J. J. (1982) Local inter- neurons in the terminal abdominal ganglion of the crayfish. J. Comp. Physiol., 149: 145-162. 4 Pearson, K.G. and Fourtner, C. R. (1975) Non- spiking interneurones in walking system of the cockroach. J. Neurophysiol., 38: 33-52. 5 Burrows, M. and Siegler, M. V. S. (1976) Trans- mission without spikes between locust interneu- rones and motoneurones. Nature, Lond., 262: 222-224. 10 11 12 Local Non-Spiking Neurons in Crayfish 49 Burrows, M. (1980) The control of sets of moto- neurones by local interneurones in the locust. J. Physiol., 298: 213-233. Harreveld, A. Van (1936) A physiological solution for fresh water crustaceans. Proc. Soc. Exp. Biol. Med., 34: 428-432. Nagayama, T., Takahata, M. and _ Hisada, M. (1983) Local spikeless interaction of motoneuron dendrites in the crayfish Procambarus clarkii Girard. J. Comp. Physiol., 152: 335-345. Wilkens, L. A. and Wolfe, G. E. (1974) A new electrode design for en passant recording, stimula- tion and intracellular dye injection. Comp. Bio- chem. Physiol., 48: 217-220. Larimer, J. L. and Kennedy, D. (1969) Innervation patterns of fast and slow muscle in the uropods of crayfish. J. Exp. Biol., 51: 119-133. Stewart, W. W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell, 14: 741-759. Goodman, C.S., Pearson,K.G. and Heitler, 13 14 15 16 byl W. J. (1979) Variability of identified neurons in grasshoppers. Comp. Biochem. Physiol., 64A: 455-462. Rall, W. (1981) Functional aspects of neuronal geometry. In ‘‘Neurones Without Spikes”. Ed. by A. Roberts and B. M. H. Bush, Cambridge Uni- versity Press, Cambridge, pp. 223-254. Burrows, M. (1979) Synaptic potentials effect the release of transmitter from locust non-spiking interneurons. Science, 204: 81-83. del Castillo, J. and Katz, B. (1954) Changes in endplate activity produced by presynaptic polari- zation. J. Physiol., 124: 586-604. Zettler, F. and Jarvilehto, M. (1971) Decrement- free conduction of graded potentials along the axon of a monopolar neuron. Z. Vergl. Physiol., 75: 402-421. King, D.G. (1977) Organization of crustacean neuropil. I. Patterns of synaptic connections in lobster stomatogastric ganglion. J. Neurocytol., 5: 207-237. ZOOLOGICAL SCIENCE 1: 50-61 (1984) Molecular Mechanism of Cell Division in Tetrahymena thermophila I. Analysis of Execution Period of a Division-Arrest ts-Mutant Ryojyl TAMURA!, MIHOKO TAKAHASHI and YOSHIO WATANABE~ Institute of Biological Sciences, University of Tsukuba, Niihari-gun, Sakura-mura, Ibaraki 305, Japan. ABSTRACT — We isolated a division-arrest ts-mutant, TU100, from Tetrahymena thermophila. Genetic analysis revealed that TU100 was a single recessive mutant and belonged to cdaC (former name, mo3) first described by Frankel et al. (1976). As TU100 was the sixth cdaC allele, we designated it as cdaC6. When a cdaC6 culture was shifted from 30°C to 38°C, the cells failed to divide and division-arrested cells accumulated to nearly 100%, but their protein, RNA and DNA syntheses and micro- and macronuclear divisions continued. CdaC6 cells exhibited temperature-induced syn- chronous rounding like wild-type cells, suggesting that there existed ts-defect in a process of cyto- kinesis. The results of temperature shift-up and pulse heat-shock experiments with single and synchronized cells of known-ages demonstrated that the execution period for the phenotypic expression of the mutant was limited within cytokinesis. Among the cells which passed through the physiological transition point and were capable of dividing normally without further protein synthesis, the cells whose division furrows were not observed during 10-min heat shock exhibited normal division immediately after the heat shock, whereas the cells which entered division or were in division stages. during the entire pulse heat shock exhibited long-term division arrest in response to the heat shock. This implys that, in response to the heat shock, the function of mutant gene product is fully reversible © 1984 Zoological Society of Japan before it is associated with division structure but becomes irreversible after the association. INTRODUCTION A ciliate Tetrahymena is known to be one of the best experimental materials for studying the mechanism of cell division in morphological, bio- chemical and physiological aspects [see, Reviews 1, 2]. Furthermore, T. thermophila has been used as a good material for genetical works and some useful technics for isolating mutants have been developed [3-5]. As a possible lead towards elucidating the relationships among a division- associated structure, its protein component and its function, investigation of cell division by the use of conditional mutants could be useful. Frankel Accepted October 7, 1983 Recieved June 29, 1983 * Present address: Department of Pharmacology, Teikyo University School of Medicine, Itabashi-ku, Tokyo 173, Japan. 2 To whom correspondence should be sent. and his colleagues isolated for the first time tem- perature-sensitive (ts) division-arrest mutants in T. thermophila, classified them into 7 complementa- tion groups (7 cda loci) and analysed the morphological, genetical and physiological charac- teristics of the mutants [6-10]. We have also isolated ts-mutants affecting cell division in T. thermophila and investigated their characteristics. Here we present a detailed study on one of our mutants, TU100 (later on cdaC6). Especially, we describe evidence that the execution period of the mutant exists within cytokinesis on the basis of analyses with single and synchronized cell cultures and that the function of the mutant gene product is defective at restrictive temperature. MATERIALS AND METHODS Cells: The strains of Tetrahymena thermophila used in this study were B III (an inbred strain), Tetrahymena Division-Arrest Mutant 51 C*, and Mpr functional heterokaryon Mpr/Mpr (6mp-S, IV). These strains were kindly provided by Dr. D. L. Nanney, University of Illinois. For the mutant complementation tests, following mu- tants, cdaA1, cdaC2, cdaD3, cdaE1, cdaF 1, cdaG1, cdaH 1 and cdal 1, were used. They were kindly gifted by Dr. J. Frankel, University of Iowa. These cells were cultivated in PYD medium (1% proteose peptone, 0.5% yeast extract, and 0.87% dextrose). In some experiments, 6mp-PYD medi- um was used as a selection medium in which 6- methyl purine was contained in PYD medium at a concentration of 25 g/ml. Mutant isolation: Mpr functional heteroka- ryons were mutagenized with 10 g/ml of N- methyl-N ’-nitro-N-nitrosoguanidine for 3 hr and kept for 12 hr in PYD medium and then conju- gated with C*. Afterwards, short-circuit genomic exclusion was induced according to the method of Bruns et a/.[4]. After treatment with 6mp-PYD medium, cloning was performed as described by Bruns et al. [5]. Each clone cultivated in micro- titerplates was replicated and one was placed at 30°C and the other at 38°C. Putative mutant clones, which showed abnormal large size or no increase in number at 38°C but normal growth at 30°C, were picked up from the master plates at 30°C and cultivated. Single cell culture: To determine the tempera- ture-sensitive period (execution period) of TU100, the capillary culture procedure of Prescott [11] was followed. Synchronous rounding: Exponentially growing TU100 or wild-type cells were transferred to an amino acid-deprived synthetic medium (Kidder’s basal medium 2A minus amino acids [12]) and subjected to synchronizing temperature-treatment (7 temperature cycles each consisting of 1/2 hr at 34°C followed by 1/2 hr at 42.5°C) which was nearly the same with the treatment for synchro- nous division of 7. thermophila described in Holz et al. [13]. For details of the phenomenon referred to as synchronous rounding, see our previous papers [12, 14-18]. Synchronous division: If a ts-mutant, such as TU100, cultivated in PYD medium was directly subjected to the synchronizing temperature-treat- ment, many division-arrested cells were observed at the end of the heat treatment. Therefore, we devised the induction system of synchronous division by introducing the conversion from the rounding cycle to the division cycle. The proce- dure is as follows. Exponentially growing TU100 or wild-type cells were transferred to the amino acid-deprived synthetic medium (described above), kept for 12 hr and then an equal volume of 2 x PYD medium was added. Four hr later, the cul- ture was subjected to synchronizing temperature- treatment (the same treatment as that described in synchronous rounding). Nutrient replenish- ment at 4 hr before the heat treatment was timely for the conversion from the rounding to the divi- sion cycle. RESULTS General features of a division-arrest mutant We isolated several putative ts-mutants affecting cell division from about 510° homozygous cell clones (see Materials and Methods). One of the mutant strains (designated as TU100) was selected and analysed. The genetic analysis revealed that the mutant character was controled by a single recessive gene, because all of F, progeny of the crosses with wild type (inbred strain B III) showed nearly wild-type phenotype and F, progeny from the crosses between F, progeny segregated into wild type and the typical mutant with a ratio of 3: 1 (data not shown). However, within 20 cell divisions after conjugation the heterozygous clones frequently became a mixture of cells expressing wild-type and the mutant phenotypes. We then performed the complementation tests of TU100 (homozygote crossed with B III three times) with the stocks of several kinds of cda (cell- division-arrest) mutants previously isolated by Frankel’s group [6-10], since some properties of TU100 (briefly described later) appeared to be very similar to those of the cdaC mutant. The result clearly indicated that TU100 failed to com- plement with cdaC, but complemented with any other cda mutants, such as cdaH and cdal. The same result was obtained by Frankel’s group (personal communication). According to his communication, 5 independent cdaC alleles, cdaC | 52 R. TAMURA ef al. 10ra 10T b through cdaC5 (former name, mo3* through mo3* [6]), are extant in his laboratory, so that TU100 would thus be the sixth cdaC allele and could be designated cdaC6. Three of Frankel’s cdaC alleles show cold-sensitivity [6], but our cdaC6 does not show cold-sensitivity (15°C) like the other two cdaC mutants (cdaC2 and cdaC4). Since the general characterization of cdaC mutant has been made by Frankel’s group [6— 1 1 10], here we describe it briefly. When a cdaC6 ye 100r g mass culture was shifted from 30°C (a permissive temperature) to 38°C (a restrictive temperature), the great majority of cells failed to multiply and the proportion of division-arrested cells gradually 50 Fic. 1. Cell multiplication (panels a and b) and divi- sion-arrested cell accumulation (panels c and d) after the temprature shift-up. Abscissa: time after temperature shift-up (hr); ordinate: cells/ml in panels a and b, and frequency of division-arrested cell (%) in panels c and d. Panels a and c, wild 0 10 20 0 10 20 type; panels b and d, cdaC6. O, 30°C; @, 38°C. Fic. 2. Tandem chains of cdaCé6 at a restrictive temperature. Tetrahymena Division-Arrest Mutant 38) increased and finally attained nearly 100% (Fig. 1). In a dividing mutant cell, the division furrow constriction ceased soon after the temperature shift-up and elongation along the longitudinal axis occurred. Therefore, division-arrested cells were easily distinguished from normally dividing cells. About 3 hr (corresponding roughly to the generation time) after the temperature shift-up, mutant cells tended to become tandem 4-cell chains (Fig. 2). This may reflect the fact that each half of a division-arrested cell continues to grow and enters the next cell division but the division furrow constriction fails to occur at 38°C as in the case of the first cell division. The tandem 4-cell chains usually contained 4 macro- and 4 (or more) micronuclei and 4 normal oral apparatus (al- though macronuclear division tended to be some- what delayed). In the cdaC6 mutants, syntheses of protein, RNA and DNA continued to occur 3 6 Fic. 3. DNA synthetic activities of cdaCé6 cells after temperature shift-up. Abscissa: time after tempe- rature shift-up (hr); ordinate: cpmx10°*. The activities were measured by cumulative incorpora- tion of 1’#Ci/ml of *H-thymidine into acid-in- soluble fraction. O, 30°C; @, 38°C; —— cdaC6; ---— wild type. 40 20 0 15 30 45 60 75 Fic. 4. Synchronous rounding of cdaC6 cells. Abs- cissa: time after EHT (min); ordinate: rounding index (%). @, cdaC6; O, wild type. even at the restrictive temperature (as a representa- tive example, DNA synthetic activities in cdaC6 mutants at 30°C and 38°C are shown in Fig. 3). To determine whether the cdaC6 mutant is a cell cycle-arrest mutant or a cytokinesis-arrest mutant, we subjected the mutant cells to the tem- perature treatment for synchronous rounding. As shown in Figure 4, the mutant cells exhibit synchronous rounding just as wild-type cells do, in spite of the fact that cdaC6 mutant is ts-mutant and the progression of its normal division is com- pletely arrested by the exposure to a restrictive temperature (above 38°C). The result strongly suggests that cdaC6 is not a cell cycle-arrest mutant but a cytokinesis-arrest mutant. Execution period analysis with single cell culture To investigate the execution period of cdaC6, temperature shift-up and 10-min pulse heat-shock experiments were carried out with known-aged single cells cultivated in microcapillaries in which the mean generation times of wild-type and cdaC6 cells at 30°C were 172.3 min and 174.9 min, re- spectively. In shift-up experiments, almost all mutant cells failed to divide and showed typical division arrest except that the cells exposed to 38°C 54 R. TAMURA ef al. EME Mme io Fic. 5. Effects of the restrictive temperature on cdaC6 single cells of known-ages. Abscissa: cell age or stage; ordinate: frequency of normally dividing cells (%). Single cells of varying ages or stages were exposed to 38°C continuously or for a 10-min pulse. Percentages of cells that completed division normally following temperature shift-up and 10min pulse heat shock were plotted as a function of the age or stage at which the cell was exposed to 38°C. Division stages are classified into 5 stages (E, M*, M, M2, L). AA, wild type: O@, cdaC6; open symbols, 10-min pulse heat shock; closed symbols, temperature shift-up. For each point, 50-300 single cells were examined. at the late stage of normal division or later could divide normally (Fig. 5). From the results, the end of the execution point was determined to be the late stage of the division. Next, the beginning of the execution period was investigated in the heat-shock experiments. Cells at various stages in the cell cycle exhibited mainly two kinds of responses to 10-min pulse heat shock (38°C) depending on cell stages: One was the so-called ‘delayed division’ (we use the term here- after for the type of cell division in which the forthcoming division is delayed but the cell division itself is normal) and the other was division arrest unique to the mutant. As for the former, nearly the same ‘set-back’ curves (the notion of ‘set-back’ was proposed for the first time by Zeuthen [19] and later on substantiated by Frankel [20]) were obtained in both cdaC6 and wild-type cells (Fig. 6). The maximum delay of division was exhib- ited by 120-min-aged cells and the delay tended to decrease sharply in 150-min-aged cells. Such a delayed division has been known to be a temper- ature-sensitive response of normal Tetrahymena cells, so that the execution period for the pheno- 0 30° "60 90720 Fic. 6. Delayed divisions following 10-min pulse heat shock on varying aged-single cells of cdaC6. Abs- cissa: age since division (min); ordinate: excess 150 delay of division (min). Set-back times of wild- type (a) and cdaCé6 (b) cells are shown as a function of the cell age. One dot corresponds to a single cell, and zero time in ordinate to the generation time of non-treated single cell cultures. typic expression of the ts-mutant does not seem to exist in the stage earlier than the critical point with respect to delayed division, that is, the “physiological transition point’ (PTP). On the other hand, when dividing cells were exposed to the heat shock, they showed pronounced division arrest even at a very early dividing stage. We therefore assumed that the execution period begins at a certain point in the period between the PTP Tetrahymena Division-Arrest Mutant 55 and the onset of division. For investigating this in detail, 10-min pulse heat shocks were given to 150-min-aged cells (the 150-min cells may consist of cells in different physiological states because of the considerable variation in generation times). By their responses to the heat shock, 150-min cells were clearly clas- sified into 4 groups (A-D) as follows. When a division furrow was not observed during the entire 10-min shock, the treated cells either (A) exhibited delayed division, or (B) completed normal divi- sion soon after the heat shock. (C), When a division furrow was not observed at the start of 10-min shock but a shallow furrow was found at the end of the shock, such cells exhibited the division arrest. (D), When a shallow furrow was appreciable at the start of the heat shock, the TABLE 1. Recovery periods of division-arrested cells No. of single Period A (min) Period B (min) cells 1 120 ND 2 135 250 3 135 250 4 70 DIS 5 — 261* 6 —- ope ‘| 70 ND 8 38 135 9 148 250 10 102 260 11 114 104 12 i Fi 246 13 124 237 14 113 132 15 100 105 16 102 167 17 — 200* 18 105 310 19 70 ND Period A: period between the end of heat shock and the time when a cell resorbed an appreciable division furrow. Period B: period between the beginning of regression of the division furrow and completion of new division. ND: not divided. * divided without prior regression. treated cells always resulted in division arrest. Among the 4 groups, group A appears to contain the cells treated before the PTP and groups B, C and D contain the cells treated after the PTP. The groups clearly reflect the cell’s stages, so that we can arrange them in the order of A, B, C, D as stages of cell cycle progression. Of 41 cells ex- amined, 8, 9, 5 and 19 cells belonged to the stages A, B, C and D, respectively. The existence of stage B cells is especially important for defining the beginning of the execution period. If we assume the PTP and the onset of division (gener- ation time minus division time) to be in the vicinities of 145 min and 160 min, respectively, the stage B cells should be present in the inter- space, which means that the beginning of the execution period presumably corresponds to the onset of division furrow constriction. Events following division arrest induced by 10- min pulse heat shock were observed at 10- to 15- min intervals under a microscope. As shown in Table 1, division arrest continued for 103.6--28.9 min, after which the division furrows regressed. After these cells spent a further long time (207-4 69 min), they recommenced seemingly normal division. Execution period analysis with synchronized cell culture Using a devised induction system for synchro- nous division of the ts-mutant (see Materials and Methods), the execution period of cdaC6 cells was analysed. As shown in Figure 7, synchronized cdaC6 cells were exposed to a 10-min pulse heat shock (42.5°C) at 0, 15, 30, 40, 50 and 60 min after the end of the heat treatment (EHT). When the cells were exposed to the pulse heat shock at 0, 15 and 30 min after EHT (curves II, III and IV), the division was delayed gradually as the exposures were pushed later, but in all cases normal synchro- nous division was observed. However, little or no division delay was observed when the cells were exposed at 40 min (curve V), 50 min (curve VI) and 60 min (curve VII) after EHT, while division arrest coincidentally occurred to a great or mod- erate extent. In both cdaC6 and wild-type cells, the PTPs existed in the vicinity of 35 min after EHT, suggesting that the execution period for the 56 R. TAMURA ef al. 0 60 Fic. 7. Effects of 10-min pulse heat shock on synchronous cultures of cdaC6. Abscissa: time after EHT (min); ordinate: division index (%). Curve I represents a non-treated control (0). The pulse heat shock was Started at 0 (curve II, A), 15 (curve III, Vv), 30 (curve IV, 4), 40 (curve V, @), 50 (curve VI, @) and 60 (curve VII, @) min after EHT. mutant phenotype expression does not exist before the PTP. With regard to division arrest, its existence is suggested by the 3curves (V, VI, VII) of Figure 7. However, the curves were somewhat curious, be- cause curves V and VII were very similar to each other but dissimilar from curve VI. We can easily understand the shape of curve VII, if we assume that cells at a very late dividing stage can complete division normally even at 42.5°C, but the remaining dividing cells are prevented from pro- gression of division by the heat shock. Similarly, the cell population at 50 min after EHT should mainly include cells in the early and middle divid- ing stages and not include many cells in the late dividing stage, since curve VI did not show an obvious initial fall soon after the heat shock. To validate our assumption, we investigated dividing cells at various stages in normal synchronous culture (curve I of Fig. 7) with respect to frequency of appearance of division stages (Fig. 8). The 120 180 histograms at 60 and 50 min after EHT accord with the above-mentioned explanations for curves VII and VI, respectively. At 60 min after EHT, about 1/4 of the dividing cells could divide nor- mally (curve VII of Fig. 7) and the value corre- sponded to the ratio of D6, D7, D8 cells to total dividing cells (Fig. 8), suggesting that the end of the execution period is D6. However, we suppose that the exact end point is somewhat later (pre- sumably D7), because there is a few min lag in heat conduction to the culture medium. In the histogram of dividing cells at 50 min after EHT, the sum of D6—D8 corresponds to only a few per cent (Fig. 8), so that the majority of the dividing cells could not divide during and after the pulse — heat shock (curve VI of Fig. 7). However, divi- sion index appeared to be increased by the heat shock. This suggests that considerable numbers of cells that had no appreciable division furrow at the beginning of the shock were recruited into division arrest by the heat shock. A diagramma- Tetrahymena Division-Arrest Mutant =))/ 65 ain ee Wn aa 99 a 40 Fic. 8. Histograms showing the distributions of divid- ing cells at various stages of synchronous culture of cdaC6. Abscissa: 8 dividing stages (D,—D;); ordinate: numbers of dividing cells. Cell samples were the same as those of curve I of Figure 7. Num- bers on the right show sampling times (min after EHT). In each sample, all cells in a fixed culture volume were investigated (about 690 cells except 65-min sample). In the histograms, only normally dividing cells are plotted with real numbers. A scale mark on the ordinate represents 10 cells. tic explanation of this is given in Figure 9. Stage C cells indicated in Figure 9 are the cells which have no furrow but are situated within 10 min before the onset of furrowing. Therefore, these cells may be arrested as dividing cells with shallow furrows at the end of the 10-min heat shock. If so, division-arrested cells present in the cell popu- lation which was exposed to the heat shock at 40 min after EHT (curve V of Fig. 7) may include considerable numbers of division-arrested cells derived from such stage C cells (see Figs. 8 and 9). However, we observed that in the cell population exposed to the heat shock at 40 min after EHT a considerable number of cells can divide normally soon after the heat shock (curve V of Fig. 7). These cells, named as stage B cells in Figure 9, may correspond to cells having the capacity for completing division normally soon after the heat shock. They appear to have already passed beyond the PTP at the beginning of the heat shock and are easily distinguished from stage A cells situated before the PTP (Fig. 9). The result of 10-min pulse heat-shock experi- D1 D2 D3 D4 D5 D6 D7 D8 C DI 0203 D405 BC010203 00009098888; P\ G2 ES D8 Di D2 D3 D4D5 D6 D7 ti Fic. 9. Diagram for explanation of the execution period of cdaC6. The main points of Figures 7 and 8 are shown schematically. Numbers on curves in the right side represent the starting times when synchronous cultures were exposed to 10- min heat shock. In the left, cell stages included in each sample are shown schematically. Thick bars represent a stage at which pulse heat shock brings about division arrest. Arrows in the lower left indicate capacity of cells to divide normally after the heat shock (right direction) and capacity of cells to be set-back the cycle (left direction). Cell stages are expressed as A—D, in the order of cell cycle. 58 R. TAMURA ef al. ment and its explanation lead to the conclusion that the execution period is indeed a period rang- ing from the onset of furrowing to a very late stage of division. This conclusion is highly consistent with that in the experiments with single cell culture. Figure 7 also shows that after the long division arrest (40-60 min) the proportion of cells having distinct furrows tended to decrease synchronously (curves V, VI, VII). This may be due to regression of old furrows (non-functional furrows) in the division-arrested cells, because cell numbers of such populations little increased. At 125 min after EHT, seemingly normal division began to appear but the percentages of these cells were not high, and even after that time division occurred at a low rate and asynchronously. We then attempted to ascertain whether a re- aVIl strictive temperature exerts a harmful influence upon the synthesis of mutant gene product or the function of it. For this purpose, 25 yvg/ml of cycloheximide was added to the synchronized cdaCé6 cells at 0, 25 and 40 min after EHT. Virtu- ally no protein synthesis occurred during the inhibitor treatment (data not shown). As shown in Figure 10, synchronous division was completely inhibited when cycloheximide was added to the culture before the PTP (curves II, HI), whereas it was virtually not inhibited when the inhibitor was added after the PTP (curve IV). The same response has been well-known to occur in syn- chronized normal Tetrahymena cells [12, 20, 21]. Our result clearly shows that the cells which passed through the PTP have already synthesized sufficient proteins, including mutant gene product, to fully 0 60 division of cdaCé6. synchronous culture with no drug. Abscissa: time after EHT (min); ordinate: division index (%). Curves II, III and IV; synchronous cultures treated with cyclohex- 120 180 Fic. 10. Effects of cycloheximide (25 yg/ml) on normal and 10-min pulse heat-shocked synchronous Curve I; normal imide at 0, 25 and 40 min after EHT, respectively. Curve V; synchronous culture exposed to 10-min pulse heat shock at 50 min after EHT. Curves VI, VII and VIII; heat-shocked cultures (the same as cureve V) treated with cycloheximide at 60, 80 and 105 min after EHT, respectively. Tetrahymena Division-Arrest Mutant 59 sustain the oncoming division. Hence it follows that the temperature shock (42.5°C) given at dividing stage of cdaC6 cells exerts a harmful effect on the function of the mutant gene product but not on its synthesis. We also examined effects of cycloheximide on the process of recovery from the pulse heat shock- induced division arrest in cdaC6 (Fig. 10). In the experiment, the synchronized population was exposed to 10-min shock at 50 min after EHT and then aliquots of the culture were exposed to 25 pg/ml of cycloheximide at 60, 80 and 105 min after EHT (curves VI, VII, VIII). When the in- hibitor was given at 60 and 80 min after EHT, the first part of the synchronous division (represent- ing normally dividing cells presumably derived from the stage D6—-D8 and the stage B cells of Figure 9) was virtually not inhibited but the second part of synchronous division (representing cells that underwent division arrest) remained for a long time (more than 1 day), unlike the curve with no cycloheximide (curve V). When cycloheximide was given at 105 min after EHT (cruve VIIJ), partial recovery from division arrest occurred. These results strongly suggest that after the PTP new protein synthesis is not needed for normal division but is absolutely essential for the recovery from division arrest induced by the heat shock. DISCUSSION Previously we have reported that there exists a contractile ring composed of microfilaments and several kinds of ring-associated structures in a furrow region of a dividing Tetrahymena cell [22]. The contractile ring of Tetrahymena is very similar to those of other higher animal cells, which strongly suggests that a common molecular mechanism of cytokinesis is underlying among animal cells. Therefore, we have considered that if we can isolate conditional division-arrest mutants in Tetrahymena, we will be able to scrutinize the ultrastructural and biochemical differences be- tween ts-mutants and wild-type cells and be able to connect the structure and biological function of a division-related substance. If so, Tetrahymena could become one of the best model cells for study- ing molecular mechanism of cell division. For this purpose, we performed the isolation of divi- sion-arrest ts-mutants in TZ. thermophila and succeeded in isolating several strains of mutants. Then, we investigated some characteristics of these mutants. In our preliminary experiments, we have been aware of the fact that one of the mutants, tentatively named as TU100, is a very interesting ts-mutant because the temperature-sensitive period (execution period) of this mutant is limited within cytokinesis and the exposure of a dividing mutant cell to a restrictive temperature brings about im- mediate division arrest. While we were investi- gating the execution period of TU100 in detail, Frankel et a/. published two papers [9, 10] report- ing that among 7 different division-arrest mutants (7 cda loci) isolated by them, 3 cda loci showed complete division arrest at restrictive temperatures within one cell cycle and the 3 cda mutants (cdaC, cdaA and cdaH) possessed different execution periods: the execution periods of cdaC, cdaA and cdaH were present during division, before divi- sion and in an early stage of the cell cycle, respec- tively [9, 10]. Similarity between TU100 and cdaC in terms of the execution period urged us to perform the complementation test of TU100 with cdaC. As a result, it has become clear that the mutant gene of TU100 belongs to the cdaC gene locus. Thus, we designated TU100 as cdaC6 (see Results). In this paper, we found that cdaC6 exhibited synchronous rounding (the phenomenon was first found by one of the authors (Y. W.) in 1963) in an amino acid-deprived synthetic medium just as wild-type cells did, when the cdaC6 cells were exposed to ordinary synchronizing temperature shocks (Fig. 4). Although no division is seen in this synchronous rounding system, it has been known that rounding occurs in place of division [12, 14] and that timing and degree of synchrony [12, 14, 15] and the time sequence of the underly- ing changes in synchronous rounding (for example, protein SH-change [14] and oral primordium development [23]) correspond exactly to those in synchronous. division. In both synchronous rounding and division, a common phasing mech- anism (set-back of the cell cycle) is surely involved [12, 15, 18, 23]. The progression of the basic cell cycle in cdaC6 appears to be the same as that in 60 R. TAMURA ef al. wild type. Moreover, high-temperature shock does not induce rounding arrest in cdaC6. Hence, it follows that the mutant must have a ts-defect in the furrowing process for cytokinesis, but not in a certain process of the basic cell cycle. The execution period for mutant phenotype expression of cdaC6 has been analysed mainly by pulse heat-shock experiments using single cells of known-ages (Figs. 5 and 6) and synchronized cells of known-ages (Figs. 7, 8 and 9). From the ana- lyses, it was demonstrated that the execution period of cdaC6 existed between the onset and the very late stage of cell division. This result coincides well with that of Frankel’s group using single cdaC2 cells of known-ages [9, 10]. In studying the execution period of cdaC6, we can newly distinguish and characterize 4 different cell stages showing different responses to 10-min heat shock when 150-min-aged single cells or 40- min-aged synchronized cells after EHT are exposed to the pulse heat shock. The 4 cell stages are as follows; stage A, cells situated before the PTP that exhibit delayed division in response to the heat shock; stage B, cells after the PTP that exhibit normal division soon after the heat shock; stage C, cells after the PTP that have not yet reached the onset of furrowing at the start of heat shock and become arrested in division in response to the heat shock; stage D, cells in division stages that exhibit division arrest in response to the heat shock. Cells at stages B, C, D could divide nor- mally at permissive temperatures under complete inhibition of protein synthesis by cycloheximide (Fig. 10), indicating that proteins, including mu- tant gene product, necessary for sustaining the oncoming division have been synthesized suffi- ciently in these cells. It is, therefore, natural to consider that the function, but not synthesis, of the mutant gene product is temperature-sensitive. The finding of stage B cell seems to be utmost important. Such cells can divide immediately and normally even if they are exposed to a pulse heat shock, suggesting that the temperature-dependent conformational change of the mutant gene product in this stage is reversible. On the contrary, in stages C and D, cells exhibit division arrest in response to the pulse heat shock, and the recovery from the long-term arrest depends absolutely on new protein synthesis (Fig. 10), which suggests that the temperature-dependent conformational change of the mutant gene product in these stages is irreversible. The discrepancy in reversibility of the mutant gene product is presumably due to the difference of conformational states of the product: We speculate that in stage B the product exists in a free form whereas in stages C and D the product exists in an associated form, linked with the division structures (machinery for cytokinesis). In the latter case, division structures themselves presumably fail to perform their function under a restrictive temperature. For restoration of the heat-damaged division structures, cells must re- move the defective division structures with accompanying new protein synthesis, and remodel new division structures. We always observed that the heat shock brought about a very rapid cessa- tion of division furrow constriction at any dividing stage except the very later stage. This may reflect that a certain structure present in common to all the division structures found during nearly entire dividing stages is seriously affected by the heat shock. The correspondence of the mutant gene product with a division structure will be described in the succeeding paper [24]. In the present experiment, we succeeded in the induction of synchronous division by supplying nutrient medium to the amino acid-starved cells at 4 hr before the start of the synchronizing tem- perature-treatment. The devised synchronous division system of a division-arrest ts-mutant may become a powerful means for analysing biochemi- cal properties of the mutant. ACKNOWLEDGMENTS We wish to express our sincere thanks to Dr. J. Frankel, University of Iowa, for his invaluable advice, for his kind performance of the complementation tests between TU100 and his cda mutants, and for his kindness that he gave us his cda mutants including unpublished ones to perform the same tests in our laboratory. This work was partly supported by grants from the Ministry of Education of Japan to Y.W. (5221202) and from the projects of University of Tsukuba to Y. W. REFERENCES 1 Zeuthen, E. (1964) The temperature-induced di- 10 11 12 13 Tetrahymena Division-Arrest Mutant 61 vision synchrony in Tetrahymena. In ‘Synchrony of Cell Division and Growth’’. Ed. by E. Zeuthen, John Wiley, New York, pp. 99-156. Zeuthen, E. and Rasmussen, L. (1972) Synchro- nized cell division in protozoa. In “Research in Protozoology”. vol. 4, Ed. by T. T. Chen, Per- gamon Press, New York, pp. 9-145. Orias, E. and Bruns, P. J. (1976) Induction and isolation of mutants in Tetrahymena. In ‘“‘Methods of Cell Biology”. vol. 13, Ed. by D. M. Prescott, Academic Press, New York and London, pp. 247-282. Bruns, P.J., Brussard, T.B. and Kavka, A. B. (1976) Isolation of homozygous mutants after induced self-fertilization in Tetrahymena. Proc. Natl. Acad. Sci. USA, 73: 3243-3247. Bruns, P.J. and Sanford, Y. M. (1978) Mass isolation and fertility testing of temperature-sen- sitive mutants in Tetrahymena. Proc. Natl. Acad. Sci. USA, 75: 3355-3358. Frankel, J., Jenkins, L.M., Doerder, F.P. and Nelsen, E. M. (1976) Mutations affecting cell division in Tetrahymena pyriformis. I. Selection and genetic analysis. Genetics, 83: 489-506. Frankel, J., Jenkins, L.M. and DeBault, L. E. (1976) Causal relations among cell cycle processes in Tetrahymena pyriformis: An analysis employ- ing temperature sensitive mutants. J. Cell Biol., 71: 242-262. Frankel, J.. Nelsen, E.M. and Jenkins, L. M. (1977) Mutations affecting cell division in Tetra- hymena pyriformis, syngen 1. II. Phenotypes of single and double homozygotes. Dev. Biol., 58: 255-275. Frankel, J., Mohler, J. and Frankel, A. K. (1980) Temperature-sensitive periods of mutations affect- ing cell division in Tetrahymena thermophila. J. Cell Sci., 43: 59-74. Frankel, J., Mohler, J. and Frankel, A. K. (1980) The relationships between the excess-delay phe- nomenon and temperature-sensitive periods in Tetrahymena thermophila. J. Cell Sci., 43: 75-91. Prescott, D. M. (1957) Change in the physiological state of a cell population as a function of culture growth and age (Tetrahymena geleii). Exp. Cell Res., 12: 126-134. Watanabe, Y. (1963) Some factors necessary to produce division conditions in Tetrahymena pyri- formis. Japan. J. Med. Sci. Biol., 16: 107-124. Holz, G.G., Scherbaum,O.H. and Williams, N. E. (1957) The arrest of mitosis and stomato- 16 17 18 19 20 21 ZZ 23 24 genesis during temperature-induction of synchro- nous division in Tetrahymena pyriformis, mating type 1, variety 1. Exp. Cell Res., 13: 618-621. Tamura, S., Toyoshima, Y. and Watanabe, Y. (1966) Mechanism of temperature-induced syn- chrony in Tetrahymena pyriformis. Analysis of the leading cause of synchronization. Japan. J. Med. Sci. Biol., 19: 85-96. Watanabe, Y. (1971) Mechanism of synchrony induction. I. Some features of synchronous round- ing in Tetrahymena pyriformis. Exp. Cell Res., 68: 431-436. Watanabe, Y. (1971) Mechanism of synchrony in- duction. II. Synthesis and turnover of biomole- cules during the induction phase of synchronous rounding in Tetrahymena pyriformis. Exp. Cell Res., 68: 437-441. Watanabe, Y. (1971) Mechanism of synchrony induction. III. Changes of water-soluble and water-insoluble protein fractions involved in syn- chronous rounding in Tetrahymena pyriformis. Exp. Cell Res., 69: 324-328. Watanabe, Y. (1971) Mechanism of synchrony induction. IV. Accumulation of a special protein in the induction process of synchronous rounding in Tetrahymena pyriformis. Exp. Cell Res., 69: 329-335. Zeuthen, E. (1958) Artificial and induced perio- dicity in living cells. Adv. Biol. Med. Phys. 6: 37-73. Frankel, J. (1962) The effects of heat, cold, and p- fluoro-phenylalanine on morphogenesis in syn- chronized Tetrahymena pyriformis GL. Compt. Rend. Trav. Lab. Carlsberg, 33: 1-52. Rasmussen, L. and Zeuthen, E. (1962) Cell divi- sion and protein synthesis in Tetrahymena as studied with p-fluoro-phenylalanine. Compt. Rend. Trav. Lab. Carlsberg, 32: 333-358. Yasuda, T., Numata,O., Ohnishi, K. and Watanabe, Y. (1980) A contractile ring and cor- tical changes found in the dividing Tetrahymena pyriformis. Exp. Cell Res., 128: 407-417. Frankel, J. (1970) The synchronization of oral development without cell division in Tetrahymena pyriformis GL-C. J. Exp. Zool., 173: 79-100. Yasuda, T., Tamura, R. and Watanabe, Y. (1984) Molecular mechanism of cell division in Tetra- hymena thermophila. U1. Ultrastructural changes found in a division-arrest ts-mutant. Zool. Sci., 1: 62-73. ZOOLOGICAL SCIENCE 1: 62-73 (1984) © 1984 Zoological Society of Japan Molecular Mechanism of Cell Division in Tetrahymena thermophila II. Ultrastructural Changes Found in a Division-Arrest ts-Mutant TOMOYOSHI YASUDA, RYoJI TAMURA! ? and YOSHIO WATANABE!’? Laboratory of Technology, National Institute of Health of Japan, Shinagawa-ku, Kamioosaki, Tokyo 141, and ‘Institute of Biological Sciences, University of Tsukuba, Niihari-gun, Sakura-mura, Ibaraki 305, Japan. ABSTRACT — A division-arrest ts-mutant of Tetrahymena thermophila, cdaC6, having the execution period for the mutant phenotype expression within the dividing stage was investigated ultrastructurally following the time after transferring the dividing cells from 30°C (a permissive temperature) to 40°C (a restrictive temperature). The primary drastic change was exhibited on ‘lateral stripes’ which seemed to fasten contractile ring microfilaments: An extraordinary array of the lateral stripes on microfilaments was observed at 5 to 6 min after the temperature shift-up. The result implies that the mutant gene product might correspond to a protein component of the lateral stripe or a regulator protein relevant to the dissociation of the lateral stripes crossbridging microfilaments. Following the extraordinary array of the iateral stripes, the penetration of two types of longitudinal microtubular bands into the mid-division plane and the regression of contractile ring structure occurred in the division-arrested cells. From the detailed observation, these events appear to be the secondary changes in response to the temperature shift-up. INTRODUCTION The results of our physiological studies on a division-arrest ts-mutant, cdaC6 (former name, TU100), of Tetrahymena thermophila have sug- gested that (i) cdaC6 is not a cell cycle-arrest mutant but a cytokinesis-arrest mutant, (ii) the execution period for the mutant phenotype ex- pression of cdaCé6 is limited only within cytokinesis, (iii) the mutant gene product of cdaC6 shows a functional defect in response to a restrictive temperature, (iv) the mutant gene product is pre- sumably associated with division structures concurrently with their formation and, therefore, the division machinery itself incurs an irreversible damage when a cdaCé6 dividing cell is exposed to a restrictive temperature, (v) the mutant gene product Accepted October 7, 1983 Received June 29, 1983 2 Present address: Department of Pharmacology, Teikyo University School of Medicine, Itabashi-ku, Tokyo 173. 3 To whom correspondence should be sent. has a concern with a certain structure common to the division machinery present in nearly entire dividing stages [1]. On the other hand, we found for the first time Tetrahymena’s contractile ring structure, like that of a higher animal cell, in the furrow region of a dividing 7. pyriformis [2]. We observed several division-associated structures in a Tetrahymena’s contractile ring, such as three kinds of filaments running parallel to the division furrow, a tadpole- like structure, lateral stripes, and two kinds of linkers that connect between lateral stripes and the epiplasmic layer [2]. We have also showed that drastic ultrastructural changes occur in the cortex of the fission zone accompanying contraction of the division furrow [2]. From the physiological study of the mutant [1] together with the ultrastructural study of normally dividing Tetrahymena cells [2], it is possible to consider that the mutant gene product of cdaC6 plays a crucial role in the contraction of the contractile ring of a dividing cell and some ultra- structural changes in the division structures, in- Tetrahymena Division-Arrest Mutant 63 cluding a contractile ring, of a dividing cdaCé6 cell must have been brought about in response to the temperature shift-up (40°C). Identification of the mutant gene product with a certain division- associated structure is necessary for elucidating the molecular mechanism of cell division, so that we have undertaken the ultrastructural study on dividing cdaC6 mutant cells responding to the temperature shift-up. In the present paper, we describe evidence that, as a primary response of a dividing cdaC6 cell to a restrictive temperature, lateral stripes which seem to fasten contractile ring microfilaments are extraordinarily arrayed on the contractile ring microfilaments. We also discuss the relationships between the mutant gene product of cdaC6 and lateral stripes, and the function of lateral stripes in the process of division furrow constriction. MATERIALS AND METHODS Cells used in this study were of inbred strain B of Tetrahymena thermophila and a cdaC6 mutant stock (homozygote crossed with strain B three times) of 7. thermophila. Cultivation of these cells was performed as described previously [1]. For the electron-microscopic observation, cells were fixed with a 1: 1 (v/v) mixture of 3% gluta- raldehyde and 2% OsO, [3] in 0.07 M phosphate buffer (pH 7) for 30 min. Afterwards, they were treated with a 1: 1 (v/v) mixture of 2% tannic acid and 3% glutaraldehyde in the phosphate buffer for 30 min, and dehydrated in a graded series of acetone. These cells were transferred to Epon 812 [4] and embedded in a plastic dish (Permanox Contour Dish, 60 x 15 mm, LUX Scientific Corpo- ration, Newbury Park, Calif., U.S.A.). A cell of an appropriate dividing stage embedded in the resin plate was selected and trimmed to cut in a selected orientation. The ultrathin sections were prepared with an LKB-8800 microtome and stained with uranly acetate and lead citrate [5] as described previously [2]. Electron micrographs were taken with a Hitachi H-500. RESULTS When cdaC6 mutant cells were cultivated at 30°C, they exhibited normal division just as wild type cells did. A transverse section at the mid- division plane of a cdaC6 cell normally dividing at 30°C revealed that just beneath the epiplasmic layer a number of contractile ring microfilaments were running parallel to the division furrow and the thickness of the contractile ring was about 0.1-— 0.15 wm (Figs. 1 and 2). We always prepared serial sections of a furrow region and confirmed that at the mid-division plane the contour of cell surface was a smooth round shape and the con- tractile ring microfilament zone existed distinctly (see the upper and right sides of the cell in Fig. 1). Within a contractile ring, a cluster of lateral stripes (3-8 stripes) was sporadically seen in a normally dividing cell (arrows in Fig. 1). Such an aspect of the contractile ring structure observed in cdaC6 mutants cultivated at 30°C was identical with that in an inbred wild-type cell and also with that in T. pyriformis described previously [2]. When cdaC6 mutant culture was transferred from 30°C to 40°C, the mutant cells in dividing stages resulted in division arrest immediately after the transfer [1]. Figure 3 shows a _ transverse section at the mid-division plane of a dividing cdaCé6 cell at 6 min after the temperature shift-up. As compared with an image of the mutant cell dividing normally at 30°C (Figs. 1 and 2), lateral stripes were seen everywhere at regular intervals of about 80 nm on the contractile ring microfilaments (Figs. 3 and 4). This extraordinary array of the lateral stripes has not so far been observed at any stage of normal division of 7. pyriformis [2]. How- ever, spacing of the lateral stripes seen in the heat- treated mutant cell nearly coincided with that of a normally dividing 7. pyriformis [2]. The very similar images to those presented in Figures 3 and 4 were obtained in the case of 5-min treated mutant cells (data not shown). Since there is 2- to 3-min lag in heat conduction to the culture me- dium, it can be said that the extraordinary array of the lateral stripes is a very rapid response to the restrictive temperature. At 5 to 6 min after the temperature shift-up, microfilaments themselves remained unchanged. On the other hand, we also observed the division furrow region of a wild-type cell exposed to 40°C for 6 min as a control. As seen in Figure 5, the extraordinary array of lateral 64 T. YASUDA et al. Fic. 1. A transverse section at the mid-division plane of a cdaC6 cell normally dividing at 30°C (a permissive temperature). CR: contractile ring, LS: lateral stripes (arrows), LM;: so-called longitudinal microtubules, LLM,: longitudinal microtubules from apical couplet. Tetrahymena Division-Arrest Mutant Fic. 2. An enlarged image of the square region of Fig. 1. J ial a * », * 65 66 T. YASUDA et al. WH: Mili Fic. 3. A transverse section at the mid-division plane of a dividing cdaCé cell at 6 min after tem- perature shift-up to 40°C (a restrictive temperature). Note the extraordinary array of lateral stripes on the contractile ring microfilaments. EDZ: electron-dense zone. Fic. 4. An enlarged image of the square region of Fig. 3. Fic. 5. A transverse section at the mid-division plane of a dividing wild-type cell at 6 min after temperature shift-up to 40°C. Tetrahymena Division-Arrest Mutant Fic. 6. A transverse section at the mid-division plane of a dividing cdaC6 cell at 10 min after temperature shift-up. Note the contractile ring in the process of regression (rCR). EDZ: electron-dense zone. . 67 T. YASUDA et al. 68 Pde Wey ie owe & Fic. 7. An enlarged image of the square region of Fig. 6. Tetrahymena Division-Arrest Mutant OL eR LAV SS ap alae ; oes Pya dip ve Si ney a eal EER Ais 8B Fic. 8. A: An enlarged image of electron-dense zone observed in a transverse section of the mid- division plane of a 10-min heat-treated cdaC6 cell. Electron-dense zone (EDZ) and regressed contractile ring (rCR) are seen. B: Electron-dense zones consisting of so-called longitudinal microtubular bands (LM,) and microtubule bands starting from apical couplet (LM,). LM; and LM, are shown schematically by tracing the contours of the microtubules of the image A. 69 70 T. YASUDA et al. stripes was not seen at all in the wild-type cell whose division was capable of completing normally at 40°C. When a dividing cdaCé6 cell was exposed to 40°C for 8-10 min, we could distinguish its dividing image (so-called ‘division arrest’) from normal division image at a light microscopic level, since the elongation along the longitudinal axis occurred with no progression of the division furrow con- traction. Figure 6 shows a transverse section at the mid-division plane of a cdaC6 division-arrested cell which was exposed to 40°C for 10 min. Sucha cell preserved a vestigial contractile ring structure in its division furrow region: most parts of the contractile ring were in the process of disap- pearance, yet in some parts microfilaments arrayed extraordinarily with lateral stripes were seen (Figs. 6 and 7). These striped microfilaments were frequently curved toward inner cytoplasm by the appearance of dense materials of epiplasm, which looked as if they lost the contractile activity (for example, the rihgt upper surface of the cell in Fig. 7). The other characteristic change was observed in 8- to 10-min heat-treated mutant cells: Electron- dense zones appeared in the vicinity of epiplasmic layer. These zones began to appear at 6 min after the heat treatment and they expanded side- ways and sometimes fused together at 8 to 10 min after the heat treatment. The place where the electron-dense zone appeared at the beginning seemed to correspond to a ridge between ciliary meridians. In an enlarged micrograph (Fig. 8), the electron-dense zone was found to consist of epiplasm and microtubules arranging in a row. These microtubules must have penetrated into the division furrow region mainly from the anterior part of the posterior presumptive daughter cell. There we could observe two types of microtubular bands: A microtubular band (about 10 tubules) which penetrated from a ridge between ciliary meridians was present between inner alveolar membrane and epiplasmic layer, and another microtubular band (several tubules) which pene- trated from a ciliary meridian was present between epiplasmic layer and contractile ring. The former appears to be longitudinal microtubular bands as judged by their orientation [6-9], and the latter may correspond to the microtubular bands starting from the anterior basal bodies of the apical couplets (data not shown). From the observation on the division-arrested Fic. 9. A transverse section of the mid-division plane of a dividing cdaCé6 cell treated with both heat-shock (40°C) and 25 »g/ml of cycloheximide for 10 min. Note the regressed contractile ring (rCR). Tetrahymena Division-Arrest Mutant 71 cells, it is suggested that the function of contractile ring structure of cdaC6 cell ceases soon after the temperature shift-up and the division structure disappears at the time when wild-type cells culti- vated under the same conditions or the mutant cells at a permissive temperature complete normal division. In order to ascertain whether the dis- appearance of contractile ring structure in the division-arrested cells requires new protein syn- thesis, we carried out an experiment under protein synthesis inhibition. From the data of the previous paper [1], division arrest induced by 10- min pulse heat shock is shown to continue for a very long time under the presence of 25 g/ml of cycloheximide. Under the same conditions, the contractile ring structure of the division-arrested cell showed a vestigial change at 10 min after the heat treatment (Fig. 9). This suggests that dis- appearance of contractile ring structure occurs without any additional protein synthesis and undergoes the same process as that of a dividing wild-type cell. Long-term division arrest induced by both heat shock and inhibition of protein synthesis may be due to the microtubule-epiplasm configurations in the fission zone: the cell shape may remain rigid by a close attachment between epiplasm and the ends of elongated microtubules. DISCUSSION In the present paper, we demonstrate that the primary ultrastructural defect of a division-arrest ts-mutant, cdaC6, is expressed on the lateral stripes. An extraordinary array of the lateral stripes seen in the mutant (Figs. 3 and 4) is a very rapid response of the cell to a restrictive tem- perature (40°C). It seems to occur within only a few min, as we take the heat conduction to the culture into account. Such an_ ultrastructural change has not so far been observed in cdaC6 cells at 30°C (Figs. 1 and 2), wild-type cells at 40°C (Fig. 5), and T. pyriformis W [2], so that the change in lateral stripes appears to be unique to the cdaC6 mutant at a restrictive temperature. Ina previous study, we have found that there exist the contractile ring microfilaments in a dividing 7. pyriformis and some regions of the filaments have a striped pattern [2]. Therefore, we named the striped structures as ‘lateral stripes’. Later on, Jerka-Dziadosz recon- firmed the presence of a contractile ring consisting of microfilaments in 7. thermophila [3]. However, she failed to detect lateral stripes on the contractile ring filaments. In order to know the reason of the discrepancy, we used in this study a fixative very similar to her fixative (see Materials and Methods). But, we were always able to observe the lateral stripes in serial sections of dividing Tetrahymena cells. Therefore, why she could not find out the lateral stripes is unknown. From the previous observation on the lateral stripes [2], we have implied their function as follows. (i) The lateral stripes presumably fasten the contractile ring microfilaments, since the features of a cluster of the lateral stripes (ordinarily, 3-8 stripes) are much the same in both tangential and transverse sections. (ii) The lateral stripes may fix a microfilament bundle at their arrayed region, since intervals between stripes are always about 80 nm whenever we observe them at any dividing stages (or presumably even during the contraction of contractile ring). (iii) The lateral stripes may function in the transmission of con- tractile force generated from the contractile ring to the membrane-lining epiplasmic layer, since we detect linkers that connect between epiplasmic layer and the lateral stripes, and sometimes the lateral stripes and linkers appear to mediate the subsidence of the epiplasm into cytoplasm accom- panying the contraction of contractile ring. We have further speculated that the lateral stripes crossbridge microfilaments to fasten the filaments and transmit the contractile force to the cell surface, but the crossbridge is transient: The function of lateral stripes is presumably very dynamic and the association and dissociation of lateral stripes with microfilaments may occur repeatedly in the process of the contraction of division furrow. In a dividing cdaC6 cell, temperature shift-up brings about immediate arrest of the constriction of division furrow. This may be due to the extraordinary array of lateral stripes on the con- tractile ring microfilaments: Entire microfila- mentous machinery is fixed by the extraordinary association of lateral stripes and the resulting machinery may not work at all. Perhaps, a restri- ctive temperature does not exert any influence upon 72 T. YASUDA et al. the association of lateral stripes with micro- filaments, but exerts a very great influence upon the dissociation of them. Under these circumstances, we are tempted to speculate that cdaC6 mutant gene product might be a protein component of the lateral stripe itself or a regulator protein relevant to the dissociation between lateral stripes and microfilaments. Biochemically, the lateral stripes might cor- respond to a kind of high molecular weight actin- binding proteins, such as HMWP from BHK cells [101], ABP from macrophages [11] and filamin from smooth muscle [12, 13]; since the striped pattern of microfilaments in Tetrahymena ((2] and Figs. 3 and 4) bears a strong resemblance to the striped pattern detected in the thin sections of the actin-HMWP gel [10] (although spacing of lateral stripes of Tetrahymena (84 nm) differs considerably from the spacing of HMWP on F-actin (34 nm)). Temporal sequence following the extraordinary array of lateral stripes is the apperance of electron- dense zones in the vicinity of epiplasmic layer at the mid-division plane (Figs. 6, 7 and 8) and the gradual regression of the contractile ring structure (Figs. 6, 7 and 9). In a Tetrahymena cell, so-called ‘fission zone’ is formed prior to the constriction of division furrow. At the zone, discontinuity of the ciliary meridians clearly occurs. Ultrastructurally, within the fission zone, basal bodies and their associated micro- tubular system are known to regress, so that microtubules are not prominent at the mid- division plane during normal cell division. Frankel et al. [14] reported that even in cdaC mutants at restrictive temperatures the fission zone was formed before entering division. In the cdaC mutant, contraction of contractile ring ceases in response to the restrictive temperature, whereas other temperature-insensitive events appear to progress on an ordinary cell cycle schedule. One of them is the pointing of the anterior parts of both presumptive daughter cells. The elongation of longitudinally orientated two kinds of microtubular bands seems to be responsible for this event (Fig. 8). In normally dividing Tetrahymena cells, such mi- crotubules elongate near the mid-division plane at a late dividing stage, but do not penetrate into the mid-division plane (see Figs. 1 and 2). Jerka- Dziadosz also observed the longitudinal micro- tubules near the contractile ring [3]. The elongation of longitudinal microtubular bands in a division-arrest Tetrahymena mutant, cdaA2 (former name, moJ”), in the recovery from the heat shock has been reported by Ng [8]. According to him, the elongation mainly occurs in the longi- tudinal microtubules of the anterior part of the posterior presumptive daughter cell. This holds true for our mutant, cdaC6. From the exami- nation of serial sections of dividing cdaC6, not only longitudinal microtubules but also another type of microtubules starting from the anterior part of apical couplet of the posterior presumptive daughter cell were shown to penetrate into the mid- division plane in response to temperature shift-up (Fig. 8). The ends of the elongated microtubules seemed to be fixed at the mid-division plane with the materials like those of epiplasm. Therefore, the attached region becomes somewhat thickened and is recognized as an electron-dense zone. These epiplasmic and microtubular configurations may sustain the division shape of a division-arrested cell even after its contractile ring has already disappeared. More detailed study on the micro- tubular elongation of cdaC6 mutant during the division arrest will be published elsewhere. The regression of division structures observed during division arrest of cdaC6 mutant (Figs. 6 and 7) is also considered to be temperature-insensitive and to progress on an ordinary schedule as if the cells have not been exposed to a restrictive tem- perature. It has been known that events underlying normal division process do not require any additional protein synthesis if the cells have already passed through the physiological transition point. Therefore, we tested the effect of cyclo- heximide on the regression of division structure during division arrest of cdaC6 mutant. The result showed that cycloheximide did not exert a disturbing influence on the regression (Fig. 9), suggesting that the regression occurred on the schedule of ordinary division. In the previous paper, we showed that division-arrested figure of cdaC6 remained unchanged for a long time under cycloheximide treatment [1]. Thus, it is possible to consider that new protein synthesis is needed for the disorganization of the epiplasmic and Tetrahymena Division-Arrest Mutant 73 microtubular configurations sustaining the division- arrested figure. In the present paper, a possible role of the lateral stripes is emphasized. We are now trying to isolate the cdaC6 mutant gene product for eluci- dating the relationships between the product and the lateral stripes. ACKNOWLEDGMENTS We would like to thank Dr. Satoshi Nakazawa, National Institute of Health, for his much help in the course of the study. This work was partly suppoted by grants from the Ministry of Education of Japan to Y. W. (521202) and from the projects of University of Tsukuba to Y. W. REFERENCES 1 Tamura, R., Takahashi, M. and Watanabe, Y. (1984) Molecular mechanism of cell division in Tetrahymena thermophila. 1. Analysis of execu- tion period of a division-arrest ts-mutant. Zool. Sci., 1: 50-61. 2 Yasuda,T., Numata,O., Ohnishi, K. and Watanabe, Y. (1980) A contractile ring and cor- tical changes found in the dividing Tetrahymena pyriformis. Exp. Cell Res. 128: 407-417. 3 Jerka-Dziadosz, M. (1981) Cytoskeleton-related structures in Tetrahymena thermophila: Micro- filaments at the apical and division-furrow rings. J. Cell Sci. 51: 241-253. 4 Luft, J.H. (1961) Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. 5 Venable, J. H. and Coggeshall,R. (1965) A 10 11 | WA 14 simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Pitelka, D. R. (1961) Fine structure of the silver- line and fibrillar systems of three tetrahymenid ciliates. J. Protozool., 8: 75-89. Allen, R. D. (1967) Fine structure, reconstruction and possible functions of components of the cortex of Tetrahymena pyriformis. J. Protozool., 14: 553-565. Ng, S. F. (1978) Directionality of microtubule assembly: An in vivo study with the ciliate Tetra- hymena. J. Cell Sci., 33: 227-234. Satir, B. H. and Wissig, S. L. (1982) Alveolar sacs of Tetrahymena: Ultrastructural characteristics and similarities to subsurface disterns of muscle and nerve. J. Cell Sci., 55: 13-33. Schloss, J. A. and Goldman, R. D. (1979) Isola- tion of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells. Proc. Natl. Acad. Sci. USA, 76: 44844488. Hartwig, J. H. and Stossel, T. P. (1975) Isolation and properties of actin, myosin and a new actin- binding protein in rabbit alveolar macrophages. J. Biol. Chem., 250: 5696-5705. Shizuta, Y., Shizuta, H., Gallo, M., Davies, P., Pastan, I. and Lewis, M.S. (1976) Purification and properties of filamin, an actin binding protein from chicken gizzard. J. Biol. Chem., 251: 6562— 6567. Wang, K. (1977) Filamin, a new high-molecular- weight protein found in smooth muscle and non- muscle cells. Purification and properties of chicken gizzard filamin. Biochemistry, 16: 1857-1865. Frankel, J.,. Nelsen, E.M. and Jenkins, L. M. (1977) Mutations affecting cell division in Tetra- hymena pyriformis, syngen 1. II. Phenotypes of single and double homozygotes. Dev. Biol., 58: 255-275. ZOOLOGICAL SCIENCE 1: 74-81 (1984) Atypical Phenylketonuria due to Biopterin Deficiency: Diagnosis by Assay of an Enzyme Involved in the Synthesis of Sepiapterin from Dihydroneopterin Triphosphate SHIN’ ICHI YOSHIOKA, MASAHIRO MASADA, TAKEO YOSHIDA, TAKASHI MIZOKAMI, MIKI AKINO! and NoBUTAKE MATSUO? Department of Biology, Tokyo Metropolitan University, Tokyo 158, Japan, and !Department of Pediatrics, Tokyo Metropolitan Children’s Hospital, Kiyose, Tokyo 204, Japan ABSTRACT — Assay procedures for two enzymes involved in the synthesis of sepiapterin from dihydroneopterin triphosphate in peripheral erythrocytes from patients with biopterin deficiency are described. One enzyme, sepiapterin-synthesizing enzyme-1, which catalyzes in the presence of Mg?t the conversion of dihydroneopterin triphosphate to an intermediate designated compound X, was assayed by determining pterin formed from compound X under acidic condition. The other enzyme, sepiapterin-synthesizing enzyme-2, which catalyzes the NADPH-dependent reduction of compound X to sepiapterin, could not be assayed directly, because compound X is unavailable. However, with the use of purified enzyme-1 from silkworm to generate compound X from dihydroneopterin tri- phosphate, it was possible to assay for enzyme-2 activity in erythrocytes. Analytical results demonstrate that enzyme-1 activity in the patients was decreased by 50-60% compared to that in controls, whereas no significant difference was detected in enzyme-2 activity © 1984 Zoological Society of Japan between patients and controls. INTRODUCTION Recently, several lines of evidence have been accumulated showing that L-erythro-7, 8-dihydro- biopterin (abbreviated as H,biopterin) is synthe- sized from GTP by way of D-erythro-7, 8-dihydro- neopterin 3’-triphosphate (H.neopterin-PPP) and sepiapterin [1-9], and the following reaction sequence has been proposed [7]: GTP——H,neopterin-PPP (1) H_,neopterin-PPP——>compound X (II) compound X+ NADPH+H*—— sepiapterin-+ NADP* (IIT) sepiapterin + NADPH + H*——> H,biopterin-+- NADP* (IV) H,biopterin could be further reduced by dihydro- folate reductase (EC 1.5.1.3) [10, 11] to tetrahy- drobiopterin which participates as the natural Accepted August 9, 1983 Received June 8, 1983 * To whom correspondence should be addressed. cofactor in the enzymatic hydroxylation of aromatic amino acids [12]. Reactions (I) and (IV) are catalyzed by GTP cyclohydrolase I and sepiapterin reductase (EC 1.1.1.153), respectively. Reaction (II), the pro- duction of an intermediate designated compound X [7] from Hneopterin-PPP, is catalyzed by a heat-stable and Mg’*-requiring enzyme fraction. Reaction (IID), the production of sepiapterin from compound X, is catalyzed by a heat-labile and NADPH-dependent enzyme fraction. These two fractions were partially purified from an extract of chicken kidney [7]. Concerning the pterin metabolism, atypical phenylketonuria (PKU) due to biopterin deficiency, so far reported, appears to be characterized as follows: (a) an abnormally higher ratio of neopterin to biopterin in urine compared with that in controls and other types of PKU [13, 14]; (b) elevated urinary excretion of 3’-hydroxysepiapterin, pre- sumably formed from an H,neopterin derivative [15]; (c) lowering of serum phenylalanine levels Atypical Phenylketonuria is) when sepiapterin and/or H.biopterin are admini- stered to these patients [16]. In view of the proposed reaction sequence of H,biopterin synthesis described above, these biochemical characteristics suggest that the GTP cyclohydrolase step and the steps leading from sepiapterin to the production of tetrahydrobiopterin in these patients might be normal. Therefore, it is possible that a defect may lie between H,neopterin-PPP and sepiapterin, i.e., at either reaction (II) or (III). Such a defect could account for biopterin deficiency in these patients. Another type of PKU variant has been recently described [17] and in this type there is a deficiency in both biopterin and neopterin, presumably due to a defect in reaction (I) (the GTP cyclohydrolase step). In a previous paper [18], the enzymes involved in reactions (II) and (III) were tentatively desig- nated sepiapterin-synthesizing enzyme-1 (SSE-1) and -2 (SSE-2), respectively, and assay methods for SSE-1 and for overall synthesizing activity of sepiapterin and/or biopterin from H,neopterin- PPP were described. Using these methods, we have found activities in several rat tissues including kidney, pineal gland, liver and adrenal. It was particularly noted that low but significant activities were detectable in rat erythrocytes. Similarly, a preliminary experiment showed that the methods with modification were also applicable to analysis of these reactions in human blood. In the present paper we demonstrate that SSE-1 activity in peripheral erythrocytes from two patients with biopterin deficiency is decreased by 50-60% of that in controls. In addition, levels of biopterin and neopterin in blood from the patients and controls are presented. MATERIALS AND METHODS Patients Two cases of biopterin deficiency were studied. They were described in an abstract form previously [19]. The clinical features of the patients are summarized as follows. Case N.S. This boy is the first child of unrelated Japanese parents. He was born at term after an uncom- plicated pregnancy and delivery. At 3-4 months of age he was found to be diffusely hypotonic and developed frequent episodes of impared alertness. At 3 years of age he was noted to have elevated serum phenylalanine levels (4-6 mg/dl). Despite a low phenylalanine diet, he developed very poorly and was subsequently referred to our service for further investigation. At 5 years of age he was diagnosed as having biopterin deficiency and was placed on 120 mg of 5-hydroxytryptophan (5-HTP), 100 mg of L-dopa and 10 mg of carbidopa daily. At 8 years of age he remains markedly retarded and is functioning motorwise below 12 months of age. Case K.S. This girl, younger sister of case N.S., was born uneventfully at 41 weeks of gestation with a birth weight of 3573 g. At 2 months of age, she was noted to be unusually irritable and frequently displayed opisthotonic posturing in response to loud noises. At 7 months of age she was found to have serum phenylalanine levels of 8-20 mg/dl on a regular formula. At 12 months of age, she was started on a low phenylalanine diet without benefit. At 3 years of age she was diagnosed as having biopterin deficiency and placed on 100mg of 5-HTP, 100 mg of L-dopa and 10 mg of carbidopa daily. At 5 years of age she was started on tetra- hydrobiopterin, 20 mg/day, which was generously provided by Drs. A. Niederwieser and B. Schircks, Zurich, Switzerland. She has responded fairly well to the above regimen. At 6 years of age she commands three word sentences and begins to walk. Materials H,neopterin-PPP was prepared from GTP using GTP cyclohydrolase I from Escherichia coli [18]. Biopterin and neopterin were generous gifts from Dr. S. Matsuura of Nagoya University, sepiapterin was isolated from the mutant sepia of Drosophila melanogaster [20] and pterin was synthesized by a published method [21]. GTP was a product of Yamasa Shoyu Co. and assay kits for hemoglobin and creatinine were obtained from Wako Pure Chemical Co. Other chemicals were of analytical grade from commercial sources. Analysis for biopterin and neopterin levels in human blood Blood plasma and erythrocytes were separated by 76 S. YOSHIOKA ef al. centrifugation of heparinized whole blood at 800 x¢ for 5min. The erythrocytes were hemo- lyzed by adding 9 vol. of 5mM potassium phos- phate buffer (pH 7.4). The hemolysate was used as the erythrocyte fraction. In contrast to the hemolysate prepared from rat blood [22], human hemoglobin could not be removed from _ the hemolysate by mechanical agitation. To 60041 of blood plasma 100 wl of 20% trichloroacetic acid (TCA) were added and the mixture was centrifuged. The supernatant fluid (200 yl) was treated with an equal volume of iodine solution (1g of I, and 2g of KI in 100ml of distilled water) for 60 min at room temperature. Excess iodine was destroyed with ascorbic acid and the solution was applied to a Dowex 50W column (H*, x8, 200-400 mesh, about 100 yl bed volume). After washing the column with distilled water, pterins were eluted with 3 ml of 1 NNH,OH. The eluate was concentrated to dryness and the residue was dissolved with 500 wl of distilled water. An aliquot (100 yl) was analyzed by high performance liquid chromatography (HPLC). The erythrocyte fraction (1200 wl) was deproteinized by adding 200 wl of 20% TCA and the supernatant fluid (700 pl) was treated as above. Biopterin and neopterin in urine samples were determined by HPLC. Urine samples diluted 10-fold with distilled water were treated by the same procedure as in the case for blood plasma. Analysis of pterins with HPLC Pterins were analyzed by a HPLC procedure [23] which was modified [18] using a model 635 Hitachi Liquid Chromatograph equipped with a continuous flow fluorometer (Hitachi 650—10S) and a Whatman 10 » ODS reverse phase column (4.6 x 250 mm). Enzyme preparation from the erythrocyte fraction The erythrocyte fraction prepared as described above was fractionated with ammonium sulfate between 30 and 80% saturation. The precipitate was dissolved in a small amount of 25 mM tri- ethanolamine-HCl buffer (pH 7.4) and the solution was applied to a Sephadex G-25 column (1.8 x 14cm) equilibrated with the same buffer. The column was eluted with the buffer and the void volume fraction was used as the enzyme source. All procedures were performed at 4°C. To determine SSE-1 activity, the enzyme pre- paration was treated at 80°C for 1 min in order to destroy SSE-2 activity and phosphatase activity which could reduce the actual concentration of substrate, H.neopterin-PPP. However, when the enzyme preparation was heated, about 50% of SSE-1 activity coprecipitated with the bulk of coagulated hemoglobin. Therefore, after heat- treatment the enzyme suspension was _ finely suspended by homogenization in a small Teflon homogenizer and an aliquot of the suspension was used for assay. Assay for SSE-\ activity The assay for SSE-1 activity is based on the observation that compound X is decomposed to pterin and pyruvic acid under acidic condition [7, 18]. The reaction mixture was prepared to contain, in a final volume of 300 yl: triethanol- amine-HCl buffer (pH 7.4), 25 mM; H.neopterin- PPP, 30 uM; MgsSO., 5mM and _ heat-treated enzyme. The reaction was started by the addition of substrate. The reaction mixture without Mg”* served as the control. After incubation at 37°C for 60min in the dark, the reaction was terminated by adding 50 wl of 20% TCA and the mixture was centrifuged. To 100 yl of the supernatant fluid 5 wl of iodine solution was added and the mixture was allowed to stand for 5 min at room temperature. Excess iodine was destroyed by adding ascorbic acid and the solution was diluted to 500 wl with distilled water. Pterin in aliquot (50 wl) was analyzed by HPLC. One unit of SSE-1 was defined as that amount of enzyme which produces 1 nmol of pterin under the assay condition described. Specific activity was ex- pressed as unit/g hemoglobin of whole blood. Assay for SSE-2 activity An assay method for activity of sepiapterin synthesis from H,neopterin-PPP has been des- cribed previously [18]. However, since the SSE-1 activity of enzyme preparations from human blood is So low, it is not possible to determine sepiapterin formed by the HPLC procedure. Furthermore, the low fluoresence intensity of sepiapterin con- tributed to this difficulty. Also compound X which has been proposed as the substrate for SSE-2 is not available. Although the structure for compound X is yet to be proven and it is unknown Atypical Phenylketonuria ad whether SSE-1 from human tissues is identical to that found in other animals, we used a purified SSE-1 preparation from silkworm as a means generating the putative intermediate compound X from H,neopterin-PPP. We then utilized com- pound X as the substrate for SSE-2 from human erythrocyte preparations. SSE-1 was partially purified from fat bodies of silkworm, Bombyx mori. All subsequent pro- cedures were performed below 4°C. The buffer solutions used contained 10mM_ 2-mercap- toethanol except for the homogenizing buffer. Frozen fat bodies (100 g) from Sth instar silk- worm larvae were thawed, homogenized with 200 ml of 100mM potassium phosphate buffer (pH 7.0) in a Waring blendor and centrifuged at 15,000xg for 60min. After the supernatant solution was passed through a layer of glass wool, the filtrate was fractionated with ammonium sulfate between 40 and 70% saturation. The precipitate was dissolved in a small amount of 25 mM triethanolamine-HCl buffer (pH 7.4) con- taining 50 mM KCl and dialyzed overnight against the same buffer. The dialyzed solution was applied to a DEAE-Sephadex A-50 column (5 x 45 cm) equilibrated with the same buffer. Proteins were eluted by 2 litre of linear gradient of KCl (50-600 mM) in 25mM_ triethanolamine-HCl buffer (pH 7.4). The flow rate was maintained at 40 ml per hour and 5-ml fractions were collected. Each fraction was assayed for SSE-1 activity as described above. The active fractions (fraction Nos. 230-330) were combined and concentrated by precipitation with ammonium sulfate at 70% saturation. The precipitate was dissolved in a small amount of 150mM_ potassium phosphate buffer (pH 7.4) and dialyzed overnight against the same buffer. The dialyzed solution was chro- matographed on a hydroxylapatite column (2.4 x 25cm) equilibrated with the same buffer. After the column was thoroughly washed with the equilibrating buffer, proteins were eluted by 400 ml of linear gradient of potassium phosphate buffer (pH 7.4) from 150 to 500 mM. _ The flow rate was 24 ml per hour with a fraction size of 3ml. The peak fractions (fraction Nos. 135-145) were com- bined and dialyzed overnight against 25 mM triethanolamine-HCl buffer (pH 7.4) containing 50 mM KCl. The dialyzed solution (180 units/ml; specific activity, about 1300 units/mg protein) was used as the SSE-1 preparation. Since the pre- paration was still slightly contaminated with SSE-2, prior to use the preparation was heated at 80°C for 1 min to destroy residual SSE-2 activity. The reaction mixture for assay of sepiapterin- synthesizing activity was prepared to contain, in a final volume of 300 wl: triethanolamine-HCl buffer (pH 7.4), 25 mM; H.neopterin-PPP, 30 uM; MgsoO., 5mM; NADPH, 1 mM; silkworm SSE-1, 5 units and human enzyme preparation. The reaction mixture without NADPH served as the control. After incubation at 37°C for 60 min in the dark, the reaction was terminated by adding 4 vol. of ice-cold ethanol, centrifuged and 1 ml of the supernatant fluid was concentrated to dryness. The residue was dissolved with 200 wl of distilled water and 20 wl of the solution was subjected to HPLC analysis. One unit of sepiapterin-synthe- sizing activity is expressed as that amount of enzyme which produces | nmol of sepiapterin under the assay condition described. The activity was determined by using varied amounts of the enzyme preparation (expressed as mg hemoglobin) in the presence of a constant amount of silkworm SSE-1. The activity was plotted against mg vie |) L =| x > E C ~~ i Ss ; 5 al < © 0.5 or Ew WwW 1 Z, 3 mg Hemoglobin Fic. 1. Calibration curves for assay of sepiapterin- synthesizing activity. Assays were carried out in the presence of a constant amount (5 units) of silkworm SSE-1 and the indicated amounts of enzyme preparation (expressed as mg hemoglobin) from human erythrocyte fraction. The synthesiz- ing activity was plotted against enzyme amount. Details in the text. (@), adult controls. (0), patient (N.S.). (A), control child (Y.N.) with infectious mononucleosis. 78 S. YOSHIOKA et al. hemoglobin, and the specific activity was obtained from the slope of the linear portion of the plot (see Fig. 1). RESULTS AND DISCUSSION Analysis for biopterin and neopterin in blood of controls and patients with biopterin deficiency Analytical results for biopterin and neopterin levels in blood are summarized in Table 1. For comparison, these levels in urine samples from the patients, their parents and two children with infectious mononucleosis are provided in Table 2. The urinary values in these individuals were checked by comparing with those given in the literature [24]. Corresponding to high levels of neopterin and low levels of biopterin found in urine of the patients (N.S. and K.S.), the ratios of neopterin to biopterin (NP/BP) in both blood plasma and erythrocytes were also apparently higher than those in the two groups of controls. The NP/BP ratios in the plasma and erythrocytes of the mother of the patients also seemed higher than that found in the controls. In the case of the father, the ratios in plasma and erythrocytes TABLE 1. patients (pmols/ml, Mean+SE) appear to be normal, although the value in plasma appears slightly higher than that in adult controls. The ratios in urine of the parents were normal (Table 2), when compared with those reported [24]. There was no significant difference related to age in the NP/BP ratios among blood samples from con- trols examined (in plasma, r= —0O.37; in ery- throcytes, r=0.05). When studied during the first few weeks of their illness, two control children (Y.N. and S.K.) who had infectious mononucleosis showed high NP/BP ratios in both their erythrocytes and plasma that were comparable with those observed in our patients. By contrast, levels of these pterins in urine were normal. Calibration of synthesizing activity of sepiapterin As described under methods, the sepiapterin- synthesizing activity of erythrocyte fractions was assayed in the presence of a constant amount of SSE-1 from silkworm larvae. In Figure 1, activity is plotted against the amount of the enzyme preparation (expressed as mg of hemoglobin) from blood of one patient (N.S.) and three controls (see legend). In each case a linear relation was observed at least up to about 1.5 mg hemoglobin/ Blood levels of neopterin (NP) and biopterin (BP) in controls and biopterin-deficient Plasma Erythrocytes Subjects (n) NP BP NP/BP NP BP NP/BP Controls Adults (5) sae ll 6.5+1.4 0.9 14.3+3.2 2a IS) 7: (23-60 years) (1.3-7.6)* (2.3-11.2) (0.6—-1.3) (5.2-22.7) (2.9-9.8) (0.6-4.9) Children (7) SI(9 SE oS) 6.4+1.2 1.4 17.4+2.1 17.4+6.1 1.0 (0-13 years) (4.5—20.1) (2.6-12.0) (0.9-2.8) (4.0-25.5) (3.6-53.4) (0.2-5.4) Child ve NE=* 45.7 4.0 11.4 62.9 10.4 6.0 (4 years) Child S. K.** wll 1.8 12.3 32.8 4.6 Tail (12 years) Patient N. S. 47.9 1.0 47.9 53.9 6.4 8.4 (8 years) Patient K. S. 20.0 Dall 9.5 51.1 2.8 18.3 (6 years) Mother LV il7/ 2.4 4.9 33.0 5.1 6.5 (38 years) Father 10.8 A\ 2.3 10.6 Si 3.3 (38 years) * Range. ** Infectious mononucleosis. Atypical Phenylketonuria 79 TABLE 2. Urinary levels of neopterin (NP) and biopterin (BP) in biopterin-deficient patients, their parents and control children with infectious mononucleosis (#mols/mol cre- atinine) Subjects* NP BP NP/BP Patient N. S. 5797 102 56.8 Patient K. S. 1226 125 9.8 Mother 500 2000 0.3 Father 277 382 0.7 Gmia’yY. N.** 446 661 0.7 Child S. K.** 351 330 iI * Subjects are the same as in Table 1. ** Infectious mononucleosis. reaction mixture. The value for specific activity of sepiapterin-synthesizing activity (units/mg hemo- globin) was obtained from the slope of the straight line portion of each calibration curve. Although specific activity thus obtained does not imply the actual sepiapterin-synthesizing activity of the enzyme preparation, the value could be useful for comparing activities of the enzyme preparations from different subjects. As seen in Figure 1, no significant difference was found in specific activities of the erythrocyte preparations from the patient and adult controls, whereas the specific activity in the child with infectious mononucleosis was apparently lower. Since compound X was unavailable, SSE-2 activity could not be assayed directly. However, since the amount of silkworm SSE-1 added was in large excess compared with the endogenous SSE-1 in enzyme preparations from human blood, the amount of compound X formed (determined as pterin, see method) from H.neopterin-PPP in the reaction mixture (data not shown) is due primarily to the silkworm enzyme and not to the SSE~-1 activity of human enzyme preparation. Further- more, biopterin formation was negligible because sepiapterin reductase in human erythrocyte is very low [22]. From these observations the specific activity of sepiapterin synthesis, obtained as described above, could be regarded as a relative measure of the specific activity of SSE-2 in ery- throcyte enzyme preparations. This activity is denoted as ‘SSE~2’ activity. TABLE 3. SEE-1 and ‘SSE-2’ activities in erthrocyte fractions from controls and biopterin-deficient patients Subjects* SSE-1 ‘SSE-2’ (units/z Hb) (units/mg Hb) Controls Adults 6.44+0.37** 0.30-40.03 (5.59—7.69)*** (0.26—0.40) Children 6.95 +£0.52 0.33+0.02 (4.97—8.30) (0.25—0.40) Child Y. N. 2.60 0.17 Child S. K. SILI) 0.20 Patient N. S. 2.66 0.28 Patient K. S. 3.49 0.34 Mother 4.29 0.29 Father 6.18 0.36 * Subjects are the same as in Table 1. ** Mean-+sE. ate Range: SSE-1 and ‘SSE-2’ activities in the erythrocyte fractions from the patients and controls A summary of assay results for SSE-1 and ‘SSE—2’ activities is presented in Table 3. ‘SSE-—2’ activity of each subject in the table was obtained by the graphical procedure depicted in Figure 1. It is apparent from the table that SSE-1 activities in the patients are decreased by 50-60°% compared with those in controls, while no significant dif- ference in ‘SSE-2’ activities is detectable between the patients and controls (see also Fig. 1). The SSE-1 activity did not appear to be related to age of the controls examined (r=—0.16). SSE-1 activity in the mother of the patients seemed some- what lower than that obtained in controls. This finding may correspond to the slightly high NP/BP ratio in the mother’s erythrocytes and plasma (Table 1). However, both enzyme activities in the samples from the father were in the normal range. More blood samples from patients with biopterin deficiency and their parents are necessary for analysis of correlation between the enzyme acti- vities and the NP/BP ratio in blood. Genetic analysis of the disease also must await further studies. As shown in Table 3, two children with infectious mononucleosis were peculiar in enzyme activities. 80 S. YOSHIOKA et al. Both SSE-1 and ‘SSE~2’ activities were apparently decreased in these cases. This may account for the high NP/BP ratio in their blood (Table 1). It can be concluded from the present study that biopterin deficiency in the patients examined is the result of a defect in the conversion of H.neopterin- PPP to compound X in the synthesis of sepiapterin. This may, in turn, aid verification of the validity of the proposed reaction sequence for H,biopterin synthesis described above. In order to delineate further the nature of the defect in patients with biopterin deficiency, it would be of prime im- portance to assess SSE-1 and SSE-2 activities in other tissues such as cultured skin fibroblasts, liver and central nervous system of the patients. We anticipate that depressed SSE-1 activity will be uniformly demonstrable in tissues of the patients and the technique will likely find application in the clinical assessment of children with hyperpheny!- alaninemia. ACKNOWLEDGMENTS We are grateful to Dr. Shin’ichiro Esumi of Kaken Chemical Co. for the generous supply of E. coli. Thanks are also extended to Dr. Tetsuo Shiota of the University of Alabama in Birmingham for reading the manuscript. REFERENCES 1 Fukushima, K., Eto, I., Saliba, D. and Shiota, T. (1977) The enzymatic synthesis of Crithidia active substance(s) and a _ phosphorylated pD- erythroneopterin from GTP or GDP by liver prep- arations from Syrian golden hamsters. Biochem. Biophys. Res. Commun., 65: 644-651. 2 Fan, C. L. and Brown, G. M. (1976) Partial puri- fication and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster. Biochem. Genet., 14: 259-270. 3 Fukushima, K., Richter, W.E., Jr. and Shiota, T. (1977) Partial purification of 6-(D-erythro-1’, 2’, 3’-trihydroxypropyl)-7, 8-dihydroneopterin tri- phosphate synthetase from chicken liver. J. Biol. Chemine252); 550-5755: 4 Eto, I., Fukushima, K. and Shiota, T. (1976) Enzymatic synthesis of biopterin from D-erythro- dihydroneopterin triphosphate by extracts of kidneys from golden hamsters. J. Biol. Chem., 251: 6505-6512. 5 Fan, C.L., Krivi, G. G. and Brown, G. M. (1975) The conversion of dihydroneopterin triphosphate 10 11 1 13 14 15 16 17 to sepiapterin by an enzyme system from Dro- sophila melanogaster. Biochem. Biophys. Res. Commun., 67: 1047-1054. Krivi, G. G. and Brown, G. M. (1979) Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepia- pterin from dihydroneopterin triphosphate. Bio- chem. Genet., 17: 371-390. Tanaka, K., Akino, M., Hagi, Y., Doi, M. and Shiota, T. (1981) The enzymatic synthesis of sepiapterin by chicken kidney preparations. J. Biol. Chem., 256: 2963-2972. Yoshida, T., Masada, M., Tomino, S. and Akino, M. (1979). Biosynthesis of sepiapterin. Zool. Mag., 88: 559. Hausermann, M., Ghisla, S., Niederwieser, A. and Curtius, H.-Ch. (1981) New aspects in biopterin biosynthesis in man. FEBS Letters, 131: 275-278. Kaufman, S. (1967) Metabolism of the phenyl- alanine hydroxylation cofactor. J. Biol. Chem., 242: 3934-3943. Nagai (Matsubara), M. (1968) Studies on sepia- pterin reductase: further characterization of the reaction product. Arch. Biochem. Biophys., 126: 426-435. Kaufman, S. and Fisher, D. B. (1974) Pterin- requiring aromatic amino acid hydroxylases. In ‘““Molecular Mechanisms of Oxygen Activation’’. Ed. by O. Hayaishi, Academic Press, New York., pp. 285-365. Nixon, J. C., Lee, C.-L., Milstien, S., Kaufman, S. and Bartholomé, K. (1980) Neopterin and biopterin levels in patients with atypical forms of phenylketonuria. J. Neurochem., 35: 898-904. Niederwieser, A., Curtius, H.-Ch., Gitzelmann, R., Otten, A., Baelocher, K., Blehova, B., Berlow, S., Grobe, H., Rey, F., Schaub, J., Scheibenreiter, S., Schmidt, H. and Viscontini, M. (1980) Excre- tion of pterins in phenylketonuria and phenyl- ketonuria variants. Helv. Paediat. Acta, 35: 335- 342. Niederwieser, A., Matasovic, A., Curtius, H. -Ch., Endres, W. and Schaub, J. (1980) 3’-Hydro xysepiapterin in patients with dihydrobiopterin deficiency. FEBS Letters, 118: 299-302. Curtius, H. -Ch., Niederwieser, A., Viscontini, M., Otten, A., Schaub, J., Scheibenreiter, S. and Schmidt, H. (1979) Atypical phenylketonuria due to tetrahydrobiopterin deficiency. Diagnosis and treatment with tetrahydrobiopterin, dihydrobiop- terin and sepiapterin. Clin. Chim. Acta, 93: 251- 262. Niederwieser, A., Standemann, W., Wang, M., Curtius, H. -Ch., Atres, M. and Cardesa-Garcia, J. (1982) Hyperphenylalaninemia with neopterin 18 19 20 21 Atypical Phenylketonuria 81 deficiency- A new enzyme defect presumably of GTP cyclohydrolase. Eur. J. Pediat., 138: 97. Yoshioka, S., Masada, M., Yoshida, T., Inoue, K., Mizokami, T. and Akino, M. (1983) Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues. Biochim. Biophys. Acta, 756: 279- 285. Kondo, T., Matsuo, N., Seki, T., Tsuchiya, Y., Fukashima, O. and Kumagai, M. (1979) Biopterin deficiency in a Japanese sibship. Acta Paediat. Jap., 83: 1399. Fukushima, T. and Akino, M. (1969) Nuclear magnetic resonance studies of some biologically active dihydropterins. Arch. Biochem. Biophys., 128: 1-5. Mowat, J.H., Boothe, J. H., Hutchings, B. L., Stockstad, E. L. R., Waller, C. W., Angier, R. 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ZOOLOGICAL SCIENCE 1: 82-88 (1984) Changes in the Distribution of Calcium in the Frog, Rana nigromaculata, following Ultimobranchialectomy and Calcitonin Administration CHITARU OGURO, MASAKO FUJIMORI and YUICHI SASAYAMA Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan ABSTRACT — In the frog, Rana nigromaculata, the calcium content of different tissues and organs, and the effects of ultimobranchialectomy (UBX) and salmon calcitonin (SCT) on the calcium dis- tribution were studied to clarify the function of the ultimobranchial gland (UBG) and its target organs, if any. The calcium contents of the lime sac and femur were comparatively very high in comparison with about 30 different tissues and/or organs, the former being 2.3 and the latter 1.9 10° wg per g fresh weight. The vertebra and cartilage contained 1.0 10° and 9.5 x10? yg/g, respectively. Among the soft tissues the calcium content of the skin was very high. Other tissues contained much less calcium. Fifteen and 35 days after UBX the calcium content of the lime sacs was decreased to 56% and 29%, respectively, of that of the control animals. On the other hand, no changes in calcium content were found in any tissues other than the lime sacs, including bone, cartilage and skin. Ad- ministration of SCT resulted in a remarkable increase in *°Ca uptake by the lime sac, while no changes in *°Ca incorporation were induced in other tissues. The *°Ca activities in the lime sacs of SCT- treated animals were 30 and 50 times as high as those of the control animals 15 and 35 days after UBX, respectively. The possible role of the UBG (or CT) in calcium uptake or deposition in anuran © 1984 Zoological Society of Japan amphibians is considered. INTRODUCTION The occurrence of a substance that is immunore- active to an anti-porcine calcitonin antibody in anuran amphibians was first reported in Rana temporaria and Rana pipiens [1]. The presence of a physiologically active CT in the UBG has re- cently been demonstrated in some frogs and toads using a rat bioassay [2, 3]. Robertson [4-6] has reported in a series of studies on the morphology and function of the UBG in R. pipiens. Ultimo- branchialectomy (UBX) caused hypercalcemia with a latent period and increased urinary calcium excretion and osteoclastic activity. In bullfrog tadpoles, UBX resulted in a marked hypercalcemia Accepted August 19, 1983 Received July 9, 1983 in a high calcium medium and UBG implantation or treatment with salmon calcitonin (SCT) pre- vented the development of the hypercalcemia [7, 8]. Recently, it was reported that administration of a UBG extract brought about a significant hypocalcemia in specimens of R. nigromaculata whose parathyroid glands had been partially re- moved to lower the hypercalcemic potency which might have antagonized the hypocalcemic effect of the UBG extract [9]. These facts suggest a possible role for CT in the regulation of serum calcium concentration and calcium metabolism in anuran amphibians. How- ever, nothing is known about the mechanism responsible for the acute decrease in serum calcium concentration following administration of UBG extract. It has been reported that in anuran larvae and Calcitonin Function in Frog 83 adults the size of the paravertebral lime sacs (lime sacs), which accumulate a calcium salt, decreased markedly after UBX [10, 11]. However, quan- titative analyses of the calcium content in the lime sacs as well as in the other portions of the amphib- ian body have not been carried out. Knowledge of the distribution of calcium in the body is necessary if the function of the UBG with regard to calcium regulation is to be substantiated. It is also not known whether the lime sac is the only target or whether this decrease in size, and hence probably a decrease in calcium content, after UBX occurs also in the other parts of the body. Con- sequently, to specify the target site of CT there is a need for a thorough examination of calcium distribution in frog tissues and its changes after CT administration. In the present study, the calcium content was first determined in about 30 different tissues and/or organs of R. nigromaculata. Then the effects of UBX on calcium distribution were investigated and finally the effects of SCT on the distribution and incorporation of calcium in different parts of the body were studied using *°Ca as a tracer. MATERIALS AND METHODS Adults of the frog, Rana nigromaculata Hallo- well, of both sexes (20-40 g bw) were used in the present study. They were collected in the suburbs of Toyama City, central Japan, during the spring to summer period and used for experiments as soon as collected. Aged tapwater (Ca, 1.7; Mg, 0.6; Na, 1.0; K, 0.2 mg/100 ml) was used for keep- ing the animals. They were force fed pieces of porcine liver twice a week in the long-term experi- ments. For the determination of the calcium content in a tissue or organ, frogs were anesthetized with tricaine methanesulfonate and dissected under a binocular dissecting microscope. Pieces of each tissue or organ were weighed in pre-weighed tubes and dissolved with a mixture of metal-free nitric acid and perchloric acid (11: 2). The dissolved material was diluted appropriately and the calcium concentration was determined by an atomic ab- sorption spectrophotometer (Hitachi, 180~70). The calcium content of each tissue or organ was calculated from the weight of the material, the dilution rate and the calcium concentration of the diluted solution. The sciatic artery of anesthetized animals was cannulated using polyethylene tubing (PE 10, Clay Adams) in order to administer SCT (dissolved in 0.6% NaCl adjusted to pH 4.6 by HCl), vehicle (CT solvent) or *°Ca. For the determination of *°Ca incorporation into the tissues, pieces of each tissue or organ were removed from the anesthetized animals, weighed and dissolved with a tissue solubilizer (NCS, Amersham). When dissolution was complete, a small amount of HCI was added. The bones, cartilage and lime sac were first dissolved with a small amount of HC! and later the same amount of NCS as was used for the dissolution of the soft tissues was added. A liquid scintillation counter (Aloka, LSC-653) was then used to determine the *°Ca activity in these solutions. R. nigromaculata posesses a pair of UBGs. They are situated on the glottic constrictor muscles at both sides of the glottis. After anesthesia, frogs were placed on an operation board and the mouth was held open by a set of retractors. The UBGs were then removed through a cut made in the buccal floor. Blood and urine were collected from the aortic trunk and the urinary bladder, respectively, directly into glass capillaries. After removing particulate material by centrifugation, the *°Ca activity was determined as described above. The significance of difference between values was calculated using Student’s f-test. Differences were considered significant when P<0.05. Details of each experiment will be described in each section of the results as an experimental protocol. RESULTS 1. Distribution of calcium in different tissues and/or organs Experimental protocol: Animals (6 females, 4 males) soon after collection from the field were anesthetized and about 30 different tissues and/or organs were removed. The subsequent procedure 84 C. OcuRO et al. for measuring calcium is described above. Results: The results are expressed in terms of ug calcium per g tissue fresh weight (Table 1). From the results obtained, it is known that a por- tion has a specific value in its calcium content, although individual variation is fairly large. How- ever, the sexes were not found to differ in the cal- cium content of the tissues they have in common. TABLE 1. Calcium content in tissues and/or organs of Rana _ nigromaculata. Values are shown in g/g fresh weight tissue/organ number mean _ standard error Ventral skin posterior 10 4,119.5 150.4 middle 10 4,226.1 152.9 anterior 10 4,271.9 187.4 Dorsal skin posterior 10 6,122.8 511.0 middle 10 6,643.0 360.2 anterior 10 7,188.2 334.7 with dermal glands 10 4,966.5 Kodo Stomach 10 65.3 2.0 Duodenum 10 56.1 4.3 Lower intestine 8 65.7 3)55) Rectum 10 61.1 4.9 Liver 10 305 W633 Gall bladder 10 379.6 56.6 Pancreas 10 81.2 5.9 Lung 10 Ihsod) 2.9 Aorta 10 143.3 11.5 Ventricle 10 54.9 3.9 Spleen 10 33.6 35) Muscle 10 154.5 24.2 Brain 6 642.7 77.9 Nerve trunk 10 60.4 9.3 Kidney 9 48.5 Jol) Testis 4 30.2 4.5 Ovary 6 Ses) 4.6 Oviduct 5 304.8 34.3 Fat body 9 14.8 1.8 Lime sac 10 233,220.0 10,349.1 Femur 10 135,39331"" 3512910 Vertebra 10 102,362.5 2,309.3 Cartilage 9 953.0 250.3 The calcium contents of the lime sacs and femur were the highest among the tissues and/or organs examined in the present study; the former con- taining 2.3 and the latter 1.9x10° wg/g. The tissue containing the next highest amount was the vertebra which contained 1.010° wg/g. The skin contained an unusually high amount of cal- cium (4.1-7.2 x 10° wg/g) among the soft tissues and this value exceeded that of the cartilage (9.5 x 10° ng/g). Other tissues all had lower values, ranging from 14.8 in the fat body to 379.6 mg/g in the gall bladder (containing bile). High value in the brain seems to be caused by a contamination of the endolymphatic sacs around the brain. From the proportional weight and calcium con- tent of each tissue or organ, the ratio of its calcium content to the total calcium content was estimated. Thus the capacity of a tissue or organ for calcium was known. The bones in the limbs contain the largest proportion of calcium and the vertebrae and lime sacs follow in order. The skin holds a large amount of calcium, following the lime sacs, due to its large proportional weight and fairly high calcium content. On the other hand, the muscle follows the skin due to its massiveness despite its rather low calcium content per unit weight. 2. Effects of ultimobranchialectomy on calcium distribution Experimental protocol: After anesthesia, bi- lateral UBX was performed (n=S, for either post- operative day). The sham-operated control animals (n=10, for either postopearative day) were treated similarly but the UBGs were untouched. All animals were kept in tapwater and fed on porcine liver as described before. Fifteen or 35 days after UBX, they were dissected into different tissues and organs as in the preceding experiment and the calcium contents were determined for each. Results: Fifteen days after UBX, the calcium content of the femur, vertebra and cartilage were 2.1 10°, 1.0 10° and 7.5 x 10’ yg/g, respectively. These values are statistically not different from those of the controls and the normal animals, although the former showed a tendency to increase slightly (NS). Furthermore, no difference was observed in the calcium content of the soft tissues between the UBX and control animals. The Calcitonin Function in Frog 85 only portion that changed in calcium content was the lime sac, of which the calcium content in UBX frogs was 56% of that of the controls (P<0.001). This decrease in calcium content of the lime sacs was more pronounced 35 days after UBX, al- though no other tissues changed substantially in calcium content. The calcium content in the lime sacs was only 29% of the control value 35 days after UBX (P<0.001), contrasting with the other portions. Figure 1 shows the calcium content in some calcium-rich tissues for the comparison be- tween the normal and UBX values. These facts strongly suggest that the UBG functions chronically in calcium deposition in the lime sacs but not in the other tissues even in those rich in calcium such as bones and skin. pS CALCIUM CONTENT uc/e FRESH WEIGHT Bllikei _ ee Y 15 DAYS 35 DAYS NORMAL AFTER UBX Fic. 1. The effect of UBX on the calcium content in some calcium-rich tissues in Rana nigromaculata. Each column shows the mean for 10 normal or 5 UBX animals. The vertical bar at the top of each column indicates the standard error of the mean. S833, lime sac; [[[jji], femur; 6293, vertebra; MM, cartilage; GZ, skin (dorsal). * signifi- cantly different (P<0.001) from the control. 3. Effects of salmon calcitonin on the distribution of calcium as determined by *°Ca incorporation Experimental protocol: Frogs were ultimobran- chialectomized bilaterally and kept for 15 or 35 days thereafter. At the end of either term, they were divided into 2 groups. Animals in one group (n=6) received SCT (50 mU/50 1/10 g bw) and animals in the other group (n=6) received the vehicle, both through an arterial cannula. Thirty minutes thereafter, they received *°Ca (8 Ci/50 jul/ 10 g bw) through the same cannula. Two hours after *°Ca administration, all animals were anes- thetized and dissected as shown in Table 1 for the determination of *°Ca activity. Since the individual variation in the specific *°Ca activity was high, the results are expressed as ratios of specific *°Ca activity of each tissue to that of the muscle in the same individual. Results: No differences were found between the animals receiving SCT and the control animals in most tissues including the bones, cartilage and skin (Fig. 2). The lime sac was the only excep- 15 DAYS AFTER UBX 35 DAYS AFTER UBX "SCq ACTIVITY RATIO TO MUSCLE ww = © a =z Ww ea ae — a) (6 WwW =< = Yu LIME SACh =z o — <= << i ” = a | a« =< =< ” vu a °o a DORSAL SKIN Bees Fic. 2. Comparison of **Ca uptake by some calcium- rich tissues of UBX Rana nigromaculata after SCT or control saline administration. {_J, saline administered; 888%, SCT administered. Each column shows the mean for 6 animals and the vertical bar at the top of each column indicates the standard error of the mean. * significantly different (P<0.05) from the control. 86 C. OGuRO ef al. tion. The ratio of the *°Ca activity of the lime sac to that of the muscle was remarkably high in SCT-treated animals (30 times as great as that in the control animals) 15 days after UBX (P<0.05). Thirty-five days after UBX, the *°Ca activity of the lime sac of SCT-treated animals was 543 times that of the muscle. On the other hand, that of the control animals was only 11 times high. Thus the value in SCT-treated animals was 50 times that of the control animals (P<0.05). This demon- strated that SCT enhanced the acute uptake of *°Ca by the lime sacs but it had no effect on *°Ca uptake by the other tissues. Figure 2 illustrates the comparison of the ratios of *°Ca activity of some major calcium-rich tissues to that of the muscle. DISCUSSION The present study comprehensively maps for the first time the distribution of calcium in the anuran amphibian body. The results clearly show that differences in calcium content in different tissues and/or organs are remarkable. It is understand- able that the calcium content of the bones is very high since the bones are generally believed to be the main site of calcium deposition. However, it has been suggested that the bones of anuran am- phibians are not a depot tissue for utilizable calcium due to the lack of osseous tubercula [11]. On the other hand, the calcium content of the muscle was low and similar low values were obtained for other soft tissues except for the lime sac. The calcium content of the liver of R. temporaria has been reported 50-70 g/g on a fresh weight basis [12]. This value is slightly higher than that obtained in R. nigromaculata in the present study. Among the soft tissues, a comparatively high calcium content was found in the gall bladder. Excess calcium might be excreted into the gut through the bile. The physiological significance of the high calcium content in the gall bladder must be clarified in future studies. The calcium content of the amphibian skin has been reported in relation to skin permeability, sodium transport, calcium exchange or morphol- ogy [13-16]. Very high calcium content was re- ported for the skin of Bufo marinus and Rana catesbeiana [15, 16]. In the present study also, it was observed that the calcium content of the skin of R. nigromaculata was extremely and unexpectedly high. The calcium content of the dorsal skin of the present species is 0.8°% on a fresh weight basis and this value is 330 times as much as in the muscle. This is even higher than that of the car- tilage. The ventral skin contains less calcium than does the dorsal skin. According to Baldwin and Bentley [16], the skin of B. marinus and R. pipiens contains 26,000 and 8,000 yg calcium per g tissue weight, respectively. Although the portion of the skin they used in their studies was not specified, the latter value is in the same range as that of the dorsal skin of the present species. The total weight of the skin comprises about 7% of the body weight. Thus a frog weighing 30g may contain more than 15 mg of calcium within the skin alone. The extremely high calcium content of the skin must have a role in the physiological or morpho- logical functions of these animals. However, the biological significance of the high content of cal- cium in skin is not known at present. The highest calcium content in terms of fresh weight was found in the lime sac. The lime sacs appear during premetamorphic development and accumulate a calcium salt which is utilized for skeletal ossification during metamorphosis when the larvae do not feed [17, 18]. By visual observa- tion, it has been recognized that in R. pipiens and R. catesbeiana tadpoles UBX results in a marked regression of the lime sacs [5, 10, 11]. However, the important question of whether the lime sac provides the only calcium which is regulated by UBX, or whether the calcium content of still other tissue changes after UBX, has remained unan- swered. In the present study, it was demonstrated that the only organ undergoing changes in calcium content after UBX is the lime sac. The calcium content of the bones, cartilages and all other tis- sues and/or organs did not change significantly in response to UBX. Calcium content of the skin was not modified either. The amount of calcium deposited in the lime sacs decreased to 56% of that of the control animals 15 days after UBX. This decrease was even more remarkable (29% of the control) 35 days after UBX. These facts indicate that regulation of the calcium content of all cal- Calcitonin Function in Frog 87 cium-containing tissues and/or organs except the lime sacs is not directly under the control of the UBG, or that the control is very minor if any. It has been reported that the frog UBG contains human CT-like molecules which are immunore- active to anti-human CT antibodies [19]. Admin- istration of a UBG extract from R. nigromaculata, R. rugosa and B. bufo japonicus to rats caused hypocalcemia and hypophosphatemia. The pat- terns of these responses were very similar to those caused by SCT [2, 3]. An electronmicroscopic study on the UBG of R. nigromaculata revealed the presence of C-cells that contained probable CT granules that decreased after a calcium load [20]. Taking these facts into consideration, it is con- cluded that the anuran UBG secretes CT which promotes calcium deposition in the lime sacs. In the present study, this effect was shown through a long term experiment. The acute effect of SCT on calcium incorporation into the the lime sacs was then studied using *°Ca as a tracer. The results indicate very clearly that exogenous CT enhanced the uptake of *°Ca by the lime sacs. This effect of CT was greater in animals 35 days after UBX than in those 15 days after UBX. Thus the effect of CT seemed to be strengthened when endogenous CT had been absent for a longer time. Thirty- five days after UBX, the lime sacs of the animals receiving SCT accumulated 50 times as much *°Ca as those of the animals receiving control saline. No other portions of the body, including the bones, cartilage or skin significantly responded to SCT administration. This was anticipated from the results of UBX and hence confirmed the UBG control of acute uptake of calcium by the lime sacs. It has long been known that in mammals an acute decrease in serum calcium concentration after CT administration is mainly caused by a change in calcium kinetics in the bones, such as inhibition of calcium release and decrease of osteolytic osteolysis by CT [21-23]. However, it is highly probable that in R. nigromaculata the decrease in serum calcium concentration after the adminis- tration of UBG extract [9] is achieved, at least partly, by the acute uptake of calcium from the blood by the lime sacs but not by the bone. ACKNOWLEDGMENTS The present study was supported in part by Grants- in-Aid from the Ministry of Education, Science and Culture of Japan (56340038, 57480024). REFERENCES 1 Van Noorden, S. and Pearse, A. G. E. (1971) Im- munofluorescent localization of calcitonin in the ultimobranchial gland of Rana temporaria and Rana pipiens. Histochemie, 26: 95-97, 2 Oguro, C. and Uchiyama, M. (1980) Comparative endocrinology of hypocalcemic regulation in lower vertebrates. In ‘“‘Hormones, Adaptation and Evolution”. Ed. by S. Ishii, T. Hirano and M. Wada, Japan Sci. Soc. Press, Tokyo/Springer- Verlag, Berlin, pp. 113-121. 3 Oguro, C., Nagai, K. -I., Tarui, H. and Sasayama, Y. (1981) Hypocalcemic factor in the ultimo- branchial gland of the frog, Rana rugosa. Comp. Biochem. Physiol., 68A: 95-97. 4 Robertson, D.R. (1968) The ultimobranchial body of Rana pipiens. VII. Cellular response in denerved glands in autoplastic transplants. Zeitschr. Zellforsch., 90: 273-288. 5 Robertson, D. R. (1969) The ultimobranchial body of Rana pipiens. X. Effect of glandular extirpation on fracture healing. J. Exp. Zool., 172: 425-442. 6 Robertson, D. R. (1970) The ultimobranchial body in Rana pipiens. XI. Response to increased dietary calcium — Evidence of possible physiological function. Endocrinology, 85: 1041-1050. 7 Sasayama, Y. and Oguro, C. (1976) Effects of ultimobranchialectomy on calcium and sodium concentrations of serum and coelomic fluid in bullfrog tadpoles under high calcium and high sodium environment. Comp. Biochem. Physiol., 55A: 35-37. 8 Sasayama, Y. (1978) Effects of implantation of the ultimobranchial glands and the administration of synthetic salmon calcitonin on serum Ca concen- trations in ultimobranchialectomized _ bullfrog tadpoles. Gen. Comp. Endocrinol., 34: 229-233. 9 Oguro, C. and Sasayama, Y. (1982) Endocrinology of hypocalcemic regulation in anuran amphibians. In “Proc. [X Internat. Symp. Comp. Endocrinol.” Ed. by B. Loft, Hong Kong Univ. Press, Hong Kong, in press. 10 Robertson, D. R. (1971) Cytological and physi- ological activity of the ultimobranchial glands in the premetamorphic anuran Rana _ cateshbeiana. Gen. Comp. Endocrinol., 16: 329-341. 11 Robertson, D. R. (1972) Calcitonin in amphibians and the relationship of paravertebral lime sacs with 88 13 14 15 16 17 18 C. OcurRO et al. carbonic anhydrase. In ‘Calcium, Parathyroid Hormone and the Calcitonins’. Ed. by R. V. Talmage and P.L. Munson, Excerpta Medica, Amsterdam, pp. 21-28. Pasanen, S. and Koskela, P. (1974) Seasonal changes in calcium, magnesium, copper and zinc content in the liver of the common frog, Rana temporaria L. Comp. Biochem. Physiol., 48A: 27— 36. Taylor, R.E., Jr., Taylor,H.C. and _ Barker, S. B. (1966) Chemical and morphological studies on inorganic phosphate deposits in Rana catesbeiana skin. J. Exp. Zool., 161: 271-286. Elkan, E. (1968) Mucopolysaccharides in the anuran defense against desiccation. J. Zool., 151: 19-53. Zadunaisky, J. A. and Lande, M. A. (1972) Cal- cium content and exchange in amphibian skin and its isolated epithelium. Am. J. Physiol., 222: 1309-1315. Baldwin, G. F. and Bentley, P. J. (1981) A role for skin in Ca metabolism of frogs. Comp. Biochem. Physiol., 68A: 181-185. Guardabassi, A. (1960) The utilization of. the calcareous deposits of the endolymphatic sacs of Bufo bufo bufo in the mineralization of the skeleton. Investigations by means of Ca‘*t®. Zeitschr. Zellforsch., 51: 278-282. Pilkington, J.P. and Simkiss, K. (1966) The mobilization of calcium deposits in the endo- 19 20 21 22 23 lymphatic sacs of metamorphosing frogs. J. Exp. Biol., 45: 329-341. Perez-Cano, R., Galan, F. G., Girgis, S. I., Arnett, T. R. and MacIntyre, I. (1981) A human calcitonin- like molecule in the ultimobranchial body of the amphibia (Rana pipiens). Experientia, 37: 1116— 1118. Suzuki, K., Yoshizawa, H.., Yoshihara, M., Sasayama, Y. and Oguro, C. (1982) Ultrastruc- tural studies on the ultimobranchial glands in some lower tetrapods. In ‘““Comparative Endocrinology of Calcium Regulation’. Ed. by C. Oguro and P.K. T. Pang, Japan Sci. Soc. Press, Tokyo, pp. 115-119. Aliapoulios, M. A., Goldhaber, P. and Munson, P. L. (1966) Thyrocalcitonin inhibition of bone resorption induced by parathyroid hormone in tissue culture. Science, 151: 330-332. Bélanger, L. F. and Rasmussen, H. (1968) Inhi- bition of osteolytic osteolysis by thyrocalcitonin and some growth factors. In ‘“‘Parathyroid Hor- mone and thyrocalcitonin (Calcitonin)”. Ed. by R. V. Talmage and L. F. Bélanger, Excerpta Medi- ca, Amsterdam, pp. 156-168. Raynolds, J. J. and Minkin, C. (1970) Bone studies in vitro: use of calcitonin as a specific inhibitor of bone resorption. In “Calcitonin 1969”. Ed. by S. Taylor, R. Fraser, G.F. Joplin and E.D. Williams, W. Heinemann Medical Books, London, pp. 168-174. ZOOLOGICAL SCIENCE 1: 89-99 (1984) A Golgi-Electron Microscopic Study of Anterior Preoptic Neurons in the Bullfrog and the Toad AKIHISA URANO Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Saitama 338, Japan ABSTRACT — The structures of certain neurons in the anterior part of the preoptic nucleus (APON), which is a presumed center for anuran mating behavior, were studied using Golgi-electron microscopic and scanning electron microscopic techniques. The examined locus contains cerebrospinal fluid (CSF)-contacting neurons and blood capillary (BC)-contacting neurons. Gold-toned CSF-contacting neurons were examined by scanning electron microscopy after removal of surrounding tissues with hydrochloric acid and collagenase. The somata of these neurons bear debris of nerve terminals on their surfaces, while their processes protrude into the preoptic recess. It is probable that these neurons receive neuronal inputs on their somata and detect ventricular hormonal inputs on their intravent- ricular end bulbs. Further, when unstained semithin sections of gold-toned neurons were examined by scanning transmission electron microscopy, it was found that the BC-contacting neurons sent their dendrites laterad toward the white matter. There, many axon terminals form synapses on the dendritic spines of these neurons. The BC-contacting neurons probably detect changes in titers of blood-born hormones, and receive neuronal inputs through dendritic synapses. Thus, the present study shows that a portion of the APON neurons have the proper anatomical features for integrating © 1984 Zoological Society of Japan both neural and hormonal signals concerned with the initiation of sex behavior. INTRODUCTION The preoptic area plays an important role in the evocation of sexual behavior in many verte- brate species [1-6]. In the anuran brain, this region is concerned with male mate calling [2] and female orientation to conspecific mating calls [7]. Because of its conspicuousness, the neuro- endocrine control mechanisms of mate calling have been studied more extensively than female sexual behavior; e.g., mate calling has been induced by electrical stimulation of the anterior part of the preoptic nucleus (APON) [2]. In- tracranial implantation of testosterone into this area enhanced mate calling which was evoked acoustically by play-back of tape-recorded con- specific mating calls in Rana pipiens [8]. Wada and Gorbman [9] further localized the rostro- ventral part of the APON (rv-APON) as the best locus for evoking male mate calling. This area Accepted August 26, 1983 Received July 11, 1983 contained many testosterone accumulating neurons in Xenopus laevis [10] and Rana pipiens [11]. These facts suggest that the rv-APON is testo- sterone sensitive, and that the activity of the rv-APON neurons is responsible for mate calling which has been evoked acoustically. This hypo- thesis is supported by the fact that electrical activity of many rv-APON units was excited by auditory stimulation in Rana pipiens, and that injections of pituitary homogenate significantly increased the percentage of rv-APON units excited by pipiens mating calls [12]. The previous studies mentioned above thus indicate that neuronal activity of the rv-APON is controlled by both neural and hormonal input signals. However, it is not clear whether or not individual rv-APON neurons have appropriate anatomical features for receiving these two different kinds of signals. In this study, the structures of rv-APON neurons, which were determined by scanning electron microscopic and Golgi-electron microscopic techniques, are described in relation to the above question. 90 A. URANO Such information would permit further under- standing of the neural and hormonal control mechanisms of sexual behavior in anurans. MATERIALS AND METHODS Adult bullfrogs (Rana catesbeiana) and toads (Bufo japonicus) of both sexes were used as exper- imental animals. They were purchased from an animal supplier between April and October, and were kept in plastic basins in a room at a reg- ulated temperature (18-21°C) with controlled lighting (12L-12D) for 1 week to 3 months before use. They were fed live crickets during this time. Prior to the electron microscopic studies, Nissl- stained paraffin sections and hematoxylin-eosin stained celloidin sections were prepared as ref- erence sections. The fixation procedure for brain tissues was the same in the various electron microscopic techniques utilized in this study. The animals were anesthetized by injection of MS222 (tricaine methanesulfonate, 0.1 mg/g B.W.) into the dorsal lymph sac. After brief transcardial perfusion with frog Ringer’s solution, they were perfused with 30 ml of a fixative which contained 2% glutaraldehyde, 2°%% paraformaldehyde and 0.03% calcium chloride in 0.08 M cacodylate buffer (pH 7.2). The brains were removed, fur- ther fixed overnight by immersion in the same fixative at 4°C, and were washed in 0.15 M ca- codylate buffer for 15 to 20 min. Scanning electron microscopy (SEM) of the APON ventricular wall Serial transverse sections (200-300 wm) of the fixed brains were cut at the level of the preoptic area on a vibratome. Each section was halved along the midline, and then postfixed in a 2% osmium tetroxide in 0.1M_ cacodylate buffer (pH 7.2) for 1 hr. They were washed twice in cacodylate buffer for 15 min each, immersed twice in 1% tannic acid in deionized water (DW) for 30 min each, and were washed again in the buffer for 10min to remove free tannic acid. Thereafter, the sections were dehydrated through graded ethanols and isoamyl acetate, dried by the critical point method, mounted on specimen holders, and were sputtered with platinum- palladium. Then, the ventricular wall of the preoptic recess was observed using JEOL 100CX- ASID4 in the SEM mode. Nomencratorial identification of the observed loci was performed by examining the transverse plane of the APON in the same specimen. Scanning electron microscopy of freeze-fractured brains After fixation followed by washing in cacodylate buffer, the whole brains were dehydrated through graded ethanols. They were then frozen by immersion in liquid nitrogen, and were fractured at the level of the APON with a razor blade. The fractured brain tissues were dried by the critical point method, mounted on specimen holders, sputtered with platinum-palladium, and were Observed by scanning electron microscopy. Rapid Golgi-staining and gold-toning For Golgi-staining and gold-toning, the rapid Golgi method of Peters [13] was adopted with a slight modification. In brief, the whole brain which was fixed and washed as mentioned above was immersed in a osmium-dichromate solution including 0.2% osmium tetroxide and 2.4% potassium dichromate in DW. A week later it was rinsed in 0.75% silver nitrate and stored in a fresh solution of silver nitrate for 2 days. After silver impregnation, the tissue block was trans- ferred through a graded series of glycerol, super- ficially embedded in 7% agar, and was cut at 200 um on a vibratome. Sections were trans- ferred back through a descending series of glycerol, rinsed in 0.05°% hydrogen tetrachloroaurate, and gold-toned in a fresh gold chloride solution at 0°C for 40min. They were then immersed in a 0.05% oxalic acid solution at 0°C for 6 min to reduce the gold chloride to metallic gold, rinsed in DW, and were deimpregnated in a 1% sodium thiosulphate solution at room temperature for 90 min. Afterward, the sections were rinsed in DW at room temperature. Golgi-scanning electron microscopic study The gold-toned vibratome sections, in which many liquor contacting cells were stained, were digested first with 8 N hydrochloride (60°C, 1 hr) and then by treatment with collagenase (Sigma type 2, 1 mg/ml, 37°C, 4hr) to remove tissues surrounding the gold-toned neurons. Then, the sections were washed in phosphate buffer (0.05 M, A Golgi-EM Study of Anuran APON Neurons 91 pH 7.2), and processed further for SEM observa- tion by a procedure similar to the one mentioned above. Golgi-scanning transmission electron microscopic (STEM) study After gold-toning, the thick vibratome sections were immersed in 2° osmium tetroxide in 0.1 M cacodylate vuffer (pH 7.2) for 30min at room temperature, rinsed in 0.15 M cacodylate buffer, and dehydrated through graded ethanols. They were then embedded in Epon-Araldite mixture in flat trays so that the thick sections could be studied under the light microscope. Stained neurons were drawn with camera lucida, sectioned with glass knives at 0.1-0.2 wm, and were mounted on collodion carbon-coated slot grids. These semi- thin sections were then observed with the JEOL 100CX-ASID4 in the STEM mode _ without uranium- and lead-staining. Frequently, 1 micron sections were cut and stained with toluidine blue for light microscopy to determine the exact location of the gold-toned neurons in the semithin sections. RESULTS The anterior part of the preoptic nucleus surrounds the preoptic recess as a densely packed mass of neurons (Fig. 1). This region can be divided into the laminar periventricular part, the non-laminar medial part, and the lateral part of the white matter. Along the dorsal-ventral axis, the APON can be divided into the dorsal part where neurons containing melanin pigments are abundant and the ventral part where such neurons are few. Scanning electron microscopic observation The SEM study showed the presence of many trv-APON neurons which contact either the cere- brospinal fluid (CSF) or the blood capillaries (BC) of the brain. They are referred to as the CSF- contacting neurons and the BC-contacting neurons, respectively. The ventricular wall of the dorsal part of the APON is ciliated as are the surfaces of the ven- tricular walls in many other brain loci; however, the wall of the ventral part is sparsely ciliated (Figs. 2-4). The sparsely ciliated area includes almost all the portions of the ventricular wall of the rv-APON. The dominant surface structures of this area are bulbous protrusicns of two types: large bulbous protrusions (2.85-+0.095 4m in di- ameter, mean-+-S.E., N=49) often with a wrinkled surface, and small bulbous protrusions (0.67-+ 0.026 wm, N=112) usually with a smooth surface (Fig. 3). A comparison of the magnitudes of protrusion sizes with those in STEM photographs of the rv-APON indicates that the large bulbous protrusions are cytoplasmic extensions of ep- endymal and hypendymal! cells, and that the small bulbous protrusions are dendritic end bulbs of CSF-contacting neurons. Although seasonal changes in the surface structures of the APON ventricular wall were not examined in this study, large bulbous protrusions were numerous in the toad killed in the summer. Examination of cross sections of fractured bullfrog APON showed the presence of neurons located very close to blood capillaries (Fig. 5). Such neurons were frequently found in the non- laminar medial part of the rv-APON near the boundary of the laminar periventricular part. They appear to contact the blood capillaries with their somata or proximal dendrites (Fig. 6). Occasionally some rv-APON neurons are apposed to a soma or a dendrite of another rv-APON neuron (Fig. 6). This observation suggests that the rv-APON neurons have somato-somatic or dendro-somatic synapses in this region. Light microscopy of Golgi-stained APON neurons The periventricular part of the APON is mainly composed of unipolar and bipolar cells in both bullfrogs and toads. The CSF-contacting neu- rons mainly belong to the bipolar neurons (Fig. 7). Various types of CSF-contacting neurons in the rv-APON are illustrated at the lower left of Figure 11. Their processes, antipodal to the CSF-con- tacting dendrites, bifurcate occasionally. Poorly arborizing processes of unipolar neurons generally run toward the white matter lateral to the neu- ronal mass of the APON (Fig. 11). The medial part of the rv-APON includes multipolar and pyriform cells in addition to unipolar and bipolar cells (Figs. 8 and 1i). Their processes are usually running mediad or laterad perpendicular to the ventricular wall. Although Fic. FIG. Fic. FIG. Fic. ee : a ae : 3 é terior part (APON) and the posterior part (PPON). OC, optic chiasma; POR, preoptic recess. Hematoxylin-eosin stained horizontal section. Scale, 200 ~m. 2. Surface structure of the ventricular wall of the preoptic recess at the level of the APON in the toad. RD, rostro-dorsal ciliated part; RV, rostro-ventral sparsely ciliated part. SEM-photo- graph; scale, 20 ym. 3. Large (LBP) and small (SBP) bulbous protrusions of the ventricular wall of the rv-APON. SEM-photograph; scale, 2 um. 4. The ciliated dorsal part of the ventricular wall of the APON. Numerous microvilli are also shown. SEM-photograph; scale, 4 ym. 5. A cross section of the fractured bullfrog APON. Note the presence of neurons (*) located very close to blood capillaries (BC). SEM; scale, 10 »m. 6. Higher magnification of the fractured APON. A portion of neurons (x) located very close to blood capillaries are apposed to a soma or a dendrite of another neuron (arrows). SEM; scale, 4 pm. A Golgi-EM Study of Anuran APON Neurons 93 POR, preoptic recess. Fic. 7. A bipolar CSF-contacting neuron in the rv-APON of the bullfrog. Rapid Golgi-stained specimen; scale, 20 ym. ax, axon; d, dendrite; Fic. 8. An ordinary pyriform cell (ord-n) in the medial part of the ry-APON. A cerebrospinal fluid- contacting neuron (csf-n) is also shown. staining; scale, 20 um. Rapid Golgi staining; scale, 20 ym. Fic. 9. Presence of rv-APON neurons (bc-n) located very close to a blood capillary (BC). Rapid Golgi Fic. 10. A pyriform APON neuron (be-n) whose proximal dendrite seems to contact a blood capillary (BC). ax, axon; d, dendrite. arborization is rather poor, the dendrites which proceed toward the white matter usually bifur- cate several times, and intermingle with the processes of medial amygdala neurons (Fig. 12). A considerable number of rv-APON neurons in the medial part are located very close to blood capillaries (Figs. 9 and 10). Their processes behave similarly to those of the ordinary neurons mentioned above. Golgi-SEM study Observation of stripped rv-APON neurons with SEM revealed a tendency for CSF-contacting neurons in the medial part to bear many debris, but for those in the periventricular part to have only a few on their somata and processes. Figure 13 shows two CSF-contacting neurons whose surrounding tissues were removed. In spite of Rapid Golgi-stained specimen; scale, 20 um. the violent digesting procedure, gold-toned neu- rons kept their shape almost intact. One of them, the bipolar neuron whose soma is located at some distance from the ventricular wall, sends processes towards the ventricular wall and the white matter. Higher magnification of this neu- ron shows that the surfaces of the soma and the proximal dendrite are studded with many debris (Fig. 14) as are ordinary neurons (Fig. 15). The sizes of the debris are compatible with those of the nerve endings. On the contrary, the other neuron in Figure 13 whose soma lies in the periventricular part has a smooth surface. Figure 16 shows a higher magnification of another bipolar CSF-contacting neuron which is also located in the periventricular part adjacent to the ventricular wall. The soma and processes of this neuron 94 A. URANO Til eae Fic. 11. Camera lucida-drawing of Golgi-stained cells in the APON of the bullfrog. Various types of CSF-contacting neurons in the rv-APON are illustrated at the lower left. ep, ependymal layer; lat, lateral part of the APON; m, medial part of the APON;; pv, periventricular part of the APON; POR, preoptic recess. 12 Fic. 12. Drawing of Golgi-stained neurons in the amygdala medialis (Am) and the APON. Proces- ses of Am and APON neurons intermingle with each other in the white matter where the fibers of the medial forebrain bundle (MFB) run through. AC, anterior commissure; LFB, lateral forebrain bundle. Scale, 100 ym. are fairly smooth, and bear a few debris. Golgi-STEM study The main advantage of the use of the Golgi-elec- tron microscopic method is that the overall shapes of neurons can be determined light-microscopi- cally before thin-sectioning. However, the method has a serious limitation, in that, it is unpredictable which neurons will be impregnated by the rapid Golgi staining. Another problem is that, with- out a diamond knife, serial sectioning of single gold-toned neurons is practically impossible. In spite of these limitations, I have succeeded in observing a single BC-contacting neuron at various levels along its dendrites and soma. This gold-toned rv-APON neuron which is fairly large and bipolar contacts a blood capillary with an intervening basement membrane (Figs. 17 and 20). One of the dendritic processes proceeds toward the white matter, and bears many spines in this region (Fig. 17). Various types of nerve terminals and processes are main _ structural components of this region. Many nerve terminals are connected with the dendrite and spines of the BC-contacting neuron in which fine gold particles are deposited (Fig. 18). The proximal part of this dendrite, which is located in the medial part of the rv-APON, forms a gap junction with the soma of another neuron (Fig. 19). This junction lacks intercellular space and synaptic density. Further, the axon which contains electron dense granules emerges out of the same dendrite. Axo-axonic, axo-dendritic and dendro-dendritic synapses are observed near this axon emerging point. Since the soma contains well developed rough endoplasmic reticulum, Golgi apparatus, and 200nm electron dense granules (Fig. 20), this BC-contacting neuron should be peptidergic. Examination of several other neurons, whether gold-toned or not, confirms that the fine struc- ture of the BC-contacting neuron described above is general in the rv-APON. DISCUSSION The present study in the bullfrog and the toad showed that the APON, a presumed center for anuran mating behavior, contains CSF- and BC-contacting neurons especially in its ventral A Golgi-EM Study of Anuran APON Neurons 95 Fic. 13. SEM-photograph of rv-APON neurons whose surrounding tissues were removed. a, a bipolar CSF-contacting cell located at a distance from the preoptic recess; b, a CSF-contacting cell near the ventricular wall. Scale, 5 um. Fic. 14. Higher magnification of the bipolar CSF-contacting cell seen in Fig. 13 (cell a). on the surfaces of the soma and the proximal dendrite (arrows). SEM-photograph of an ordinary rv-APON neuron whose surface bears debris (arrows) pro- Fic. 15. bably of nerve terminals. Fic. 16. Scale, 2 um. Higher magnification of a CSF-contacting cell whose soma lies near the ventricle. Note debris Scale, 2 um. Note rather few debris on the surfaces of the soma and the dendrite (arrow). part. These neurons form synapses with nerve terminals of other neurons. Occasionally, dendro- dendritic synapses and gap junctions are found mainly in the non-laminar medial part. Since the cytoarchitecture of the APON is similar in bullfrogs and toads, the presence of CSF- and BC-contacting neurons in the APON seems to be a common feature of this nucleus among many anuran species. The CSF-contacting neurons The presence of CSF-contacting cells in the preoptic and infundibular been reported in many amphibian species [14, 15]. A topographic SEM study on the specialization of the wall of the third ventricle in Rana temporaria showed that the most ventral part of the wall of recesses has the preoptic recess is studded with numerous large bulbous protrusions [16]. These protru- sions were divided into two types: intraventricular dendritic end bulbs of secretory neurons and large cytoplasmic extensions of ependymal cells. These observations by other authors coincide well with the present result that the dominant surface structure of the ventricular wall of the rv-APON is bulbous protrusions, and that two types of protrusions are discriminable. In the APON of the Xenopus brain, sex-steroid concentrating cells were localized throughout the dorsal-ventral extent in the rostral part; however, such cells were found ventrally in the caudal part [10, 17]. This distributional pattern of steroid concentrating APON neurons corresponds Sex- 96 A. URANO Fic. 17. Drawing of an overall shape of a large BC-contacting neuron in the rv-APON which is success- fully Golgi-stained and examined at various levels along its dendrites and soma. The STEM photographs corresponding to level A to C are Fig. 18, 19 and 20, respectively. Fic. 18. STEM-photograph of the gold-toned spined dendrite (SD) of the neuron seen in Fig. 17 (at level A). Note various types of nerve terminals around this dendrite in the white matter. sp, spine. Dark deposits are metallic gold. Scale, 2 um. Fic. 19. Proximal dendrite (PD) of the neuron seen in Fig. 17 (at level B). This dendrite forms gap junctions (arrow heads) with other dendrite (D) and soma, and also synapses (arrows). ax, axon. Scale, 1 ym. Fic. 20. Soma of the neuron seen in Fig. 17 (at level C). This neuron contacts an endothelial cell (ET) of a brain blood capillary (BC) only with an intervening basement membrane (arrows). DG, dense electron granule; ER, rough endoplasmic reticulum; GA, Golgi apparatus. Scale, 1 ~m. to the localization pattern of ventricular bulbous changes in ventricular surface structures of the protrusions described in this study, although the median eminences of rats [18], the structures of species of experimental animals are different. CSF-contacting neurons also can be modified by Since castration or estrogen treatments induced sex-steroid hormones in amphibians. A Golgi-EM Study of Anuran APON Neurons 97 The CSF-contacting neurons in the medial part of the APON bear many debris on their somata and proximal dendrites after digestion treatments. Since the debris and nerve terminals have similar sizes, and the adhesion between two synaptic membranes withstands many drastic treatments [19], these debris may be the nerve terminals which form synapses on the CSF-contacting neurons. Then, neuronal activity of these cells can be modulated by various signals transmitted at the synapses on their surfaces. On the other hand, Smoller [20] suggested that the dendrites projecting into the preoptic recess from preoptic neurosecretory cells are equipped to serve both secretory and sensory functions. The CSF- ventricular system is thought to distribute bio- logically active molecules within the brain, since many authors have found various hormonal sub- stances in the CSF, such as luteinizing hormone- releasing hormone and thyrotropin-releasing hor- mone [21], oxytocin and vasopressin [22], melatonin [23], and so on. The concentrations of these hormones in the CSF are either increased or decreased according to various physiological conditions. Therefore, the CSF-contacting neu- rons whose dendritic processes protrude into the preoptic recess may detect changes in ventricular hormonal status through their intraventricular end bulbs. The BC-contacting neurons Blood capillaries in the vertebrate brain are generally surrounded by astrocytic endfeet with an intervening basement membrane, so that brain neurons, even hypothalamic neurosceretory cells in fish, are separated from the vascular endothelium [24]. The present finding that the peptidergic rv-APON neurons contact blood capillaries only with an intervening basement membrane is thus incompatible with the general concept of the relations between brain neurons and capillaries. However, the presence of neurosecretory cells which directly contact blood capillaries was pre- viously shown in the toad preoptic nucleus by ordinary transmission electron microscopy [25]. Moreover, in this study, the presence of BC- contacting neurons was exhibited by use of two different electron microscopic techniques, the SEM of freeze-fractured brains and the Golgi- STEM method. These facts strongly support the claim that the present finding of the presence of BC-contacting neurons was not artifactual. In addition, the Golgi-cSTEM study showed that the same gold-toned cell contained characteristic electron dense granules and Golgi apparatus, contacted a blood capillary, and formed synapses with various nerve terminals. This BC-contacting cell therefore can not be an astrocyte, but a peptidergic neuron. The single BC-contacting neuron that was observed in this study forms various synapses, such as axoO-spinous, axo-dendritic, axo-axonic and dendro-dendritic chemical synapses and a gap junction between its own dendrite and a soma of another neuron. Since Halpern [26] noted that terminal degeneration by the tel- encephalic lesions was located along the lateral edges of cell masses in the frog hypothalamus, the nerve terminals which contact the spined dendritic process in the white matter may have a partly telencephalic origin. A previous horse- radish peroxidase study in Rana pipiens has shown that the brain loci which send axons to the APON are the limbic area including the amygdala pars medialis, the magnocellular part of the preoptic nucleus, several thalamic nuclei, the reticular formation, and some _ tegmental nuclei (Urano and Gorbman, unpublished). The BC-contacting neurons may receive various neu- ronal signals of both extrahypothalamic and intranuclear origin in addition to humoral signals conveyed through the BC-contacting surfaces of their plasma membranes. Physiological significance of the CSF- and BC- contacting neurons Seasonal changes found in concentrations of various hormones in toad plasma including sex- steroids showed intimate relations to breeding activity [27, 28]. Further, electrical activity of APON units was modulated by injections of pituitary homogenate in Rana pipiens [12], and an intraventricular injection of luteinizing hor- mone-releasing hormone induced changes in amplitudes and frequency of electroencephalogram in hibernating toads (Urano, unpublished data). These facts strongly indicate that blood-born hormones and hormones in the CSF act on 98 A. URANO the APON neurons to modulate their electrical activity, although direct experimental evidence is scarce. The present study gives anatomical evidences that the CSF- and BC-contacting neurons have appropriate structures to accept such hormonal influences. Thus, they could integrate neuronal and hormonal signals which concern initiation of sex behavior, since Schmidt [29] proposed the APON as the triggering center for mate calling. The BC-contacting neurons can have another physiological role in sex behavior. Sex-steroids may be conveyed rapidly through the thin base- ment membranes to the BC-contacting neurons. The activational effects of sex-steroids then first Operate on these cells to alter the neuronal cir- cuitry of the APON, and induce seasonal changes or sexual dimorphism in the APON volume which has been found in the toad brain (Takami and Urano, in preparation). ACKNOWLEDGMENTS I would like to thank Mr.S. Kondo, Mitsubishi Kasei Institute of Life Sciences, for his kind help and valuable advice, and Mr. P. R. Ross for reading the manuscript. 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(1964) Electronenmikroskopisch Untersuchungen am Nucleus Praeopticus der Krote (Bufo vulgaris formosus). Z. Zellforsch., 63: 208-225. Halpern, M. (1972) Some connections of the telencephalon of the frog, Rana pipiens. An experimental study. Brain Behav. Evol., 6: 42-68. Inoue, M., Takada, K., Ishii, S. and Kikuyama, S. (1981) Seasonal changes in reproductive organ weights and plasma and pituitary prolactin levels in the toad. Zool. Mag., 90: 599. Inoue, M., Takada, K. and Ishii, S. (1982) Sea- sonal changes in plasma androgen concentrations and reproductive behavior in toads. Zool. Mag., 91: 568. Schmidt, R. S. (1976) Neural correlates of frog calling. Isolated brainstem. J. Comp. Physiol., 108: 99-113. ZOOLOGICAL SCIENCE 1: 100-105 (1984) © 1984 Zoological Society of Japan Influence of Vasoactive Intestinal Peptide and Urotensin II on the Absorption of Water and NaCl by the Anterior Intestine of the Tilapia, Sarotherodon mossambicus JAMES R. MAINOYA! and Howarp A. BERN2 Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720, U.S.A. ABSTRACT — The effects of vasoactive intestinal peptide (VIP) and Gillichthys urotensin II (UII) on the absorption of water, sodium and chloride have been examined in vitro using the anterior segment of the tilapia intestine. VIP significantly inhibited water and ion absorption by intestine from freshwater (FW)- and seawater (SW)-adapted fish; in fact, secretion of water and sodium was induced in the SW fish. UII, at the single dose used, significantly increased intestinal absorption in SW tilapia (but not in FW tilapia) but failed to prevent the inhibitory action of VIP in FW or SW fish. INTRODUCTION Vasoactive intestinal peptide (VIP) is a 28-amino acid polypeptide originally isolated from porcine intestine [1, 2] and is also present in neurons of both the central and peripheral nervous system [3, 4]. VIP possesses a variety of physiological as well as pharmacological actions which include: vasodi- lation and hypotension, bronchodilation, glycogen- olysis and hyperglycemia, gastrointestinal smooth muscle relaxation, inhibition of gastric secretion, stimulation of water and bicarbonate secretion by the pancreas, central nervous system arousal, and prolactin secretion [5—7; cf. 8 for review]. It is generally accepted that the intestine of vertebrates constitutes a major site of ionic as well as osmotic regulation and is influenced both by environmental and endogenous (hormonal) factors. Available evidence suggests that VIP affects mammalian intestinal fluid transport through an inhibition of sodium absorption and stimulation of chloride secretion [9-15] and also by vascular changes [16]. This peptide has also been shown to Accepted August 26, 1983 Received June 15, 1983 ‘ Present address: Department of Zoology, Uni- versity of Dar-es-Salaam, P.O. Box 35064, Dar- es-Salaam, Tanzania. * To whom requests for reprints should be addressed. stimulate chloride secretion in the rectal glands of dogfish [17, 18], a finding which suggests that VIP may have an important role to play in the regula- tion of water and electrolyte transport at sites other than the intestinal epithelium. Thus, the recent findings that VIP stimulates chloride secretion by the gill opercular membrane of tilapia [19] and electrogenic sodium reabsorption in Gillichthys urinary bladder [20] are consistent with the suggested possible osmoregulatory function of this peptide in vertebrates. Recently, we showed that Gillichthys urotensin II (UII), a dodecapeptide analogue of somatostatin [21], stimulated intestinal (anterior) absorption of water and NaCl in seawater (SW)- but not in fresh- water (FW)-adapted tilapia [22]. UII stimulated active Na and Cl absorption across the posterior intestine of 5°% SW-adapted Gillichthys but not in SW-adapted fish [23]. Somatostatin itself was without effect [23]. Somatostatin has also been shown to inhibit VIP-stimulated intestinal water and chloride secretion in mammals [cf. 23 for literature]. In the present study, we have in- vestigated the ability of partially purified Gillichthys UII to alter the effects of VIP on the absorption of water and NaCl by the anterior segment of the tilapia intestine. VIP and Teleost Intestinal Absorption 101 MATERIALS AND METHODS The present study was performed on tilapia (Sarotherodon mossambicus) of both sexes with body weights ranging from 30 to 50g. The fish were obtained from a breeding stock maintained in a FW pond at the University of California, Berkeley. The FW or artificial seawater (ASW) in which the fish were kept was maintained at a temperature of 26+1°C. The ASW used in these experiments was prepared according to recom- mendations of the Marine Environment Company (San Francisco, CA). Some FW tilapia were initially placed in 33% ASW where they were maintained for more than two weeks prior to being transferred to 100° ASW, where they were again maintained for more than two weeks prior to being used. Subsequent to decapitation of the fish, the body cavity was opened to expose the long but coiled small intestine. A 7-cm long intestinal segment was carefully removed from the region posterior to the pyloric bulb and using Krebs-bicarbonate- Ringer (KbR) solution, its contents were rinsed out. A noneverted sac was made by tying a ligature at one end of the intestinal segment with a silk thread. The empty sac was blotted lightly on moist filter paper and then weighed using a Mettler-H balance. By syringe, 0.3 ml of KbR solution was introduced into the sac. This volume was found to be just enough to distend the sac without causing differences in hydrostatic pressure between the mucosal and serosal fluids. The open end of the sac was then ligatured and, after blotting, the sac was then reweighed to determine the initial amount of mucosal fluid added. Each sac was subsequently incubated at 27°C for 1 hr in a 50-ml Erlenmeyer flask containing 15ml KbR. During the incubation period the flasks were agitated at 60 oscillations/min while the tissue was being aerated continually with 95%0.: 5%CO:. At the termination of incubation, each sac was again reweighed and both mucosal and serosal fluids were collected for determinations of sodium and chloride concentrations. The concentration of sodium in fluid samples was measured using an Atomic Absorption Spectrophotometer (Perkin-Elmer Model 290B) and that of chloride using a Buchler-Cotlove Chloridometer. Mucosal water and ion transfers were expressed as ml water or Eq ion/g wet weight/ hr in a 7-cm sac. Significance of the data was analyzed by the Student f-test. Hormones VIP was obtained from Peninsula Laboratories (Belmont, CA). Weighed amounts were dissolved in 0.9°% NaCl solution and then diluted with KbR to the desired concentration of 1.0 g/ml. Partial- ly purified UII prepared from Gillichthys mirabilis urophyses was also dissolved in 0.9% NaCl solution and added to the KbR with or without VIP to give a concentration of 20mU/ml. The UII pre- paration was a fraction prepared from a Bio-Gel P-6 column as part of a single peak of activity as assayed on the trout posterior intestine [24]. No appreciable peptide contaminant was evident; ions and small molecules, however, would be present. Hormones were added to both mucosal and serosal fluids. RESULTS Table 1 summarizes the results of an experiment designed to investigate the nature of the interaction between VIP and Gillichthys UII on intestinal absorption in FW tilapia. VIP 1.0 wg/ml caused a dramatic reduction in water (P<0.001), sodium (P<0.001) and chloride (P<0.01) absorption. Intestinal sacs incubated with UII exhibited transport rates which were not different from those of the control group. Furthermore, intestinal sacs incubated in the presence of both VIP and UII had absorption rates which were not statistically dif- ferent from those obtained with VIP alone, and therefore were also significantly lower than rates obtained with 20 mU/ml UII alone. Table 2 shows the effects of VIP or Gillichthys UII, or both, on intestinal absorption in ASW- adapted tilapia intestine. VIP significantly sti- mulated intestinal secretion of water (P<0.01) and sodium (P<0.01), and inhibited chloride (P< 0.001) absorption. However, unlike its lack of effect on the FW tilapia intestine, 20 mU/ml UII significantly increased water (P<0.02) and sodium (P<0.02) absorption, while having no effect on chloride absorption. The addition of UII to the TABLE |. J. R. MAINOYA AND H. A. BERN Effects of vasoactive intestinal peptide (VIP) and urotensin II (UII) on water and NaCl absorption in freshwater tilapia anterior intestine VIP or UII Mucosal water Mucosal sodium Mucosal chloride concentration transfer transfer transfer in medium (ml/g wet wt/hr) (uEq/g wet wt/hr) (vEq/g wet wt/hr) Controls 0.43 +0.05 53.0+6.3 45.9+5.6 (7) (7) (7) 1.0 ng/ml 0.14+0.02*** 14.5+4.2*** 19.7+2.4** VIP (7) (7) (7) 20 mU/ml 0.48 +0.07 75.4+15.9 69.0+ 16.6 UIL (6) (6) (6) 1.0 ug/ml VIP + 0.19+0.03***t 1274S .2tttte 22 322608 20 mU/ml (6) (6) (6) UII Mean values significantly different from control values: * P<0.05, ** P<0.01, *** P<0.001; from 20 mU/ml UII values: * P<0.05, ** P<0.01. TABLE 2. Effects of vasoactive intestinal peptide (VIP) and urotensin II (UII) on water and NaCl absorption in seawater-adapted tilapia anterior intestine VIP or UII Mucosal water Mucosal sodium Mucosal chloride concentration transfer transfer transfer in medium (ml/g wet wt/hr) (uvEq/g wet wt/hr) (wEq/g wet wt/hr) Conirols 0.06+0.02 —4,4+3.0 38.4+2.5 (5) (5) (5) 1.0 ng/ml —0.04-£0.01** —22.9+3.8** 14.743.1*** VIP (7) (7) (7) 20 mU/ml 0.18-0.03* 16.5+5.9* 39.3+3.3 UII (6) (6) (6) 1.0 »g/ml VIP + —0.04+0.03*t*+* —18.3+3.0**t*+ 10.0+4.0**#*tt 20 mU/ml (7) (7) (7) UII Mean values significantly different from control values: * P<0.05, ** P<0.01, *** P<0.001; from 20 mU/ml UII values: +***t P<0.001. VIP-containing media failed to alter the VIP- induced responses in the ASW tilapia intestine. DISCUSSION Available information indicates that several gut hormones, such as secretin, gastrin, glucagon, gastric inhibitory peptide (GIP) and VIP, inhibit absorption or cause net secretion of water and electrolytes in the mammalian intestine [5, 9, 10, 25-27]. VIP has been reported to inhibit sodium absorption and stimulate chloride secretion [9]. The results of the present study indicate that VIP inhibited water, sodium and chloride absorption in the anterior intestine of tilapia adapted to either FW or SW environment. Regiona! differences in intestinal transport exist, and the posterior intestine of tilapia functions differently from the anterior portion, as has been discussed earlier [28]. Although several gut peptide hormones such as secretin, glucagon and to a lesser extent GIP share structural homology with VIP and may or may not stimulate intestinal secretion of water and electro- lytes in vitro [29, 31], only VIP produced a significant increase in chloride secretion in the elasmobranch rectal gland [17]. Furthermore, in both the mammalian gut as well as in the dogfish rectal gland, the VIP-stimulated chloride secretion VIP and Teleost Intestinal Absorption 103 was inhibited by somatostatin — a tetradecapetide of hypothalamic/pancreatic/gut origin [17, 32]. We have recently reported that the response of tilapia anterior intestinal absorption to two teleost urophysial peptides, urotensin I (UI) and UII, depended on acclimation of the fish to SW or FW. UI inhibited intestinal absorption of water and NaCl in FW fish but had no significant effect in SW-adapted fish [22]. Although UII is a dode- capeptide analog of somatostatin [21], the effects of somatostatin on tilapia anterior intestinal sac preparations were opposite to the effects of partially purified Gillichthys UII: somatostatin inhibited absorption of water and NaCl in FW fish but had no effect on SW fish (Mainoya, unpublished). Somatostatin alone does not appear to have significant effects on electrolyte transport by the dogfish rectal gland [17, 18]. Somatostatin inhibits Cl secretion or stimulates ion absorption across the mammalian intestine [see 18 for references]; UII has no such effect on rat colon Cl secretion [23]. Thus somatostatin and UII would appear to have different effects on various transporting epithelia. An influence of VIP on electrolyte transport by teleost osmoregulatory structures other than the gut has been suggested. For example, tilapia opercular membrane chloride secretion is sti- mulated by both VIP and glucagon and inhibited by somatostatin and UII [19, 20]. However, in the Gillichthys urinary bladder, VIP stimulated electrogenic sodium reabsorption, as did UII [20]. Dockray [33] reported the presence of a VIP- like substance in purified extracts of the small intestine of a teleost fish. VIP is present in the dogfish Scyliorhinus canicula, the ballan-wrasse Labrus berggylta, and the bib Trisopterus luscus [34]. In the dogfish the highest concentration of VIP was found in the hindgut, whereas in the two teleosts examined the highest levels of VIP were found in the foregut. In the elasmobranch where a physiological action of VIP on the rectal gland has been demonstrated, an increase in intraluminal gastrointestinal osmolality elevated plasma VIP levels [18, 35]. Moreover, in the dogfish feeding on marine fauna or ingestion of seawater may release VIP from its stores in the intestinal tissue into the plasma [17, 18]. Factors similar to those which stimulate release of VIP in elasmobranchs and mammals [6, 36] may also regulate VIP release in teleosts. Whereas the stimulatory effects of VIP on chloride secretion in the teleost gill opercular membrane and in the elasmobranch rectal gland may contribute significantly to ionoregulation, the VIP stimulation of intestinal water and electrolyte secretion could play an as yet undefined role in osmoregulatory adjustments to hypertonic media but also in absorptive processes (the intestinal secretion of water may aid in establishing a more isotonic milieu which favors efficacious nutrient absorption — cf. 2). Although VIP and UII have separate effects on tilapia anterior intestinal absorption, the dose of UII used was unable to prevent the effect of VIP in either FW or ASW tilapia. ACKNOWLEDGMENTS This work was supported by NIH Fogarty Fellow- ship FP5 TWO 2738 to J.R.M. and by National Science Foundation Grants PCM 78-10348 and PCM 81-10111. 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ZOOLOGICAL SCIENCE 1: 106-113 (1984) Effects of Angiotensins and Other Vasoactive Substances on Drinking in the Eel, Anguilla japonica TETSUYA HIRANO and SANAE HASEGAWA Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan ABSTRACT — Effects of angiotensins and other vasoactive substances on water intake were examined in esophagus-cannulated eels in fresh water. Intra-arterial injection of human angiotensin I or II at doses of more than 5 ng/100 g induced drinking and also a rise in dorsal as well as ventral aortic blood pressure within a few minutes. Initial drinking was followed by inhibition of drinking, the duration of which was in parallel with that of the pressor effect. Epinephrine, norepinephrine and carp urophysial extract caused a dose-dependent increase in dorsal and ventral aortic pressure and also inhibition of water intake; no initiation of drinking was observed. Inhibition of drinking was also observed when oxytocin or arginine vasotocin was injected at doses causing an increase in ventral but not dorsal aortic pressure. On the other hand, drinking was induced immediately after injection of hypotensive substances such as acetylcholine and histamine. Substance P and eel calcitonin had no appreciable effect on either drinking or blood pressure. The fact that an increase (or a decrease) in ventral aortic blood pressure invariably causes inhibition (or initiation) of drinking suggests that a baroreceptor(s) or volume receptor(s), which is probably located on the afferent side of branchial artery, is involved in the control of drinking in the eel. © 1984 Zoological Society of Japan INTRODUCTION Regulation of water intake is one of the essential Osmoregulatory processes for euryhaline teleosts such as the eel, which migrate between fresh water and seawater. In fresh water, they are continually faced with the need to dispose of water that enters through the body surface, and they drink little water. In seawater, on the other hand, body water is lost by high salinity. In order to com- pensate for the water loss, they drink the sur- rounding seawater and absorb water together with monovalent ions from the intestine [1, 2]. Using esophagus-cannulated eels, Hirano [3] has shown that water-replete eels in fresh water can be made to drink by cellular or extracellular dehydration, and that expansion of extracellular space or distentsion of the stomach results in interruption of the copious drinking of the seawater eel; thus drinking in the eel can be explained in terms of the traditional concept of homeostasis as in higher Accepted September 2, 1983 Received July 8, 1983 vertebrates [3]. In higher vertebrates, angiotensin II is a potent dipsogen, involved probably in thirst caused by extracellular dehydration [4, 5]. In previous papers, we have shown that intra-arterial injection of Asn’-Val° angiotensin II amide and eel angioten- sins provoke drinking in water-replete freshwater eels [6, 7]. Following injection of the angioten- sins, eels started drinking for a few to 15 min depending on the doses injected, and the initial drinking was invariably followed by inhibition of the drinking. The threshold dose of angiotensins required to induce drinking was 5 ng/100g, and this dose is known to cause an increase in blood pressure in the eel [8, 9]. In the present study, effects of angiotensins and other vasoactive sub- stances on induction or inhibition of drinking were examined in freshwater-adapted eels. MATERIALS AND METHODS Cultured Japanese eels, Anguilla japonica, weighing about 200 g were obtained from a com- mercial source. They were kept in freshwater Angiotensin and Drinking in Eel 107 aquaria at 18°C for at least 2 weeks before use. For measurement of water intake, the esophagus was cannulated with a vinyl tube (o.d.=2.0 mm) as described previously [3]. The cannula was connected to a drop counter for continuous recording of the drinking rate. Swallowed water was not re-introduced into the stomach. Blood pressure of the dorsal aorta was measured through a cannula (PE 50, Clay Adams) into the pneumogastric artery [7]. In some preparations, the ventral aorta was also cannulated following the method described by Kirsch [10] with some modification. Eels were anesthetized in a solution containing 0.1 °%% each of tricaine methane sulfonate (Sigma) and NaHCO,;. The ventral aorta was exposed by a longitudinal incision (1.5cm in length) along the abdominal skin. A cannula (PE 10) was filled with heparinized saline (100 IU/ml) and inserted into the ventral aorta through a hole made by the tip of an electrode of the high- frequency coagulation unit (Mizuho, Micro-1D). The cannula was fixed to the body wall as shown in Figure 1. The incision was then stitched with silk suture. <= PE 10 tubing bw + VA BA Fic. 1. Cannulation of the ventral aorta. VA: ven- tral aorta, BA; bulbus arteriosus, h; polyethylene hook, bw; body wall. After cannulation, the eel was placed in a plastic trough adjusted to the size of the fish. Well- aerated water, which was filtered and thermore- gulated at 18°C, was continually circulated through the trough. The cannulae were connected to a pressure transducer (San-ei, MPU-0.5-290) and a recorder (Rikadenki, M-13). Injections of angi- otensins and other substances were intitiated more than 18 hours after the operation. In general, the experiments were repeated using at least five different preparations and representative results are presented. Human angiotensins (Asp'-Ile* angiotensins I and II, and human angiotensin III) and substance P were obtained from Protein Research Founda- tion, Osaka. Epinephrine (Sigma), norepinephrine (Sankyo), acetylcholine Cl (Tokyo Kasei), histamine (Sigma), arginine vasotocin (Sigma) and oxytocin (Sigma) were purchased commercially. Eel calcitonin was kindly suplied by Dr. H. Yama- uchi of Toyo Jozo Co., Tokyo. They were all dissolved in 0.9% NaCl, and 0.05 ml/100 g of the solution was injected intra-arterially after appro- priate dilution. The carp urophysial extract was prepared by homogenizing the acetone-dried urophyses in 0.25% acetic acid. The homogenate was heated for 10 min in a boiling water bath and centrifuged. The supernatant was injected after dilution with 0.9% NaCl. RESULTS Injection of human angiotensins I and II into freshwater eels at a dose of 50 ng/100 g induced drinking and also a rise in dorsal aortic blood pressure within a minute or two. The drinking was inhibited after 5 minutes and the inhibition lasted for about 20 minutes. Angiotensin I was as potent as angiotensin II in its dipsogenic and pressor effects and also in the inhibitory effect on drinking, whereas angiotensin III was far less effective (Fig. 2). In this particular preparation, angiotensin I did not induce drinking. Initial induction of drinking after angiotensins I and II was observed in more than 70% of the prepara- tions, and inhibition of drinking after a rise in blood pressure was observed in all the preparations. Dipsogenic effect of angiotensin III was equivocal, although inhibition of drinking was consistently observed in association with its pressor effect. As shown in Figure 3, the increase in blood pressure was dependent on the dose of angiotensin II, and the duration of inhibition of water intake increased 108 T. HIRANO AND S. HASEGAWA ay a Se : Se Steels: 122 = = = : = | Aili bdacle ’ £40 == =| {S| : 3 = SES = no = es —-|—— ® == = | =. = = || a 90 = T efits 8 eh Be | = ee I. va] = j= Al fi 00ng- 9 fall 100 ng— ob SS - oe | i = \--}-|I eee (oe a EEE 30min Fic. 2. Effects of human angiotensin I (A I), angio- tensin II (A ID) and angiotensin III (A III) on drinking and dorsal aortic blood pressure of a freshwater-adapted eel (body weight, 200g). Each spike represents 0.03 ml of swallowed water. i) S) = 25 100 § E = a fe) o =] 5 20 80 o o a g 2 15 com x) a a = 40 40 = o o = 2 2 20 § g = = 3. 0 0 2 Angiotensin I (ng/100g) Fic. 3. Relationship between vasopressor response to angiotensin II (open circles) and duration of drinking inhibition (closed circles) in freshwater eels. Vertical bar represents standard error of the mean. Number of injections is shown in paren- theses. in parallel with its pressor effect. Injection of epinephrine or norepinephrine also caused a dose-dependent increase in blood pressure and inhibition of water intake. However, indu- tion or acceleration of drinking was not observed after epinephrine injections (Fig. 4). Acid extract of the carp urophysis also caused a rise in blood pressure and an inhibitory effect on drinking. Re- flecting the long-lasting pressor effect, the duration of drinking inhibition after the urophysial extraxt was longer than after injections of angiotensins or BR oO i) (eo) Blood Pressure (mmHg) | i | ' a a Fic. 4. Effects of norepinephrine (nEP) on drinking and dorsal aortic blood pressure in a freshwater eel (body weight, 190g). Each spike represents 0.03 ml of swallowed water. (mmHg) © , Jo aS 40} 20 { 20}! 3 | Ach Hist b GI {an | ot —4- Yygh2ng | Y2UK500Ng. mi Pees H ii fo | typaty Lary i Ae ea fa TC th ft | I | alan 30min 3min Fic. 5. Effects of carp urophysial extract, acetylcho- line (Ach), histamine (Hist) and eel calcitonin (CT) on drinking and dorsal aortic blood pressure of freshwater eels, weighing 200 g (A) and 195 g (B). Each spike represents 0.03 ml of swallowed water. epinephrine (Fig. 5A). On the other hand, hy- potensive substances such as acetylcholine and histamine immediately induced drinking, although the drinking lasted only for less than a minute Angiotensin and Drinking in Eel 109 Oo (2) Blood Pressure (mmHg) £ fo) ‘All 100ng) batt 30min 30min Fic. 6. Effects of angiotensin II (A II) norepinephrine (nEP) and oxytocin (OXT) on drinking and dorsal and ventral aortic blood pressure of freshwater eels, weighing 190 g (A) and 180 g (B). Each spike represents 0.03 ml of swallowed water. (Fig. 5B). Synthetic eel calcitonin (50-100 mU/ 100 g) and substance P (25-2500 ng/100 g, data not shown) were without effect either on blood pressure or on drinking rate. As shown in Figure 6A, when 50ng/100¢g of angiotensin II was injected through a cannula in the ventral aorta, there was an immediate increase in blood pressure at both ventral and dorsal aorta, and drinking was induced for the first 5 minutes followed by an inhibition of the drinking. Nor- epinephrine also caused an increase in both ventral and dorsal aortic pressure, whereas acetylcholine decreased both ventral and dorsal aortic pressure. On the other hand, when arginine vasotocin or oxytocin was injected at a dose of 25 ng/100 g, only ventral aortic blood pressure increased for about 2 hours, without causing appreciable change in the dorsal aortic pressure. Nevertheless, drinking was inhibited during the rise in ventral aortic pressure (Fig. 6B). DISCUSSION It has been well established in mammals that angiotensin II is involved in the thirst of extracel- lular dehydration [4, 5]. When administered peripherally or intracranially, angiotensin II induces drinking also in several avian and reptilian species [11-16] but not in amphibians [6, 16]. In spite of the fact that the renin-angiotensin system makes it first appearance in bony fish [17, 18], little work has been devoted to clarify its roles in fishes. In the previous studies, we have shown that the water-replete eel in fresh water drinks in response to angiotensin II (Asn'-Val° angiotensin II amide, Hypertensin) injected into the pneumo- gastric artery. In seawater, where the eel drinks continually, angiotensin II accelerates the rate of drinking [6, 7]. Angiotensin II has also been shown to stimulate drinking in euryhaline killifish [19] and flounder [20]. Kobayashi and his col- leagues have recently surveyed th dipsogenic action of angiotensin II (Hypertensin) in 20 species of freshwater fishes and 17 seawater fishes. They suggested that drinking response to angiotensin II is characteristic of fishes which encounter or tolerate water more hypertonic than in the normal habitat (including the eel), and that drinking in- duced by angiotensin II may be a compensatory emergency reaction to dehydration stress [21]. In the present study, Asp*-IIe° angiotensins I and II (human angiotensins) induced immediate drinking in water-replete freshwater eels, followed by a period of drinking inhibition during their pressor effect. Effects of angiotensin III were appreciably less marked. In accord with this observation, angiotensin I is as effective as angi- otensin II in causing water-replete rats and pigeons to drink water, whereas the (2-8) heptapeptide, ‘angiotensin III’, although retaining considerable dipsogenic activity, is less active than angiotensin II [12, 22]. As in the case of mammals, dip- sogenic and pressor actions of angiotensin I seem to be mediated also in the eel through angiotensin II, since no effect of angiotensin I was seen after administration of a converting enzyme inhibitor (50 vg/100g of inhibitor C, Protein Research Foundation, unpublished observation). The naturally occurring angiotensin I in the Japanese eel has recently been identified as Asp’-Val*-Gly’ angiotensin [23]. The fact that Hypertensin and human angiotensin II are equipotent in dip- sogenic and pressor effects in the eel seems to indicate that the dipsogenic as well as the myotropic receptors of the eel are unable to distinguish bet- ween the Asn‘ and Asp‘ analogues or between the Val’ and Ile° analogues of angiotensin II. In mammals, a variety of pharmacological agents are known to stimulate drinking. Intracranial injection of acetylcholine or carbachol induces “iG T. HIRANO AND S. HASEGAWA drinking in the rat, while epinephrine or norepi- nephrine causes mainly eating [4, 5]. Sub- cutaneous injection of histamine has been reported to cause a rapid decrease in the blood pressure and also stimulate drinking and plasma renin activity in the rat [24, 25]. However, the histamine- induced drinking is not exclusively a _ renin- dependent phenomenon, since it is only partly reduced after nephrectomy [25]. In the rat, angiotensin is known to facilitate the release of acetylcholine, although the angiotensin-induced drinking is relatively unaffected by atropine [26]. In mammals and birds, angiotensin II is known to induce drinking through direct action on the brain, although the possibility that a dipsogenic action of angiotensin is mediated through activation of a peripheral receptor cannot be ruled out [5]. Thus, both neural and endocrine mechanisms seem to be involved in controlling water intake in higher vertebrates. It remains to be clarified whether angiotensin II exerts its dipsogenic effect in the eel by acting on the brain or on a peripheral receptor, or by modulating the release of acetylcholine or histamine. Angiotensin II has been shown to interact with the peripheral sympathetic system by facilitating release and stimulating synthesis of norepinephrine [18]. In the American eel, A. rostrata, the pressor response to angiotensin II is reduced considerably by administration of adrenergic blocking drugs [27]. It is unlikely, however, that the pressor effect and inhibitory effect on drinking observed after angiotensins in the eel are entirely due to catecholamine release, since intra-arterial] infusion of phentolamine (100 »g/100 g.hr) into the eel resulted in only 30-40% reduction of the pressor activity, while having no appreciable effect on the dipsogenic action of angiotensin II (unpublished observation). When injected into the brain, substance P causes drinking, although less effec- tively than angiotensin II or eledoisine, in the pigeon, whereas it is not effective in the rat or even in the pigeon when given systemically [28]. Rece- ntly, salmon calcitonin has been shown to reduce feeding in the rat, apparently by a direct action on the central nervous system [29]. The inhibition of feeding was accompanied by diuresis and an increase in drinking. As shown in the present study, however, substance P and eel calcitonin had no acute effect on drinking behavior or on blood pressure of the eel. The decline or inhibition of water intake after the initial burst of drinking observed after injection of angiotensins into the freshwater eel could be caused by temporary satiety mechanisms as occurs in mammals [30]. However, this possibility seems to be ruled out, since the inhibition of drinking was invariably observed after epinephrine, norepine- phrine, carp urophysial extract and neurohy- pophysial peptides, in association with their pressor effect. No dipsogenic activity was seen after these pressor substances, angiotensins being the only exception. Epinephrine has also been shown to reduce the drinking rate in seawater- adapted mullet, Mugil capito, when injected intra- peritoneally at 50-100 g/100g fish [31]. Al- though blood pressure was not measured, the doses used could be pressor in the mullet. In the present study, on the other hand, vasodepressor substances such as acetylcholine and histamine induced drinking, indicating clearly that changes in arterial blood pressure are closely related to the control mechanisms of water intake in the eel. In contrast to well-studied thirst mechanisms caused by hypovolemia and involvement of the renin-angiotensin system, little attention has been paid to inhibition of drinking in response to hypervolemia even in mammals [5]. For terrestrial vertebrates, constantly faced with the danger of dehydration, hormonal as well as neural mecha- nisms controlling water intake in response to hypovolemic thirst may be of utmost importance. On the other hand, for euryhaline fishes such as the eel, inhibition of drinking when entered hypotonic (hydrating) fresh water, may be as critical as induction of drinking in hypertonic (dehydrating) seawater. In mammals, it is known that hypovolemia is detected by receptors in low pressure areas of the circulation such as in the wall of the large vessels entering the heart and in the wall of the heart itself, especially the atria. Im- pulses from these receptors are carried by the vagus nerves and exert an inhibitory effect on thirst neurons [4, 5]. Arterial baroreceptors may also play a part. In the dog, an increase in left atrial pressure reflexly reduces the rate of renin secretion Angiotensin and Drinking in Eel se via vagal afferent and renal sympathetic efferent pathways [32]. The toadfish, Opsanus tau, has been shown to respond to decreases in blood pressure and/or blood volume by an increase in plasma renin activity [33]. However, the location of baroreceptor or volume receptor and the pathways through which the stimulus is transmitted are not known in fishes. We have reported earlier that systemically administered angiotensins in the eel continued to cause drinking after destruction of the forebrain and midbrain, whereas vagotomy abolished the response [6, 7]. Vagotomy has also been shown to interfere with seawater adaptation of the eel [34, 35]. In fishes, the vagus and also the glos- sopharyngial nerves mainly innervate the gills. The branchial vascular circulation of teleost fishes is equipped with an arterio-arterial pathway al- lowing blood to flow from the afferent to efferent arteriole via the respiratory secondary lammellae. In the eel, both afferent and efferent arterioles are linked via arteriovenous anastomoses to a central venous sinus which drains into the branchial vein. Catecholamines, angiotensins, and pressor princi- ples in the urophysial extract exert most of their pressor effect by peripheral vasoconstriction in the eel [8, 9, 36, 37]. In addition, catecholamines have been shown to decrease branchial vascular resistance by opening the distal lamellae to blood perfusion. Acetylcholine, on the other hand, closes the sphincter muscles guarding the entrance to the blood vessels in the lamellae and blood passes through the central compartments of the filament [38]. Vasodilatation occurs also in the peripheral vasculature. In the present study, oxytocin and arginine vasotocin caused an increase Only in ventral aortic pressure, without significantly affecting dorsal aortic pressure. The neurohypo- physial hormones seem to increase branchial vascular resistance of the eel, thus forcing blood to be channelled into the central venous sinuses, in addition to a reflex vasodilation in the systemic vasculature [36, 39]. The fact that the increase in dorsal aortic pressure invariably inhibited drinking in the eel seems to indicate the presence of baroreceptor(s) on the afferent side of the branchial arteries. Little is known about the site of action of the nerve supply on the various effectors of the complex vasculature of the fish gills [40]. Further studies must be conducted to clarify the nerve supply on the baroreceptor‘(s) involved in the control of water intake in the eel. 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Endocrinol., 27: 52-61. Steen, J.B. and Kruysse, A. (1964) The res- piratory function of teleostean gills. Comp. Biochem. Physiol., 12: 127-142. Chan, D.K.O. and Chester Jones, I. (1969) Pressor effects of neurohypophysial peptides in the Angiotensin and Drinking in Eel 113 eel, Anguilla anguilla L., with some reference to control of the branchial vascular resistance of the their interaction with adrenergic and cholinergic Atlantic cod, Gadus morhua. J. Comp. Physiol., receptors. J. Endocrinol., 45: 161-174. 129: 179-183, 40 Pettersson, K. and Nilsson, S. (1979) Nervous ZOOLOGICAL SCIENCE 1: 114-119 (1984) © 1984 Zoological Society of Japan The Mode of Ecdysteroid Accumulation in Ovaries of Bombyx mori during the Pupal and Pharate adult Period KUNIO WATANABE and EIJI OHNISHI! Biological Institute, Faculty of Science, Nagoya University Chikusa-ku, Nagoya 464, Japan ABSTRACT — By combining liquid chromatography with radioimmunoassay, a procedure was developed to estimate the following ecdysteroids separately and simultaneously; ecdysone, 20- hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 2-deoxyecdysone and 2, 22-dideoxy-20-hydroxy- ecdysone. By use of this procedure, mode of accumulation of these ecdysteroids in ovaries of the silkworm, Bombyx mori, was studied during the pupal and pharate adult period. At the early stage, ecdysone and 20-hydroxyecdysone appeared, and then 3 ecdysteroids of 2-deoxy type accumulated. The results are discussed in relation to the possible origins of these ecdysteroids. INTRODUCTION Since the reports on the discovery of the molting hormone activity in the eggs and ovaries of the silkworm, Bombyx mori, a number of papers ap- peared reporting the accumulation in eggs and ovaries of ecdysteroids of various molecular species [1, 2, 3]. From the ovaries of Bombyx mori, we have isolated and identified 5 ecdysteroids: viz., 2-deoxyecdysone, ecdysone, 2-deoxy-20-hydroxy- ecdysone, 20-hydroxyecdysone and 2, 22-dideoxy- 20-hydroxyecdysone [4, 5, 6, 7]. Together with One more ecdysteroid of unknown structure, these ecdysteroids seem to be the major constituents of the ovarian ecdysteroids of this insect. Since ecdysteroids of 2-deoxy type could not be found in the sources of post-embryonic stages and occur- rence of these ecdysteroids suggested the biosyn- thetic pathway peculiar to the ovary, we have proposed to use the term “ooecdysteroids” to characterize these compounds [8]. In the present article, a procedure is described to estimate 5 ooecdysteroids of Bombyx mori separately and simultaneously by combination of high performance liquid chromatography (HPLC) with radioimmunoassay (RIA). By use of this Accepted August 17, 1983 Received June 25, 1983 1 To whom all correspondence should be addressed. procedure, pattern of the ecdysteroid accumulation during the growth and maturation of the ovary was studied. MATERIALS AND METHODS Materials Larvae of a hybrid, between Japanese 106 and Daizo, were reared on mulberry leaves under LD 12: 12 photoperiod at 25°C. Pupal and pharate adult period lasted about 9 days under these con- ditions. Age of the animals at the time of pupal ecdysis was designated as day 0. Ovaries were dissected out from animals of day 2, day 4, day 6, and day 8. After washing thoroughly with saline solution, they were pooled and weighed. The materials were then frozen with liquid nitrogen, crushed to powder with a mortar and a pestle, and lyophilized. The lyophilized powder was stored in a deep freezer until extracted. Partial purification and high performance liquid chromatography Procedure of the extraction and partial purifi- cation of the ecdysteroids from ovaries were essentially the same as described previously [5, 6], but some simplification was made. The lyo- philized powder was extracted with 80% aqueous ethanol and the extract was concentrated under reduced pressure. The concentrate was dissolved Ecdysteroids in Bombyx Ovary 115 in ethanol and the precipitated materials were removed by filtration. The filtrate was concen- trated and delipidated by partitioning between petroleum ether and 70% aqueous methanol. The methanol layer was concentrated and the concentrate was applied on the silicic acid column. Free ecdysteroids were eluted from the column with a mixture of benzene and methanol (9: 1, by volume), while the conjugated forms were re- covered by elution with methanol. The methanol eluate was treated with snail juice to liberate free ecdysteroids and the treated samples were again applied on the silicic acid column as mentioned above. Ecdysteroids were further purified on the preparative thin-layer chromatography (TLC). The materials adsorbed on the plate (Merck pre- coated plate, 60 PF.,;, 20x20; solvent system, chloroform-—96°% aqueous ethanol 4:1, by volume) from R,;-values between 0.3 and 0.75 were eluted with ethanol. The eluate was applied to HPLC, employing a reverse phase column (Wakogel ODS-10K, 4.0 x 500 mm) and a solvent system of methanol -— water (50: 50, by volume). The effluent was collected at every 1 minute and the fractions were subjected to RIA. Radioimmunoassay Preparation of antisera and RIA were performed according to the method of Borst and O’Connor [9]. Standard curve was constructed with 20- hydroxyecdysone. Concentrations of ecdysteroids were adjusted by measuring the absorbance at 243 nm in ethanol, taking a molecular extinction coeffi- cient as 11,600. Chemicals The sources of the chemicals were as follows: [?H]-Ecdysone (ca. 34 Ci/mmol) from Drs. D. S. King and J. B. Siddall of Zoecon Corp., 2-deoxy- ecdysone and 2-deoxy-20-hydroxyecdysone from Dr. D.H.S. Horn of CSIRO, Melbourne, 20- hydroxyecdysone from Dr. T. Takemoto of Tokushima Bunri University, and ecdysone from Simes, Milan. 2, 22-Dideoxy-20-hydroxyecdysone was purified from ovaries of the silkworm in our laboratory. RESULTS RIA of ooecdysteroids Since the affinity of ooecdysteroids for antiserum is different from that for ecdysone, calibration curves for 2-deoxyecdysone, 2-deoxy-20-hydroxy- ecdysone and 2, 22-dideoxy-20-hydroxyecdysone were constructed (Fig. 1). Concentrations of these ecdysteroids were adjusted by use of the optical density at 243nm in ethanol using a molecular extinction coefficient of 11,600. The affinity of these ecdysteroids to the antiserum could be expressed by the amount of the particular ecdysteroid required to displace 50 per cent of [*H]-ecdysone bound to the antiserum. If the affinity of the antiserum for 20-hydroxyecdysone is designated as 1, values for other ecdysteroids were estimated to be | for ecdysone, 1/3 for 2- deoxyecdysone and 2-deoxy-20-hydroxyecdysone and 1/15 for 2, 22-dideoxy-20-hydroxyecdysone. For quantification of these ecdysteroids, these values were used for the calculation. Pattern of ecdysteroid accumulation in ovary during pupal and pharate adult development Growth and maturation of the ovary of Bombyx mori are induced by the stimulating action of the ecdysteroid at the early phase of pupal develop- ment [10]. After the initiation of the development, the ovary acquires the biosynthetic capacity of its own ecdysteroids and accumulates them [11]. Just before the eclosion of the moth, most of the oocytes complete maturation and the moth lays eggs shortly after the imaginal ecdysis. In order to study the mode of accumulation of ooecdysteroids, the ovaries were dissected out from staged animals at intervals of 2 days from 2 days after the pupal ecdysis until a day before the eclo- sion (day 8). After extraction and partial puri- fication, the composition and relative amounts of the ooecdysteroids were analyzed by the procedure described under Materials and Methods. From the ovaries of day 2 animals, ecdysone and 20-hydroxyecdysone were found in both free and conjugated ecdysteroid fractions (Fig. 2). However, ecdysteroids of 2-deoxy type, which are rather abundant in ovaries of later stages, could not be detected at this stage. From the ovaries of day 4 animals, a prominent peak of 2-deoxy-20-hydroxyecdysone appeared in the free ecdysteroid fraction (Fig. 3A). The peak of this compound was also observable in the 116 K. WATANABE AND E. OHNISHI 10 100 Concentration of ecdysteroid (ng) 100 Ae) ee eS e) A @) e 5 & U = J) ae 0.1 1.0 Fic. 1. Calibration curves for the following ecdysteroids: 20-hydroxyecdysone (O), ecdysone (A), 2-deoxy-20-hydroxyecdysone (@), 2-deoxyecdysone (A), and 2, 22-dideoxy-20-hydroxyecdysone (QO). amounts of these ecdysteroids. 300 200 Ecdysteroids(ng) 100 10 20 30 Fraction number Fic. 2. of HPLC were as follows: 120 kg/cm7?. ment. A: free ecdysteroid fraction. B: chromatogram of the conjugated forms, although it was not so conspicuous as that of the free ecdy- steroid fraction. 2-Deoxyecdysone and 2, 22- dideoxy-20-hydroxyecdysone were detected in comparable amounts in both free and conjugated ecdysteroid fractions. Identity of these ecdy- HPLC pattern of the ecdysteroids obtained from ovaries of day 2 animals. column was monitored by UV-absorption at 254 nm (curves). 1 min and the content of ecdysteroids in each fraction was estimated by RIA (histograms). The curves were obtained by competition in RIA system of *H-ecdysone with varying 300 N (2) (o) Ecdysteroids(ng) ° (o} 10 20 30 Fraction number Effluent from the Fractions were collected at intervals of Conditions Solvent system, methanol-water 50: 50 (v/v); flow rate, 1 ml/min; Pressure, conjugated ecdysteroid fraction, after snail juice treat- Histograms indicate the contents of 20-hydroxyecdysone (a) and ecdysone (b). ecdysteroid content in ng per g wet weight of ovaries. Ordinate: Abscissa: fraction number. steroids was confirmed by co-chromatography with the authentic samples in separate runs. In the chromatograms of the later stages, RIA-posi- tive substance could be detected after the peak of 2-deoxy-20-hydroxyecdysone (designated as d in the chromatograms). The chemical nature of Ecdysteroids in Bombyx Ovary way 300 -_o -_- fea) a = c ~~ ~~" s ¥% 200 ra) Te) - = © o a rf > > M4 U0 Ww 2 100 10 20 30 40 50 60 Fraction number Fraction number Fic. 3. HPLC pattern of the ecdysteroids obtained from ovaries of day 4 animals. Procedure and conditions of HPLC were the same as indicated in the legend to Fig. 2. A: free ecdysteroid fraction. B: conjugated ecdysteroid fraction, after snail juice treatment. Histograms indicate the contents of 20-hydroxyecdysone (a), ecdysone (b), 2-deoxy-20-hydroxyecdysone (c), unknown ecdysteroid (d), 2-deoxyecdysone (e), and 2, 22-dideoxy-20-hydroxyecdysone (f ). 300 300 a -_—-_ oO fe.) c = ~~ 7) W 200 UD 200 = ro ° iS s 7 =, > v 100 bs oj uw 100 Fraction number Fraction number Fic. 4. HPLC pattern of the ecdysteroids obtained from ovaries of day 6 animals. Procedure and conditions of HPLC were the same as those indicated in the legend to Fig. 2._ For the explanation of symbols, see the legend to Fig. 3. 300 we. _ a fe) © AS Da mi 0 200 3 ; 2 — — = £2 = % 2 3 Y 100 Vv Ww WwW Fraction number Fraction number Fic. 5. HPLC pattern of the ecdysteroids obtained from ovaries of day 8 animals. Procedure and conditions of HPLC were the same as those indicated in the legend to Fig. 2. For the explanation of symbols, see the legend to Fig. 3. 118 K. WATANABE AND E. OHNISHI Ecdysteroids(ng/gww) Days after pupal ecdysis Fic. 6. Summary of the changes in ecdysteroid accu- mulation during the maturation of ovary. Con- centrations of 5 ooecdysteroids were calculated from the histograms shown in the preceding figures. A: free ecdysteroid fraction. B: conjugated ecdysteroid fraction, after snail juice treatment. Symbols of ecdysteroids are: 20-hydroxyecdysone. (O), ecdysone (@), 2-deoxy-20-hydroxyecdysone (A), 2-deoxyecdysone (4), 2, 22-dideoxy-20-hy- droxyecdysone ((). this material is not known. In the chromatograms of ecdysteroids of day 6 animals, the peak of 2-deoxy-20-hydroxyecdysone dominated further, whereas those of ecdysone and 20-hydroxyecdysone decreased (Fig. 4A). 2-Deoxyecdysone and 2, 22-dideoxy-20-hydroxy- ecdysone were detected in nearly comparable amounts. In the conjugated ecdysteroid fraction of this stage, the relative amount of ecdysone increased as compared with that of day 4 animals. The chromatographic profile of the ecdysteroids at this stage was much the same as compared with the earlier stages, except that the content of ecdy- sone and 20-hydroxyecdysone in the free ecdy- steroid fraction almost disappeared. Pattern of the changes of ecdysteroids during the pupal and pharate adult period is summarized in Figure 6, where the contents of individual ecdy- steroids are expressed in ng per g wet weight of ovaries. Since the weight of ovaries increases greatly (about 3 hundredfold) during the matura- tion, the total amount of the accumulated ecdy- steroids in the ovary of individual animal increases to a quite large extent. The unknown ‘“‘d’’, which is not shown in the figure, amounted to 25 ug per g wet weight of ovaries on day 8. DISCUSSION Our antiserum, raised against 20-hydroxyecdy- sone bound to bovine serum albumin, showed the following relative affinities when the value of ecdysone was designated as 1. 2-Deoxyecdysone and 2-deoxy-20-hydroxyecdysone 1/3, 2, 22- dideoxy-20-hydroxyecdysone 1/15. This fact in- dicates that OH-group at C-2 position has a significant effect on the affinity, whereas that at C-20 is insignificant. Further, OH-group at C-22 iS more essential than that at C-2 for the binding. In our previous paper [6], we showed in the chromatograms the fifth peak eluted from the reversed phase column at the retention time about twice that of 2-deoxyecdysone and commented that it might be an unknown ecdysteroid. Although further analysis revealed that it is indeed ecdy- steroid of unknown structure, it is not included in the present study, since it reacted rather poorly with our antiserum in the RIA system. The major components of matured ovarian ecdysteroids consisted of five ecdysteroids together with the unknown one mentioned above. The result is essentially the same as that reported ear- lier [6]. However, minor differences were found in the relative abundance of these ecdysteroids. Previous study showed that the contents of 2- deoxyecdysone and _ 2, 22-dideoxy-20-hydroxy- ecdysone were not so different from that of 2- deoxy-20-hydroxyecdysone, in both free and con- jugated ecdysteroid fractions. They occurred roughly in the similar molecular ratio. In the present study, however, the content of 2-deoxy- 20-hydroxyecdysone was estimated to be several Ecdysteroids in Bombyx Ovary 119 times higher than those of the other two 2-deoxy type ecdysteroids. One of the reasons for this difference might be attributable to the difference in the source of the materials. In our previous study, commercial strains (mostly Taihei-Choan) were the source of materials, but we used a hybrid strain between Japanese 106 and Daizo in the present study. In most chromatograms of ecdysteroids derived from ovaries of later pupal stages, a small peak of RIA-positive substance was detected just after the peak of 2-deoxy-20-hydroxyecdysone. Since the content of this material in ovaries was low, analysis on the chemical nature was not conducted. Since the ecdysteroids of 2-deoxy type could be considered to be the precursors of ecdysone and/or 20-hydroxyecdysone [6], these are expected to appear in the ovary before the accumulation of ecdysone and 20-hydroxyecdysone. Contrary to this expectation, ecdysone and 20-hydroxyecdysone were detected in the ovaries of day 2 pupae and the ecdysteroids of 2-deoxy type appeared on day 4. This fact can be reconciled if we assume that ecdysone and 20-hydroxyecdysone, found at the early stage, are originated in the prothoracic glands and transported into the ovary. The following lines of evidence favor this hypothesis. In Bombyx mori, ovary is induced to develop by the action of exogenous ecdysteroids [10]. Trans- port of exogenous ecdysteroids into ovaries was demonstrated by organ culture system [12]. A considerable portion of the injected [*H]-ecdysone could be recovered from the pupal ovary as ecdy- sone or derivatives of ecdysone [13]. Contrary to the origin of these ecdysteroids of day 2 animals, ecdysteroids accumulated at later stages are con- sidered to be synthesized almost exclusively in the ovary itself [13]. Further experiments supporting this hypothesis are now in progress. ACKNOWLEDGMENTS We wish to express our thanks to Drs. D.S. King and J. B. Siddall of Zoecon Corporation for generous gift of [H]-ecdysone, to Dr. T. Takemoto of Toku- shima Bunri University for the sample of 20-hydro- xyecdysone, to Dr. D.H.S. Horn of CSIRO, Mel- bourne for samples of 2-deoxyecdysone and 2-deoxy- 20-hydroxyecdysone and to Mr. K.Soma for his technical assistance. A part of this work was supported by the grant-in-aid (No. 00548016) from the Ministry of Education, Science and Culture. REFERENCES 1 Ohnishi, E., Ohtaki, T. and Fukuda, S. (1971) Ecdysone in the eggs of the silkworm, Bombyx mori. Proc. Japan Acad., 47: 413-415. 2 Hanaoka, K. and Ohnishi, E. (1974) Changes in ecdysone titre during pupal-adult development in the silkworm, Bombyx mori. J. Insect Physiol., 20: 2375-2384. 3 Hoffmann, J. A., Lagueux, M., Hema, G,, Charlet, M., and Goltzene, F. (1980) Ecdysone in reproductively competent female adults and in embryos of insects. In ‘Progress in Ecdysone Research” Ed. by J. A. Hoffmann, Elsevier/North Holland Biomed. Press, Amsterdam, New York and Oxford. pp. 431-465. 4 Ohnishi, E., Mizuno, T., Ikekawa, N., Awata, N. and Sakurai, S. (1977) Occurrence of a-ecdysone in the developing embryos of the silkworm, Bombyx mori. J. Insect Physiol., 23: 317-319. 5 Ohnishi, E., Mizuno, T., Chatani, F., Ikekawa, N., and Sakurai, S. (1977) 2-Deoxy-a-ecdysone from ovaries and eggs of the silkworm, Bombyx mori. Science, 197: 66-67. 6 Ohnishi, E., Mizuno, T., Ikekawa, N., and Ikeda, T. (1981) Accumulation of 2-deoxy-ecdy- steroids in ovaries of the silkworm, Bombyx mori. Insect Biochem., 11: 155-159. 7 Ikekawa, N., Ikeda, T., Mizuno, T., Ohnishi, E. and Sakurai, S. (1980) Isolation of a new ecdy- steroid, 2, 22-dideoxy-20-hydroxyecdysone, from ovaries of the silkworm, Bombyx mori. Chem. Comm., 448-449. 8 Ohnishi, E. (1981) Ecdysteroids in the silkworm, Bombyx mori. Sericologia, 21: 14-22. 9 Borst, D.W. and O’Connor, J.D. (1974) Trace analysis of ecdysteroids by gas-liquid chromato- graph, radioimmunoassay and bioassay. Steroids, 24: 637-656. 10 Chatani, F. and Ohnishi, E. (1976) Effect of ecdysone on the ovarian development of Bombyx silkworm. Develop. Growth Differ., 18: 481-484. 11 Ohnishi, E. and Chatani, F. (1977) Biosynthesis of ecdysone in the isolated abdomen of the silk- ‘worm, Bombyx mori. Develop. Growth Differ., 19: 67-70. 12 Ogiso, M. and Ohnishi, E. (1982) Incorporation and metabolism of ecdysone in cultured ovaries of the silkworm, Bombyx mori (in Japanese). Do- butsugaku Zasshi, 91: 540. 13 Ohnishi, E. and Watanabe, K. (1983) Biosyn- thesis and accumulation of ecdysteroids in the ovary of the silkworm, Bombyx mori. Proc. 9th Intern. Symp. Comp. Endocrinol., Hong Kong Univ. Press (in press). ZOOLOGICAL SCIENCE 1: 120-125 (1984) © 1984 Zoological Society of Japan Acceleration by Vitamin A of the Permanent Proliferation of Mouse Vaginal Epithelium Induced by Neonatal Treatment with Progestin NosBoruU TAKASUGI and TRETsuO KATo! Department of Biology, Yokohama City University, Kanazawa-Ku, Seto 22-2, Yokohama 236, Japan ABSTRACT — In 210-day-old mice which had received daily injections of 100 ug 17a-hydroxypro- gesterone caproate (17-OHP) for 5 days starting on the day of birth, permanent proliferation occurred in the vaginal epithelium when ovariectomized at 180 days of age, but not in those ovariectomized at 30 days. Neonatal injections of 200 IU vitamin A acetate (VA) given simultaneously with 17-OHP caused more progressive proliferation of the vaginal epithelium, increasing the number of both cell layers and downgrowths, and the mitotic rate as compared with the epithelium of ovariectomized mice treated neonatally with 17-OHP alone. This finding suggests that VA accelerates the permanent proli- feration of the vaginal epithelium induced by 17-OHP when given simultaneously with the progestin. INTRODUCTION Female rats and mice treated neonatally with appropriate doses of estrogen or androgen exhibit the persistent anovulatory syndrome, in which the vaginal epithelium is persistently proliferated and cornified by continuously secreted ovarian estrogen due to permanent functional alteration of the hy- pothalamo-hypophysial system [1-5]. In contrast, Ovary-independent (estrogen-independent) persi- stent changes in the vaginal epithelium occur in Ovariectomized mice treated neonatally with relatively large doses of estrogen or androgen [6, 7]. The vaginal changes are irreversible, frequently culminating in vaginal tumors at old ages [8,9]. In mice given neonatal injections of progestin, permanent proliferation and cornification also take place in the vaginal epithelium, resulting in hyperplastic epithelial lesions including the down- growths and carcinomas [10-15]. On the other hand, it is demonstrated that in neonatally estrogen- or androgen-treated, ovari- ectomized mice, the occurrence of permanent Accepted September 8, 1983 Received June 8, 1983 ' Present address: Department of Microbiology, Tokyo Dental College, Chiba 260, Japan. proliferation and cornification in the vaginal epithelium is prevented by vitamin A given simul- taneously with the sex hormones [16-21]. In the present study, however, it was found unexpectedly that vitamin A accelerates the permanent pro- liferation of the vaginal epithelium in adult ovariectomized mice treated neonatally with 17a- hydroxyprogesterone caproate (17-OHP) when given simultaneously with 17-OHP, the results being reported herein. MATERIALS AND METHODS Mice of the C57BL/Tw strain were maintained at temperature of 22—-25°C under 12hr L-12hr D daily illumination. Daily dose of 100 wg 17a- hydroxyprogesterone caproate (17-OHP) (Schering AG., Berlin) in 0.02 ml sesame oil were sub- cutaneously injected into the female mice for 5 days starting within 24 hours after birth, and the mice were ovariectomized at ages of 30 and 180 days of age. Another group of female mice received 5 daily injections of a mixture of 100 vg 17-OHP and 200 IU vitamin A acetate (Sigma Co., St. Louis) in 0.02 ml oil beginning on the day of birth, and were ovariectomized at 180 days. Female mice given neonatal injections of 0.02 ml sesame oil alone Vitamin A and Hyperplastic Vaginal Epithelium 121 and ovariectomized at 180 days served as controls. When the animals of all groups attained the age of 210 days, they were sacrificed 5 hours after a single subcutaneous injection of 1 mg colchicine/ 20g body weight. At autopsy, vaginae were fixed in Bouin’s solution and sectioned longitudi- nally at 8m. Serial sections were stained with Delafield’s hematoxylin and eosin. The number of dividing cells at metaphase per 1,000 basal cells of the epithelium was counted in five randomly selected sections of the proximal (upper 2/5), middle (middle 1/5) and distal (lower 2/5) parts, respecti- vely. The upper and middle parts are derived from Miillerian duct and the lower part from urogenital sinus [22]. The uterine horns were cut transversely. In vaginae, the number of hyperplastic epithelial downgrowths was counted in the serial sections, on which the lumen was discernible. The number of cell layers of the epithelium was counted in five flat portions of the proximal, middle and distal parts, separately. RESULTS Changes in the vaginal epithelium of mice treated neonatally with 17a-hydroxyprogesterone caproate (17-OHP) or with 17-OHP plus vitamin A Female mice which had received injections of 100 wg 17a-hydroxyprogesterone caproate for 5 days starting on the day of birth (17-OHP mice) began to show persistent cornification of the vaginal epithelium within 90 days of age. Mice treated neonatally with a mixture of 100 ng 17- OHP and 200 IU vitamin A acetate (17-OHP-VA TABLE |. plus VA-treated ovariectomized mice Number of cell layers Neo-Oil Neo-17- Neo -17- Neo-17- HP OHP OHP-VA 180d OVX 30d OVX 180d OVX 180d OVX (A) (B) (C) (D) Fic. 1. The number of cell layers of the vaginal epithelium in 210-day-old, ovariectomized mice treated neonatally with 17-OHP or with 17-OHP plus VA. P: proximal, M: middle, D: distal, 30d OVX: ovariectomy at 30 days of age, 180d OVX: ovariectomy at 180 days of age. Vertical bar indicates standard error of the mean. For other abbreviations, see Table 1. Each datum was obtained from 5 individuals. Student’s ¢ test: M D P<0.001 P<0.001 P=<0.001 NS P (B) vs (C) P<0.001 (C) vs (D) NS Persistent vaginal cornification and weight of uterus in neonatally 17-OHP- or 17-OHP Age at the start Age at Age at Weight of uterus at Groups Neonatal of persistent vagi- ovariectomy autopsy autopsy (age, 210 days) treatments nal cornification mg/20 g body (days) (days) (days) weight A Oil 180 210 10.6 +0.65* B 17-OHP 30 210 5.6+0.75 Cc 17-OHP 75-90 180 210 16.0+1.88 D 17-OHP-VA 70-80 180 210 16.0+-1.76 * mean-+standard error, 17-OHP: 17a-hydroxyprogesterone caproate, VA: Vitamin A ace- tate. Difference in uterus weight (Student’s ¢ test): A vs B, P<0.02; A vs C, B vs C, A vs D, P< 0.001. Each datum was obtained from 5 individuals. P22. N. TAKASUGI AND T. KATO mice) also started to show persistent vaginal cornification by 80 days of age (Table 1). How- ever, the persistently appearing cornified cells in the vaginal smears were no longer observable in groups of 17-OHP and 17-OHP-VA mice when Ovariectomized at 180 days. Uteri of 17-OHP mice which were ovariectomized (OVX) at 30 days of age (30 d.) were smaller in weight than those of the oil controls ovariectomized at 180 days (180 d.). In both 210-day-old OVX (180 d.) 17-OHP and 17-OHP-VA mice, the uterine weight was increased as compared with that in the age-matched OVX controls (Table 1). The ovaries removed at 180 days of age contained varying sizes of normal and atretic follicles and hypertrophied interstitial cells with or without a few small clumps of atrophic luteal tissue. In 210-day-old, OVX (180 d.) 17-OHP mice, all parts of the vaginal epithelium consisted of 2-6 layers of proliferated prickle cells and a layer of basal cells (Fig. 3), which were significantly greater in number than those in OVX (30 d.) 17-OHP mice and the OVX controls (Fig. 1). The superficial layer of the epithelium was neither cornified nor mucified in OVX (180 d.) 17-OHP and 17-OHP-VA mice. Vaginae of OVX (30 d.) 17-OHP mice were lined with an atrophic epithelium of 2 cell layers, which was similar to that in the OVX controls (Fig. 2). In the group of 210-day-old, OVX (180 d.) 17-OHP-VA mice, the proximal and distal parts of the epithelium were proliferated, being similar to Fic. 2. The middle part of vagina of a 210-day-old, neonatally oil-injected mouse ovariectomized at 180 days of age (180d.). The epithelium is atrophic. x 260. Fic. 3. The middle vaginal part in a 210-day-old, neonatally 17-OHP-treated OVX (180d.) mouse. The epithelium is proliferated. 260. Fic. 4. The middle vaginal part in a 210-day-old, OVX (180d.) mouse treated with 17-OHP and vitamin A acetate (VA). The epithelium is proliferated. Note numerous dividing cells at metaphase. Fic. 5. x 260. The middle vaginal part in a 210-day-old, neonatally 17-OHP plus VA-treated OVX (180d.) mouse. Note a downgrowth of the prolifereated epithelium into the connective tissue stroma. x 260. Vitamin A and Hyperplastic Vaginal Epithelium TABLE 2. 123 Mitotic rates (%) in the vaginal and uterine epithelia and the number of vaginal epithe- lial downgrowths in 210-day-old, ovariectomized mice treated neonatally with 17-OHP or with 17-OHP plus VA Age at Mitotic rate in the No. of Mitotic rate Neonatal ovari- vaginal epithelium vaginal in the Groups ___ treatments ectomy epithelial uterine (days) down- epithelium Proximal Middle Distal growths A Oil 180 0.12+0.010 0.15+0.019 0.16+0.025 0 0.06+0.007 B 17-OHP 30 0.13+0.004 0.22+0.033 0.18+0.009 0 0.05 +0.009 Cc 17-OHP 180 0.31+0.053 0.37+0.042 0.34+0.064 1.8+0.66 0.05+0.018 D 17-OHP-VA 180 0.86+0.261 1.78+0.259 1.72+0.269 3.64040 0.04+0.009 A vs C P<0.02 P<0.002 P<0.05 NS Student’s ¢ test B vs C P<0.01 P<0.01 P<0.02 NS C vs D P<0.05 P<0.001 P<0.002 P<0.05 NS Each datum was obtained from 5 individuals. those in OVX (180 d.) 17-OHP mice. However, the middle part of the epithelium was markedly proliferated, consisting of 4-10 cell layers. The number of the cell layers of OVX 17-OHP-VA mice was greater than in the same part of OVX (180 d.) 17-OHP mice (Figs. 1 and 4). Downgrowths of the proliferated vaginal epi- thelium into the stromal tissue were observed in 210-day-old, OVX (180 d.) 17-OHP and 17-OHP- VA mice (Fig. 5), while the hyperplastic down- growths were never encountered in OVX (30 d.) 17-OHP mice as well as in the OVX controls. The epithelial downgrowths in OVX (180 d.) 17-OHP- VA mice were increased in number as compared with those in OVX (180 d.) 17-OHP mice (Table 2). Uteri of both 210-day-old, OVX (180 d.) 17-OHP and 17-OHP-VA mice were atrophic, having the epithelium composed of a single layer of flattened cells. No difference was found in the uterine histology between OVX (30 d.) 17-OHP and OVX (180 d.) 17-OHP mice, although weight of uterus was smaller in the former than that in the latter (Table 1). Mitotic activity in the vaginal and uterine epithelia of ovariectomized mice treated neonatally with 17-OHP or 17-OHP plus vitamin A In vaginae of 210-day-old OVX (30 d.) 17-OHP mice, the proximal, middle and distal parts of the epithelium showed low mitotic rates similar to those in the OVX controls. However, a high mitotic rate was obtained in the vaginal epithelium NS: not significant. of OVX (180 d.) 17-OHP mice (Table 2). In contrast, mitotic rate in the epithelium of OVX (180 d.) 17-OHP-VA mice indicated much higher value in all parts of the vagina than did the groups of OVX (30 d. and 180 d.) 17-OHP mice (Table 2). Mitotic rates in the uterine epithelium of OVX (30 d. and 180 d.) 17-OHP and OVX (180 d.) 17- OHP-VA mice were approximately the same as in the OVX controls. DISCUSSION It has been established that neonatal treatment with relatively large doses of natural and synthetic estrogens such as estradiol-17§8 and diethylstil- bestrol (DES) causes permanent proliferation and cornification in the vaginal epithelium of mice ovariectomized at different early postnatal ages [for reviews, see 6, 7]. The permanent vaginal changes result in hyperplastic epithelial lesions including vaginal carcinomas in old mice [8, 9]. On the other hand, cervicovaginal lesions (adenosis and clear-cell or squamous-cell carcinomas) happen in human offspring of mothers given DES in the trimester of pregnancy for the prevention of pre- mature birth [23, 24]. Neonatal treatment with androgen or progestin caused the permanent changes in mouse vaginal epithelium [10—-13, 25]. However, the permanent vaginal changes were not observed in neonatally progesterone-treated mice when ovariectomized at 124 N. TAKASUGI AND T. KATO 60 days of age [26]. Kohrman [14] reported that neonatal injections of 17a-hydroxyprogesterone caproate (17-OHP) induced the permanent vaginal changes in 30% of ovariectomized mice examined at 60 days. In the present study, permanent pro- liferation of the vaginal epithelium was induced by neonatal 17-OHP injections in 210-day-old mice when ovariectomized at 180 days of age, but not in those ovariectomized at 30 days. This finding indicates that ovariectomy performed at an early postnatal period abolishes the occurrence of permanent vaginal changes in 17-OHP mice, suggesting the necessity of a relatively long period of stimulation by ovarian hormones, probably estrogen, for the induction of the permanent vaginal changes by 17-OHP. The previous studies revealed that the occurrence of permanent proliferation and cornificantion of the vaginal epithelium in neonatally estrogenized or androgenized mice was prevented by injections of vitamin A (VA) given simultaneously with neonatally administered sex hormones [16-21]. On the contrary, the present study demonstrated that mitotic rate and the number of both cell layers and hyperplastic epithelial downgrowths increased in the vaginal epithelium of 17-OHP-VA mice ovariectomized at 180 days, suggesting an acceleration by VA of the 17-OHP-induced, per- manent epithelial proliferation. Inasmuch as a relatively long period of the presence of ovaries is required for the induction of the permanent vaginal changes in 17-OHP mice, mechanism of the cellular alterations in the vaginal epithelium of neonatally progestinized mice appears different from that in neonatally estrogenized or androgenized mice. Accordingly, the mode of action of VA on the vaginal epithelium may be different between the progestinized mice and the estrogenized or andro- genized ones. It may be suggested, therefore, that neonatally administered 17-OHP and VA co- operatively increase the cellular responsiveness of the vaginal epithelium to the later stimulation of ovarian hormones, resulting in an acceleration of the permanent proliferation. It has been reported that 17-OHP has approxi- mately the same order of activity as progesterone, but the effective duration is 4-6 times longer than progesterone [27]. Johnson et al. has proposed 17-OHP for use in the prevention of premature birth [28]. In Japan, 17-OHP has been used with or without progesterone or estradiol derivatives for the prevention of threatened abortion [29]. On the basis of the present and previous studies [6-9, 23, 24], however, the possibility may be suggested that administration of 17-OHP in combination with vitamin A to the pregnant women enhances the risk of the hyperplastic vaginal lesions in their offspring, since in mouse vaginal epithelium, neoplastic or preneoplastic lesions have been produced by neonatally administered progestin [10-15]. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to N. T. REFERENCES 1 Takewaki, K. (1962) Some aspects of hormonal mechanism involved in persistent estrus in the rat. Experientia, 18: 1-6. 2 Barraclough, C. A. (1967) Modifications in repro- ductive function after exposure to hormones during the prenatal and early postnatal period. In ‘“‘Neuroendocrinology” Ed. by L. Martini and W. F. Ganong, Academic Press, New York, Vol. 2, pp. 61-99. 3. Takasugi, N., Kimura, T. and Mori, T. (1970) Irreversible changes in mouse vaginal epithelium induced by early postnatal treatment with steroid hormones. In ‘“‘Postnatal Development of Pheno- type”. Ed. by S. Kadzda and V.H. Denenberg, Academia, Prague, pp. 229-251. 4 Arai, Y. (1973) Sexual differentiation and devel- opment of the hypothalamus and steroid-induced sterility. In ‘“‘Neuroendocrine Control”. Ed. by K. Yagi and S. Yoshida, Univ. Tokyo Press, Tokyo, pp. 27-55. 5 Flerké, B. (1975) Perinatal androgen action and the differentiation of the hypothalamus. In “Growth and Development of the Brain”. Ed. by M.A. B. Brazier, Raven Press, New York, pp. 117-137. 6 Takasugi, N. (1976) Cytological basis for perma- nent vaginal changes in mice treated neonatally with steroid hormones. Int. Rev. Cytol., 44: 193- 224. 7 Bern, H. A. and Talamantes, F.J. (1981) Neo- natal mouse models and their relation to disease 10 11 12 13 14 15 16 17 18 Vitamin A and Hyperplastic Vaginal Epithelium in the human female. In “‘Developmental Effects of Diethylstilbestrol (DES) in Pregnancy’’. Ed. by A.L. Herbst and H. A. Bern, Thieme Strat- ton, New York, pp. 129-147. Dunn, T. B. and Green A. W. (1963) Cysts of the epididymis, cancer of the cervix, granualr cell myoblastoma, and other lesions after estrogen injection in newborn mice. J. Nat. Cancer Inst., 31: 425-45S. Takasugi, N. and Bern, H.A. (1964) Tissue changes in mice with persistent vaginal cornifi- cation induced by early postnatal treatment with estrogen. J. Nat. Cancer Inst., 33: 855-865. Takasugi, N. (1964) Persistent changes in vaginal epithelium of ovariectomized mice induced by early postnatal injections of testosterone or progesterone. J. Fac. Sci., Univ. Tokyo, Sec. IV, 10: 397-401. Jones, L.A. and Bern, H. A. (1977) Longterm effects of neonatal treatment with progesterone, alone and in combination with estrogen, on the mammary gland and reproductive tract of female BALB/cfC3H mice. Cancer Res., 37: 67-75. Ainslie, M.D. and Kohrman, A. F. (1977) The effect of 17a-hydroxyprogesterone caproate on vaginal development in mice: A_ preliminary report, abstracted. J. Toxicol. Environ. Health, 33/539. Jones, L.A. and Bern, H.A. (1979) Cervico- vaginal and mammary gland abnormalities in BALB/cCrgi mice treated neonatally with pro- gesterone and estrogen, alone or in combination. Cancer Res., 39: 2560-2567. Kohrman, A. F. (1978) The newborn mouse as a model for study of the effects of hormonal steroids in the young. Pediatrics, 62: 1143-1150. Jones, L. A. and Pacillas-Verjan, R. (1979) Trans- plantability and sex steroid hormone responsive- ness of cervicovaginal tumors derived from female BALB/cCrgl mice neonatally treated with ovarian steroids. Cancer Res., 39: 2591-2594. Mori, T. (1968) Effects of neonatal injections of estrogen in combination with vitamin A on the vaginal epithelium of adult mice. Annot. Zool. Japon., 41: 113-118. Mori, T. (1969) Further studies on the inhibitory effect of vitamin A on the development of ovary- independent vaginal cornification in neonatally estrogenized mice. Proc. Japan Acad., 45: 115-120. Yasui, T. and Takasugi, N. (1977) Prevention by vitamin A of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice. An electron microscopic study. Cell Tiss. Res., 179: 475-482. 19 20 21 ae 23 24 25 26 Pf | 28 29 125 Yasui, T., Iguchi, T. and Takasugi, N. (1977) Blockage of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice by vitamin A; Parabiosis and transplantation studies. Endocr. Japon., 24: 393-398. Iguchi, T. and Takasugi, N. (1979) Blockade by vitamin A of the occurrence of permanent vaginal changes in mice treated neonatally with Sa- dihydrotestosterone. Anat. Embryol., 155: 127- 134. Tachibana, H. and Takasugi, N. (1980) Resto- ration of normal responsiveness of vaginal and uterine epithelia to estrogen in neonatally estrogenized, A-vitaminized adult mice. Proc. Japan Acad., 56: 162-166. Forsberg, J.-G. (1963) Derivation and differen- tiation of the vaginal epithelium. Thesis, Univ. Lund, 1-197. Scully, R. E. and Welch, W. R. (1981) Pathology of the female genital tract after prenatal exposure to diethylstilbestrol. In ‘“‘Developmental Effects of Diethylstilbestrol (DES) in Pregnancy”. Ed. by A. L. Herbst and H. A. Bern, Thieme Stratton, New York, pp. 26-45. Herbst, A. L. (1981) The epidemiology of vaginal and cervical clear cell adenocarcinoma. In “Developmental Effects of Diethylstilbestrol (DEs) in Pregnancy”. Ed. by A. L. Herbst and H. A. Bern Thieme Stratton, New York, pp. 63- 70. Takasugi, N. (1979) Development of permanently proliferated and cornified vaginal epithelium in mice treated neonatally with steroid hormones and the implication in tumorigenesis. Nat. Cancer Inst. 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ZOOLOGICAL SCIENCE 1: 126-131 (1984) Mesencephalic Central Gray as Supraspinal Neural Substrates for Lordosis Reflex: Deprivation of Serotonergic Influence by p-Chlorophenylalanine KOREHITO YAMANOUCHI, YUMIKO NAKANO!, MASARU FUKUDA! and YASUMASA ARAI Departments of Anatomy and ‘Obstetrics and Gynecology, Juntendo University, School of Medicine, Hongo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT — Functional relationship between the mesencephalic central gray and serotonergic neurons in regulation of female sexual behaviors was examined in estradiol benzoate (EB)- pro- gesterone (P) primed ovariectomized rats. The mesencephalic dorsal central gray was destroyed bilaterally by a radiofrequency lesion generator. The females with central gray lesions (CGL) showed extremely low sexual receptivity, compared to sham operated females (CGL sham), both lordosis and soliciting behaviors being inhibited. One week after the first behavioral test, CGL females were divided into two groups. In one group, CGL females were injected with 100 mg/kg p-chloro- phenylalanine (PCPA, an inhibitor of serotonin synthesis) daily for 4 days simultaneously with EB—P priming (CGL-PCPA group). The rest of CGL females were treated with saline and EB—-P (CGL— saline group). CGL-saline females showed still low sexual receptivity in respone to EB—P priming. In contrast, CGL—PCPA females showed significantly higher sexual receptivity, compared to CGL-— saline females. The degree of lordotic response and soliciting behavior in these rats was statistically comparable to that of CGL sham group. Since PCPA was effective in facilitating the display of lordosis in the females with CGL, the focus of serotonergic influence on lordosis may locate outside © 1984 Zoological Society of Japan the midbrain central gray. INTRODUCTION There is a considerable body of evidence in- dicating that neural substrates for the supraspinal lordosis reflex exist in the lower brain stem. Electrical stimulation of the mesencephalic central gray facilitates the display of lordosis, whereas destruction of the same region suppresses lordosis response [1, 2]. In the pontine level, the lesions in the periventricular gray and adjacent area also caused a severe loss of lordosis but not of soliciting behavior [3, 4]. Other than these neural structures, midbrain or medullary reticular for- mation [1, 2, 5], the peripeduncular area [6, 7] and the lateral vestibular nucleus [8] have been reported to the involved in lordosis regulation. Accepted August 20, 1983 Received June 25, 1983 Furthermore, monoaminergic neuronal system is thought to be closely associated with lordosis mediating system [9]. Most of the cell bodies of these monoaminergic neurons are distributed in the central gray and tegmental area in the lower brain stem [10, 11]. Especially an inhibitory role of serotonergic neuronal system in expression of lordosis has been emphasized, because activation of serotonergic neurons by p-chloroamphetamine [12, 13] or stimulation of serotonin receptor by ergot alkaloids [12, 14] inhibits lordotic behavior, conversely the suppression of serotonin synthesis by p-chlorophenylalanine (PCPA) facilitates the display of lordosis in female rats [12, 15, 16, 33]. In the present study, in order to clarify the functional relationships between serotonergic neu- rons and the mesencephalic central gray which is one of the important components of the supraspi- nal lordosis regulating system, effect of treatment Midbrain Central Gray, Serotonin and Lordosis 127 with PCPA was examined in estrogen—progesterone primed ovariectomized rats following lesioning the dorsal central gray. MATERIALS AND METHODS Female Wistar rats (220-250 g) were housed under a controlled photoperiod (14 :10 hr, light: dark) and temperature (24-25°C). Twenty-one rats were ovariectomized and subjected to the behavioral test to confirm the lordosis facili- tating effect of PCPA [12, 15, 16, 33]. Two weeks after the ovariectomy, all females were treated with a subthreshold dose (0.5 wg) of sesame oil) estradiol benzoate (EB, dissolved in 0.1 ml daily for 3 days and 0.5 mg progesterone (P, in 0.1 ml oil) 4-6 hrs before the behavioral test on the fourth day. Simultaneously, varying doses (50-200 mg/kg, see Table 1) of PCPA (p-chlorophenylalanine methylester hydrochloride, Sigma, dissolved in 0.2-0.4 ml saline) or saline (0.2 ml) were injected intraperitoneally daily for 4 days during the period of EB—P priming. In the behavioral test, each female was placed in an observation cage with one or two vigorous males. The ratio of lordosis response to 10 mounts times 100 (lordosis quotient, LQ) was recorded in each rat. In addition, the incidence of soliciting behaviors (ear wiggling and hopping) was also recorded. Twenty-three females were ovariectomized and subje-ted to brain surgery. The dorsal central gray in the midbrain was destroyed bilaterally by radiofrequency lesion generator (Radionics Inc., Barlington, MA) under ether anaesthesia in 14 females (central gray lesion, CGL group) TABLE 1. (see Fig. 1). The electrode was lowered to the dorsal central gray according to Szentagothai et al. [17] with the stereotaxic coordinates: 8 mm posterior to the bregma, 6mm lower from the brain surface and | mm lateral to the midline and then current was applied. Nine females in which the electrode was lowered without current served as sham-operated control (CGL sham group). Two to three weeks after the operation, all fe- males were treated with 2 “g EB daily for 3 days and 0.5 mg P on the fourth day and tested. One week after the first test, CGL females were divided into two groups. Eight CGL females received 100 mg/kg PCPA intraperitoneally daily for 4 days simultaneously with EB-P priming (the same dose as in the first test) (CGL—PCPA group). The rest 6 CGL rats were treated with saline and EB-P (CGL-saline group). CGL sham females were also treated with EB—P alone in the second test. After the behavioral tests, each brain was examined histologically in order to determine the localization of lesions. LQs were analyzed by the analysis of variance (F test) and then Student’s t test or Chochran-Cox methods. For comparison of incidence of behaviors among the groups, y° test with Yates’ correction was used. RESULTS Effects of PCPA on lordosis response in EB-P primed ovariectomized rats are summarized in Table 1. High scores of LQ could not be obtained by treatment of 0.5 ~g EB-P in saline-injected ovariectomized rats, because of subthreshold dose Effect of p-chlorophenylalanine (PCPA) on female sexual behavior in estradiol benzoate (EB)-progesterone (P) primed ovariectomized rats Lordosis Ear wiggling Hopping ory ars rine LOLS. E incidence incidence Saline 5 5/5 64.0+6.8 3/5 1/5 50 mg/kg PCPA 5 4/5 62.0+.15.9 4/5 4/5 100 mg/kg PCPA 6 6/6 91.7+4.0* 4/5 5/5 200 mg/kg PCPA 5 5/5 86.6+6.2* 4/5 4/5 All females were treated with 0.5 »g EB daily for 3 days and 0.5 mg P on the fourth day. PCPA or saline (0.2 ml) was injected intraperitoneally daily for 4 days during EB-P priming. * P<0.05 vs saline group. 128 K. YAMANOUCHI ef al. of EB. The females injected with 50 mg/kg PCPA also showed low sexual receptivity. When the dose of PCPA was increased to 100 mg/kg, however, the mean LQ increased significantly compared to that of controls (P<0.05). Further increase in LQ was not seen in the females treated with 200mg/kg PCPA. They became rather aggressive against males, but most of them showed ear wiggling and hopping. Figure 2 illustrates effects of mesencephalic cen- tral gray lesions on female sexual behaviors in EB-P and PCPA treated females. In the first test, the mean LQ of CGL sham females was 65.6+12.5 in response to 2 ug EB-P. Most of oO CGL Sham CGL-Saline [J caL-pcra 100 50 LQ (lordosis quotient) 0 Incidence Lordosis 8/9 9/9 0/6 1/6 Ear wiggling 4/9 6/9 0/6 0/6 0/8 2/8 Hopping 4/9 1/9 0/6 0/6 0/8 3/8 Fic. 2. Lordosis and soliciting behaviors in females with mesencephalic central gray lesions (CGL) or sham operation (CGL sham) in the first (left column in each group) and second (right column) tests. Vertical bar at the top of the column indi- cates S.E. In the first test, all females were treated with 2 ug estradiol benzoate (EB) daily for 3 days and 0.5 mg progesterone (P) on the fourth day. In the second test, simultaneously with EB-P pri- ming (the same dose as in the first test), 100 mg/kg p-chlorophenylalanine or saline was injected daily for 4 days in 8 and 6 CGL females, respectively. Sham females received EB-P priming alone. 2/8 6/8 Fic. 1. Cross sections of the midbrain from a female with large central gray lesions (Rat No. 64) at 4 different levels between just posterior to the me- dial geniculate body and inferior colliculus. The hatched areas show the lesions. This female did not showed female sexual behavior (LQ=0) fol- lowing the treatment with 2 wg estradiol benzoate (EB) daily for 3 days and 0.5 mg progesterone (P) on the fourth day. However, daily injections with 100 mg/kg p-chlorophenylalanine for 4 days during the period of EB-P priming effectively potentiated the display of lordosis, the LQ of this rat being 80. CP: cerebral peduncle, IC: inferior colliculus, IP: interpeduncular nucleus, MGB: medial geni- culate body, P: pons, SC: superior colliculus, SCP: superior cerebellar peduncle, III: nucleus of the oculomotor nerve. Midbrain Central Gray, Serotonin and Lordosis 129 them exhibited soliciting behaviors such as ear wiggling and hopping. In contrast, only 2 out of 14 CGL females displayed lordosis and the mean LQ was 1.4+1.0. None of them showed soliciting behaviors. In the second test, CGL sham females showed high scores of LQ (Mean; 87.8+9.8). However, CGL-saline females still showed a low incidence of lordosis, the mean LQ (6.7+6.7) being not significantly different from that in the first test (0.0+0.0). In contrast to the CGL-saline females, PCPA effectively facilitated lordosis in CGL females. Six out of 8 PCPA treated CGL females displayed lordosis. The mean LQ (60.0+ 13.6) was significantly higher than that in the first test (2.5+1.6) and that of the CGL-saline group (P<0.01). Ear wiggling and hopping were observed in 2 and 3 out of 8 rats, respectively. Histological examination showed that the cen- tral gray lesions were located between the level just posterior to the medial geniculate body and the level of the dorsal tegmental nucleus (Fig. 1). The dorsal part of the mesencephalic central gray and adjacent area were destroyed. The lesions invaded to the ventral part of the central gray in 8 CGL females. In the rest 6 CGL females, the ventral part of the central gray and dorsal teg- mental region seemed to be intact. DISCUSSION It has been suggested that the supraspinal mech- anisms for lordosis reflex in the lower brain stem are controlled by the forebrain inhibitory and facilitatory influences [4, 18, 19]. The inhibitory signals for female sexual behaviors are thought to be delivered from the septal area and preoptic area, because lesions in the preoptic area [13, 20— 22] or by transection of the dorsal afferents of the preoptic area [19, 23] facilitated lordosis and soliciting behaviors. On the other hand, island isolation of the ventromedial hypothalamus [18, 24, 25] or lesions in the ventromedial nucleus [4, 26-29] suppressed both lordosis and soliciting behaviors. The ventromedial hypothalamus is considerd to be the origin of female sexual be- havior facilitating signals. Our observation, that destruction of the dorsal central gray in the midbrain effectively sup- pressed the display of lordosis and _ soliciting behaviors, confirms the concept that the dorso- lateral central gray is an important supraspinal neural structure for lordosis reflex [30]. Since electrical stimulation to the ventromedial hypo- thalamus could not facilitate lordotic response in the females with dorsolateral central gray lesions [2], the neural elements in this area may participate in neural circuit for reflex arc of lordosis. In the present study, however, treatment of CGL females with PCPA could effectively re- store lordotic activity. Since the treatment with 100 mg/kg PCPA daily for 3 days depletes brain serotonin to 11% of the control level [31], the facilitatory effect of PCPA on lordosis observed in our females with or without CGL seems to be due to deprivation of serotonergic inhibiting influence on the lordosis mediating system. The fact that PCPA could induce lordosis response in the absence of the neural substrates of the midbrain central gray suggests that this neural substrate may not be included in the minimal neural requirement for the display of lordosis. In addition, serotonergic inhibition does not seem to be focused on this area. Another important point in the present study concerns the impairment of soliciting behaviors. In CGL females, both lordosis and _ soliciting behaviors were inhibited. This suggests that the midbrain central gray plays a critical role in expression of both female sexual behaviors. There is suggestive evidence that regulatory mechanism involved in soliciting behaviors is also associated with that of lordosis at the fore- brain level [23, 24]. However, a clear disso- ciation between the display of lordosis and so- liciting behavior has been reported in the females with pontine central gray lesions [3, 4]. A similar dissociation phenomenon has been found when the ventral noradrenergic fiber bundle was inter- rupted at the pontine level [32]. In these females, lordotic response was completely suppressed, but soliciting behaviors were still observed. 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ZOOLOGICAL SCIENCE 1: 132-147 (1984) New and Unrecorded Oribatid Mites from Amami-Ohshima Island, Southwest Japan JUN-ICHI AOKI Institute of Environmental Science and Technology, Yokohama National University, Yokohama 240, Japan ABSTRACT — Soil inhabiting oribatid mites collected on Amami-Ohshima Island, Southwest Japan, contained one new genus, Defectamerus, and twenty new species and subspecies. These are Euphthiracarus aggenitalis, Allodamaeus striatus, Defectamerus crassisetiger, Nellacarus lunaris, Liacarus montanus, Cultroribula angulata, Austroceratoppia japonica, Nippobodes brevisetiger yuwanensis, Acrotocepheus curvisetiger, Allosuctobelba bicuspidata, A. tricuspidata, Dometorina tuberculata, Symphauloppia plana, Scheloribates decarinatus, Ischeloribates lanceolatus, Perschelori- bates clavatus torquatus, Peloribates levipunctatus, P. moderatus, Galumna granalata and Pergalumna © 1984 Zoological Society of Japan amamiensis. Other three species, Pedrocortesia hardyi (BALOGH), Microtegaeus borhidi BALOGH et MAHUNKA and Parakalumma robustum (BANKS), are reported for the first time from Japan. INTRODUCTION Amami-Ohshima is an island of special interest in zoogeography, being located nearly on the borderline between the subtropical and warm- temperate zones of Japan. Aoki [1-6] recorded 28 species of oribatid mites from the island, but they are apparently a small portion of the rich oribatid fauna. Before an adequate discussion is made on the characteristic feature of the oribatid fauna, it is necessary to describe a number of new and unrecorded species found in the island. All the type-specimens of the new taxa described here will be deposited in the collection of the National Science Museum (Nat. Hist.), Tokyo. COLLECTING DATA Soil and litter samples were all collected on Amami-Ohshima Island (Lat. 28°20’ N and Long. 129°30’ E) at the following 12 stations. Collector: J. Aoki. AMA-1 : Ayamaru-Misaki in Kasari-cho, 8— II-1980. AMA-2 : Tonzaki in Tatsugo-mura, 8—II-1980. Accepted August 17, 1983 Received June 24, 1983 AMA-3 : S of Ohtana in Yamato-mura, 9-II-— 1980. AMA-4 : Near Fukumoto in Yamato-mura, 9-II-1980. AMA-6 : N of Mt. Eboshi in Setouchi-machi, 9-II-1980. AMA-8 : Sancho Park of Koniya in Setouchi- machi, 9-IJ-1980. AMA-9 : Uken-son, 9-II-1980. AMA-10: Near the summit of Mt. Yuwan, 10- II-1980. AMA-11: W of the summit of Mt. Yuwan, 10— II-1980. AMA-12: Mt. Yuwan, 10—II-1980. AMA-14: E of Nishinakama in Sumiyo-mura, 10-II-1980. AMA-15: Near Fukumoto in Yamato-mura, 9-II-1980. Euphthiracarus aggenitalis sp. 0. (Fig. 1) | Length of notogaster 460-480 wm, height of notogaster 320-330 wm, length of aspis 255-260 ym. [Aspis] Two lateral carinae on each side; the upper one strong, the lower one weak and indis- tinct. Setae Je and in sparsely barbed. Seta ro nearly smooth, with tip curved inward. Their relative lengths: in>/e>ro; mutual distance: in- New Oribatids from Amami-Ohshima 133 in>ro-ro> le-le. Sensillus weakly thickened api- cally in a fusiform and slightly barbed. Prodorsal surface finely and densely punctured. [Noto- gaster] Thirteen pairs of notogastral setae thin, only weakly barbed, shorter than ro. Notogastral surface finely punctured as on aspis. [Genito- anal region] Nine pairs of genital setae; g, and g, thick and long, g; and g, medium-sized, g; and g. minute. Median aggenital seta (ag.) very thick and long, thicker than in, almost reaching insertion of g2; on the contrary, ag, minute, being about 1/8 as long as ag». Two pairs of anal setae and 4 pairs of adanal setae nearly of equal length; setae an strongly curled at tip. [Legs] Monodactyle. [Type-series] Holotype (NSMT-Ac 9552): AMA- 2; 2 paratopotypes: AMA-2. The new species is easily distinguishable from any other species of the genus Euphthiracarus by the great difference in length between aggenital setae (ag, and ag.) and finely pitted body surface. Allodamaeus striatus sp. Nn. (Fig. 2) 690-740 x 410-430 wm. [Prodorsum] Setae /e long, inserted rather dorsally and about 2.3 x as long as their mutual distance. Setae in minute and, in dorsal view, only their setal pores discerni- ble. Sensillus with a barbed head which appears to be weakly or only slightly swollen according to aspects, because it is somewhat flattened distally. On the posterior part of prodorsum between bothridia found a short median and a pair of lat- eral arched rigdes. [Notogaster] Almost circular in dorsal view, showing a weak unevenness on the posterior margin. Indistinct ribbon-shaped striae running longitudinally on the dorsal side. Only two pairs of short setae found near the posterior margin; the anterior setae 1.5 as long as the posterior ones. [Ventral side] Genital plate with 5 setae; the anterior 4 setae situated with shorter intervals. Anal plate with 2 setae. Distance ad,- ad, about 2.5 as long as ad,-ad;. [Type-series] Holotype (NSMT-Ac 9519): AMA-8; 6 paratopo- types: AMA-8. The new species is closely related to A. adpressus Aoki et Fujikawa, 1971 [7], but the latter is different from the new species in the posterior two pairs of notogastral setae strikingly different in length from each other. Tarsus I is longer than tibia I and tarus IV is subequal in length to tibia IV in A. adpressus, while tarsus I is subequal! to tibia I and tarsus IV is shorter than tibia IV in A. striatus. RLN (relative length to notogaster) of the segments (Fe, Ge, Ti and Ta) of legs I and IV — I (61, 14, 35 and 41) and IV (55, 16, 39 and 39) in A. adpressus; I (70, 18, 42 and 41) and IV (70, 18, 51 and 42) in A. striatus. Pedrocortesia hardyi (Balogh, 1968), comb. n. (Fig. 3) Pedrocortesella hardyi Balogh, 1968, p. 262, p1. 2, figs. 9-10. [8] 540-560 x 300-320 ~m. [Prodorsum] Prodorsal surface wholly covered with foveolae of variable shape; they are oval, elliptical or sometimes slit- like. A ridge running in anterior direction from bothridium. Another ridge found in more lateral position. Setae ro and /e long and incurved. Seta in minute. Sensillus gradually thickened apically and strongly barbed. [Notogaster] Median part of notogaster weakly swollen longitudinally along the anterior half of median line. Notogastral margin fairly uneven, especially in the posterior part; a pair of knots at the posterior end are marked. All the surface covered with foveolae which are never complete circles, but broken circles, arches or slits. Three pairs of lyrifissures are detected; im located posteromedially of latero- abdominal gland opening. Notogastral setae difficult to see; only 5 pairs of setae are detected posteromarginally. [Ventral side] Seven pairs of genital and 2 pairs of anal setae found; an, and an, Situated very close together. A pair of ag- genital setae located on the level of posterior margin of genital opening. Only 2 pairs of adanal setae. An arched ridge found in front of genital opening. Genital, anal, ventral and epimeral plates all covered with foveolae as on prodorsum and notogaster. [Legs] Heterotridactyle. [Mate- rial examined] 10 exs.: AMA-1. The Japanese material is well in accord with the original description, except for the absence of adanal fissures and the presence of 2 pairs of adanal setae. Distribution: New Guinea and Japan. 134 J. AOKI Roo aaa ta Goa Wario (718 >) ORCS Qe YxCC® [ eG CGI IO WP (eey ( 5 Ss Gee (OPS utYic¢ c ok (cus (Ae, Cre ME tare Sus © u Q9°¢ C6) WUC > KC OC Se CGD i Gee) AK SOC SH) ah (ie See 7 Y “Se ( err. ~ A~/) aS Nh t Sad cana y, h . NE ee lt ape P ~ ay Noa Zs aS x Ctr aN New Oribatids from Amami-Ohshima 135 Defectamerus gen. n. [Diagnosis] Notogaster with only 8 pairs of setae. Bothridia not so near to each other. Setae in strong. Rostrum with 2 incisions. Notogaster with humeral projections (cristae). Genital aper- tures with 6 pairs of setae. Legs monodactyle. Type-species: Defectamerus crassisetiger sp. n. By the number of notogastral setae the new genus is easily distinguishable from the two known genera of the family Ameridae, Amerus and Cristamerus, which have 10 pairs of setae on notogaster. Defectamerus crassisetiger sp. Nn. (Fig. 4) 610-660 x 380-450 wm. [Prodorsum] Rostrum with 2 incisions; the median projection between them usually with a small indentation. Seta ro long, with a fine tip. Setae /e and in strong, slight- ly roughened; /e arising from an apophysis on an arch-shaped ridge which is provided posteriorly with a triangular projection; in inserted on an oval apophysis; relative lengths of the prodorsal setae: in>le>ro. Bothridium well separated from each other; each bothridium with a sharp lateroposterior projection and a short and weak anterior ridge. Sensillus strong, as thick as seta in, finely barbed. Seta ex fine and short. [Notogaster] Dorsosejugal suture lacking; two weak transverse lines are interior structures. Humeral projection with a short protrusion on the median side. Only 8 pairs of notogastral setae are present; fe, r. and r; markedly longer and thicker than the remaining setae; their relative lengths —r.2:r,: te=1: 1.6: 2.5—2.8; ta, te, p:, p2 and p; short and fine; setae ms and r; lacking. [Ventral side] Genital plate with 6 setae, the anteriormost one being the long- est. Three pairs of adanal setae thicker than genital, aggenital or anal setae. Adanal fissure aligned parallel to the lateral margin of anal open- ing, being remote from the margin for a distance longer than its own length. A _ dark-colored preanal plate exists. Both genital and anal aper- tures surrounded each by an obscure shield-shaped rim. Epimeral chaetotaxy: 3-1-3-3. [Legs] Total chaetotaxy (Fe—Ge-Ti-Ta): I(44-6-22), I1(44-5-16), III(2—2-4-15), IV(2-2-4-11). Sole- nidial chaetotaxy: I(0—1—2-2), II(0—-1-1-2), III(O- 1-1-0), IV(O-O-1-0). Setae p of legs II-IV short, thick and _ conical. [Type-series] Holotype (NSMT-Ac 9532): AMA-2; 12 paratopotypes: AMA-2. Nellacarus lunaris sp. n. (Fig. 5) 260-270 x 180-200 wm. [Prodorsum] Rostrum pointed at tip. Anterior tip of lamella with a short dent on each side and a shallow concavity between the dents, in which a sword-shaped lamellar seta is inserted. Median margins of lamellae irregularly undulating posteriorly, forming a spindle-shaped interspace between lamellae. In the posterome- dian part of each lamella found a crescent-shaped structure. A short, fine seta in located near the basal part of lamella, outside the crescent. Seta ro inserted each on a distinct apophysis and strongly curved inward, so that they are crossing each other. Sensillus bending posteriorly, being densely barbed on the outer side. [Notogaster] Humeral projection with some longitudinal striae and small granules on the outer side. The poste- rior margin has 3 small indentations, one medially between setae p, and the other two each in front of seta p;. Ten pairs of notogastral setae short and fine. [Ventral side] Genital opening pentag- onal, with 6 pairs of setae, of which one pair of setae are inserted close to the lateral margins; the anteriormost pair of genital setae much longer than the remaining setae. Adanal fissure very long, situated close and parallel to the lateral margin of anal opening. [Type-series] Holotype (NSMT- Ac 9514): AMA-2; 7 paratopotypes: AMA-2, 2 paratypes: AMA~-9. Nellacarus hellenicus Mahunka, 1977 [9], from Greece seems to be most similar to N. lunaris sp. Fic. 1. 1d. Aspis. Fic. 2. Allodamaeus striatus sp. n. Fic. 3. Pedrocortesia hardyi (Balogh, 1968). Fic. 4. Defectamerus crassisetiger gen. et sp. n. 2a. Tarsus and tibia of leg IV, Euphthiracarus aggenitalis sp. n. 1a. Sensillus, 1b. Ano-genital region, Ic. Notogastral seta, 2b. Genital plates. 4a. Ventral side. 136 n., but it differs from the new species in (1) the shorter lamellar setae, (2) the humeral projections with toothed tip and without granular structure, (3) the absence of large crescent-shaped structures in the basal part of lamellae, and (4) the small body size (230-240 pm). Microtegaeus borhidi Balogh et Mahunka (Fig. 6) Microtegaeus borhidi Balogh et Mahunka, 1974 [10], Pe seis 2: 225x150 wm. [Prodorsum] Median field of prodorsum coarsely reticulated. Two pairs of obscure small knobs found posteriorly. Most part of lamella finely granulated. Setae ro and le smooth, the former being thicker than the latter. Seta in minute. Sensillus provided apically with dense papillae. [Notogaster] Wholly covered with polygonal reticulation as on prodorsum. Ten pairs of minute notogastral setae exist; the poste- riormost 3 pairs not visible from above. Humeral projections weakly developed. [Ventral side] Ven- tral plate with polygonal surface structure except for the 8-shaped area around genital and anal Openings. Genital and anal plates smooth, the former with 5 pairs and the latter with 2 pairs of setae. Epimeral plates very finely granulated; some light spots found in part. [Legs] Mono- dactyle. [Material examined] 5 exs.: AMA-2, 1 ex.: AMA-3, 1 ex.: AMA-4. Distribution: Cuba and Japan. Liacarus montanus sp. n. (Fig. 7) 700-830 « 448-535 wm. [Prodorsum] Rostrum with a triangular tooth on each side. Lamellar cusp bearing 2 apical teeth; the outer tooth longer than the median one. Interspace between lemellae narrow, but at the basal part it forms a rounded space of a characteristic shape. Setae ro, le and in smooth; their relative lengths — ro: le: in=2: 3: 5. Sensillus bearing a spindle-shaped head with a short sharp apical tip; the basal 1/3 of sensillus J. AOKI hidden under notogastral shield. Lateral part of prodorsum in the posterior half covered with dense granules. [Notogaster] Anterior margin weakly concave medially. The posteriormost setae Pp: 0.7-0.8 x as long as their mutual distance. [Ventral side] Genital plate with 5 setae. Adanal fissure iad aligned obliquely, being located at a level posterior to an,. Sternal ridge found only in epimerata I and II. [Legs] MHeterotridactyle. [Type-series] Holotype (NSMT-Ac a AMA-— 11, 1 paratype: AMA-12. The new species is readily distinguishable from any other congener by its peculiar shape of the basal part of lamellar interspace. Cultroribula angulata sp. n. (Fig. 8) [Prodorsum] Rostrum tricuspid- ate. Seta ro less than half as long as seta Je. Seta in are absent. Distal 2/3 of sensillus broadened, being provided apically with 6-8 long spines. [Notogaster] Dorsosejugal suture nearly straight. Humeral projection markedly developed, wtih a rounded anterior corner and a lateral angulation. Nine pairs of minute notogastral setae discernible; setae r; not detected. [Ventral side] Genital aperture square-shaped, as long as wide, with 4 pairs of setae. Anal aperture a little longer than wide, being somewhat wider than genital aperture. Interspace between both the apertures 4/9 as long as genital one. Sternal ridge developed only in the posterior half of epimerata I. [Legs] Monoda- ctyle. [Type-series] Holotype (NSMT-Ac 9530): AMA-8; 1 paratopotype: AMA-8. The new species is quite different from any other congener in the large and angular humeral projec- tions and the sensilli with many long spines apically. 170 x 100 pm. Austroceratoppia japonica Sp. n. (Fig. 9) 470-480 « 290-300 wm. Hammer [11] estab- lished the genus Austroceratoppia with A. dentata from Java as the type-species. She compared her Fic. 5. Nellacarus lunaris sp.n. 5a. Ventral side. Fic. 6. Microtegaeus borhidi Balogh et Mahunka, 1974. Fic. 7. Liacacrus montanus sp. n. Fic. 8. Cultroribula angulata sp.n. 8a. Ventral side. Fic. 9. Austroceratoppia japonica sp.n. 9a. Ventral side, 9b. Rostrum and rostral seta. New Oribatids from Amami-Ohshima | 137 138 species with Ceratoppia crassiseta Balogh et Mahunka, 1967, from Vietnam and showed several differences between them. Although the Vietnam- ese species was not included in her genus, it should be a member of Austroceratoppia, because it bears all of its characteristic features. The third mem- ber of the genus, A. japonica sp. n., is distinguish- able from Austroceratoppia crassiseta, comb. n., and A. dentata by its markedly long adanal setae, especially ad,, which are 3.6—-4.2 x as long as their mutual distance and often subequal in length to notogastral setae. The shape of median rostral projection (Fig. 9b) is also peculiar and different from any of those in the other two species. The number of lateral rostral teeth is 4 on each side. Setae ro 1.2 and seta Je 1.5x as long as their mutual distances, respectively. Lamellar cusp bearing 2 teeth. Anterior margin of notogaster weakly arched. [Type-series] Holotype (NSMT- Ac 9577): AMA-~10; 8 paratopotypes: AMA-8. Nippobodes brevisetiger yuwanensis subsp. n. (Fig. 10) 515-655 x 340-438 ym. The new subspecies differs from the nominate one from the Ohsumi Islands only in the shape and length of body setae. In N. brevisetiger yuwanensis, notogastral setae are barbed and longer (p,=11.9-12.2% of body length), while in N. brevisetiger brevisetiger Aoki, 1981 [12], they are smooth and shorter (p,=7.7% of body length). Setae ms are 18.5-21.3 % of body length in the new subspecies. The lengths of pro- dorsal setae are similar between the two forms, but the setae are all distinctly barbed in the new sub- species, while they are all smooth in the nominate subspecies. [Type-serjes] Holotype (NSMT-Ac 9555): AMA-14; 4 paratopotypes: AMA-14. Acrotocepheus curvisetiger sp. n. (Fig. 11) J. AOKI 1110-1150 x 450-500 wm. [Prodorsum] Lateral side with a narrow spa. /. Lamellae almost parallel and fairly close to each other, the anterior tip ending in a small sharp point (Fig. 11a). Margin of ventral bothridial plate broadly rounded. Lat- eral prodorsal condyle lobe-shaped; median pro- dorsal condyle small and triangular. Seta ro distinctly and densely barbed on the outside, 22—25 in RLN. Seta /e strongly curled at tip; /e and in almost glabrous, 27-29 in RLN, only slightly and sparsely barbed; in blunt at tip, while Je sharply pointed. Sensillus weakly clavate, the apex being not completely rounded, but with a very short point (Fig. 11b). [Notogaster] Lateral notogastral con- dyle bearing a distinct median projection which is separated from the main part by a clear indentation on the anterior margin; interspace between them 12-13 in RLN. Ten pairs of notogastral setae rather blunt at tip, weakly and sparsely barbed (Fig. llc and 11d), 25-34 in RLN; the posterior pairs (r- and p-serieses) sometimes strongly curled or bent. Lyrifissure im situated close and posterior to lateroabdominal gland opening; a weakly curved, longitudinal ridge found just inside gla. Notogastral surface covered by indistinct foveolae. [Ventral side] Genital plates distinctly darker in color than ventral plate, bearing 4 pairs of long setae. Three pairs of adanal setae long, curled and more distinctly barbed than notogastral setae. Ventral plate foveolate. Most part of epimerata covered by somewhat dark-colored, pebble-like structures. [Legs] Monodactyle. Type of ultimate setae: L-S-S-S. Solenidia w, and mw, on tarsus I and ¢, on tibia I similar in length and shape, being curved downward at tip. Genua J and II each with a very strong bristle. [Type-series] Holotype (NSMT-Ac 9574): AMA-10; 2 para- topotypes: AMA-—10. The most characteristic feature of A. curvisetiger is the strongly curved setae on the posterior half Fic. 10. Nippobodes brevisetiger yuwanensis subsp. n. Fic. 11. Acrotocepheus curvisetiger sp. n. gastral setar,, 11d. Notogastral seta ry. Fic. 12. Allosuctobelba bicuspidata sp. n. Rostrum and rostral setae. Fic. 13. Allosuctobelba tricuspidata sp. n. Fic. 14. Dometorina tuberculata sp. n. Fic. 15. Symphauloppia plana sp.n. 15a. Sensillus, lla. Tip of lamella in lateral view, 12a. Ventral side, 13a. Sensillus, 10b. Notogastral seta p>. 11b. Sensillus, 11c. Noto- 10a. Rostral seta, 12b. Sensillus and bothridium, 12c. 13b. Ventral side. 15b. Ventral side. 139 New Oribatids from Amami-Ohshima 140 J. AOKI of notogaster. Lateral notogastral condyles of A. consimilis Balogh, 1970 [13], seem to be most similar in shape to those of the new species, but the former is distinguishable from the latter in the far longer body setae. The only known species of Acrotocepheus in Japan, A. gracilis Aoki, differs from the new species in the short notogastral setae (RLN: 7-14). Allosuctobelba bicuspidata sp. n. (Fig. 12) 424 470 x 260-290 wm. [Prodorsum] Rostral margin with a pair of projections rather blunt at tip and without lateral teeth (Fig. 12c). Seta ro almost smooth, being inserted on a median ele- vation. Anterior part of prodorsum with a flat marginal area on each side. Setae /e smooth, 3 X as long as their mutual distance, each being in- serted on a distinct rounded chitinous knob. Seta in less than half as long as Je. Sensillus bearing a fusiform head with a short pointed tip and minute barbation (Fig. 12b). [Notogaster] Ten pairs of notogastral setae rather strong and sharply pointed at tip; most of the setae with an indistinct chitinous ring around their insertion pores; seta ti located at a level a little anterior to fe; setae r, close to each other. [Ventral side] Genito-anal chaetotaxy: 6-1-2-3. Adanal setae distinctly longer than the remaining genito-anal setae. Anal seta an, in- serted near the mid-distance along the length of anal plate and close to an. Epimeral setal formula: 3-1-2-3. Apodemata IV well developed, running along circumgenital ridge. [Legs] Mono- dactyle. [Type-series] Holotype (NSMT-Ac 9572): AMA-11; 1 paratype: AMA-12. Allosuctobelba tricuspidata sp. n. (Fig. 13) 545 x 330 ym. [Prodorsum] Rostral tip narrow, with a projection on each side. No lateral teeth. Seta ro strongly curved inward and distinctly barbed on the outside. Setae Je smooth, 2.3 x as long as their mutual distance, being inserted each on a thick chitinous ridge. A pair of longitudinal, curved ridges well developed. Bothridium with a small posterior lobe. Sensillus bearing a fusiform head with a long pointed tip minutely barbed (Fig. 13a). [Notogaster] Ten pairs of notogastral setae long and flagelliform; seta fi inserted at a level posterior to fe; seta r; inserted near the posterior margin, not anterior to pi; setae ms and r3 Situated nearly on the same level. [Ventral side] Genito-anal chaetotaxy: 6-1-2-3. Anal setae (an, and an2) inserted close to lateral margin of anal plate. Adanal fissure iad located at a level near to the anterior margin of anal opening and aligned obliquely. Apodemata IV running along circumgenital ridge, its anterior part ex- tending forward to be connected with apo.sj; in the area between these anterior extentions of apo.4 found no distinct sternal ridge. [Legs] Monodactyle. [Type-series] Holotype (NSMT- Ac 9570): AMA-6; 9 paratypes: AMA-9. Key to the Three Japanese Species of the Genus Allosuctobelba 1. Sensillar head with a short pointed tip. Rostral setae almost smooth. Anal setae inserted close to lateral margin of anal plate. Body length: 424-470 um SACL ee A. bicuspidata sp. n. — Sensillar head with a long pointed tip. Rostral setae barbed. Anal setae inserted nearly on the mid-line of anal plate 2. Rostral margin with 1 projection on each side. Notogastral setae long and flagelliform. Seta ti inserted at a level posterior to fe. Body length: 545 wm ............ A. tricuspidata sp. n. — Rostral margin with 2-3 teeth on each side. Notogastral setae rigid. Seta fi inserted in anterior level to te. Body length: 410-460 wm bash sialon dks tet catered erent A. grandis (Paoli) Dometorina tuberculata sp. n. (Fig. 14) 350-375 x 190-217 wm. [Prodorsum] Rostral tip rounded. Setae ro, le and in long and thin, min- utely and sparsely barbed; setae in 1.5 x as long as ro and twice as long as their mutual distance. A curved prolamellar ridge not reaching setal in- sertion of ro, but ending at its posterolateral part. Basal part of lamella producing a small angulation on the outer margin. Bothridium completely con- cealed under notogastral shield, only exposing globular head of sensillus. [Notogaster] Elongate oval, 1.4 as long as wide. Two distinct indents New Oribatids from Amami-Ohshima 141 found on the posterior end of notogaster. Ten pairs of notogastral setae short and fine; setae p, inserted near the bottom of the posterior notches. [Ventral side] Genito-anal chaetotaxy: 4 1-2-3. Seta ad; nearly level with the anterior margin of anal opening. [Legs] Each legs provided with 3 strong claws, of which the median one is the strongest. The number of setae on femora I-II- III-IV: 5—S—3-2. Keels very poorly developed on femora II-IV. [Type-series] Holotype (NSMT- Ac 9505): AMA-9; 3 paratopotypes: AMA-9. The new species is readily distinguishable from the other species of Dometorina by the indentation on the posterior end of notogaster. Symphauloppia plana sp. n. (Fig. 15) 300-330 x 128-140 wm. [Prodorsum] Lamellae almost straight and of the same thickness through- out their length, being weakly converging -in the anterior direction. Setae ro and /e as long as their mutual distances. Seta in a little shorter than /e. Distal part of sensillus strongly swollen, minutely barbed and directed upward. Bothri- dium with a narrow lateral lobe of rounded triangular shape. A thin ridge connecting inser- tion pore of in and dorsosejugal suture. [Noto- gaster] Arched dorsosejugal suture very weak and indistinct. Anterolateral margin of notogaster very weakly undulating, producing two slight protrusions; just inside the anterior protrusion found a small protuberance. Four pairs of areae porosae small and circular. Twelve pairs of notogastral setae short and fine; they become comparatively longer in the posterior part of notogaster. [Ventral side] Genito-anal chaetotaxy: 3-1-2-3. Seta ad, located at a level between an, and an., ad, nearly on the level of anterior margin of anal opening, and ad, far anterior to the margin. Apodemata apo. 2 and apo. sj distinct; apo. 3 short. Epimeral setae 3a arranged longitudinally. [Legs] Heterotridactyle. [Type-series] Holotype (NSMT- Ac 9558): AMA-15; 7 Paratopotypes: AMA-15. In addition to the type-species, Sympahuloppia interrupta (Jeleva, 1962) [14] from Bulgaria, Para- phauloppia cordylinosa Higgins et Woolley, 1975 [15] from U.S.A. should be included in the genus Symphauloppia, because it has more than 10 pairs of notogastral setae (the authors mentioned 11 pairs in their description, but they showed 12 pairs in their figure) and 3 pairs of genital setae. Sym- Phauloppia cordylinosa (Higgins et Woolley), comb. n., differs from the new species in the barbed prodorsal setae and the stouter notogaster. S. interrupta is distinguishable from the new species by the interrupted dorsosejugal suture, the distinctly barbed sensilli and the reticulate structure around genital opening. The. anterior position of setae ad, and the presence of humeral protuberances must be features peculiar to the new species. Scheloribates decarinatus sp. n. (Fig. 16) 332-428 x 230-293 wm. [Prodorsum] _— Rostral tip rounded. Transverse prolamellar line and translamellar line are present; the latter with a median V-shaped incision. Seta /e and in nearly equal in length. Sensillus bearing a head weakly barbed and with a rounded apex in most cases. [Notogaster] Ten pairs of setal pores and 4 pairs of sacculi found on rather broad notogaster. Seta ms and sacculus S, very close together. A small round spots resembling areae porosae found posterolateral to seta fi, posterior to Sa. [Ventral side] Genito-anal chaetotaxy: 4-1—2-3; ad; preanal in position. Apo.3 short, not meeting apo.sj. [Legs] All legs tridactyle. The number of setae on femora I-II-III-IV: 5-4-3-2. Keel developed moderately on femur II and weakly on femora III and IV (Fig. 16c-e). A dark ridge found on the basal half of femora I and II. Trochanter III with 2 setae inserted close to each other; trochanter IV with a single seta, distal margin bearing 2 sharp dorsal teeth and | ventral tooth (Fig. 16e). [Type- series] Holotype (NSMT-Ac 9562): AMA-15; 19 paratopotypes: AMA-15. Scheloribates decarinatus is very similar to S. praeincisus (Berlese, 1910) [16], especially the form A of Corpuz-Raros [17] from the Philippines. All the forms of S. praeincisus described by Hammer [18] and by Corpuz-Raros [17] have prolamellar ridges, while the new species is completely lacking in the prolamellar ridges (Fig. 16f ). The presence of area porosa-like spot behind Sa and the two sharp dorsodistal teeth on trochanter IV may also be characteristic of the new species. 142 J. AOKI New Oribatids from Amami-Ohshima Ischeloribates lanceolatus sp. n. (Fig. 17) 314-350 x 197-210 um. [Prodorsum] ___ Pro- lamellar ridges and a faint prolamellar transverse line are present. Translamellar line very short. Setae ro, /e and in all weakly barbed; their relative lengths — ro: Je: in=1: 1.2: 1.4; in 1.7 x as long as their mutual distance. Sensillus with a slender lanceolate head bending backward and barbed; the pointed apex short; the organ appears to be evenly swollen on both sides in dorsal view (Fig. 17 and 17a, left). [Notogaster] Anterior margin arched. Ten pairs of minute notogastral setae and 4 pairs of sacculi are present. [Ventral side] Genito-anal chaetotaxy: 4-1-2-3. Seta ad; located anterior to anal opening. Fissure iad situated near the anterior corner of anal opening. Epimeral chaetotaxy: 3—1-3-3. [Legs] The num- ber of setae on femora I[-II-III-IV: 5-—5-—2-2. Trochantera III and IV each with a single seta. Keel developed most markedly on femur II (Fig. 17c), fairly well on femur III, and narrowly on femur IV; femur I without keel. [Type-series] Holotype (NSMT-Ac 9508): AMA-9; 12 para- topotypes: AMA-9. In the shape of sensilli, the arrangement of setae ms, r; and p;, and the shape and size of body, Ischeloribates rustenburgensis (Pletzen, 1963), comb. n., from South Africa is similar to the new species. The African species differes from the Japanese one in having kidney-shaped Sa, a narrow keel on femur II and 3 setae on femur III [19]. Perscheloribates clavatus torquatus subsp. n. (Fig. 18) 395-430 « 240-290 wm. [Prodorsum] Rostrum drawn out into a distinct snout, the tip not pointed, but rounded. Setae ro, Je and in weakly barbed and sharply pointed at tip; in slightly larger than 143 le; ro about 5/8 as long as /e. Sensillus directed anterolaterad at an angle of 45° to axis; peduncle strongly twisted in dorsal aspect (Fig. 18a); the head elongate oval, with minute barbs. [Noto- gaster] Ten pairs of notogastral setae very minute and hardly visible. In dorsal view anterior end of pteromorpha somewhat protruding; lateral outline of pteromorpha has a weak concavity; lateral margin in laterodorsal view weakly undu- lating (Fig. 18, left); obscure striation found on pteromorpha. [Ventral side] Genito-anal cha- etotaxy: 4-0-2-3; ag are absent; ad; situated nearly on the level of anterior margin of anal opeining. Epimeral ridges bo.2 connected to sternal ridge; bo.sj, bo.3 and apo.sj connected to circumgenital part; apo.2 and apo.3 short. [Legs] Monodactyle. Femora II-IV and trochanter III each with a keel; keel on femur II triangular and most conspicuous; femora I and II with a strong ridge on ventral side; femora I-IV with 5, 4, 3 and 2 setae, respectively; trochanter III with 2 setae and trochanter IV with 1 seta. [Type-series] Holotype (NSMT-Ac 9525): AMA-4, 6 para- topotypes: AMA-+4. The nominate subspecies Perscheloribates clavatus clavatus Hammer, 1973 [20] differs from the new subspecies in (1) the short sensilli with smooth head and peduncle not twisted, (2) the presence of aggenital setae and (3) the small body size (375 ym). Peloribates levipunctatus sp. n. (Fig. 19) 290-350 « 200-270 pm. [Prodorsum] __ Pro- dorsal surface glabrous, without foveolate sculp- ture. Rostral tip extending beyond the outline of prodorsal margin (Fig. 19d). A_ transverse line found in the anterior part of prodorsum. Setae ro, le and in weakly barbed; ro short, just reaching, or only slightly extending beyond, Fic. 16. Scheloribates decarinatussp.n. 16a. Sensillus, 16b. Femurl, 16c. Femur II, 16d. Trochanter and femur III, 16e. Trochanter and femur IV, Fic. 17. Ischeloribates lanceolatus sp. n. and femur III, 17e. Trochanter and femur IV. Fic. 18. Perscheloribates clavatus torquatus subsp. n. Femur II, 18d. Trochanter and femur III, Fic. 19. Peloribates levipunctatus sp. n. 19d. Tip of rostrum, 19e. Sensillus. 19a. Notogastral setae ps, 16f. Dorsolateral view of prodorsum. 17a. Sensilli, 17f. Epimeral and genital region. 17b. Femur I, 17c. Femur II, 17d. Trochanter 18a. Sensillus and bothridium, 18b. FemurlI, 18c. 18e. Trochanter and femur IV. 19b. Genital plate, 19c. Anal plate, 144 rostral tip; /e 1.78 and in 2.04x as long as ro. Sensillus with a long slender peduncle and an elongate oval head with barbation. [Notogaster] Small foveolae found on notogaster, but they are very obscure. No foveolae found on _ ptero- morphae. On the basal part of pteromorpha found a rectangular area with fine irregular margin including lyrifissure ia. Notogastral setae long, weakly barbed, blunt at tip; their RLN: 22-34; setae c;, C2 and /aa little longer than the remainder; setae dp and h; the shortest; setae of ps-series (Fig. 19a) sometimes slightly thickened in apical portion. A network-like structure around seta da distinct. [Ventral side] Ventral plate sculptured with small obscure foveolae as on notogaster. Anal and genital plates smooth, with neither foveolae nor punctures. Genito-anal chaetotaxy: 5—1-2-3; all the setae fine and short. Adanal fissure close and parallel to the lateral margin of anal opening. Mentum smooth. [Legs] Tridac- tyle. [Type-series] Holotype (NSMT-Ac 9539): AMA-2; 9 paratopotypes: AMA-2. Having notogastral setae partly showing a tendency of being slightly swollen at the tip, P. - levipunctatus resembles the next species, P. moder- atus, but it is distinguishable from the latter (1) the longer notogastral setae, (2) the shorter rostral setae, (3) the small and obscure foveolae on noto- gaster and ventral plate, and (4) the smooth pteromorphae, genital and anal plates. Peloribates moderatus sp. n. (Fig. 20) 280-340 x 200-230 pm. [Prodorsum] Pro- dorsal surface sculptured by foveolae and small punctures; the foveolae disappear only in the part along the anterior margin of notogaster. Setae ro, le and in weakly barbed and pointed at tip; Je and in subequal in length, being equal to, or somewhat longer than, their mutual distance; they are about 1.3 x as long asro. Sensillus with a long slender peduncle and an elongate oval head 20a. Notogastral seta ps,, J. AOKI which is distinctly barbed and not so strongly swollen; minute barbs also found on the distal half of the peduncle. [Notogaster] Longer than wide. Surface of notogaster and pteromorphae sculptured as on prodorsum. Notogastral setae moderately long, their RLN: 15-25; setae c., la, ps. and ps; a little longer than the remaining setae; seta dp and h, the shortest; all notogastral setae weakly barbed and blunt at tip; some of them, especially setae of ps-series, seem to be slightly thickened in apical portion (Fig. 20a). [Ventral side] Ventral plate sculptured as on notogaster. Anal plates also sculptured so, but foveolae are smaller. On genital plates only small punctures are found. Genito- anal chaetotaxy: 5—1—2-3; all the setae fine and short. Adanal setae ad; situated on, or slightly posterior to, the level of the anterior margin of anal opening. Mentum finely and densely pun- ctured. [Legs] Tridactyle. [Type-series] Holo- type (NSMT-Ac 9544): AMA-2; 29 paratopotypes: AMA-2. Three Peloribates-species from the Philippines, P. varisculptus Corpuz-Raros, 1981, P. buntotanus Corpuz-Raros, 1981, and P. pilipinus Corpuz- Raros, 1981 [21], as well as one Japanese species, P. nishinoi Aoki, 1977 [22], have notogastral setae swollen at the tip. Notogastral setae of the two new species also have such a tendency, but are not so markedly swollen as in the species mentioned above. Sensilli of the two new species are most similar to those of P. varisculptus, but not so strongly clavate as in the remaining three species. P. yvarisculptus has longer notogastral and inter- lamellar setae, all of which are swollen at the tip. Paralamellobates schoutedeni (Balogh) (Fig. 21) Oribatella schoutedeni Balogh, 1959, p. 106, figs. 50-51 [23]; Hammer, 1971, p. 33, fig. 36 [18]. Paralamellobates schoutedeni: Balogh, 1972, p1.57 [24]; Harada & Aoki, 1978, p. 159 [25]. 252-265 x 173-180 wm. The Japanese speci- 20b. Sensillus, 20c: Genital plate, 21a. Ventral side. 22a. Sensillus. Fic. 20. Peloribates moderatus sp. n. 20d: Anal plate. Fic. 21. Paralamellobates schoutedeni (Balogh, 1959). Fic. 22. Parakalumma robustum (Banks, 1895). Fic. 23. Galumna granalata sp.n. 23a. Pteromorpha. Fic. 24. Pergalumna amamiensis sp. n. 24a. Genital plate, 24b. Anal plate, 24c. Sensillus. New Oribatids from Amami-Ohshima 145 146 J. AOKI mens generally accord well with the original description of the species from Angola. In the Japanese specimens, however, setae Je are rough- ened and setae in extend well beyond the rostral tip, while in the Angolan specimens /e are glabrous and in nearly reaching the rostral tip. The speci- mens from the Fiji Islands reported by Hammer [18] seem to have features similar to the Japanese ones. The species was reported once before from Oki Island of Japan, having been only included in a table. This is the second report of the species with the figures and some comments. [Material examined] 3 exs.: AMA-15. Distribution: Angola, the Fiji Islands and Japan. Parakalumma robustum (Banks) (Fig. 22) Oribata robusta Banks, 1895, p. 7[26]. Parakalumma robustum: Jacot, 1929, p. 8, pls. 1-3 [27]. 850-930 x 650-730 wm. The Japanese materials are well in accord with the North American ones described by Banks [26] and redescribed in detail by Jacot [27]. But the Japanese specimens are somewhat larger in body size than the American ones (750 wm by Banks and 750-840 x 525-585 ym by Jacot). Relative lengths of prodorsal setae —ro: le: in=1: 2: 2.8. [Material examined] 3 exs.: AMA-10. New to Japan. Distribution: North America and Japan. Galumna granalata sp. n. (Fig. 23) 310-330 x 250-260 ym. [Prodorsum] Seta le short and fine. Seta ro also fine, indiscernible in dorsal view. Seta in minute. Sensillus bearing a weakly clavate head; peduncle sigmoid in basal part. [Notogaster] Almost circular in dorsal view. Four pairs of areae porosae circular, but they appear to be oval in dorsal view; Aa the largest of them. Lyrifissure im located between setae fi and ms. Median pore (mp) is present. Ptero- morpha (Fig. 23a) showing distinct median groove and radiating pattern; besides the pattern present numerous (more than 20) small grain-shaped structure; each “grain” well chitinized, pointed at tip and projection from the surface of pteromorpha. [Ventral side] Setae in genito-anal region very minute. Genito-anal chaetotaxy: 6-1-2-3. Adanal seta ad; situated near the level of mid- distance along the length of anal opening. Area porosa postanalis is present. [Type-series] Holo- type (NSMT-Ac 9583): AMA-10; 14 paratopo- types: AMA-10. The most characteristic feature of G. granalata is the presence of distinct grain-shaped projections scattered on pteromorphae, by which the new species is easily distinguishable from any other species of the genus Galumna. Pergalumna amamiensis sp. n. (Fig. 24) 540 x 390 wm. [Prodorsum] Whole surface densely and finely punctured. Setae ro, le and in comparatively strong, only ro being slightly roughened and the remaining setae smooth; in> le>ro in length. Sensillus glabrous, almost of the same thickness throughout its length, very slightly thickned distally. [Notogaster] Anterior margin slightly curved. Area porosa Aa wedge- shaped, the lateral end a little projecting anteriorly; A,-A; oval. Lyrifissure im situated median or lateral to the line fti-ms. Median pore exists. Pteromorpha covered with fine striae and granules. [Ventral side] Genital plate with punctures of irregular shape which have a tendency to fuse with one another longitudinally; several longitudial wrinkles found near the median margin of the plate. Of 6 genital setae g; and g. inserted on the anterior margin and g, on the posterior margin. Anal plate sparsely covered with punctures distributed not uniformly. Adana] fissure iad located at a level posterior to Ad;. Area porosa postanalis elliptic. [Type] Holotype (NSMT-Ac 9587): AMA-10. Pergalumna elongata Engelbrecht, 1972 [28] from South Africa is similar to the new species in the shape of areae porosae, but its sensilli, lamellar and interlamellar setae are barbed and the body size is larger (648 um in body length). Sensilli of P. reniformis Hammer, 1968 [29] from New Zealand are very similar in shape to those of the new species, but the New Zealand species has minute lamellar and interlamellar setae and shorter Aa. REFERENCES 1 Aoki, J. (1965) A preliminary revision of the 10 11 12 13 14 New Oribatids from Amami-Ohshima family Otocepheidae (Acari, Cryptostigmata). I. Subfamily Otocepheinae. Bull. Natl. Sci. Mus. Tokyo, 8: 259-341. Aoki, J. (1967) A preliminary revision of the family Otocepheidae (Acari, Cryptostigmata). Il. Subfamily Tetracondylinae. Bull. Natl. Sci. Mus. Tokyo, 10: 297-359. Aoki, J. (1970) New and interesting species of oribatid mites from Kakeroma Island, Southwest Japan. 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Balogh, J. and Mahunka, S. (1974) A foundation of the oribatid (Acari) fauna of Cuba. Acta Zool. Acad. Sci. Hung., 20: 1-25. Hammer, M. (1979) Investigations on the oribatid fauna of Java. Biol. Skr. Dan. Vid. Selsk., 22 (9): 1-79, 47 pls. Aoki, J. (1981) Discovery of the second species of the genus Nippobodes from Ohsumi Islands (Acari: Oribatei). Bull. Biogeogr. Soc. Jap., 36 (4): 29-33. Balogh, J. (1970) New oribatids (Acari) from Ceylon. The scientific results of the Hungarian Soil Zoological Expeditions. Opusc. Zool. Bu- dapest, 10: 33-67. Csiszar, J. and Jeleva, M. (1962) Oribatid mites (Acari) from Bulgarian soils. Acta Zool. Acad. Sci. Hung., 8: 273-301. 15 20 21 22 23 24 ZS 26 27 28 29 147 Higgins, H.G. and Woolley, T. A. (1975) New mites from the Yampa Valley (Acarina: Cryp- tostigmata; Oribatulidae, Passalozetidae). Great Basin Naturalist, 35: 103-108. Berlese, A. (1910) Brevi diagnosi di generi e specie nuovi di Acari. Redia, 9: 77-111, pls. 1-8. Corpuz-Raros, L. A. (1980) Philippine Oribatei (Acarina). V. Scheloribates Berlese and related genera (Oribatulidae), Kalikasan. Philipp. J. Biol., 9: 169-245. Hammer, M. (1971) On some oribatids from Viti Levu, the Fiji Islands. Biol. Skr. Dan. Vid. Selsk., 16 (6): 1-60, 35 pls. Pletzen, R. van (1963) Studies on South African Oribatei (Acarina). Acarologia, 4: 690-703. Hammer, M. (1973) Oribatids from Tongatapu and Eua, the Tonga Islands, and from Upolu, western Samoa. Biol. Skr. Dan. Vid. Selsk., 20 (3): 1-70, pls. 1-29. Corpuz-Raros, L. A. (1981) Philippine Oribatei (Acarina). IV. The genus Peloribates Berlese (Oribatuloidea, Haplozetidae). Philipp. Ent., 4: 435-456. Aoki, J. (1977) Two new _ Peloribates-species (Acari, Oribatida) collected from lichens growing on tomb stones in Ichihara-shi, Central Japan. Annot. Zool. Japon., 50: 187-190. Balogh, J. (1959) Oribates (Acari) nouveaux d’Angola et du Congo Belge (lére serie). Publ. cult. Co. Diam. Ang., Lisboa, (48): 91-108. Balogh, J. (1972) The Oribatid Genera of the World. Akademiai Kiado, Budapest. pp. 1-188, pls. 1-71. Harada, H. and Aoki, J. (1978) Oribatid mite communities under different vegetations in Oki Island. Bull. Inst. Environm. Sci. Technol. Yoko- hama Natl. Univ., 4: 155-164. Banks, N. (1895) On the Oribatoidea of the United States. Trans. Amer. Ent. Soc., 22: 1-16. Jacot, A. P. (1929) American oribatid mites of the subfamily Galumninae. Bull. Mus. Comp. Zool., 69: 3-37, pls. 1-6. Engelbrecht, C. M. (1972) Galumnids from South Africa (Galumnidae, Oribatei). Acarologia, 14: 109-140. Hammer, M. (1968) Investigations on the oribatid fauna of New Zealand with a comparison between the oribatid fauna of New Zealand and that of the Andes Mountains, South America. Part III. Biol. Skr. Dan. Vid. Selsk., 16 (2): 1—96, pls. 1-33. ZOOLOGICAL SCIENCE 1: 148-150 (1984) [COMMUNICATION ] © 1984 Zoological Society of Japan Chorion Glycoprotein-Like Immunoreactivity in Some Tissues of Adult Female Medaka TATSUO HAMAZAKI, IcHIRO IucHiI! and KENJIRO YAMAGAMI Life Science Institute, Sophia University, Kioicho, Chiyoda-ku, Tokyo 102, and !Zoological Institute, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT — An immunoreactivity to anti-chorion glycoprotein antibody, as determined by immuno- diffusion and indirect immunofluorescence methods, was found to be localized not only in the chorion, cytoplasm and follicle cells of growing oocytes, but also in some other tissues such as germinal epithelium of ovary, parenchymal cells of liver and blood plasma in the spawning female medaka, Oryzias latipes. Egg yolk itself showed only a very weak reactivity and a probable vitellogenin band on polyacrylamide gel disc electrophoresis of the spawning female blood plasma did not react with the antibody at all. This reactivity was absent in tissues of the non-spawning female as well as the male fish. Therefore, the occurrence of the reactive principle seems to be related to oogenesis. Teleostean egg envelope (chorion) is a thick non-cellular structure which is composed of various layers and performing, after being hard- ened on fertilization, a role of protecting an embryo against biological as well as physical damages. According to recent concept, the chorion is composed of substances derived from both fol- licle cells and oocyte itself [1, 2]. Especially, the inner layer of chorion, which constitutes a major part of chorion, is reported to be synthesized in oocyte [3]. In the course of our immunological analyses of choriogenesis in medaka, we found substance(s) reactive to anti-chorion glycoprotein antibody not only in ovary but also in some other Accepted August 5, 1983 Received June 30, 1983 tissues of adult female fish. Some evidences suggest that this substance(s) is related to oo- genesis. The present report will describe a preliminary information of the immunoreactivity. MATERIALS AND METHODS Throughout the experiments, egg chorion, embryos and adult tissues of orange-red variety of random-bred medaka, Oryzias latipes, were used. Embryos or adult fish were cultured as described elsewhere [4]. Preparation of anti-chorion glycoprotein antibody Chorion glycoprotein used as immunogen was obtained from the hatching enzyme (PII-0.3 en- zyme) [4, 5] digests of egg chorion. A major glycoprotein, PI, was obtained by fractionating the chorion digests with Sephadex G-75, and PI was further fractionated through Sephadex G-200 into two glycoprotein fractions, Fl and F2, as reported previously [6]. Rabbit antisera against Fl and F2 were ob- tained by injecting 5 mg of Fl or F2 subcutane- ously once a week. Anti-Fl and -F2 IgG’s were prepared by precipitating IgG fraction from respective antisera by adding ammonium sulfate to 33% saturation. A preliminary immunological characterization proved that a major component of anti-Fl antibody and that of anti-F2 antibody were identical, while anti-F1 antibody additionally possessed a specific minor antibody component. In the present experiment, anti-Fl antibody was Chorion-like Immunoreactivity in Medaka 149 employed as a probe, as this antibody was con- sidered to maintain all components reactive to major chorion glycoproteins. and two-dimensional immuno- Tissue extraction diffusion Blood plasma of adult fish was obtained by cutting the tail in the isotonic saline [7]. The suspension was centrifuged at 3,000rpm for 15 min to remove blood cells and the supernatant was concentrated with a collodion bag (SM 132-00, Sartorius GmbH, G6ttingen) at 4°C. Other organs such as liver and ovary were homogenized in phosphate buffered saline (PBS, pH 7.2), centrifuged at 15,000 rpm for 20 min at 4°C and the supernatant was concentrated as described above. Two-dimensional immunodiffusion analy- sis was carried out following the method of Ouchterlony [8]. One percent agarose in PBS was used for diffusion matrix and the diffusion was performed in a moist chamber at 4°C. Immunohistochemistry Detection of chorion glycoprotein-like immuno- reactivity in various tissues of adult medaka was carried out by an indirect immunofluorescence method; specimens were fixed in 95% ethanol or 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C for 4-6 hr, dehydrated through a grad- ed alcohol series, embedded in paraffin and were cut at 4m thickness. Deparaffinized sections were treated with 1: 50 dilution of rabbit anti-Fl IgG in a moist chamber at 37°C for 40 min, rinsed thoroughly with PBS and allowed to react with 1:50 dilution of fluorescein isothiocyanate- conjugated goat anti-rabbit IgG (Miles-Yeda Ltd., Rehovot). Fluorescence was observed in Olympus BHS-RF-A fluorescence microscope. RESULTS AND DISCUSSION Immunodiffusion As shown in Figure 1, a continuous distinct precipitation line was observed between anti-Fl antiserum and PI, liver extract, blood plasma and ovary extract of spawning female, but not be- Fic. 1. adult medaka. A, anti-Fl antiserum; B, b, blood plasma of a spawning female and a male fish, respectively; spawning female and a male fish, respectively; Indirect immunofluorescence staining of ovary section of a spawning medaka. 2, early vitellogenic oocyte (ca 200 4m); FG. 2. oocyte (ca 80 wm in diameter); (ca 500 wm); 4, fully grown oocyte (ca 1,000 ~m) Fic. 3a and b. Immunodiffusion analyses of chorion glycoprotein-like immunoreactivity in some tissues of PI, PI obtained from hatching enzyme digests of chorion; L, 1, liver extracts of a O, ovary extract of a spawning female. 1, Young 3, vitellogenic oocyte x 60. Indirect immunofluorescence staining of liver sections of adult male and female fish. Some reactivity still remains in the endothelium of blood vessels of non-spawning female. M, male; sF, spawning female; nF, non-spawning female x 45, 150 T. HAMAZAKI et al. tween the antibody and male blood or liver. Essentially the same results were obtained when anti-F2 antiserum was used as antibody. A very weak precipitation line formed between the anti- body and the concentrated yolk (not illustrated). Immunohistochemistry Figure 2 shows the fluorescence in ovary sec- tion of a spawning female. Cytoplasm of young oocyte, smaller than 80 ~m in diameter, shows no reactivity, while that of early vitellogenic oocyte, 100-125 wm in diameter, is stained in- tensely. Chorion and follicle cells around the oocyte also show strong yellow fluorescence. However, cytoplasm of vitellogenic oocyte, 300- 600 um in diameter, shows a very weak reac- tivity, though the chorion is still stained intensely. In the fully grown oocyte, 800-1,000 wm in diameter, most parts of the thickened chorion are not so intensely stained, except for inner and outer peripheral portions, where intense yellow fluorescence is observed. A relatively low im- munoreactivity of thick chorion is _ probably attributable to concealment of the immunore- active sites in the chorion architecture, because the fluorescence of chorion section increased remarkably on partial digestion with hatching enzyme (Hamazaki ef al., unpublished). Oo- plasm and follicle cells of the fully grown oocyte show weak fluorescence. Some other tissues than follicles in ovary, such as germinal epithe- lium and interstitial cells are also stained. It is Of some interest to note that any part of the ovary did not fluoresce when the anti-Fl IgG which was previously absorbed with liver of spawning female was used. As shown in Figure 3a, the section of liver of the spawning female manifests intense fluores- cence which is localized within each parenchymal cell, while no fluorescence is recognized in the male liver. Almost no or very weak fluorescence is observed in the non-spawning female. In the case of blood, blood plasma, not ceils, of the spawning female represented intense fluorescence, while that of male or nonspawning female did not (not illustrated). A high immunoreactivity in these tissues of spawning females was decreased gradually as they ceased laying eggs, although the endothe- lium of hepatic blood vessels, blood plasma or germinal eipthelium in the ovary remained to be somewhat reactive for a while even after hepatic cells had become unreactive (Fig. 3b). Thus, all tissues of female fish lose the reactivity in the midst of off-breeding season. These findings suggest that this chorion glycoprotein-like immuno- reactivity in female fish is closely related to oo- genesis. Preliminary immunoelectrophoretic an- alyses revealed that the immunoreactive sub- stance(s) derived from different tissues of spawning females showed an identical relative mobility (Rm) on polyacrylamide gel disc electrophoresis, which was evidently distinct from the Rm value of an unreactive band of probable vitellogenin found in blood plasma of the spawning female. Studies on the nature of the reactive substance(s) and its role in oogenesis are currently under way. REFERENCES Laale, H. W. (1980) Copeia, 1980: 210-226. Tesoriero, J. (1977) J. Ultrastr. Res., 59: 282-291. Tesoriero, J. (1978) J. Ultrastr. Res., 64: 315-326. Iuchi, I., Yamamoto, M. and Yamagami, K. (1982) Develop. Growth Differ., 24: 135-143. 5 Yamagami, K. (1972) Develop. Biol., 29: 343-348. 6 lIuchi,I. and Yamagami, K. (1976) Biochim. Biophys. Acta, 453: 240-249. 7 Yamamoto, T. (1944) Annot. Zool. Japon., 22: 109-136. 8 Ouchterlony, O. (1962) In ‘Progress in Allergy.”’ Ed. by P. Kallos and B.H. Waksman, Karger, Basel/New York, Vol 6, pp. 30-154. BRwWN eS iii INSTRUCTIONS TO AUTHORS ZOOLOGICAL 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 one of the Associate Editors in advance. Articles of less than 5 printed pages will be published free of charge. Charges will be made for extra pages (7,000 yen/page). A Communication cannot ex- ceed 3 printed pages. No charge will be imposed for invited reviews. No free reprints of Articles and Communications are available. To the author of an invited review fifty reprints are pro- vided gratis. Submission of papers from non- members of the Society is welcome. Page charges (7,000 yen/page) will be made to nonmembers. A. 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(1981) Photoreac- tivation of ultraviolet light-induced damage in cultured fish cells as revealed by increased colony forming ability and decreased content of pyrimidine dimers. Photochem. Pho- tobiol., 33: 313-316. 3 Hubel,O. and Wiesel, T.N. (1968) The functional architecture of the striated cortex. In “Physiological and Biochemical Aspects of Nervous Integration”. Ed. by F.D. Carlson, Prentice-Hall, New Jersey, pp. 153-161. 4 Campbell, R. C. (1974) Statistics for Biolo- gists, Cambridge University Press, London, 2nd ed., pp. 59-61. Titles of cited papers may be omitted in Reviews and Communication. The source of reference should be given following the commonly accepted abbreviations for journal titles (e.g., refer to ‘Internationa] List of Periodical Title Abbrevi- ations’). 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Those who submit reviews should consult with one of the Associate Editors in advance. Articles of less than 5 printed pages will be published free of charge. Charges will be made for extra pages (7,000 yen/page). A Communication cannot ex- ceed 3 printed pages. No charge will be imposed for invited reviews. No free reprints of Articles and Communications are available. To the author of an invited review fifty reprints are pro- vided gratis. Submission of papers from non- members of the Society is welcome. Page charges (7,000 yen/page) will be made to nonmembers. A. SUBMISSION OF MANUSCRIPT The manuscript should be submitted in triplicate, one original and two copies, each including all illustrations. Rough copies of line drawings and graphs may accompany the manuscript copies, but the two copies of continuous-tone prints (photo- micrographs, etc.) should be as informative as the original. 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This section should clearly describe the objectives of the study, and provide enough background information to make it clear why the study was undertaken. Lengthy reviews of past literature are discouraged. 3. Materials and Methods. This section should provide the reader with all the information that will make it possible to repeat the work. For modification of published method- ology, only the modification needs to be described with reference to the source of the method. 4. Results. Results should be presented referring to tables and figures, without discussion. 5. Discussion. The Discussion should include a concise statement of the principal findings, a discussion of the validity of the observations, a discussion of the findings in the light of other published work dealing with the same subject, and a discussion of the significance of the work. Redundant repetition of material in Introduction and Results, and extensive discussion of the literature are discouraged. 6. Statistical analysis. Statistical analysis of the data using appropriate methods is mandatory and the method(s) used must be cited. 72 References: References should be cited in the text by an Arabic numeral in square parentheses and listed at the end of the paper in numerical order. For example: 1 Takewaki, K. (1931) Oestrus cycle of female rat in parabiotic union with male. J. Fac. Sci. Imp. Univ. Tokyo, Sec. IV, 2: 353-356. 2 Shima, A., Ikenaga, M., Nikaido, O., Takabe, H. and Egami, N. (1981) Photoreac- tivation of ultraviolet light-induced damage in cultured fish cells as revealed by increased colony forming ability and decreased content of pyrimidine dimers. Photochem. Pho- tobiol., 33: 313-316. 3 Hubel,O. and Wiesel, T.N. (1968) The functional architecture of the striated cortex. In “Physiological and Biochemical Aspects of Nervous Integration”. Ed. by F.D. Carlson, Prentice-Hall, New Jersey, pp. 153-161. 4 Campbell, R. C. 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JAMARIN U(unsterilizable) Readily soluble. Concentration up to _ triple strength of sea water can be prepared. Per cent of gastrulation in the sea _ urchin, Anthocidaris crassispina mache Vins ate wem aE eC w nod aime 98% 2. JAMARIN S(sterilizable) Can be autoclaved. Per cent of gastrulation in the sea_ urchin, Anthocidaris crassispina::**+++**1+s0t teeter 96.3% JAMARIN U JAMARIN S 3. Modified JAMARINs Ca-free JAMARIN, Ca, Mg-free JAMARIN Sulfate-free JAMARIN etc., in compliance with the requests of customers. Yamarin Applied fields of JAMARIN Bacteria: Separation and culture of marine bacteria. Isolation of antibiotics from marine bacteria. Algae : ‘Polarity regeneration in Bryopsis. PH and temperature studies on the pro- toplast formation in Bryopsis maxima. Reassembly of dissociated components in Bryopsis. \solation of enzymes in carbohydrate metabolism in Platy- monas Sp. Nitrate and Nitrite metabolism in the red alga, Porphyra yezoensis. Evolution of ethylene in marine algae. Invertebrates : Regulation of gene expression in sea urchin embryos. Isolation of the mito- tic apparatus from sea urchin embryos. Meiotic division in starfish oocytes. Branchial regeneration in Polychetes. Strobilation of Aurelia aurita. Phototactic responses of a flatworm, Convoluta sp. Price (Postage : excluded). | Domestic | Overseas | | Customers | Customers | [2 @ pack ¥ 500 |USS 2.50. | 5 2 pack | ¥ 800 |US$ 4.00| | 20 £ pack ( ¥2,000 | USS 10.00 | Inquiries invited. Jamarin Laboratory 11-5, Shigino-Nishi 2 chome, Johto-ku, Osaka, 536 Japan. Phone: 06-961-6007 Cable: JAMARINLAB OSAKA ZOOLOGICAL SCIENCE VOLUME 1 NUMBER 1 FEBRUARY 1984 CONTENTS EDITORIAL — “Zoological Science’, the new official publication of the Zoological Society me lanan AN. BRAmn cobs ctteacatoncs ceeceesanee REVIEWS Kawamura, T.: Polyploidy in amphibians ... Sakai, H., H. Murofushi and G. Matsumoto: High molecular weight proteins of nerve cells inducing cross-linking or bundling of cyto- skeletal proteins eer eeceseseseeereseeeereseeesoeosceseoeeee ORIGINAL PAPERS Physiology Hiramoto, Y., M.S. Hamaguchi, Y. Nakano and Y. Shoji: Colcemid UV-microirradi- ation method for analyzing the role of microtubules in pronuclear migration and chromosome movement in sand-dollar eggs Gomi, S.and H. Sugi: Factors influencing the force-extension relation of the series elasticity in glycerinated rabbit psoas muscle fibers ... Hisada, M., M. Takahata and T. Nagayama: Structure and output connection of local non-spiking interneurons in crayfish Cell Biology Tamura, R., M. Takahashi and Y. Watanabe: Molecular mechanism of cell division in Tetrahymena thermophila. 1. Analysis of execution period of a division-arrest ts- 1111112 51 | ae SD EM 3 Sen Sa Mas ap rte fut Yasuda, T., R.Tamura and _ Y. Watanabe: Molecular mechanism of cell division in Ultrastru- ctural changes found in a division-arrest ts- eeeceecaee Tetrahymena thermophila. II. SEINE GAEL sis sicisss dv vcjcles veida'n ws Boe RCOEE Con EE ER ERSTE Biochemistry Yoshioka, S., M. Masada, T. Yoshida, T. Mizokami, M. Akino and N. Matsuo: A- typical phenylketonuria due to biopterin deficiency: Diagnosis by assay of an enzyme involved in the synthesis of sepiapterin from dihydroneopterin triphosphate .................. 16 29 35 50 62 Hamazaki, T., I.Iuchi and K. Yamagami: Chorion glycoprotein-like immunoreactivity in some tissues of adult female medaka (COMMUNICATION )).2...2. 22.0220 sense 148 Endocrinology Oguro, C., M. Fujimori and Y. Sasayama: Changes in the distribution of calcium of the frog, Rana nigromaculata, following ultimo- branchialectomy and calcitonin admini- stration Urano, A.: A Golgi-electron microscopic study of anterior preoptic neurons in the bullfrog and the toad ........... Mainoya, J. R. and H. A. Bern: Influence of vasoactive intestinal peptide and urotensin II on the absorption of water and NaCl by the anterior intestine of the tilapia, Sarotherodon mossambicus Hirano, T. and S. Hasegawa: Effects of angio- tensins and other vasoactive substances on drinking in the eel, Auguilla japonica Watanabe, K. and E. Ohnishi: The mode of ecdysteroid accumulation in ovaries of Bombyx mori during the pupal and pharate adult peiod Takasugi, N. and T. Kato: Acceleration by vitamin A of the permanent proliferation of mouse vaginal epithelium induced by neo- natal treatment with progestin Behavior Biology Yamanouchi, K., Y. Nakano, M. Fukuda and Y. Arai: Mesencephalic central gray as supraspinal neural substrates for lordosis reflex: Deprivation of serotonergic influ- ence by p-chlorophenylalanine Taxonomy New and unrecorded oribatid mites southwest Aoki, J.: from Amami-Ohshima Island, Japan Printed by Daigaku Letterpress Co., Ltd. Hiroshima, Japan HSON, TOOLOGICAL SCIENCE tinuation of the “Zoological Magaz!ne” and the “Annotationes Zoologicae Japonenses— ZOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Nobuo Egami (Tokyo) Toshin-building, Hongo 2—27-2, Bunkyo-ku, Associate Editors: Tokyo 113, Japan. (03)-814—-5675 Susumu Ishii (Tokyo) Officers: Seiichiro Kawashima (Hiroshima) President: Hideshi Kobayashi (Funabashi) Koscak Maruyama (Chiba) Secretary: Makoto Asashima (Yokohama) Assistant Editors: Treasurer: Kiyoaki Kuwasawa (Tokyo) Takeo Machida (Hiroshima) Librarian: Shun-Ichi Uéno (Tokyo) Kazuyoshi Tsutsui (Hiroshima) Editorial Board: Howard A. Bern (Berkeley, endocrinology) Aubrey Gorbman (Seattle, endocrinology) Robert B. Hill (Kingston, physiology) Yukio Hiramoto (Tokyo, cell physiology) Yukiaki Kuroda (Mishima, cell genetics) Hiromichi Morita (Fukuoka, sensory physiology) Tokindo S. 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New subscriptions and renewals begin with the first issue Of the current volumes. 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 1984, The Zoological Society of Japan q 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 1: 151-160 (1984) © 1984 Zoological Society of Japan REVIEW The Cause and Consequence of Unequal Cleavage in Sea Urchins KATSUMA DAN! Tokyo Metropolitan University, Tokyo 158, Japan The 4th cleavage of sea urchin embryos is unequal, giving rise to macromeres and micro- meres. Besides, it has also been established that this micromere formation is a crucial step of differentiation to segregate a spicule-forming population of the pluteus larva. In spite of the fact that these phenomena are illustrated in practically all the textbooks of embryology, hardly any analysis has been made to elucidate the underlying mechanism of un- equal divisions. CAUSES FOR UNEQUAL CLEAVAGE IN SEA URCHINS In 1979, the author became aware of the fact that the initial step for the inequality in the blasto- mere size between macromere and micromere is due to the migration of the nuclei toward the vegetal pole in the 4 lower cells of the 8-cell stage. This is the case in Hemicentrotus plucherrimus as well as in Clypeaster japonicus [1]. An example of Clypeaster case is shown in Figure 1. More recently, it was further found that this migration of the nuclei is a highly organized movement. Sections for transmission electron microscopy (TEM) reveal that free or outer sur- faces of blastomeres are underlain by a row of vesicles, while the vesicles are rare or absent along the contact surfaces between the blasto- meres (Fig. 2). However, in the 4 vegetal cells of the 8-cell stage, there are small gaps in the vesi- Received September 6, 1983 1 Address for correspondence: 3-19-8 Kami-Yoga, Setagaya-ku, Tokyo 158, Japan. cular rows near the vegetal pole (Fig. 3). The resting nuclei of the lower 4 cells migrate down toward respective gaps of the blastomeres, al- ways headed by one of the 2 centrioles (Fig. 4) [2]. The author thinks this is the first event in the unequal division in sea urchins. In the 4 upper cells (future mesomeres), no gaps are found and the nuclei and the spindles remain at the cell centers and they cleave equally (Fig. 2). The second event, although this is somewhat hypothetical at present, is the attachment of the leading centriole to the gap cortex. In the polar body formation of Crepidula [3] and in ganglion cell formation in the neuroblast of the grass- hopper Chortophaga [4], both of which divide unequally, attachment of a spindle pole to the cortex was fully proven because they were shown to resist mechanical interventions. However, such adequate proof is not yet available for sea urchin eggs, although some circumstantial evidences are at hand. As the result of the attachment of one centriole, when a spindle is formed, it points directly toward the gap and takes an extremely eccentric position within the blastomere (Fig. 3). Result is the formation of micromeres. Furthermore, when a micromere is_ being pinched off, it looks as if the micromere creeps out through the gap. An important consequence is that the formed micromeres have only a few vesicles on the surface, while the free surface of the macromeres is covered by the cortex lined with the vesicles as mesomeres (Fig. 5) [2]. There are a few reports pointing out a difference in agglutinability by Concanavalin A_ between micromere progenies and mesomere and macro- mere lines [5]. Obviously, such is due to a 152 K. DAN 0 min 5 min 8 min Sac 17 min 22 min 28 min 39 min Fic. 1. "Migration of vegetal nuclei in a living Clypeaster embryo as observed under a modified Nomarski arrangement. A phase turret is inserted instead of a Nomarski turret in Olympus scope changeable between Nomarski and phase contrast. Omin: immediately after the 3rd division with late telophase nuclei. 28 min: after break down of nuclear membrane. Mitotic apparatus is not visible under this optical condition. (Courtesy of Develop. Growth and Differ.) Fic. 2. A blastomere of the animal side of the 8-cell stage Hemicentrotus pulcherrimus. Note a con- tinuous row of vesicles (v) lining the free surface of the cell and central position of the mitotic apparatus (MA). 2,400. (Permission by Develop. Growth and Differ.) Fic. 3. Two vegetal blastomeres of the 8-cell stage of Hemicentrotus. Two eccentric spindles point toward gaps (g) in the vesicular row. 2,400. (Permission by Develop. Growth and Differ.) 153 Unequal Cleavage in Sea Urchins K. DAN Unequal Cleavage in Sea Urchins 133 difference in the nature of the surface between the 2 lines as seen in Figure 5. DEVELOPMENTAL HISTORY OF THE GAP Tanaka [6] photographed the vegetal view of the 16-cell stage of Hemicentrotus through a Wratten filter No 49 (to make specific pigment granules of Hemicentrotus visible) and showed that the micromeres have less granules than the other cells. He further traced the beginning of the sparsely pigmented areas and found that they appear at the 4-cell stage and are carried over to the lower 4 cells at the 8-cell stage. Quite similarly to Hemicentrotus or rather more clearly than Hemicentrotus, in Woods Hole sea urchin, Arbacia punctulata, conspicuous red pig- ment granules which can be seen with ordinary light microscope are completely absent around the vegetal pole at the 8-cell stage. Result is that when micromeres are formed, they look white in contrast to red color of the remaining blastomeres [7]. In Arbacia, too, the pigment gaps appear at the 4-cell stage as in Hemicentrotus. Belanger and Rustad [8] pushed the analysis a step further in Arbacia. When cleavage is com- pletely suppressed by colchicine, they found that even on such uncleaved eggs, the pigment-free areas appeared on time with the 4-cell stage of the control. They followed the movement of individual granules by cinematography and defi- nitely showed that the appearance of the pigment- free area is due to recession of the granules from that area and not due to loss of them. Practi- cally the same behavior has been recently reported for the brown pigment granules of Temnopleurus hardwicki by Osanai [9]. Tanaka [6] found in Hemicentrotus that the decrease of granule density around the vegetal pole was always compensated by an increase of granule density on the animal side. Since such coherent movement of the granules between the 2 hemispheres is prevented by microfilament-de- stroying agents such as cytochalasin B, dithio- threitol and tetracaine, he claims that the for- mation of the pigment gap is due to surface contraction on the animal side and stretching on the vegetal side. Incidentally, microtubule-de- stroying agents have no effect on the cortical movement. At any rate, the above facts indicate that the pigment-free area or the gap in the vesicular row is not a preformed structure but it is formed be- fore the micromere formation, precisely during the interphase of the 3rd cell cycle. As shown by the experiments of Dan and Ikeda [10], since the time of the micromere formation is strictly correlated with a definite phase of fluctuation of SH contents of the egg proteins, the time of the appearance of the pigment-free area may also be governed by the SH clock. DIFFERENTIATIVE PRECOSITY OF THE MICROMERE As is well known, after the cleavage stage, the progenies of the micromeres leave the blastular wall and arrange themselves in a characteristic pattern within the blastocoel and by the time of gastrulation, they form 2 calcarious granules which are the starting points for a pair of larval spicules [11]. To understand this seeming paradox between small size of the micromeres and their precosity in differentiative capacity, Dan ef al. [12] per- formed some analyses. In 1958 by Agrell [13] and later by Cowden and Lehmann [14] it was shown that when paraffin sections of the 16-cell stage sea urchin embryos were stained with RNA stains, micromeres always showed deeper stain- ing, suggesting that ribosomal density is higher in the micromere than in other cells. Their obser- vations were repeated by us, using Pyronin Y and azure B, combined with measurement of the stainability with microphotometry. The conclu- sion of the previous workers was confirmed. A black and white copy of original color print [12] Fic. 4. Interkinesis of the 8-cell stage of C/ypeaster japonicus. a: Migration of a resting nucleus toward the gap, escorted by a centriole. x 3,700. b: an enlarged photograph showing the centriole (c). x 10,300. (Courtesy of Develop. Growth and Differ.) . DAN K 56 ] Unequal Cleavage in Sea Urchins 157 Fic. 6. A black and white copy of an original color print of the 32-cell stage of Hemicentrotus stained with Pyronin Y. Note the Ist and 2nd micromeres are stained more heavily than other blastomeres. is shown in Figure 6. Secondly, in search for a possible explanation for the above phenomenon, the blastomeres of the 16-cell stage were examined in thin sections by TEM. It became immediately apparent that each nucleus is surrounded by a special zone which is rather ill-defined in the contour. We tentatively call this zone the “‘peri-nuclear zone” or “‘peri-nuclear core” since it wraps the nucleus concentrically. In Hemicentrotus, this core always excludes yolk granules although it retains ribo- somes and mitochondria. Owing to this sit- uation, this core looks as a brighter ring around the nucleus in the surrounding darker cytoplasm in photographs of sections. In Clypeaster, on the other hand, although the core excludes yolk granules, it is densely packed with membrane systems and presumably with ribosomes and looks darker than the cytoplasm. A_ simple way to see the peri-nuclear core is to stain 2 “m thick sections by toluidine blue which is shown in Figure 7. Thirdly, it was attempted to measure the volume of the peri-nuclear core. The volume was calcu- lated as a sphere for average radius of the core Fic. 5. Clypeaster. Differ.) A micromere is separating from a macromere. micromere is practically devoid of the vesicles except around the constriction. the cleavage furrow indicates the constricting ring (ring) Fic. 7. with cores. Clypeaster. Consecutive sections of 2 ~m thick, stained toluidine blue, showing the _peri-nuclear minus nuclear volume. A conclusion reached in Clypeaster is that the volume of the peri-nuclear core is practically constant regardless of the blastomere size and further the volume nearly equals that of the entire micromere [12]. The above finding means that micromere has the same share of the peri-nuclear core contain- ing a protein synthesizing system as other blasto- Note that the surface of the incipient Darker contour along “5,950. (Permission by Develop. Growth and 158 K. DAN meres, while its small size is due to exclusion of a large amount of the yolk granules which are in- nert in protein synthesis. As the result, within a micromere, a condition is established to secure relatively more efficient production of new proteins than other cells on the basis of total cell protein. The author thinks this explains the pre- cosity of differentiative capacity of smaller sister cells in unequal divisions in general. SUPPRESSION OF MICROMERE FORMATION Tanaka [15] was the first worker to find a method to completely suppress the micromere formation in sea urchins, using a dilute solution of Na-lauryl sulphate (20-30 ug/ml) dissolved in A convenient way to obtain the sea _ water. A Fic. 8. Defective larvae of Hemicentrotus after suppressing the micromere formation. results is to put the embryos in the detergent solution from 4-cell stage on, obviously at the time when the pigment-free area is to be formed. The result is that all the cells of the 16-cell stage are of equal size. But a further surprising re- sult is that if such treated embryos are returned to sea water and reared, they develop into de- fective larvae lacking the spicule entirely. In other words, where there is no micromere, there is no spicule. The present author repeated Ta- naka’s experiment and some of the abnormal larvae he obtained by the detergent treatment are shown in Figure 8. Supplemental observation of the present author to Tanaka’s original findings is that in the em- bryos treated by Na-lauryl sulphate, vegetal migration of the nuclei in the 4 lower cells does not take place and the nuclei remain at the cell Embryos having; A, an archenteron-like structure consisting of a few cells; B, no inner cells; C,an archenteron-like structure with randomly dispersed cells; D, intestinal epithelium-like structure. None of them has spicules. Unequal Cleavage in Sea Urchins 159 center. When these centrally located nuclei go into mitoses, the spindle acquires 2 asters of the same size and takes a horizontal direction, so that 8 daughter cells of equal size all lie on the same horizontal plane just like mesomeres; that is to say, at the 16-cell stage, 2 tiers of 8 equal cells each are formed [1]. Tanaka [16] made TEM studies of the treated embryos and found that the vesicular row lining the free surfaces was much disturbed or somewhat broken. Unfortunately, since Tanaka was not aware of the presence of the vesicular gap, we cannot obtain his specific comment on the gap. But it is highly likely that the gap may also be deranged which, in turn, obliterates the target marker of the cortex and the nuclear migration to the vegetal pole fails to occur. DISCUSSION In an orthodox embryological sense, a com- plete failure of one differentiation (here spicule formation) may sound rather shocking. But in a more modernistic sense, it is a failure of acti- vation of a small group of genes controlling the realization of such differentiation. Therefore, a real intriguing point is rather that why spicule formation disappears while the same _ vegetal cytoplasm is still remaining in the embryo. A difference between the controls and equally divided 16 cells of the treated embryos is that in the former, the micromere cytoplasm is neatly segregated into 4 micromeres, while in the latter, the same cytoplasm is not only divided among 8 cells instead of 4 but it remains mixed with the cytoplasm of the macromere. When the intestine- forming cytoplasm and spicule-forming cytoplasm are left mixed, the intestine is formed to some extent, although very defective (see Fig. 8), but the spicules are completely suppressed. In this sense, a clear-cut separation of presumptive cyto- plasms is pre-requisite for satisfactory differenti- ation. In early stages, although these presumptive areas must already exist, their boundaries are not sharp and the fringes overlapping. Only after a clear-cut segregation, characteristic chain reactions can proceed toward the activation of proper genes and eventually to the synthesis of specific pro- teins for spicule formation by the time of the mesenchyme blastula stage about 30 hr after fertilization [17-19]. Other biochemical analyses reaching a negative conclusion about this point were done too shortly after micromere separation, lacking embryologists’ patience [20, 21]. Voluminous works of H6rstadius exemplify the above situation. In his classical experiments of transplantation of micromeres, the results are un- equivocal. Here, it must be kept in mind that these experiments were performed after the sep- aration of the micromeres. However, if the vegetal side of an egg is cut earlier than the 4th cleavage, the results were very ambiguous. Micromeres are still formed by so-called ‘“reg- ulation” and spicules are formed in various degrees. In other words, in earlier stages of development, although there is a vague separ- ation of the animal and vegetal characters, such vagueness suddenly becomes definite on the micromere formation. Runnstr6m [22] wrote that the vegetalizing material distributed diffusely in unfertilized egg which gradually accumulated to the vegetal pole. Horstadius [23, 24] thought the vegetal element was localized from the beginning which was activated around the time of micromere forma- tion. In the present author’s sense, the idea of activation of Ho6rstadius is nothing but a seg- regation of this substance into the micromeres by means of unequal division (see Discussion of [12]). At one time, a biochemist friend of mine blamed me for dispensing with biochemistry by cell geom- etry. This is a serious misunderstanding. What the author means is that cleavage process, partic- ularly unequal cleavage, sets up different stages of dramas in an embryo and only on these different stages, different biochemical dramas can be played, leading to various differentiations. Stating other- wise, through well-ordered process of cleavage, final differentiation is epigenetically reached step by step through cleavages. REFERENCES 1 Dan, K. (1979) 527-535. 2 Dan, K., Endo, S. and Uemura, I. (1983) De- velop. Growth and Differ., 25: 227-237. Develop. Growth and Differ., 21: 160 Conklin, E. G. (1917) J. Exp. Zool., 22: 311-419. Kawamura, K. (1977) Exp. Cell Res., 106: 127-137. Robertson, M.S. and Oppenheimer, S. B. (1975) Exp. Cell Res., 91: 263-268. Tanaka, Y. (1981) Wilhelm Roux’ Arch., 190: 267-273. Dan, K. (1954) Embryologia, 2: 115-122. Belanger, A. M. and Rustad, R. C. (1972) Nature, New Biol., 239: 81-83. Osanai, K. (1982) Bull. Marine Biol. Asamushi, Tohoku Univ., 17: 109-116. Dan, K. and Ikeda, M. (1971) Develop. Growth and Differ., 13: 285. Okazaki, K. (1975) In “The Sea Urchin Em- bryos”. Ed. by G. Czihak, Springer Verlag, Berlin, pp. 177-232. Dan, K., Noguchi, M. and Uemura, I. (1978) In ‘“‘“Mechanism of Cell Change’. Ed. by J. Ebert and T.S. Okada, John Wiley & Sons, New York, pp. 33-48. Agrell, I. (1958) Arkiv. Zool., 11: 435-438. Station K. DAN 14 15 16 17 23 24 Cowden, R. R. and Lehman, H. E. (1963) Growth, 27: 185-197. Tanaka, Y. (1976) Develop. Growth and Differ., 18: 113-122. Tanaka, Y. (1979) Develop. Growth and Differ., 21: 331-342. Harkey, H. A. and Whiteley, A. H. (1982) De- velop. Biol., 93: 453-462. Harkey, H. A. and Whiteley, A. H. (1982) Cell Differ., 11: 325-329. Kitajima, T. and Matsuda, R. (1982) Zool. Mag., 91: 200-205. Senger, D. R. and Gross, P. R. (1978) Develop. Biol., 65: 404-415. Tufaro, F. and Brandhorst, B. P. (1979) Develop. Biol., 72: 390-397. Runnstrom, J. (1965) Arch. Zool. 239-272. Horstadius, S. (1927) Wilhelm Roux’ Arch., 112: 239-246. Horstadius, S. (1928) Acta Zool., 9: 1-195. Ital., 51: ZOOLOGICAL SCIENCE 1: 161-168 (1984) © 1984 Zoological Society of Japan REVIEW Cell Contact and the Activation of Conjugation in Paramecium AKIO KITAMURA and KOICHI HIWATASHI Biological Institute, Tohoku University, Sendai 980, Japan CONTENTS I. REPU ls SNS IR re veined Mid waits laea hiss «upheld JdnaphESd ah dL dskieepbv hate ip edcabin dea ceihe aadaks 161 te ett TH Pe ONE AON PTOCESS its occi ss eananavnssbawdys nen saemoad op esalee ene apenme Gabtwnas toamerdad 161 III. Nature of the Mating Reaction A. 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It plays an important role in fertilization as well as in developmental processes such as cell migration and sorting out during embryogenesis and differ- entiation. In the ciliated protozoan Paramecium, the contact of cells occurs only when they undergo conjugation, an event homologous to metazoan fertilization. This sexual cell contact of paramecia can easily and instantaneously be induced and also be synchronized on a large scale to enable biochem- ical studies [1-3]. Thus, Paramecium provides a good system for analysing the mechanism of sexual cell contact and of signal transduction through cell membranes into a variety of responses. In this review we will first give an overview of the interactions of cell surfaces during the initial stage of conjugation in Paramecium caudatum and of the activation-initiating mechanism involved. Received September 5, 1983 Then, discussion will be extended to the role of hydrophobic interactions in the sexual cell contact and activation for conjugation. Just as the importance of hydrophobic interactions has been reported in phagocytotic cell recognition in human neutrophils [4] and in the cellular slime mold [5], so too in the sexual cell recognition in Paramecium have these non-specific hydrophobic interaction been found to play an important role [6]. Ii. CELL CONTACT IN THE CONJUGATION PROCESS Mating reactivity in Paramecium cells appears only in the stationary phase of the sexually mature period. Cells in the immature period of the clonal life cycle or in the log-phase of growth fail to show mating reactivity [1, 2, 7]. The expression of mating reactivity in Paramecium is observed as agglutination of cells when cells of complementary mating types are mixed. This agglutination is referred to as the mating reaction [8] and marks the initial phase of conjugation. About one hour 162 A. KITAMURA AND K. HIWATASHI after initiation of the mating reaction, the aggluti- nating clumps break down into pairs joining by a direct contact of cell bodies at the region near their anterior ends (holdfast union) where some cilia degenerate during the mating reaction. Then the contact area progresses posteriorly along the ventral surfaces to complete the final stage of the cell contact (paroral union). Although the mating reaction union is highly mating type specific, the holdfast union and the paroral union are known to be mating type non-specific [2, 9]. Thus, the holdfast union may be directly induced among cells of a single mating type without prior mating agglutination by treatment with various chemicals. This is called the chemical induction of conjugation. The conditions necessary for the chemical] induction include a low concentration of calcium ions and a high concentration of potassium or some other cations in the external medium [10]. In some species of Paramecium, an additional chemical such as acriflavine is necessary [11]. Il, NATURE OF THE MATING REACTION A. Chemical Properties of Mating Substances and Ciliary Membrane Vesicles The contact of cells in the mating reaction occurs only at the tips of cilia and thus the aggluti- nating clumps are easily broken down by mechanical agitation. There is no evidence for chemotaxis or substances secreted from cells that mediate cell contact in the mating reaction [1, 3, 8, 12}. The essential role of cilia in the mating reaction is confirmed by the fact that the mating agglutination is induced among cells of a single mating type by detached cilia from cells of opposite mating type [12-15]. Only cilia from the antero- ventral side of the cell have mating reactivity [16]. Inactivation experiments with the detached cilia revealed some chemical properties of the mating substances, the molecules responsible for the mating reaction [12]. Their reactivity was dest- royed by treatment with proteolytic enzymes such as trypsin, chymotrypsin, ficin and thermolysin, but not by glycosidases or phospholipases [17-19]. Although glycoproteins play an important role in specific cell recognition and contact [20, 21], sugar residues are not involved in the mating reaction of Paramecium. This conclusion is supported by experiments in which no sugars or lectins were found to inhibit the mating reaction [17, 22]. The mating substances appear to be intrinsic proteins of the ciliary membrane since mating reactivity of detached cilia is not affected by treatment with 10mM EDTA or 2M NaCl but completely de- stroyed by treatments with various detergents [17]. The simplest system so far obtained which can induce the mating agglutination is ciliary mem- brane vesicles with a diameter of about 50 to 100 nm. These membrane vesicles can be isolated by treatment of detached cilia with a solution con- taining 2M urea and 0.1mM EDTA and are called urea-EDTA membrane vesicles (UE-MV) [23]. Another method for isolation of ciliary membrane vesicles is to treat detached cilia with a detergent, lithium diiodosalicylate (LIS). The vesicles are called LIS membrane vesicles (LIS— MV) and have a character different from UE—-MV. When LIS-MV are added to cells of opposite mating type, selfing pairs (holdfast unions) appear in about one hour without agglutinative mating reaction [24]. These results with membrane vesicles support the idea that mating substances are tightly bound to the ciliary membranes. B. Inactivation of Ciliary Movement The mating reaction, an interaction of mating substances, triggers a series of events in conjuga- tion. The earliest change observed is the inactiva- tion of ciliary movement [25]. When mating reactive cells are treated with LIS-MV from the opposite mating type, they show a marked decrease in their swimming velocity within a minute. Observation of the treated cells revealed some change in ciliary movement. Some cilia on the ventral surface of the cell stuck together at their tips, probably by adhesion of the LIS-MV. Dor- sal cilia also did not show typical metachronal waves. Their beating direction is shifted to an anterior orientation. When ciliary beat frequency is measured photometrically, some decrease in the beat frequency was observed in the dorsal cilia of the treated cells. Since frequency and direction of the ciliary beat are correlated with membrane potential [26, 27], change in membrane potential Cell Contact and Conjugation in Paramecium 163 should probably be observed when the ciliary movements are inactivated during the mating reaction. However, no change in membrane potential or resistance was observed [25]. A possible mechanism of ciliary inactivation follow- ing sexual cell contact will be discussed in Section V; C. Early Micronuclear Migration The earliest morphological change observed in the mating reaction is early micronuclear migra- tion (EMM). In stationary phase cells of P. caudatum, the micronucleus is known to sit within a concavity of the macronucleus. However, the micronucleus moves out of the macronuclear con- cavity and migrates into the cytoplasm within the first 10min of the mating reaction [28]. This phenomenon, called early micronuclear migration, is known to be a reversible step leading to meiosis. If the mating reaction is interrupted by tryp- sinization, the micronucleus moves back into the macronuclear concavity and does not undergo meiosis (Kitamura, unpublished). D. Degeneration of Cilia and Loss of Mating Reactivity A conspicuous change of the cell surface during the mating reaction is degeneration of cilia which occurs along the ventral surface where mating reactive cilia are located. The degeneration of cilia begins at anterior tips of the cells (holdfast region) about 30 min after the initiation of the mating reaction [29], and subsequent to the for- mation of holdfast union proceeds posteriorly along the suture on the ventral sides, reaching the posterior ends. This degeneration of cilia appears to be prerequisite to the formation of holdfast and paroral unions. The degeneration of cilia is thought not to be caused by detachment or lysis of the cilia but to be due to resorption of the cilia since cilia of various lengths are seen before the completion of ciliary degeneration [29]. This assumption is further supported by examination of cell free fluid when a large amount of cells of both mating type are used for mating. At the time of paroral union, cell free fluid was centrifuged and observed under the phase contrast microscope, and no detached cilia or ciliary fragments were observed. When the cell free fluid was run on SDS-polyacrylamide gel electrophoresis, no ciliary proteins were detected (Kitamura, unpublished). Loss of mating reactivity is also known to occur during the early conjugation process [30]. The loss of mating reactivity, however, is not complete until paroral union formation, since most of the holdfast union pairs and even early paroral union pairs still retain a weak mating reactivity [10, Kitamura, unpublished]. Although quantitative change in mating reactivity during the conjugation process is as yet undetectable, it is of particular interest to know the mechanism of this inactivation and its role in the activation process, because there is no case of activation for conjugation without loss of mating reactivity [12]. As mentioned in the preceding section, the mating reactivity of detached cilia is easily lost by treatment with proteolytic enzymes but not by glycosidases or other enzymes. Therefore, we wanted to know if excretion of proteases from the cell would be the probable cause of the loss of mating reactivity. Kitamura [31] tested effects of 11 different protease inhibitors on the formation of holdfast unions to see if they inhibit holdfast unions as well as the loss of mating reactivity. In the presence of those inhibitors, however, the mating clumps broke down in the normal manner to form holdfast and paroral unions. These results confirmed that extracellular proteolysis is not involved in the loss of mating reactivity during the early conjugation process. Alternatively, the loss of mating reac- tivity might be caused by some morphological or chemical modification of cilia preceding or during their degeneration, since the area of the degenera- tion on the cell surface and distribution of mating reactive cilia seem to overlap [29]. IV. ACTIVATION-INITIATING MECHANISM As mentioned in the preceding section, interac- tion of mating substances at ‘activates’ the cells of Paramecium. ciliary surfaces The activation in conjugation of Paramecium has two different aspects, nuclear activation and activation for holdfast union, though they are interrelated with each other. In P. caudatum, a good indicator of 164 A. KITAMURA AND K. HIWATASHI nuclear activation in conjugation is the early micronuclear migration (EMM). EMM is prob- ably the earliest indication of micronuclear change for meiosis and is very closely associated with the induction of conjugation. However, little is known about the mechanism of EMM except that rapid warming after cold treatment and the calcium ionophore A23187 increased significantly the frequency of EMM [32] and treatment with col- chicine inhibited the occurrence of EMM [28]. The sequence of cell contacts in conjugation necessary for the micronucleus to enter meiosis with species of Paramecium. In _ P. caudatum, for instance, maintenance of paroral union is required for the micronucleus to enter the irreversible step of meiosis [33]. It is generally accepted that degeneration of cilia and formation of cementing substances (holdfast substances) are necessary for holdfast union. Protein synthesis is involved in the process leading to holdfast union formation, since actinomycin and puromycin are known to inhibit this process [34]. However, these inhibitors also inhibit the degeneration of cilia [3], so we cannot exclude the possibility that inhibition of ciliary degeneration blocks holdfast union formation without inhibiting the formation of holdfast sub- stances. Though the two events are closely asso- ciated, they can be separated, because addition of ficin, lipase or the lectin, concanavalin A (Con A), prevented holdfast union formation without in- hibiting degeneration of cilia [3, 35]. Since the action of Con A is restricted to the first 30 min from the beginning of the mating reaction and enhanced by pretreatment with trypsin, some Con A-sensitive change in membrane fluidity might be prerequisite for the formation of holdfast sub- stances [35]. In chemical induction of conjugation, treatment with certain chemicals activates paramecia in the same sense that metazoan eggs are activated with- out sperm in artificial parthenogenesis. Since chemical treatment can induce EMM, ciliary inactivation as seen in the mating reaction and holdfast union following degeneration of cilia, chemical induction of conjugation provides an alternative system for studying the activation- initiating mechanism [1-3]. As already mentioned, varies conditions for chemical induction of conjugation are very simple, i.e. a medium with a low concent- ration of calcium and high concentration of cations such as potassium or manganese. To understand the ionic mechanism in chemical induction of conjugation, Kitamura and Hiwatashi [36] ex- amined quantitatively the ratio of Ca ions to other cations effective to the chemical induction of conjugation and confirmed Miyake’s result [10] that the concentration of K ions necessary for the induction of conjugation increases with the con- centration of Ca ions added. Furthermore, they confirmed that the effective induction of conjuga- tion takes place at a constant ratio of the K* concentration to the square root of the Ca** con- centration in the induction medium. This sug- gests an important role of ionic exchange system of the cell surface ruled by Gibbs-Donnan principle or law of mass action in the chemical induction of conjugation. The activation for conjugation might be triggered by removal of Ca ions bound to the cell surface via an exchange for other cations. The ionic conditions for chemical induction of conjugation bear a strong resemblance to the conditions for the ciliary reversal [10, 37]. They induce an anterior shift of ciliary beat direction which is known to be induced by an abrupt influx of Ca’* across the ciliary membrane [38-40]. Thus, it appears plausible that influx of Ca** across the membrane triggers the activation for con- jugation. Actually, such a hypothesis was once proposed based on the result that pawn mutants of P. tetraurelia with defective excitable membranes are not susceptible to chemical induction of conjugation [41]. In P. caudatum, however, this possibility is less probable because chemical induction is possible for similar behavioral mutants of P. caudatum that have defects in the mechanism of Ca’* influx across the ciliary membrane [42]. Even if influx of Ca’* across the membrane appeared to have no role in the activation, it would not exclude the possibility that an increase in free Ca** inside the cell play an important role in the activation of Paramecium as in fertilization of various metazoan eggs [43]. For further discussions on the role of Ca’* in activation-initi- ating mechanism in conjugation of Paramecium, refer to other reviews [32, 44]. Cell Contact and Conjugation in Paramecium 165 Vv. A MODEL SYSTEM FOR ANALYSING THE MECHANISM OF SEXUAL CELL CONTACT AND ACTIVATION Activation of Paramecium cells for conjugation is initiated by agglutinative mating reaction which depends upon the activity of mating substances on the ciliary surfaces. The mating-substance activity appears only when cells are in the stationary phase of the sexually mature period, and the same con- ditions are necessary for activation by conjugation- inducing chemicals. However, this does not necessarily mean that only cells with mating- substance activity can be activated by the con- jugation-inducing chemicals. The trypsin treat- ment which completely destroys the mating- substance activity does not affect the chemical induction of conjugation [34]. This fact suggests that the receptor site for the chemical induction of conjugation is different from the mating sub- stances or at least from their active site. A completely different method for the study of the activation-initiating mechanism was recently developed by Kitamura [6]. When mating reactive cells were placed into polystyrene Petri dishes, they quickly attached to the dish surface. Non- mating reactive cells never attached to the same surface. Observation with an interference micro- scope revealed that the cell attached exclusively at the tips of ventral cilia where mating reactivity is localized (Fig. 1). Cells attached to the poly- styrene surface irrespective of the concentration of Ca ions in the medium and even in a solution of relatively high ionic strength. On the other hand, a decrease in hydrophobicity of the dish surface decreased the attachment. When the dish surface was made hydrophilic by treating with sulfuric acid, the treated surface did not bind mating reactive cells. The attachment scarcely occurred to tissue culture Petri dishes which have surfaces less hydrophobic than those of bacteriological dishes. Thus, electrostatic interactions do not seem important, but hydrophobic interactions play an essential role in the attachment of paramecia to polystyrene. Evidence supporting the above con- clusion is obtained when non-mating-reactive cells were treated with certain hydrophobic reagents. Cells showing no mating reactivity and thus inca- Fic. 1. Photomicrograph of a cell attaching to the polystyrene surface at the tips of antero-ventral cilia (arrow). Bar, 30 um. pable of attachment to polystyrene become capable of attachment when treated with phenethylamine or benzylamine [45]. Does the fact that only mating reactive cells attach to polystyrene surfaces mean that the mating substances are the molecules acting for the attach- ment? When cells in the mating reactive stage but whose mating reactivity was completely destroyed by trypsin were tested, they showed an increased ability of attachment to polystyrene. In some mutant stocks with a special combination of genotypes [46], mating reactivity does not appear even in stationary phase of sexually mature period but conjugation can be induced chemically. When cells of these mutant stocks were tested for the attachment to polystyrene surface, they attached only during stationary phase where mating reac- These results suggest that the attachment sites in the cilia are tivity appears in wild-type stocks. different from the site of mating substance activity. A similar conclusion was obtained from immuno- logical experiments using P. multimicronucleatum [47]. In this species mating-blocking antibodies (MB~—Ab) can be obtained which prevent the mating reaction and inhibit conjugation, but do not im- mobilize cells [48]. MB-Ab are thought to be directed against a component other than the site of mating substance activity since inhibition of mating activity by the MB~Ab lacks mating-type specificity. When mating reactive cells are treated 166 with purified MB-—Ab, their attachment to poly- styrene surface was enhanced despite their loss of mating reactivity. Although the molecular mechanism of attach- ment to polystyrene is different from the interaction of mating substances during the mating reaction, these two phenomena have a common result. EMM was found in the cells attached to poly- styrene as in those in the mating reaction. The micronucleus which migrated after attachment, however, did not undergo meiosis, but returned to the macronuclear concavity about 60min after the attachment. Thus, the EMM induced by hydrophobic interaction between the cell and the polystyrene surface is reversible. Loss of mating reactivity seen in the activation process of normal conjugation was also observed in the polystyrene- attached cells. These lines of evidence suggest that some hydrophobic interaction might be working during the mating reaction in addition to 1) GP = GP 2) oe 3) GP ina Rage Ts mating—non—reactive A. KITAMURA AND K. HIWATASHI the mating substance interactions. An increase in hydrophobicity of the cell surface during the mating reaction or following chemical induction of conjugation was recently found (Kitamura, in preparation). Although mating reactive cells cannot attach to surfaces of reduced hydrophobicity such as dishes for tissue culture or their lids, once the cells have undergone the mating reaction they attach to these surfaces. This change of the cell’s ability to attach occurred within 5 min after the initiation of the mating reaction, sug- gesting a rapid change in the properties of ciliary membrane surfaces. A similar increase in the ability to attach to polystyrene was seen when mating reactive cells were treated with conjugation inducing medium. This increase in hydropho- bicity of the membrane surface might explain the ciliary inactivation seen in the mating reaction or in conjugation-inducing medium if we assume an adhesion of ventral cilia by the hydrophobic Fic. 2. Schematic presentation of relationships between mating substance and attachment site for polystyrene in the ciliary membranes. Open squares represent active sites of the mating substance. Striped portions: attach- ment sites for polystyrene. GP: glycoproteins. mating-—reactive Cell Contact and Conjugation in Paramecium 167 interaction. Miyake [10] reported that cells of the same mating type become sticky and show a temporary contact with each other in conjugation- inducing media. This report supports the above assumption. The mating substances are known to be intrinsic proteins of the ciliary membrane [2, 12, 17]. The attachment of cells to polystyrene surfaces occurs when cells are in the mating-reactive stage, but the mechanism of attachment is different from the mating substance interaction. Then, what sub- stances are involved in the polystyrene attachment of the cell? It has been suggested that the attach- ment molecules might be the receptor for chemical induction of conjugation [6], because both the attachment and chemical induction of conjugation are possible in cells in the mating-reactive stage and are not affected by trypsinization of the cells. But no direct evidence exists for such a hypothesis. Three different hypotheses for the identity of the attachment substances are possible (Fig. 2). 1) Molecules different from the mating substance are involved in the attachment, and appearance of these molecules is in some way coordinated with the appearance of the mating substances. 2) Hydrophobic components of the mating substance are involved in the attachment, and these com- ponents do not contain the mating reactive site. 3) Free lipid surfaces of the ciliary membranes are protein synthesis ; oI ae holdfast change in substance ; : | union formation mating Ge A reaction membrane fluidity at appa ta inactivation (7) increase in hydrophobicity degeneration i ae of cilia the site for the attachment, and these surfaces increase with the appearance of mating reactivity. If hypothesis 1 or 2 is the case, we might be able to isolate the mating substances or the adhesion molecules using hydrophobic affinity chromato- graphy. he possibility of hypothesis 3 can be tested by the electron microscopic observation of ciliary membrane using the freeze fracture tech- nique. These attempts are now in progress in our laboratory. VI. CONCLUSION The mating reaction triggers the sequential changes of the early stages of conjugation in Paramecium (Fig. 3). The earliest change ob- served microscopically is inactivation of ciliary movement. This ciliary inactivation is not caused by a change in membrane potential but probably is caused by an increase in hydrophobicity of the ciliary membrane. Ciliary inactivation may cor- relate with some change in properties of the ciliary membrane, such as membrane fluidity. This change of ciliary membranes may function as a trigger reaction for the formation of holdfast substances, a hypothetical cementing substance for holdfast unions. Both synthesis of the hold- fast substances and degeneration of cilia are necessary steps for holdfast union formation which parora union \ loss of ——$—$$$<—$<<——_—> mating reactivity meiosis Fic. 3. Scheme for activation in P. caudatum. . <4+ main c minuves Time scales are approximate. 168 is a major manifestation of activation in conjuga- tion of Paramecium. On the other hand, hydro- phobic interactions on the ciliary surfaces may also play an important role in the early cell contact and nuclear activation in conjugation. Early micronuclear migration (EMM), an indication of nuclear activation, is induced in cells attached to hydrophobic surfaces. Cell’s ability to attach to hydrophobic surfaces appears when cells express mating reactivity and increase during the mating reaction or in the conjugation-inducing chemicals. 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Funcet., 2: Jo "Gell ZOOLOGICAL SCIENCE 1: 169-186 (1984) Response Modification of Cricket Sensory Interneurons during Flight KENJI TOMIOKA! and TSUNBO YAMAGUCHI2 Department of Biology, Faculty of Science, Okayama University, Okayama 700, Japan ABSTRACT — Extracellular unit responses of various interneurons were recorded from the subesophageal ganglion and cervical connectives in the cricket (Gryllus bimaculatus), and their re- sponsiveness to sensory stimuli was examined in quiescence and in flight. A total of 41 interneurons were functionally identified, and they fell into the five main classes: 1) visual interneurons responding exclusively to moving objects or light-on and/or -off, 2) auditory interneurons responding to sound pulses, 3) mechanoreceptive interneurons responding to mechanical stimulation of sensory hairs on the appendages and other bodily parts, 4) multimodal interneurons with two or more sensory inputs whose modalities differ each other, and 5) activity interneurons changing their firing level under the influence of animal’s own movement or behavior. About 75 percent of these sensory interneurons showed definitely various types of flight-associated change in the responsiveness to sensory stimuli or “response modification’. The simplest type of response modification was that the response to a well-defined stimulus is suppressed or enhanced during flight. We observed the response modification even in the animal assuming the flight posture without actually flapping its wings, or in the animal whose nervous system was deafferentated. Therefore, the response modification is probably operated mainly by the central source originating in the flight motor center rather than by the peripheral or © 1984 Zoological Society of Japan reafferent source. INTRODUCTION The use of the sensory information from external sources is known to vary dependent on the animal’s own behavioral state [e.g., 1]. For instance, the tethered cricket exhibits faithfully the head roll- ing movements toward the light source to sta- bilize its flight posture as long as it keeps flying, but if it stops flying, it never does so as though it ignores the light source [2]. Furthermore, the stationary cricket suspended in air starts to fly in response to the air current directed to its head, whereas it never does so when it is glooming [3]. These facts strongly suggest that the integration and transmission of sensory information in the central nervous system must be modified with Accepted September 16, 1983 Received June 25, 1983 1 Environmental Biology Laboratory, Biological Insti- tute, Yamaguchi University, Yamaguchi 753, Japan. 2? To whom offprint requests should be sent. the behavioral context. In fact, there are some findings suggesting that the responsiveness of higher order interneurons to sensory stimuli is modified by inputs activated by the animal’s own behavior. Typical examples of these “response modification” are that in the cock- roach and cricket the responses of giant interneu- rons running through the abdominal connectives to tone pulses are almost completely suppressed or somewhat enhanced during walking [4~9], and that in the crayfish the sensory interneurons re- sponding to water vibrations are inhibited during walking [10]. Another example is that in the crayfish the visual responses of movement fibers are suppressed during eyestalk movement, but both the movement and sustaining fibers show greater responsiveness to visual stimuli when the animal is moving than when it is __ stationary [12-14]. According to the works which have dealt main- ly with the response modification of higher order 170 K. TOMIOKA AND T. YAMAGUCHI interneurons during the animal’s own movement, the excitatory or inhibitory modification could result from reafferent inputs during movement, or from descending pathways originating in the motor centers. For instance, Delcomyn and his co-worker [5, 7-9] showed that during walking the excitatory and inhibitory response modifications of cockroach giant interneurons have both major cen- tral descending and lesser peripheral ascending or reafferent components. Murphey and Palka [4] reported that during walking the inhibitory mod- ification of responsiveness of cricket giant inter- neurons originates from peripheral as well as central sources. Moreover, Orida and Josephson [6] described that in the cockroach and cricket the peripheral source is more effective than descending pathways from the thoracic ganglia in modifying the sound responsiveness of giant interneurons during walking. On the other hand, Wiersma and his co-workers [11-14] suggested that in the crayfish central nervous system the activity fibers running through the nerve cord in parallel with sensory interneurons may provide the descending pathways that mediate the excited- state influence on the responsiveness of visual interneurons. In the present experiment, the unit responses of interneurons were recorded extracellularly from the subesophageal ganglion and cervical connec- tives of crickets, and their response characteristics to sensory stimuli were analyzed in detail in a definite set of the animal’s behavioral states, i.e., quiescence and flight. We will describe that most (about 75 percent) of the identified sensory inter- neurons show the excitatory or inhibitory response modification during flight, and show that a major source of flight-associated modification may orig- inate in the flight motor center. MATERIALS AND METHODS (1) Preparation In all experiments, adult male crickets Gryllus bimaculatus obtained from laboratory culture were used. They were kept under controlled conditions of temperature (26-28°C) withan LD 12:12. Be- fore the dissection, the prothoracic legs were fixed to the lateral sides of the pronotum for the stable presentation of sound stimuli, and then the ani- mal was mounted ventral side up on a support- ing rod by the back with colophonium beeswax mixture. Held this way, most animals could begin flying or assuming the flight posture with- out flapping the wings, i.e., pseudo-flight posture™*, as soon as their heads were exposed to a wind- stream [2]. For recording from the cervical (sub- esophageal-prothoracic) connectives the recording site was exposed by removal of the ventral neck membrane, occasionally of both the ventral neck membrane and sternum extending between the left and right legs, but for recording from the subeso- phageal ganglion by removal of the mouthparts. In all cases the tracheae were left intact not to block oxygen supply to nervous tissues and muscles. Therefore, a recording electrode could be intro- duced through the intact sheath into the desired nervous tissue from the animal’s ventral side. (2) Recording methods Extracellular responses of interneurons were recorded with a glass microelectrode filled with 3M cobaltous chloride or cobaltous nitrate. Sta- ble recordings were achieved by placing a tungsten platform beneath the desired nervous tissue to im- mobilize it during the flight or respiratory move- ments of body. This platform served also as an indifferent electrode. The microelectrode poten- tials were amplified with a high input impedance preamplifier (Nihon Kohden, AVZ-9), and mon- itored on an oscilloscope (Nihon Kohden, VC~9). To record the electrical potentials of large interneurons such as the descending movement detectors (DMDs) [15-18] from a whole cervical connective, a pair of 100 ~m platinum hook elec- trodes insulated by petroleum jelly were used. Muscle potentials were recorded extracellularly with 50 wm copper wire which was insulated but for the tip and implanted into identified flight muscles [19]. These potentials were amplified with a biophysical amplifier (Nihon Kohden, AVB-9), and displayed on the oscilloscope. In most experiments, the potentials led from the * Since both the actual flight and pseudo-flight exerted the identical influences on the responsiveness of sensory interneurons, we did not discriminate be- tween them throughout the present experiment. Response Modification of Interneurons 171 interneurons were stored on magnetic tape with a four channel tape recorder (Sony Magnescale, DFR-3415). After each experiment the tape was replayed for subsequent analysis, and the re- cords were photographed with a moving film camera (Nihon Kohden, RJG—6101). In some experiments, the potentials were fed into a window discriminator (Mentor, N—750 Spike Analyser) and the output of window dis- criminator was used to produce a visual display in which each incoming spike triggered a dot on the oscilloscope screen. (3) Data analysis In order to analyse precisely the responsiveness of interneurons, the responses of each interneuron to successive sensory stimuli were summed and displayed on-line or off-line from the tape-re- corded data as latency histogram or spike dis- tribution on a histogram data processor (Nihon Kohden, DAB-5101). Before the analysis of responses, spikes were visually examined on the oscilloscope screen to determine whether spikes from one or more interneurons could be dis- tinguished by form and amplitude. If the spikes could be reliably discriminated, then they were converted into uniform pulses by means of another window discriminator built in the processor. The summing cycle of 256 bins of 10 msec width was triggered by a command pulse from a stimulator (Nihon Kohden, MSE-3) which was synchronous with a triggering pulse for visual or sound stimulus. (4) Sensory stimulation Visual stimuli were usually provided by two 9V tungsten filament microlamps (Hamai Denkyu, 9- V), each of which was located about 3 cm away from the compound eye. Stimulus presentation and duration were controlled by a relay system which was operated by the stimulator. To stimulate the DMDs, the light from a slide pro- jector was focused on the tip of a fiber optics (2mm in diameter), and then delivered to the compound eye ipsilateral or contralateral to the recording connective. The distance between the illuminating tip of fiber optics and the compound eye was lcm, so that the size of the stimulus spot subtended about 11° at the surface of the compound eye. An electromagnetic shutter was placed in the optical pathway between the slide projector and fiber optics. The shutter was operated by a pulse or pulses generated with a stimulator to control both stimulus duration and frequency. The presentation of visual stimula- tion was monitored by a solar cell, whose output was recorded on the magnetic tape simultaneous- ly with the response of interneuron. Sound stimuli were pure tone pulses synthesized electronically. The carrier frequency of each tone pulse was produced by an oscillator (Kikusui Denshi, ORC-27A). Then, the tone pulses were amplified with an audio-amplifier (Pioneer, SA-— 6600 II), and delivered through either a full- range speaker (Fostex, FE103) for frequency up to 18kHz or a super high range tweeter unit (Onkyo, TW 3001) for higher frequency range from 15 kHz up to 35kHz. The speakers were placed at the same level as the cricket, 90° to the left and right with respect to its longitudinal body axis, at a distance of 20cm. Sound pres- sure levels were measured with a sound level meter (Briiel and Kjaer, 2209) and a microphone (Briiel and Kjaer, 4135) placed at the position occupied by the cricket. All sound pressure levels were expressed in decibels relative to 0.0002 dynes/cm’ (20 Pa). Wind stimuli were given by flowing the air from a compressor through a vinyl tube (15 mm in diameter) directed to the frontal part of the head. For mechanical stimulation of the hairs on the appendages and body, they were touched manually with a thin needle. (5) Morphological examination of interneurons To confirm whether the neuron which has been encountered is an interneuron or not, the micro- electrode was left in place for about 30 min after recording to diffuse cobalt into the neuron with- out passing the current. Then, the nerve cord from the brain to the metathoracic ganglion was isolated, exposed in saline [20] containing am- monium sulphide, and fixed in Carnoy fixative. In most cases, after fixation the preparation was then intensified using a whole mount intensifi- cation procedure [21], dehydrated, cleared and mounted on a hollow slide. 172 RESULTS In the subesophageal ganglion and cervical connectives we have encountered a large variety of neurons. Cobalt staining of these neurons revealed that most of them are interganglionic interneurons connecting separate parts of the central nervous system. However, we could not succeed in staining their whole structures including the dendrites, axon terminals and somata; this failure may be ascribed to the insufficient penetra- tion of microelectrode into the axon that results in the extracellular cobalt staining of interneuron [22]. Insofar as we have examined, it was evident that most of the interneurons have rich branches extending to the ipsilateral neuropiles of the prothoracic ganglion, and poor branches in the subesophageal ganglion, and that no. great differences are visible among the pathways of interneurons of a given type through these ganglia and the branches from their axons in different individuals. In this experiment, no further ana- lysis of the morphological characteristics of inter- neurons has been carried out, because the main purposes of the work were to survey the response characteristics of them to sensory stimuli and to know the influences of behavioral switching on them. To exclude errors due to variation in the res- ponses of interneurons of a given type among different individuals, an effort was made to increase the sampling number of interneurons. TABLE 1. K. TOMIOKA AND T. YAMAGUCHI Practically, there was little interindividual vari- ation among the response characteristics of interneurons of a given type, so that the various interneurons of these animals could be compared for analysis. As the result of examination of the responsiveness of each interneuron to sensory stimuli given while the cricket was stationary or flying, we have identified a total of 41 interneurons functionally on the basis of their rather stereotyped responsiveness. They ranged from pure unimodal sensory interneurons to highly complexly respond- ing interneurons, and fell into five main classes: visual, auditory, mechanoreceptive, multimodal and activity interneurons (Table 1). About 75 percent of these sensory interneurons showed a variety of modification in their responsiveness to sensory stimuli in association with the behav- ioral switching from quiescence to flight. We will give a description of the response characteristic of each identified interneuron with special regard to the response modification. (1) Visual interneurons A total of 18 interneurons responding exclusively to visual stimuli were repeatedly recognized. A- mong them, two were the movement interneurons which respond to objects moving, with regard to the background, in their visual fields, and the remaining 16 were the on-off interneurons which respond to light-on and/or -off stimulation with the spike discharge. Interneurons of different modalities Number of interneurons Number of interneurons Class of interneuron that show the response that do not show the Totals modification response modification Visual interneurons On-off interneurons 15 1 16 Movement interneurons 1 1 ys Auditory interneurons 1 3 4 Mechanoreceptive interneurons 1 2 3 Multimodal interneurons 9 D 11 Activity interneurons 5* 0 5 Totals 32 9 4l SS RES SSS SSS SSS SSS SS * These interneurons never respond to well-defined sensory stimuli, but change their firing level under the influence of animal’s own movement. Response Modification of Interneurons 173 Movement interneurons C3* was the large interneuron found 37 times. This interneuron responded to a moving object with a long train of spikes and to light-off with a brief burst, and habituated quickly to repeated stimuli. Its visual field covered the almost whole corneal surface of the ipsilateral eye. C3 is homo- logous with the descending ipsilateral movement detector (DIMD) described in orthopteran in- sects [15-18]. The latency of off-burst was about 60 msec when the animal was stationary, but it shortened to about 50 msec as soon as the animal started to fly (Fig. 1A). A small increase in the a fom) | my aA ® | : E Pastel od) 2 = 60 ‘ wh 5 uve we =: [rae a oO ae 2 5 Bites “Li 2 Pubits = 50 a * ” Hie onl v 6107 B — = Weer: o Bisiiedlimcsee, Unie Ghiead od pubes, wed = tater anata er ve et eee 2 0 . Sha. MNT 0 50 100 Fic. 1. Latency (A) and spike number (B) histo- grams of an unilateral movement interneuron (C3). The light flash of 400 msec was presented to the ipsilateral eye every 4sec. The abscissa indicates the number of presented flash. The animal kept flying while 56 flashes were presented successively, and then it ceased to fly about the time of the 57th flash (arrow heads). Note that the latency and spike number are shorter and larger during flight than during quiescence, respectively. spike number of off-burst was also recognizable during flight (Fig. 1B). After the cessation of flight both the latency and spike number recovered gradually to the values exhibited in the stationary State. * The interneurons were designated with consecutive numbers preceded by a C as they were established. C39 is a partner of C3 and homologous with the descending contralateral movement detector (DCMD) [15-18]. This interneuron responded exclusively to a moving object or light-off stimulus in the field of the contralateral eye, habituated quickly to them. This responsiveness to visual stimuli remained unchanged when the animal started to fly. This interneuron has been found nine times. On-off interneurons CS is a bilateral on-off interneuron which responds to light-off on either side with a few spikes, while the animal is stationary. During flight, C5 kept firing spontaneously at a rather high rate, but responded to light-on and -off stimuli to either eye with a slight decrease of the firing rate of spontaneous discharge and the post- inhibitory rebound, respectively. This interneu- ron has been found only twice. A ata . . ~- --. - _ ~- — ~ = —_— a SW eee ae 500 msec Fic. 2. Responses of a bilateral on-off interneuron (C11) to successive flashes. In the upper records (A, C) the flashes were given to the ipsilateral eye, and in the lower records (B, D) to the contralateral eye. In the left records (A, B) the flashes were delivered to the eyes when the animal was stationary, in the right records (C, D) when it was flying. In each record the sweep of oscilloscope was triggered before each flash, dots represent the occurrence of spikes, and the bottom trace is the flash monitor (upward deflection, light on). Re- cords are read from bottom to top. Flash frequency was 0.4 Hz. Note that the strong response occurs with the post-excitatory rebound to flash presented during flight. 174 K. TOMIOKA AND T. YAMAGUCHI C6 responded to light-off stimulus to the ipsilateral eye with a few spikes, and to light-on stimulus to the contralateral eye with several spikes, when the animal was stationary. When it was forced to fly by the windstream on its head, C6 began to fire spontaneously in advance of the occurrence of electrical activity of the flight muscles. During flight, this interneuron responded to the ipsilateral illumination with a decrease in the firing rate of spontaneous dis- charge, and to the contralateral illumination with an increase in it. C6 may occupy its locus close to that of C40 mentioned later, because these two interneurons were often recorded in a pair in one lead. C6 has been found eight times. C10 fired a few spikes at light-on and occa- sionally at light-off to either eye in the stationary animal. However, in the flying animal it fired continuously at a low to medium frequency in the absence of visual stimuli, and responded to illumination with a burst of spikes summing with the spontaneous discharge. This interneuron has been found five times. Cll has a very similar response property to that of C10. However, compared to the contra- lateral illumination, the ipsilateral one evoked strong response (Fig. 2A, B). Once the animal started to fly, this interneuron fired tonically at a low to medium frequency and showed the sus- tained discharge and post-excitatory rebound in response to illumination on either eye (Fig. 2C, D). Even in the flying animal, the ipsilateral response was stronger than the contralateral one within the examined range of light intensity. C11 has been found four times. C18 has some similarity in the response prop- erty to C5. As shown in Figure 3A, B, while the animal was stationary, this interneuron responded to the ipsilateral illumination with small number of spikes, but not to the contralateral illumination. However, while the animal was flying, C18 fired continuously at a high frequency in the dark, but its firing was almost completely suppressed for the period of illumination on either eye (Fig. 3C, D). It has been found only twice. C19 fired spontaneously with a group of two or three spikes at the same frequency as that of the wing beat (about 30 Hz), whether the animal was 250msec Fic. 3. Responses of a bilateral on-off interneuron (C18) to successive flashes. In the upper records (A, C) the flashes were given to the ipsilateral eye, but in the lower records (B, D) to the contralateral eye. In the left records (A, B) the flashes were delivered to the eyes while the animal was quiescent, in the right records (C, D) while it was flying. Note that during quiescence C18 shows the phasic response to each flash given only to the ipsilateral eye, whereas during flight it elicits the spontaneous discharge which is inhibited by the flash given to either eye. For further explanations see Fig. 2 and text. flying or not. A slight increase in discharge frequency was observable at light-off to either eye only during flight. This interneuron has been found three times. C20 discharged tonically both during quiescence and during flight: after the initiation of flight the discharge frequency increased from 15 Hz to 20 Hz. When the light-on stimulus was delivered to the ipsilateral eye, there was a marked transient in- crease in the discharge frequency, followed rapidly by a return to the initial frequency. The light-on stimulus delivered to the contralateral eye re- sulted in a brief suppression of firing, followed by a gradual return to the level of firing in the dark. These response properties were not modified by the behavioral switching from quiescence to flight. This interneuron has been found three times. C23 discharged rarely in response to illumi- nation on the ipsilateral eye, but sometime it responded with one or two spikes with a long latency (about 90 msec). In the flying animal, Response Modification of Interneurons 175 however, this interneuron showed the spontaneous firing with a low frequency and responded to illumination on the ipsilateral eye with a strong burst of spikes with a short latency (about 50 msec). The occurrence of each spike in the burst synchronized with the wing beat. This interneuron has been found three times. C26 is an interneuron responding phasically to illumination on the ipsilateral eye while the animal is stationary. However, this interneu- ron started to fire spontaneously at the beginning of flight, and continued to fire while the animal was assuming the flight posture. Light-on and -off stimuli delivered to the ipsilateral eye during flight resulted in transient increase and decrease of the firing rate of spontaneous discharge, respectively. This interneuron has been found four times. C27 has the response property which is rather similar to that of C5, except that during quie- scence this meuron never responds to visual stimuli. It has been found three times. C29 showed the strong response with a long latency to light stimulus given to either eye in the stationary animal: the latency to the ipsi- lateral and contralateral stimuli was about 350 msec and 600 msec, respectively. In the flying animal, C29 fired continuously at a very low frequency and exhibited peculiar responses to light stimuli. That is, at light-on, there appeared immediately a slight increase in the firing frequency, followed by a rapid increase at least 600 msec after the light-on. The post-excitatory rebound was ob- served at light-off. C29 has been found nine times. C34 responded to light-off in the field of the contralateral eye with a transient burst of spikes, when the animal was stationary. During flight, ee B C 500 msec Fic. 4. Simultaneous recordings of a bilateral on-off interneuron (C40) and the ipsi- lateral hindwing depressor muscle (129a). In each record the top trace is the re- sponse of C40, the middle trace shows the electrical activity of 129a, and the bottom trace indicates the light stimulus (upward deflection, light on). The flashes were presented to the ipsilateral to flashes in the stationary animal. (A, B) Responses eye in A and to the contralateral eye in B. (C) The tonic discharge of C40 and the electrical activity of 129a was accompanied by the initiation of flight. The flight was induced by the windstream on the head. The arrow head indicates the onset of windstream. Note that the firing of C40 precedes the electrical activity of 129a. (D, E) Responses to flashes in the flying animal. The flashes were delivered to the ipsilateral eye in D and to the contralateral eye in E. 176 K. TOMIOKA AND T. YAMAGUCHI it fired spontaneously and responded to light-on with a transient frequency decrease and to light-off with a transient frequency increase. It has been found three times. C35 never responded to visual stimuli while the animal was stationary. However, after the wind was let to flow on the head to induce the flight, it began to fire spontaneously at a low frequency in advance of the actual initiation of flight. During flight, it responded rather tonically to light-on stimulus given to the contra- lateral eye. It has been found three times. C37 is an on-off interneuron which shows no spontaneous discharge and responds to light-on stimulus to the contralateral eye with a tonic burst of spikes. This response property was not modified in association with the flight beha- vior. This interneuron has been found only twice. C40 responded to the ipsilateral light-on stimulus with a long train of spikes, and to the contralateral light-off stimulus with a few spikes (Fig. 4A, B). This interneuron began to fire in advance of the initiation of electrical acitvity of the flight muscles, when the animal was stimu- lated by blowing the wind on the head to induce the flight (Fig. 4C). While it kept flying, C40 responded to the ipsilateral illumination with an increase of the firing rate of spontaneous dis- charge, and to the contralateral illumination with its decrease (Fig. 4D, E). This interneuron has been found nine times. C41 is a “‘flight-gated unilateral on-off inter- neuron”. That is, this interneuron was silent and ignored visual stimuli when the animal was stationary. However, when the animal started to fly, C41 became active just after the initiation of flight and responded to light-on stimulus given to the ipsilateral eye with a slight decrease in the firing rate of spontaneous discharge (Fig. 5A- E). C41 has been found three times. A er re ea TSE > REECE ee ee Fic. a) 500 msec Simultaneous recordings of a flight-gated unilateral on-off interneuron (C41) and the ipsilateral hindwing depressor muscle (129a). In each record, the top trace is the discharge of C41, the middle trace is the electrical activity of 129a, and the bottom trace indicates the light stimulus (upward deflection, light on). The records, A and B were obtained during quiescence, and the other records, C, D and E were obtained during flight. The light stimulus was presented to the ispilateral eye in A and D, but to the contralateral eye in B and E. In the record C, the animal was forced to fly by blowing the wind on the head. The arrow head represents wind on. Response Modification of Interneurons 177 (2) Auditory interneurons Four interneurons were identified as pure auditory interneurons. All these were the bi- lateral interneurons responding to sound stimuli presented to either side. C13 responded to sound stimuli of higher frequency (peak sensitivity, 15 kHz) and higher sound pressure (more than 100 dB) given to either side. Usually, the spike number of the ipsilateral response was more than that of the contralat- eral one. The response to sound stimuli was quite stable, irrespective of whether the animal was stationary or not. found 15 times. C16 is an unique interneuron whose response A This interneuron has been B l Be Ct OA TEE OW 6 et Wine Es characteristic is markedly modified by behav- in Figure 6A, B, when the animal was stationary, this interneuron ioral switching. As shown responded to sound stimulus presented from ei- ther side with a burst, and its peak sensitivity was at 15 kHz. to the windstream to When the animal was exposed flight, C16 started to fire rather tonically in advance of the flight muscles, followed by gradual disappearance of firing: it ceased completely from firing 30-40 sec after the initiation of flight (Fig. 6C). During flight, C16 responded to sound stimulus with a very long lasting burst. Namely, the spike dis- charge elicited by the sound stimulus lasted for several seconds, followed by gradual fade-out, induce the occurrence of electrical activity of the 1 Wi] | | tio deus ' i | {WHT HH | Hh | III Fic. 6. ipsilateral hindwing depressor muscle (129a). 900 msec Simultaneous recordings of a bilateral auditory interneuron (C16) and the In each record the top trace is the response of C16, the middle trace is the electrical activity of 129a, and the bottom trace is the sound monitor showing just only the onset of sound emission (upward de- flection, sound on). animal. tralateral side in B. C16 ceased to fire about a half minute after the initiation of flight. sponses of C16 to sound stimuli in the flying animal. delivered to the ipsilateral side in D and to the contralateral side in E. was induced by the windstream (arrow head) on the head. stimuli elicit the long-lasting responses in the flying animal. (A, B) Responses of C16 to sound stimuli in the stationary The sound stimuli were given to the ipsilateral side in A and to the con- (C) Spike discharge occurring during the initial period of flight; (D, E) Re- The sound stimuli were The flight Note that the sound Carrier frequency: 15 kHz. Sound pressure level: 80 dB. For other explanations see text. 178 K. TOMIOKA AND T. YAMAGUCHI 250 msec Fic. 7. Responses of a bilateral auditory interneuron (C17) to successive sound stimuli. In the upper records (A, C) the sound stimuli were presented to the ipsilateral side, and in the lower records (B, D) to the contralateral side. In the left records (A, B) the sound stimuli were applied to the stationary animal, in the right records (C, D) to the flying animal. In each record the sweep of oscilloscope was triggered before each sound sti- mulus, dots represent the occurrence of spikes, and the bottom trace is the sound monitor showing just only the onset of sound emission (upward de- flection, sound on). Records are read from bottom to top. Carrier frequency: 15 kHz. Sound pressure level: 80 dB. Inter-sound interval: ZA SeG: even when the duration of sound stimulus was shorter than 100 msec (Fig. 6D, E). This inter- neuron has been found five times. C17 showed the maximum spike response when the sound stimulus with carrier frequency of 15 kHz was presented from either side of the animal. Flight behavior did not give any in- fluence on its response (Fig. 7A—D). This bilat- eral auditory interneuron appears to be identical to the HF1 neuron which has been described by Rheinlaender et al. [23]. C17 has been found three times. C24 responded to rather low carrier frequency sound (peak sensitivity; 4kHz) as well as to each sound pulse of conspecific calling song. This responsiveness was not modified by flight behavior. These suggest that C24 is identical to the LF1 neuron identified by Rheinlaender ef al. [23]. C24 may occupy very close locus to C17, for a pair of these were frequently recorded in one lead. C24 has been found 16 times. (3) Mechanoreceptive interneurons Since much attention was paid to identify the interneurons responding to visual or sound stim- ulation, only three mechanoreceptive interneurons were established. C28 showed spontaneous discharge in the absence of stimulus while the animal was sta- tionary. The firing rate of spontaneous discharge increased largely as soon as the animal started to fly. However, the spontaneous discharge was in- hibited by tactile stimulation given to the ipsi- lateral cercus, irrespective of whether the animal was flying or not. This interneuron has been found three times. C31 is a mechanoreceptive interneuron which responds to tactile stimulation to either of the antennae with a phasic burst of spikes and to wind stimulation to the head with a tonic dis- charge at a low frequency. The behavioral switching did not significantly influence on the responsiveness of this interneuron. C31 has been found three times. C36 showed the spontaneous discharge, ceasing it in response to wind or tactile stimulation ap- plied to the cercus on either side. This respon- siveness was never modified by the change in behavioral state. C36 has been found four times. (4) Multimodal interneurons The interneurons now to be described differ from the above-mentioned interneurons in that they have two or more sensory inputs whose modalities are different each other, and they can also be excited by such inputs alone. A total of 11 interneurons were repeatedly recognized as mul- timodal interneurons. C2 is an interneuron which responds phasically to light-on stimulation to the ipsilateral eye, to light-off stimulation to the contralateral eye, and to wind stimulation to the frontal surface of the head, while the animal is stationary. The response characteristic of this interneuron was modified strongly in association with the ini- tiation of flight. That is, C2 started to fire spontaneously just after the initiation of flight, Response Modification of Interneurons 179 and responded to the ipsilateral illumination with an increase of the firing rate of spontaneous dis- charge and to the contralateral illumination with its decrease. The further complexity of the response modulation was that only the responsi- veness to wind stimulation remained unchanged when the animal began to fiy. This interneuron has been found five times. C4 was excited by tactile stimulation to the ipsilateral antenna with a burst and by blowing the wind on the head with a tonic discharge. However, C4 showed also the response character- istic as a “‘mechano-gated visual interneuron”: this interneuron responded to light stimulus applied to either eye with a slight decrease in the tonic discharge which appeared only while the animal was being exposed in the windstream. The change in behavioral state had not any in- fluence on these responsiveness. This _ inter- neuron has been found three times. C7 showed spontaneous discharge under any circumstances, but the firing rate was the highest in the flying state. Light or sound stimulation to either side brought about an increase in the firing rate. The response modification in asso- ciation with the flight behavior was not observed at all. C7 has been found four times. C8 responded phasically to light-off stimulus to the ipsilateral eye, to light-on stimulus to the contralateral eye, or to tactile stimulus to the abdomen, antennae or legs, in the stationary animal. In the flying animal, however, this interneuron was active continuously in the ab- sence of stimulus, and responded to the ipsilateral or contralateral illumination with a decrease or an increase of the firing rate of spontaneous discharge. Furthermore, in the flying animal C8 responded to tactile stimulus to the bodily parts mentioned above with a burst of a few spikes, so that the burst was summated with the spontaneous discharge. This interneuron has been found four times. C9 has the peculiar response property that during quiescence it responds to illumination on either eye with an increase of spontaneous firing, and also to windstream on the head with the cessation of spontaneous firing, whereas during flight it falls into a state of silence and does not respond to any stimulus. It has been found three times. C12 evoked a burst of spikes in response to light stimulus given to either eye and also to sound stimulus presented from the contralateral side, while the animal was stationary. Imme- diately after the initiation of flight, C12 began to fire tonically and showed the enhanced visual response in which a tonic burst of spikes was summated with the spontaneous firing. How- ever, the directional sensitivity to sound stimulus remained unchanged, even when the animal started to fly. This interneuron has been found three times. C14 responded to sound stimulus presented from either side with burst in both of the station- ary and flying states (Fig. 8A, B). The most effective carrier frequency for C14 was at about 15 kHz. This interneuron responded also to wind stimulation to the head with sustained discharge. However, it never responded to sound stimulus | A | i | a - SS - - 7 . - calling song 500 msec Fic. 8. Responses of a multimodal interneuron (C14) to sound and wind stimuli in the stationary animal. In each record the upper trace is the response of C1i4 and the lower trace indicates the sound stimulus (upward deflection, sound on). (A, B) The sound stimuli (artificial sound pulses or calling songs played back from the tape) were presented from the ipsilateral side in A and from the contralateral side in B. Each triangle re- presents the beginning of chirp. (C, D) While the frontal surface of the head was windswept, the sound stimuli were presented from the ipsilateral side in C and from the contralateral side in D. Note that Cl4 never responds to the sound stimulus as long as the wind blows on the head. Carrier frequency: 10 kHz. Sound pressure level: 80 dB. 180 K. TOMIOKA AND T. YAMAGUCHI 500 msec Fic. 9. Simultaneous recordings of a bilateral multimodal interneuron (C15) and the ipsi- lateral hindwing depressor muscle (129a). In each record the top trace is the response of C15, the middle trace is the electrical activity of 129a, and the bottom trace indicates the light or sound stimulus (upward deflection, stimulus on). (A—D) Records taken while the animal was stationary. (E-I) Records taken while it was flying. The light stimuli were presented to the ipsilateral eye in A and F, and to the contralateral eye in B and G. The sound stimuli (carrier frequency, 15 kHz; sound pressure level, 80 dB) were presented from the ipsilateral side in C and H, and from the contralateral side in D and I. In E, there appeared the strong spike discharge immediately after the wind stimulation (arrow head) of the head to induce the flight. Note that C15 elicits the discrete bursts synchronized with the flight muscle activity only in response to light or sound stimulus presented during flight. delivered during such exposure to the wind, irrespective of whether the animal was stationary or flying (Fig. 8C, D). Cl4 has been found four times. C15 was all but silent about any sensory stimulus given while the animal was stationary (Fig. 9A-D; in C only a spike with a long latency was evoked by sound stimulus to the ipsilateral side). How- ever, its responsiveness to sensory stimuli was modified drastically in association with the flight behavior. First, when the wind stimulation was given to the head, it fired at a high rate in advance of the occurrence of the flight motor activity (Fig. 9E), and then decreased gradually the firing rate to only the sporadic firing of single or group of spikes which were synchronized with each flight motor activity (Fig. 9F-I). Secondly, this interneuron responded to light or sound stimulus applied to either side with burst dis- charge which consisted of grouped spikes syn- chronized with each flight motor activity (Fig. 9F— I). C15 has been found only twice. C21 responded weakly to both wind stimu- lation to the head and light-off stimulation to either eye with a burst of a few spikes while the animal was stationary. This responsiveness was modified as soon as the animal began to fly: during flight, there appeared regularly the mod- erate light-off response or sometimes the moderate light on-off response to either of ipsilateral and contralateral illumination. The spontaneous dis- charge was observed only at the time when the animal moved abruptly on its own initiation. This discharge may possibly result from the excitation of some of the mechanoreceptors sensing the air current caused by the animal’s own violent movement. This interneuron has been found four times. Response Modification of Interneurons 181 C22 is a normally firing interneuron showing the strong response to ipsilateral illumination, the weak response to contralateral illumination and the sustained scant response to wind stimu- lation of the head. The asymmetrical respon- siveness to illumination was further enhanced either when the animal was exposed in the wind- stream or when it was flying. C22 -has been found only twice. C38 responded bilaterally to light-off stimulus with a few spikes only when the animal was exposed in the windstream. While it kept flying, this interneuron discharged tonically in the absence of stimulus, and responded to light-off stimulus with a transient increase of the firing rate. C38 has been found four times. (5) Activity interneurons The so-called activity fiber has been established by Wiersma and his coworkers [e.g., 14] as the fiber which shows the spontaneous firing, whose firing rate is accelerated or retarded in relation to the activity of certain part of the body, and also responds to some kinds of sensory stimulation. In the present experiment, however, the inter- neurons which shift the firing rate under the influence of an animal’s own movement or be- havior, but never respond to a_ well-defined sensory stimulation, were described as the ac- tivity interneurons. A total of five interneurons fell into this class. Cl was silent as long as the animal was station- ary. However, in the flying animal, as shown in Figure 10A, it fired with grouped firing of a few of spikes, which synchronized well with the flight motor activity. It has been found seven times. C25 fired simultaneously with the inspiratory movement of the abdomen, when the animal was stationary. This activity interneuron became si- lent as soon as the animal began to fly, and kept silence until it ceased to flap its wings. C25 has been found four times. C30 is a normally firing interneuron which falls into silence while the animal keeps flying. It has been found only twice. C32 showed a burst of spikes synchronized with the expiratory movement of the abdomen, when the animal was stationary. After the initiation of flight, it changed abruptly its firing pattern from the rhythmical discharge to the tonic one. It has been found 11 times. C33 is an activity interneuron which fires continuously with grouped firing of spikes only when the animal is flying (Fig. 10B). The ac- tivity of this interneuron was quite similar to that of Cl, except that the firing of C33 was less syn- chronized with wing beat cycle than that of Cl. This interneuron has been found seven times. (6) Influence of reafferent inputs on response modification As mentioned already, there is a_ possibility that the reafferent inputs from various mecha- noreceptors (e.g., facial wind receptors, antenna! Fic. 10. Simultaneous recordings from activity interneurons (Cl in A and C33 in B) and the ipsilateral hindwing depressor and elevator muscles (129a, larger spikes; 119, smaller spikes) during flight. In each record the top trace is the spike discharge of activity interneuron, the middle trace is the electrical activity of flight muscles, and the bottom trace is the light stimulus (upward deflection, light on). for A and 500 msec for B. Time scale: 250 msec 182 K. TOMIOKA AND T. YAMAGUCHI mechanoreceptors and wing hinge receptors) dur- ing flight may give modifier signal for the response modification to individual sensory interneuron. However, the data obtained in this experiment suggested that the reafferent inputs does not contribute in a major way to the response modifi- cation. The occurrence of spontaneous discharge and the enhancement or suppression of spike responses to sensory stimuli was still observable in all the crickets whose antennae, wings and legs were removed, who were flying without air current, and who were assuming flight posture without flapping the wings, i.e., pseudo-flight posture. Furthermore, the response modifica- tion occurred with the initiation of flight even in the animal whose antennae, wings and legs were removed, and all movable joints including the neck and abdominal segments were fixed on the supporting rod with colophonium beeswax mix- ture. In these operated animals, the activity interneurons still fired normally. Only when the electrical stimulation, of which frequency was close to that of the wing beat (about 30 Hz), was given to the contralateral meso-metathoracic connective, it resulted in shortening of the latency of C3. DISCUSSION Identification of interneurons and response mod- ification Up to date, many works have been made on the responsiveness of single neurons recorded within the central nervous system of insects [e.g., 24-26] and crustaceans [e.g., 27-31]. Most of these works have been accomplished mainly under the condition which the animals were stationary. There are, however, some reports indicating that the animal’s own behavioral state exerts various influences on the responsiveness of interneurons. For instance, of the various classes of visually responding interneurons in the crayfish optic nerve, the sustaining and move- ment fibers are influenced strongly by the excited state of animal. That is, the discharges of sus- taining fibers increase owing to the facilitatory effect of the general state of excitement [12, 13], and the responses of movement fibers are en- hanced during the defense reflex in that the response decrement to repetitive stimuli is greatly suppressed [13]. In the rock lobster, the seeing fibers seem to be clearly influenced by the “‘mood”’ of the animal in that they have a low respon- siveness when the animal is “‘drowsy’’, respond well when the animal is walking, and are actually inhibited if the animal is in a high excitement, as during a defense response [32]. In the cock- roach giant interneurons running in two groups through the ventral nerve cord, the excitability of each of these groups is modulated during walking: the dorsal group of giant interneurons is excited while the ventral group of giant interneurons is inhibited [8, 9]. The locust DCMDs are strongly inhibited during the fast phase of optokinetic nystagmus and during voluntary saccades [33]. Therefore, to elucidate the neuronal mechanism underlying various patterns of behaviors, we should pay much more attention to analysis of the responsiveness of each interneuron to stimuli under different behavioral states. We believe the work reported here is the first specificially designed to identify systematically each interneuron with due regard to the influence of behavioral states on its responsiveness. We have identified functionally a total of 41 interneurons in the subesophageal ganglion and cervical connectives of crickets. They fell into the five main classes (visual, auditory, mech- anoreceptive, multimodal and activity interneu- rons) on the basis of their responsiveness to well-defined stimuli. These results show quite convincingly both the similarity in general terms and the differences in details which are present among the arthropods [e.g., 24, 25, 27-31]. How- ever, the most intriguing finding was that about 75 percent of identified sensory interneurons mod- ify their responsiveness to stimuli in association with the flight behavior (Table 1). In percentage, the response modification which set the respon- siveness during flight was observable in about 89 percent of visual interneurons, in about 25 per- cent of auditory interneurons, in about 33 percent of mechanoreceptive interneurons and in about 82 percent of multimodal interneurons. The response modification elucidated in this Response Modification of Interneurons 183 experiment could, for convenience’ sake, be classified into three major types. The first type is the response modification which enhances the response to unimodal or multimodal sensory inputs during flight. Of 27 interneurons (except the activity interneurons) showing the response modification, 22 interneurons belonged to this type, though details differ among them. The second type is the response modification which suppresses the response to sensory inputs as long as the animal is flying. Only one interneuron (C9) showed definitely this type of response modification. The third type is the response modification which brings about the “‘flight-gated activation” by which the interneurons respond to sensory inputs only during flight. Four visual interneurons (C23, C27, C35 and C41) could be classified into this type. In addition, it should be noted that a majority of interneurons which are normally silent during quiescence elicit the spontaneous discharges during flight. These spon- taneous discharges allow them to transmit both the excitatory and inhibitory outputs to post- synaptic neurons. The firing rate of spontaneous discharges can take the form of either an increase or a decrease, which permits the interneurons to respond differentially to different stimuli or stimulus parameters. An example is seen in the discharge of C40 (Fig. 4). Furthermore, the spon- taneous discharge may contribute to the persist- ence of flight by raising the general level of ex- citability in the central nervous system, as sug- gested in the cockroach walking-activated giant interneurons [7]. This possibility was substan- tiated at least in part by the observation that the flight activity was much lower or completely lost in the animal after severance of its cervical con- nectives. The response modification of interneurons in association with the behavioral state implies that the efficacy of well-defined stimuli such as light and sound is related profoundly to the animal’s own behavioral state. This may have a great deal to do with that the crickets nearly ignore both the light and sound while they are stationary, whereas they use them reliably as important sensory cues to stabilize and control the posture while they are flying [2, 34-36]. In view of limitted numbers of neurons comprising the central nervous system, the response modifi- cation of interneurons seems to be the way of integration for insects to control various repertoire of highly developed behaviors. Possible mechanism of response modification For the neuronal mechanism underlying the response modification, there are two possible explanations. The first explanation is that the response modification is ascribable to the reaf- ferent inputs from various mechanoreceptors including the proprioceptors operating during flight. This explanation is likely to fit for the cases such as the efferent fibers of the dragonfly ocellar nerve excited by inputs from sense organs in the wing’s hinges [37], and the locust trito- cerebral commissure giant (TCG) responding to the inputs from both the compound eyes and the wind receptors on the head sensing rhythmic wind turbulance around the head created by the beat- ing of the wings and by the rhythmic head move- ments during flight [38, 39]. However, in the cricket the reafferent inputs receiving during flight are found not to contribute at least in a major way to the response modification of interneurons. In connection with these results, it is interesting that in the moth brain the acoustic neurons usually faithfully follow ultrasound inputs, and can switch into a bursting mode in which the burst rhythm can be phase-locked to the wing beat, even if the flight motor activity was not detectable [40]. The response modification of these acoustic neurons seems to be similar to that of C15 recognized in this experiment. The second explanation is that the endogenous excitation or inhibition by the corollary discharge originated in the motor center results in the re- sponse modification of sensory interneurons. This explanation has been already applied to insect interneurons [e.g., 4, 5, 7, 8, 33] and to the crayfish lateral giant escape system leading to a tail flip via the lateral giant pathway of sensory interneurons, command cell, and motoneurons [e.g., 41-44]. In the crayfish, the corollary discharge interneurons which are recruited by activity in the giant inter- neurons and/or the flexor motoneurons were found in the abdominal nerve cord [e.g., 43, 44]. In the 184 K. TomMioKA AND T. YAMAGUCHI cockroach, the interneurons defined as the move- ment-signalling (corollary discharge) interneurons which are active during a movement driven by specific muscles were recorded from the thoracic nerve connectives [45]. Furthermore, in the buc- cal roots of the marine gastropod Pleurobranchaea californicum, the descending neurons carrying a corollary of efferent activity or transmitting an exact, one to one replica of motoneuron spikes has been shown to be responsible for the co- ordination of the complex, rhythmic feeding behavior [46]. We have identified five activity interneurons which never respond to any sensory stimulus but change the firing level exclusively during flight. Two (C1, C33) of them showed the rhyth- mic bursts phase-locked to the flight muscle ac- tivity, even when the animal assumed the pseudo- flight posture (Fig. 10). Besides these, another activity interneuron (C32) fired tonically also when the animal was flying, but in this case no significant phase relationship between its firing and the flight muscle activity was recognizable. On the contrary, the remaining two activity interneurons (C25, C30) was active during quie- scence and silent during flight, as if they are counterparts of the above-mentioned activity inter- neurons. Moreover, during quiescence C25 and C30 fired in phase with the inspiratory and ex- piratory movements of the abdomen. The occur- rence of change in the firing level of activity inter- neurons did not always coincide with that of the flight muscle activity: sometimes the former ad- vanced from or lagged behind the latter. Among these activity interneurons, C32 may fall under the same category as the corollary discharge neuron which transmits an approximate image of efferent discharge, and Cl and C33 under the same category as the efference copy neurons which transmit a precise replica of motoneuron activity. However, although we do not have the direct evidence showing clearly that these activity interneurons have the same functions as the corollary discharge and efference copy neu- rons, it is most plausible at present that some of the activity interneurons, at least, have a poten- tially broader and more important function to distribute the modifier signals triggering the res- ponse modification of sensory interneurons throughout the central nervous system and to coordinate a complex, endogenous behavioral pattern. This may be emphasized by the fact that the electrical stimulation of which frequen- cy is equivalent approximately to that of the wing beat (about 30 Hz) given to the contralateral meso- metathoracic connective mimics the flight behavior, so that it decreases the latency of the ipsilateral C3. It has been, furthermore, reported that in the op- tic lobe of crickets, the visual interneurons re- sponding illumination receive via the presynaptic efferent interneurons the centrally originated inputs which are images of the flight motor activity: these inputs bring about a decrease in the latency and an increase in the flicker fusion frequency during flight [47]. Finally, we should comment upon the fact that when the locust flies there is a rapid increase in the concentration of octopamine in the hae- molymph [48]. 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ZOOLOGICAL SCIENCE 1: 187-194 (1984) © 1984 Zoological Society of Japan A Directionally Selective Binocular Unit in the Midbrain Tegmentum of 7ribolodon hakonensis MASASHI KAWASAKI and KIyosHI AOKI Life Science Institute, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102, Japan ABSTRACT — Directionally selective visual units were recorded in and around the mesencephalic posterior commissure in Japanese dace (Tribolodon hakonensis) during stimulation by a rotating striped drum. Spontaneous discharge rates of the units were increased or decreased depending on the direction of rotation of the drum. The units had movement-sensitive, binocularly-driven receptive fields with horizontal extents of 240°. The relation between the properties of the directionally selective units and optokinetic responses induced by the same stimuli is discussed. INTRODUCTION When Japanese dace (Tribolodon hakonensis) whose spinal cords have been transected to sup- press body movements are placed at the center of a rotating striped drum, they respond with distinct horizontal eye movements. Such encéphales isolés fish provide convenient preparations in which to characterise the relation between sensory input (the pattern of moving stripes) and motor output (ocular movements), and to study the intervening neuronal networks with microelectrodes. In Tribolodon, directionally selective visual units have been reported in the optic tectum [1]. Units whose activities are correlated with eye movements and are presumed to be oculomotor neurons have been recorded in the oculomotor nucleus (Kawasaki and Aoki, unpublished data). In other species of fish, directionally selective units have frequently been recorded from the optic tectum [2-4], while eye movement correlated units have been recorded from the final motor nuclei [5, 6]. In no species is there sufficient information about visual responses in the dorsal tegmentum where some part of the visuo-motor integration may occur [7]. Accepted August 27, 1983 Received July 16, 1983 In this study we demonstrate visual responses to movement stimuli in the dorsal tegmentum of curarized Japanese dace, using a pattern of moving stripes shown to induce optokinetic nystagmus in non-curarized fish. Special attention was payed to the directional selectivity of the units. The tegmental region in which directionally selective units frequently occurred was the posterior com- missure in the anterior dorsal tegmentum. The teleostean posterior commissure is a thick trans- verse bundle which interconnects the two sides of the tegmentum at its anterior border with the diencephalon and is presumably of some impor- tance in the integrative functions of the optic tectum, and of the tegmentum, in which there are various motor centers [8—11]. MATERIALS AND METHODS Japanese dace (Tribolodon hakonensis) 16 to 19cm in length were kept in a 350 liter tank at 22 to 25°C on a light-dark schedule of 12 hr light and 12 hr dark. During surgery fish were wrapped in soft paper, fixed to the surgical bed with “tear” bars, and the gills perfused with water containing MS~222 (80 mg/l). The anterior part of the brain was exposed by opening the skull with a dental drill. The fish were curarized by intra- muscular injection of tubocurarine chloride (2 to 5 188 M. KAWASAKI AND K. AOKI mg/kg body weight) and then moved to the experi- mental chamber. During the experiments the gills were perfused with oxygenated and filtered water (18 to 24°C) through a mouth piece inserted into the oral cavity. The experiments were carried out with light adapted animals. Electrophysiological recordings and _ histological verification of recording sites Elgiloy alloy electrodes [12] were used. Ex- tracellular activity was routinely recorded. Spike responses were directly analyzed by a spike analyzer (Nihonkoden ATAC-350) and a microcomputer (NEC PC-8001) or stored on magnetic tape (TEAC R-81). The electrode was inserted to as great a depth as 1500 wm from the surface of the optic tectum at about 1 mm posterior from its anterior margin. Histological verification of recording sites was made for all units using the Prussian blue method. After recordings, positive current (5 vA, 30 sec) was passed through the electrode. The brain was fixed in Carnoy’s solution for two hours, then dehydrated and embedded in paraffin wax. Dewaxed sections were immersed in 2% hydro- chloric acid containing 2°% potassium ferrocyanide for the Prussian blue reaction. The diameter of the marking spot in this method was about 70 yum. Experimental chamber and optical stimulation The experimental chamber was a transparent cylindrical plastic tank (30cm in _ diameter) equipped with a fish bed at the center. The tank was filled with water in such a way as to submerge the eyes of the fish and maintain a normal refrac- tive situation at the cornea while the cranial opening remained in air. The horizontally moving striped target was presented on a rotating drum (38 cm in diameter) concentric with the tank and with the head of the fish. Its inner surface was lined with alternating black and white stripes: The subtense of the black and white stripes at each eye was 6°. The drum subtended 40° of the visual field in the vertical plane. Because the eyes are just underneath the water surface, the vertical subtense to the target drum is doubled to 80° by a mirror effect at water-air interface. Horizontal visual field extended 300°. The inside of the drum was illuminated by an incandescent lamp located in the water just behind the fish. Lumi- nances of the black and white parts of the stimulus pattern were 9 cd/m’ and 115cd/m’*. The drum was placed on a Hi-Fi turn-table which was specially equipped with a DC motor. Angular velocities of the drum ranged from 1°/sec to 90°/sec. When testing horizontal extents of receptive fields of the units, an additional concentric drum was inserted between the innermost tank and the outer target drum. The intermediate drum acted as a mask, a restricted part (usually 30°) being trans- parent while the remainder was covered with a sheet of gray paper. By shifting the position of this “window”, the movement stimuli from the outer drum could be localised to specific regions of the horizontal visual field. Here, 0° in the horizontal visual field was defined as the rostral direction of the fish, angular directions being measured clock- wise from this line of reference. The experimental apparatus was identical with that used for the study of optokinetic nystagmus in the non-curarized encéphale isolé preparation of Tribolodon in our laboratory. RESULTS Only results for directionally selective units which responded preferentially to horizontally moving stripes are presented in this paper although several kinds of units were recorded. Some units did not show clear responses to any light stimulus tested and sometimes showed an irregular spon- taneous discharge. Others were sensitive to the movements of the drum but they were not directionally selective. The most frequently en- countered units, however, were directionally selective, and responded differently to the two directions of drum rotation. The ratio of the units which showed the directional selectivity to total number of encountered units was approxi- mately 10%. Recording sites were verified in the histological sections. Thirteen marked sites from which directionally selective units had been recorded were found in the posterior commissure or in the dorsal tegmentum close to it. Six sites were marked in the posterior commissure in regions where com- missural fibers were observed in sections stained Tegmental Units in Tribolodon 189 Fic. 1. of the Japanese dace brain. Distribution of recording sites from which the directionally selective units were recorded. Histologically verified recording sites are plotted as solid circles on transverse sections (a-f ) Recording sites were located in and around the posterior com- missure in the anterior dorsal tegmentum. Lower right: dorsal view of the Japanese dace brain and location of the transverse sections presented above. Abbreviations; OT — optic tectum, PC — posterior commissure, V — valvula cerebelli. with Cajal’s method. Other sites were located in an adjacent area of the posterior commissure in the anterior dorsal tegmentum (Fig. 1). Units either in or adjacent to the posterior commissure showed the same response characteristics. The area within which the marked sites lay extended 750 4m anteroposteriorly, 900 ~m laterally and 1500 to 2000 ~m deep from the surface of the tectum. At the center of this region, the torus longitudinalis leaves the posterior commissure. No systematic relation between either the preferred direction or the ocular dominance and the record- ing site was detected. The directionally selective units showed spon- taneous discharge when the drum was still. For 13 units, the mean of the averaged spike frequencies was 26.1 impulses/sec (SD=-+-8.9). In each unit, a spontaneous discharge rate was stable over the same length of time as the experimental procedure. In response to the drum rotation, either an increase or a decrease of the rate of discharge was observed which depended on the direction of the drum movement ina given unit. In units with a counter- clockwise (CCW) preference, for example, a CCW rotation elicited an increase in the discharge rate and a clockwise (CW) rotation elicited a decrease (Fig. 2). Discharge patterns showed no relation- ship to the periodicities of the stripe patterns. Changes in discharge rates were observed imme- diately after the onset of the drum movements. Increased or decreased levels were maintained throughout the drum rotation for several minutes. To investigate the horizontal extent of the receptive field, the stripes of the drum were screened except for a part of the visual field by an inter- mediate opaque drum which had a ‘“‘window’’. Width and height of this “‘window” were 30° and 80°. Horizontally, the position of the ““window” was shifted at intervals of 30° around the fish, while the vertical position remained fixed at the fish’s eye level. Stripes moving CCW or CW were presented through the “‘window”’ at ten different 190 M. KAWASAKI AND K. AOKI horizontal positions. It was found that all units head and eyes), the results show that the units can were binocularly driven and the horizontal extents _ be driven either by the right or left eye. The most of the receptive fields measured with these stimuli sensitive part of a receptive field was unilateral and were very large. The receptive field extended in the middle of the eye for some units (Fig. 3A), horizontally at least over 180°. In most of the or near-rostral in other units (Fig. 3B). In the units, it reached 240°. Because the visual fields former case, the visual field of one eye was alsc of left and right eyes overlap only rostrally (—30° dominant, although these units were essentially to 30°, estimated from geometrical features of the binocular. At any region of the receptive field, 001 ree mn 1) He yay SUE C CW STILL ETE Heel HUT BVA PATH HAW HTL STIL: Cc Ww STE | 50 / sec 2 sec Fic. 2. Responses of a unit to counter-clockwise (CCW) and clockwise (CW) rotation of the drum. Lower traces indicate velocity of the drum. In this CCW preferred unit, spontaneous discharge observed when the drum was still (36.2 impulse/sec) accelerated by CCW rotation (55.5 impulse/sec) and decelerated by CW rotation (17.4 impulse/sec). Velocity of CCW and CW rotation was 20°/sec. Recording site of this unit is shown in Fig. 1-d, second dot from the right. 100 4f (impulse/sec) 60 Oia =) 20 = 90 > 60 ~ 30 0 30 60 90 120 t t a 0 »-90. = 60 1-30 0 30 60 90 120 150 TARGET POSITION (deg) Fic. 3A Tegmental Units in Tribolodon 191 _- U B r~) °o Ww N = Qa E ~ — x oO ° N | - 150 ~120 = 30 - 60 ~ 30 0 30 60 90 120 ’ t ? i t t t -120 -90 -60 -30 0 30 60 90 120 150 TARGET POSITION (deg) Fic. 3B Fic. 3. Horizontal extent of the receptive fields to movement stimulus in two directionally selective untis. Horizontally moving stripes were presented through the 30° width “window”. The horizontal position of the window (target position) is measured from rostral direction (0°). Minus and plus values correspond to left and right visual field of the fish respectively. Af= (response frequency)—(spontaneous frequency; 36.2 -+5.2 impulse/sec in A, 13.5+4.0 impulse/sec in B, which were calculated from spontaneous discharge rate in each second for 5 min). (O) responses to CCW rotation, (@) responses to CW rotation. Vertical bars indicate standard deviations of the Af. Horizontal and vertical dotted lines indicate mean values and standard deviations of the Af calculated from the response to the stimulus from the whole visual field. In these cases the masking was removed and fish was able to see all inner surface of the drum except its tail region (—150° to 150°). Because the left eye is not able to see the target position from 30° to 150° and the right eye is not able to see from —150° to —30°, the units are binocularly sensitive. Recording site of unit in A is shown in Fig. |—d, second dot from the right. Unit in B is shown in Fig. 1-d, the most left dot. different directions of drum movement elicited different response changes of opposite sign: in- crease or decrease of spontaneous discharge rates. But, in some units, in the caudal region of the receptive field, the relation between the movement direction and the sign of the discharge change was inverted in a given unit. In these caudal! parts of the receptive fields, however, the degree of the discharge changes was small compared with that of the central region. When the movement stimulus was presented to the whole visual field by removing the intermediate drum, the sign of the discharge rate change was the same as in the most sensitive part of the receptive field (Fig. 3B). Commissural units were studied in further detail using various speeds of drum rotation ranging from O to 72°/sec. The width of the “‘window”’ was 90° in this case, its center being positioned at the most sensitive part of the receptive field. Up to 20°/sec of stimulus speed, the effects on the discharge frequency were stable and increased with the increase of stimulus speed. But further in- crease in stimulus speed produced no further change in response frequency and gave larger 192 M. KAWASAKI AND K. AOKI C_ : =, oD) 7) | ek £ ~~ gam =. 6h © tT oO N | 10 20 30 40 50 60 70 ANGULAR VELOCITY(deg/sec) Fic. 4. The relation between frequency shift (Af) and the angular velocity of drum move- ment in a commissural directionally selective unit. Target position was 25°-180°. CW rotation. standard deviations in response discharge (Fig. 4). DISCUSSION This study demonstrates that (i) directionally selective units exist in and around the mesencephalic posterior commissure and (1i) their receptive fields are large and binocular. These findings mean that movement direction in a very large part of the visual field is represented in the mesencephalic tegmentum as commissural single unit activity. We have recorded not only in the posterior com- missure but also in the region that surrounds it. Because directionally selective units recorded within and around the posterior commissure show similar characteristics in respect to the extent of their receptive fields and discharge patterns of response, we believe that the units recorded around the posterior commissure are closely related to the commissural fibers running within it. Oculo- motor motoneurons exist just within the posterior part of the region of our recording sites in the dorsal tegmentum; nevertheless, our units did not (O) response to CCW rotation, (@) Same unit as presented in Fig. 2. exhibit rhythmic responses that would suggest them to be motoneurons. The units may be involved in a system that controls optokinetic reactions of the fish. Because fish normally float in water in contrast to most quadrupeds, they are unable to utilize ground con- tact to monitor their own movements or postures with respect to the environment. Visual informa- tion must play an important role in controlling motor activities which adjust the position or orientation of fish in an environment. Opto- kinetic reactions in fish which are induced by movements of large visual fields have been re- ported [22]. Pursuit eye movement induced in a cylindrical drum was described by Easter [13] in goldfish. Not only the eyes but also the trunk and fins exhibit optokinetic movements [14]. Although the anatomical and electrophysio- logical relations of the commissural units to the oculomotor nucleus or the presumptive motor center [8-11, 15] remain to be demonstrated, the following points suggest that the directionally selective units of the present report participate in Tegmental Units in Tribolodon 193 a neuronal network which controls optokinetic reactions of the eyes and/or body. (i) In the identical optical stimulator used in the present study, distinct optokinetic nystagmus is elicited in an encéphale isolé non-curarized Tribolodon (Kawasaki and Aoki, unpublished data). The directionally selective pursuit eye movement in the optokinetic nystagmus is elicited wherever the “window” of the stimulator is located in the horizontal visual field. The optokinetic nystagmus has a large sensitive area throughout the visual field just as do the commissural directionally selective units. The relation between various velocities and the unit responses (Fig. 4) was almost similar to the relation between velocities of the pursuit phase of the optokinetic nystagmus and the stimulus velocity. Over 30°/sec, the velocity of the pursuit eye-movement and the stimulus velocity are not linearly related (Kawasaki and Aoki, unpublished data). (ii) Anatomically, the posterior commissure of fish is closely related to the tegmental motor nuclei and is the termination of the medial longitudinal fasciculus [16]. A recent anatomical study in the carp [17] demon- strated that the afferent fibers from the posterior commissure connect it to the nucleus motoris dorsalis tegmenti, to the corpus cerebelli and to the reticular formation from mesencephalon to lower medulla. (iii) Electrical stimulation to the anterior dorsal tegmentum induces eye movements (Kawasaki and Aoki, unpublished data). Eleven units in which discharge patterns correlated to eye movements were recorded in the dark from the dorsal tegmentum in encéphale isolé preparations of Tribolodon. After unit recordings, electrical stimulation (20 ~A, 20 Hz) was applied through the same electrode. In three of them, electrical stimulation induced conjugate eye movements. The electrode marking of these three units showed them to be located at the tegmental region ad- jacent to the posterior commissure (50-250 ~m behind the posterior margin of the posterior commissure). (iv) The existence of directionally selective units in the posterior commissure partly explains the behavioral observation of Mark ef al. [18, 19]. If transection of the posterior commis- sure is accompanied by a unilateral lesion to the adjacent tegmentum, the trunk of the fish bends to one side persistently while the eyes deviate even further in the same direction and it swims con- tinuously in circles in a horizontal plane [18]. If directionally coded impulses are conveyed through fibers in the posterior commissure to the contra-lateral oculomotor nucleus or presumptive motor centers, an asymmetrical lesion to a part of the tegmentum which normally receives directional information might result in unbalanced motor activity between the two sides. The pathway from the retina to the directionally selective units in the posterior commissure is uncertain. Recent anatomical studies demon- strated that various parts of the brain send afferent fibers directly to the posterior commissure. They are the torus longitudinalis, dorsal tegmentum, corpus cerebelli and tractus mesencephalocerebel- laris anterior [20]. The posterior commissure nucleus proved to project from the optic tectum, pretectal nuclei and bulbar reticular formation [17]. Springer and Gaffney [21] showed primary projection from the retina to the posterior com- missure. But the retina cannot be a candidate for a direct afferent source of our directionally sensitive units, because they are all binocular. We consider that the likely candidates for the immediate source of the commissural units are the pretectal nucleus, the preoptic nucleus and the optic tectum. ACKNOWLEDGMENTS We thank Dr. A. D. Blest of the Australian National University for reviewing the manuscript. This work was supported by a Grant-in-Aid for Scientific Re- search No. 57108014 and No. 56215006 from_ the Ministry of Education, Science and Culture of Japan. REFERENCES 1 Kawasaki,M. and Aoki, K. (1983) Visual responses recorded from the optic tectum of Japanese dace. J. Comp. Physiol., 152: 147-153. 2 Griisser, O.-J. and Griisser-Cornehls, U. (1973) Neuronal mechanisms of visual movement per- ception and some psychophysical and behavioral correlations. In ‘Central Processing of Visual Information”. Ed. by R. Jung, Springer, Berlin, Heidelberg and New York, pp. 333-429. 3 Guthrie, D.M. and Banks,J.R. (1978) The receptive field structure of visual cells from the optic tectum of the freshwater perch (Perca 194 Nn 11 12 fluviatilis). Brain Res., 141: 211-225. Wartzok, D and Marks, W. B. (1973) Direction- ally selective visual units recorded in optic tectum of the goldfish. J. Neurophysiol., 36: 588-604. Hermann, H. T. (1971) Eye movement correlated units in mesencephalic oculomotor complex of goldfish. Brain Res., 35: 240-244. Gestrin, P. and Sterling, P. (1977) Anatomy and physiology of goldfish oculomotor system. Il. Firing patterns and their relation to eye movements. J. Neurophysiol., 40: 573-588. Guthrie, D. M. (1981) The properties of the visual pathway of a common freshwater fish (Perca fluviatilis L.) in relation to its visual be- haviour. In “Brain Mechanisms of Behaviour in Lower Vertebrates”. Ed. by P. R. Laming, Cam- bridge University Press, Cambridge, pp. 79-112. Demski, L. S. (1982) Eye movements and related behavioral responses evoked by electrical stimu- lation of the brain in free-swimming sunfish. Brain Behav. Evol., 20: 182-195. Demski, L. S. and Bauer, D. H. (1975) Eye move- ments evoked by electrical stimulation of the brain in anesthetized fishes. Brain Behav. Evol., 11: 109-129. Demski, L.S. and Gerald, J.W. (1974) Sound production and other behavioral effects of midbrain stimulation in free-swimming toadfish, Opsanus beta. Brain Behav. Evol., 9: 41-59. Kashin, S. M., Feldman, A. G. and Orlovsky, G. N. (1974) Locomotion of fish evoked by electrical stimulation of the brain. Brain Res., 82: 41-47. Suzuki, H. and Azuma, M. (1976) A_ grass insulated “Elgiloy” microelectrode for recording unit activity in chronic monkey experiments. 13 14 115) 16 17 18 19 20 72) 22 M. KAWASAKI AND K. AOKI Electroenceph. Clin. Neurophysiol., 41: 93-95. Easter, S.S. Jr. (1972) Pursuit eye movements evoked by electrical stimulation of the brain in anesthetized fishes. Vision Res., 12: 673-688. Harden-Jones, F. R. (1963) The reaction of fish to moving backgrounds. J. Exp. Biol., 40: 437-446. Clark, S. L., Chung, M. Y., Shine, L. and Clark, M. R. (1960) Responses in free swimming fishes to electrical stimulation of the cerebellum. Am. J. Anat., 106: 121-132. Ariéns-Kappers, C. V., Huber, G. C. and Crosby, E. C. (1967) The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. Hafner, New York, pp. 903-937. Juch, P. J. W. and Luiten, P. G. M. (1981) Anat- O:ny of respiratory rhythmic systems in brain stem and cerebellum of the carp. Brain Res., 230: 51-64. Mark, R.F., Peer,O. and Steiner, J. (1973) Integrative functions in the midbrain commissures in fish. Exp. Neurol., 39: 140-156. Peter, R. E. (1977) Effects of midbrain teg- mentum and diencephalon lesions on swimming and body orientation in goldfish. Exp. Neurol., 57: 922-927. Ito, H. and Kishida, R. (1978) Afferent and efferent fiber connection of the carp torus longi- tudinalis. J. Comp. Neurol., 181: 465-476. Springer, A. D. and Gaffney, J.S. (1981) Retinal projections in the goldfish: A _ study using cobaltous-lysine. J. Comp. Neurol., 203: 401-424. Inoue, M. and Arimoto, T (1976) The role of the optomotor reaction in the schooling of fish. J. Tokyo Univ. Fish., 62: 67-81. ZOOLOGICAL SCIENCE 1: 195-204 (1984) Effects of Betaine, Taurine and Isethionate on Microtubule Assembly MAKOTO HASHIMOTO, EISUKEB NISHIDA and HIKOICHI SAKAI Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT — Two amino acid analogues, betaine and taurine, and isethionate which are major low molecular weight substances in the axoplasm of squid giant axon were studied for their pronounced effects on microtubule assembly. Betaine stabilized assembled microtubules, while both taurine and isethionate exhibited inhibitory effect on microtubule assembly, showing half maximal inhibitions at 0.5 M and 0.2 M, respectively. SDS-polyacrylamide gel electrophoresis revealed that betaine increases the contents of microtubule-associated proteins (MAPs) in the assembled microtubules while taurine causes decrease in their contents. Isethionate seemed to cause little change in the ratio of MAPs to tubulin, although less microtubules were formed with increasing isethionate concen- tration. The inhibitory effect of isethionate was overcome by stabilizing action of betaine as measured by turbidity change as well as by protein analyses after centrifugation. Upon measuring vinblastine- induced inhibition of microtubule assembly, taurine and isethionate accelerated inhibition while betaine diminished the vinblastine-induced inhibition. Like glycerol or dimethylsulfoxide, betaine initiated tubulin polymerization in the total absence of MAPs. Electron microscopic observation revealed that betaine caused polymerization of tubulin into microtubules with none of sheets or © 1984 Zoological Society of Japan ribbons, being different from the action of glycerol or dimethylsulfoxide. INTRODUCTION It was early in 1967 when the axoplasm of the squid giant axon was first shown to have high colchicine-binding activity [1]. Later, the axonal microtubule protein was purified by one cycle of temperature-dependent assembly and disassembly, thereby assembly system of the axonal tubulin being established providing the assembly character- istics quite resembling those of porcine brain tubulin [2]. The analysis of protein species in the axoplasm revealed that the axon contains tubulin in larger amount than does mammalian brain. This led to electrophysiological and bio- chemical studies of squid giant axons [3-11], which have proved that microtubules which are fully occupied with tyrosine at the N-terminus of alpha-tubulin and an axonal high molecular weight protein [2, 9] having a molecular weight of 260,000 Accepted August 5, 1983 Received June 22, 1983 (260 K protein) are inevitably required for the maintenance of the membrane excitability. Electron microscopically, the density of micro- tubules in the axon is known to be the highest at the region adjacent to the axolemma, and decreases towards the interior attaining to a steady density of 10-20 microtubules/j#m* of the cross section [6, 8,10]. This density of microtubules is not capable of accounting for the high content of tubulin in the axon. In other words, more than half of axonal tubulin must be in the unpolymerized form. The critical concentration of tubulin for poly- merization in vitro is usually around 0.2 mg/ml, this value being much lower than the possible content of unpolymerized tubulin in the axoplasm. Therefore, it is reasonable to suppose that the axoplasm contains factors that make the equilib- rium between tubulin and microtubules shift towards free tubulin molecule. Thus, the endoge- nous environment for the tubulin-microtubule equilibrium should be clarified to further under- stand electrophysiological function of microtubules 196 M. HASHIMOTO ef al. in the axon. It has well been known [12, 13] that the axoplasm of the squid giant axon contains large amounts of amino acid analogues, betaine (74mM) and taurine (107 mM), and a sulfonic acid, isethionate (165 mM), which have not been studied so far as to the effects on microtubule assembly. This paper deals with these reagents for their pro- nounced effects on tubulin polymerization. MATERIALS AND METHODS Microtubule proteins were prepared from porcine brains by two cycles of temperature- dependent assembly and disassembly [14, 15]. Tubulin was purified from the microtubule protein fraction by phosphocellulose column chromato- graphy [16, 17]. Tubulin eluted from the column in the flow through fraction was supplemented with MgCl, (1 mM) and GTP (0.5 mM), and then concentrated by using an Amicon PM 30 mem- brane. The medium for microtubule assembly con- sisted of 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1mM EGTA, 0.5mM MgCl, 0.5 mM GTP, and betaine, taurine, or isethionate at varying concentrations at a final pH of 6.8. Microtubule assembly was initiated by elevating the temperature of the protein solution from 0 to 33°C. The assembly was monitored by change in turbidity [18] measuring absorbance at 350 nm in a Gilford 260 spectrophotometer. Microtubule assembly was also quantified by centrifugation assay. After incubation at 37°C for 30min, polymerized microtubules were collected by centri- fugation at 36,000 x g for 30 min. The amount of polymerized tubulin was then calculated from the difference between the total protein and protein in the supernatant. Polyacrylamide gel electrophoresis was carried out in the presence of sodium dodecyl] sulfate for analysis of protein composition in the microtubule pellet after Laemmli [19]. Protein concentration was determined by the method of Lowry et al. [20] using bovine serum albumin as a standard. For electron microscopic observations, protein samples were mounted on Formvar-coated grids, negatively stained with 1.5% uranyl acetate and viewed with a Hitachi HU-11B electron micro- scope. RESULTS AND DISCUSSION Effect of Betaine on Microtubule Assembly Microtubule proteins were incubated with varying concentrations of betaine at 33°C. The assembly process was monitored by changes in turbidity until it attained to a steady state. The increase in turbidity in the presence of betaine represented microtubule assembly because com- plete disassembly of microtubules occurred upon cooling to O°C under this condition (data not shown). Observation by electron microscopy showed that the polymerization products in the presence of betaine were predominantly normal microtubules with a small amount of sheets (see later, Fig. 7A). Figure 1A shows apparently that betaine has an inhibitory effect on microtubule assembly with increasing betaine concentration as measured by turbidity change. However, direct measurement of the amount of pelleted microtubule proteins in the centrifugation assay revealed that betaine rather slightly promoted assembly with increasing betaine concentration (Fig. 1B). This apparently inconsistent results seem to derive from decreases in the difference of refractive indices between the medium and microtubules thereby decreasing light scattering with increasing betaine concentration, as discussed for glycerol [21]. In order to confirm this possibility, the critical concentrations for polymerization were determined in the presence or absence of 1.8 M betaine by plotting the level at the steady state of polymerization versus con- centrations of microtubule protein. Whether betaine was present or not, there was little change in the critical concentration (0.28 mg/ml). This clearly indicates that the decrease in turbidity in the presence of betaine at higher concentrations does not necessarily reflect decreases in mass concentration of assembled microtubules. In the experiment shown in Figure 2a, micro- tubules which were assembled in the presence of varying concentrations of betaine were sedimented by centrifugation and proteins in the pellets were Microtubule Assembly cn AA350 (%o) & 0 0.5 18 6 aie Concentration(M ) Fic. 1. dimetry (A) and centrifugation assay (B). 197 Pellet Protein Concentration(%.) — 1-0 Concentration(M 0 0.5 1.5 Effects of betaine, taurine, and isethionate on microtubule assembly measured by turbi- Standard microtubule assembly medium contained 2 mg/ml two-cycled microtubule proteins, 100 mM MES, 1 mM GTP, 1 mM EGTA and 0.5mM MgCl, in a final volume of 0.2 ml. To the microtubule protein solution, was added betaine (@), taurine (11), or isethionate (A) at varying concentrations indicated, and incubated at 33°C. In A, the plateau levels of turbidity increases expressed as % of control were plotted as a function of the concentration of the three reagents. trifugation at 36,000 xg and 30°C for 30 min. In B, samples were incubated at 37°C for 30 min, followed by cen- The amount of assembled microtubules was calculated by subtracting the amount of the protein in the supernatant from that of the total protein. analyzed by polyacrylamide gel electrophoresis in the presence of SDS. The increase in the amount of assembled microtubules (pelleted proteins) with increasing betaine concentration corresponds to the data shown in Figure 1B. Here it should be noted that betaine seemed to cause the ratio of microtubule-associated proteins (MAPs) to tu- bulin to increase until the concentration of betaine increased to 0.8 M. Furthermore, the lag time of microtubule assembly was significantly influenced by betaine, being shortened to half that of control at a betaine concentration of 0.8 M (data not shown). This indicates that betaine promotes nucleation of microtubule assembly. Effect of Taurine Different from the action of betaine, taurine shows inhibitory effect on microtubule assembly (Fig. 1A and B). Half-maximal inhibition oc- curred at a taurine concentration of 0.5 M. The polymers formed in the presence of taurine also represented normal microtubules as observed by electron microscopy. Moreover, the microtubules were quickly depolymerized upon cooling to 0°C. The characteristic feature of microtubules as- sembled in the presence of taurine was that the content of MAPs was greatly reduced, especially MAP 2 was almost totally deprived from micro- 198 M. HASHIMOTO ef al. a b C d 0 4:15:2 0.4 £2 eeu Fic. 2. Protein compositions of assembled microtubules in the presence of varying amounts of betaine (a), taurine (b), isethionate (c) and glutamate (d). The standard assembly media (0.2 ml) were incubated at 37°C for 30 min in the presence of each reagent at concentrations indicated, followed by centrifugation at 36,000 x g for 30 min to sediment microtubules. The microtubule pellets were dissolved in 0.4 ml of 4.6% SDS and 30 wl each was electrophoresed on 10% gels in the presence of SDS, followed by staining with 0.1% Coomassie brilliant blue R—250. tubules (Fig. 2b). This allows one to suppose that MAPs are not needed for tubulin polymerization provided taurine is present. However, poly- merization of tubulin was not initiated by taurine in such assembly media that contained 0.2 M taurine plus 0.5mM MgCl, 0.2 M taurine plus 10mM MgCl, 0.4 M taurine plus 0.5 mM MgCl, or 0.4M taurine plus 10mM MgCl. Careful observation of stained slab gel (Fig. 2b) revealed that a trace amount of MAP-1 was retained in the microtubule pellet. The ability of MAP-1 to initiate tubulin polymerization is already known [22]. Otherwise, the assembly must been promoted by taurine, although corresponding protein bands could not be detected in polyacrylamide gels. Effect of Isethionate Figure 1 shows that isethionate which resembles taurine in structure strongly suppresses micro- The inhibitory action was far tubule assembly. stronger than that of taurine. Half maximal inhibition occurred at an isethionate concentration of 0.2 M and total inhibition took place at around 0.3 M as measured by the centrifugation assay (Fig. 1B). However, the ratio of MAPs to tubulin for microtubules assembled in the presence of isethionate does not seem to change with increas- ing isethionate concentration (Fig. 2c), being different from the action of taurine (Fig. 2b). For comparison, protein compositions of micro- tubules assembled in the presence of glutamate are shown in Fig. 2d, where a little increase in the amount of assembled microtubules and _ little change in the ratio of MAPs to tubulin are shown with increasing glutamate concentration. When the concentration of glutamate was higher than 0.5 M, turbidity of the solution increased extra- ordinarily because of the formation of sheets or ribbons instead of microtubules. Microtubule Assembly 199 0 01 Isethionate (M) Assembly-supporting ability of betaine by suppressing the inhibitory action of isethionate. 02°38 Fic. 3. eee —— i] 2 * rs, 0.6M £ iS $ fe) = W E S oy o OM (e) a 205 oe ® 0 01 02-03 Isethionate (M ) In A, the standard assembly media containing varying amounts of isethionate indicated on the abscissa and betaine at the concentrations indicated on each curve were incubated at 33°C and the plateau levels of turbidity were expressed as in Fig. 1A. In B, the amounts of assembled microtubules were measured by centrifugation assay as in Fig. 1B. Action of Betaine Promoting Microtubule Assembly even in the Presence of Isethionate Foregoing results indicated that betaine is capable of stabilizing microtubules, whereas ise- thionate promotes microtubule disassembly. Their antagonistic actions are intriguing because of their coexistence in the squid giant axon. When both the reagents were contained in the assembly Fic. 4. Protein compositions of assembled micro- tubules in the co-existence of varying amounts of isethionate and betaine. Microtubules were assembled in the standard assembly media containing both isethionate and betaine at concentrations indicated under the photographs, followed by processing as in Fig. 2. a: isethionate alone. b and c: isethionate plus betaine. Isethionate 0 1 -3 Betaine 0 medium, the inhibitory effect of isethionate was largely diminished in a betaine concentration- a b Cc — ee “a & bp i 2 + ved : 2 o* ae ol 0-1 3 0.1 -3 0-6 1-2 200 M. HASHIMOTO et al. dependent fashion (Fig. 3A and B). In Figure 4 are shown protein compositions of microtubules assembled in the presence of both betaine and isethionate. It was clear that betaine retained the otherwise decreasing amounts of MAPs and tubulin by overcoming the inhibitory action of isethionate (cf. Fig. 4a with b or c). Taurine and Isethionate on Assembly Action of Betaine, Vinblastine-Sensitivity of Microtubule Microtubule assembly is suppressed by sub- stoichiometric amount of microtubule poisons. Here, vinblastine was chosen as one of the poisons and the amino acid analogues and isethionate were examined for their actions on vinblastine-induced inhibition of microtubule assembly. Figure 5 plots plateau levels of turbidity increase as a func- tion of vinblastine concentration. When taurine or isethionate was added to the assembly system, Vinblastine (uM ) Fic. 5. Effects of betaine, taurine, and isethionate on vinblastine-induced inhibition of microtubule assembly. The standard assembly media containing 1.2 M betaine (@), 0.2 M (C1) and 0.4M () taurine, or 0.1M isethionate (A) were incubated at 33°C in the presence of varying concentrations of vinblastine. The plateau levels of turbidity were plotted as a function of vinblastine concentra- tion. (X): control containing none of these three reagents. acceleration of the assembly inhibition occurred. In contrast, betaine showed an ability to protect microtubule assembly from the vinblastine-in- duced inhibition to some extent. This was probably due to the action of betaine in stabilizing microtubules. Betaine-Induced Polymerization of Tubulin Several medium conditions have so far been Ore .¢ @) 60 90 Time (min) 0 30 6 Time ( aoe oe Fic. 6. Time course of betaine- and Mg’?*-dependent polymerization of tubulin in the total absence of MAPs. In A, the assembly medium (0.2 ml) contained tubulin (2 mg/ml) purified by phosphocellulose column chromatography, 100 mM MES, 1 mM GTP, 1mM EGTA, 10 mM MgCl, and betaine at varying concentrations indicated on each curve in M. In B, the concentration of betaine was kept constant at 1.0M while the Mg’* concentra- tion was changed as indicated on each curve in mM. and phosphocellulose-purified tubulin (B) both in the presence of betaine. In A, the standard assembly medium containing 1 M betaine was incubated at 37°C for 30 min in a final volume of 0.2 ml, foliowed by processing for electron microscopy. x 50,600. In B, the same assembly medium except for purified tubulin (2 mg/ml) and Mg?* concentration (10 mM) was incubated and processed in the same way as in A. x 50,600. Note that betaine partly caused formation of sheets or ribbons when MAPs were present (A). Microtubule Assembly 201 202 M. HASHIMOTO ef al. established for initiating tubulin polymerization in the total absence of MAPs. Glycerol [23], glutamate [24], dimethylsulfoxide [25] or taxol [26] favors tubulin polymerization. In the pre- vious section, taurine was shown to be incapable of initiating polymerization of tubulin when MAPs were totally absent. However, it was newly found that betaine has an ability to induce polymerization of tubulin provided Mg ions were contained in the assembly medium at relatively high concentration. Figure 6 shows that this amino acid analogue induces tubulin polymerization in a concentration- dependent manner. In the presence of 10mM MgCl, more than 0.4 molar concentration of betaine supported polymerization. On the other hand, when betaine concentration was kept con- stant at 1.0M, the magnitude of polymerization depended on the concentration of Mg ions as in polymerization induced by glycerol. The betaine- induced polymerization was temperature-sensitive, complete depolymerization occurring quickly upon cooling to 0°C. Electron micrographs of the polymerized prod- ucts visualized that betaine favors polymerization of tubulin into microtubules, giving rise to none of aberrant polymers like sheets or ribbons (Fig. 7B). In contrast, it has been reported that glyc- erol or dimethylsulfoxide produces many of sheets composed of protofilaments [23, 25]. Therefore, it is recommended to use betaine for polymeriza- tion of tubulin in the total absence of MAPs. Furthermore, betaine was found to have advantage in that microtubules assembled with MAPs in the medium containing betaine retain more MAPs than do microtubules assembled in its absence. Glycerol is known to decrease the content of MAPs in the assembled microtubules [27]. Antagonistic effect of betaine against isethionate will favor maintenance of tubulin-microtubule equilibrium towards the polymerized form. In the squid giant axon, however, the content of isethionate (165mM) is as high as giving half maximal inhibition of microtubule assembly, being higher than that of betaine (74 mM). There- fore, the equilibrium between tubulin and micro- tubules in the axoplasm may be shifted towards unpolymerized form, resulting in that a half of tubulin is kept unpolymerizing. Moreover, con- siderably high content of K ions (344 mM) and Na ions (65 mM) will further cause increment of unpolymerized tubulin. The ionic strength for optimal microtubule assembly in vitro is well known to be around 0.1. However, for elongation of microtubules, that is, site-initiating microtubule assembly, an ionic strength of 0.25—0.3 still supports assembly [28]. In this connection, recent finding that microtubule assembly is initiated by the neurofilament [29], being contained in the squid axoplasm as well, may partly explain substantial number of microtubules in the axoplasm. The dissected squid giant axon (av. 0.6 x4 cm) contain more than 0.1 mg tubulin [2] correspond- ing to 6x 10** tubulin molecules. Single micro- tubule of 4cm long consists of 6.510’ tubulin molecules. Therefore, 0.9210’ (6x10**/6.5 x 10’) microtubules are contained in the axoplasm running parallel to the longitudinal axis of the axon. Since the cross sectional area of the axon turns out to be 0.27 10° um’, 34 microtubules/ ym? (0.92 x 10’/0.27 x 10°) should be present provided all of tubulin are in the polymerized form. However, except for the peripheral region (within 3 um from the axolemma), only less than 20 microtubules/wm* are detected in the axoplasm [8, 10]. This means that about half of tubulin molecules are in the unpolymerized form, and seems to be reasonable judging from the ionic environment of the axoplasm not so favorable for tubulin polymerization. However, in the periph- eral region of the axon, more than 90 microtubules/ ym? of the cross section are found. Since micro- tubule networks lining the axolemma confer excitability to the axon, this peripheral region should be differentiated so as to maintain a large number of microtubules irrespective of such an ionic environment. Whether this is due to the presence of abundant sites for initiating assembly or of some other devices to reduce the critical concentration for tubulin polymerization is unknown. ACKNOWLEDGMENTS We thank Mrs. S. Endo for electron microscopy. Thanks are also due to Dr. H. Kumagai for supplying phosphocellulose-purified tubulin. 10 Microtubule Assembly REFERENCES Borisy,G.G. and Taylor, E. W. mechanism of action of colchicine. colchicine-*H to cellular protein. 34: §$25-533. Sakai, H. and Matsumoto, G. (1978) Tubulin and other proteins from squid giant axon. J. Biochem., 83: 1413-1422. Matsumoto, G. and Sakai, H. (1979) Méicro- tubules inside the plasma membrane of squid gaint axons and their possible physiological function. J. Membrane Biol., 50: 1-14. Matsumoto, G. and Sakai, H. (1979) Restoration of membrane excitability of squid giant axons by (1967) The Binding of J. Cell Biol., reagents activating tyrosine-tubulin ligase. J. Membrane Biol., 50: 15-22. Matsumoto, G., Kobayashi, T. and Sakai, H. (1979) Restoration of the excitability of squid giant axon by tubulin-tyrosine ligase and micro- tubule proteins. J. Biochem., 86: 1155-1158. Endo, S., Sakai, H. and Matsumoto, G. (1979) Microtubules in squid giant axon. Cell Struct. Funct., 4: 285-293. Murofushi, H. (1980) Purification and _ chara- cterization of tubulin-tyrosine ligase from porcine brain. J. Biochem., 87: 979-984. Matsumoto, G., Murofushi, H., Endo,S. and Sakai, H. (1982) Microtubules composed of tyrosinated tubulin are required for membrane excitability in squid giant axon. In ‘“‘Biological Functions of Microtubules and Related Struc- tures”. Ed. by H. Sakai, H. Mohri and G.G. Borisy, Academic Press, Tokyo, pp. 391-404. Murofushi, H., Minami, Y., Matsumoto, G. and Sakai, H. (1983) Bundling of microtubules in vitro by a high molecular weight protein prepared from the squid giant axon. J. Biochem., 93: 639-650. Matsumoto, G.., Murofushi, H.., Endo, S., Kobayashi, T. and Sakai, H. (1983) Tyrosinated tubulin necessary for maintenance of membrane excitability in squid giant axon. In “Structure and Function in Excitable Cells’. Ed. by D.C. Chang, I. Tasaki and W. J. Adelman, Jr., Plenum Pub. Corp., New York, pp. 471-483. Matsumoto, G., Ichikawa, M., Tasaki, A., Muro- fushi, H. and Sakai, H. (1983) Axonal micro- tubules necessary for generation of sodium current in squid giant axons. I. Pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein. J. Membrane Biol., 77: 77-91. Deffner, G. J. and Hafter, R. E. (1960) Chemical investigations of the giant nerve fibers of the squid. IV. Acid-base balance in axoplasm. Biochim. 13 18 20 21 22 23 24 25 203 Biophys. Acta, 42: 200-205. Deffner, G. J. (1961) The dialyzable free organic constituents of squid blood; A comparison with nerve axoplasm. Biochim. Biophys. Acta, 47: 378-388. Shelanski, M. L., Gaskin, F. and Cantor, C. R. (1973) Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci., USA, 70: 765-768. Nishida, E., Kumagai, H., Ohtsuki, I. and Sakai, H. (1979) The interaction between calcium- dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. I. Effect of calcium-dependent regulator protein on the calcium sensitivity of the microtubule assembly. J. Biochem., 85: 1257-1266. Weingarten, M. D., Lockwood, A. H., Hwo, S.-Y. and Kirschner, M. W. (1975) A protein factor essential for microtubule assembly. Proc. Natl. Acad. Sci., USA, 72: 1858-1862. Nishida, E., Kotani, S., Kuwaki, T. and Sakai, H. (1982) Phosphorylation of microtubule-associated proteins (MAPs) controls both microtubule as- sembly and MAPs-actin interaction. In “Biological Functions of Microtubules and Related Structures”. Ed. by H. Sakai, H. Mohriand G. G. Borisy, Academic Press, Tokyo, pp. 285-295. Gaskin, F., Cantor, C. R. and Shelanski, M. L. (1974) Turbidimetric studies of the in vitro as- sembly and disassembly of porcine neurotubules. J. Mol. Biol., 89: 737-758. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. 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: 265-275. Keates, R. A. B. (1980) Effects of glycerol on microtubule polymerization kinetics. Biochem. Biophys. Res. Commun., 97: 1163-1169. Kuznetsov, S. A., Rodionov, V.I., Gelfand, V. I. and Rosenblat, V.A. (1981) Microtubule- associated protein MAPI promotes microtubule assembly in vitro. FEBS Letters, 135: 241-244. Lee, J. C. and Timasheff, S. N. (1975) The recon- stitution of microtubules from purified calf brain tubulin. Biochemistry, 14: 5183-5187. Hamel, E. and Lin, C.M. (1981) Glutamate- induced polymerization of tubulin: Characteristics of the reaction and application to the large-scale purification of tubulin. Arch. Biochem. Biophys., 209: 29-40. Himes, R.H., Burton, P.R. and Gaito, J. M. (1977) Dimethyl sulfoxide-induced self-assembly of tubulin lacking associated proteins. J. Biol. 204 26 Chem., 252: 6222-6228. Schiff, P. B., Fant, J. and Horwitz, S. B. (1979) Promotion of microtubule assembly in vitro by taxol. Nature, 277: 665-667. Scheele, R. B. and Borisy, G.G. (1976) Com- parison of the sedimentation properties of micro- tubule protein oligomers prepared by two different procedures. Biochem. Biophys. Res. Commun., 28 29 M. HAsHIMOTO ef al. 70: 1-7. Nishida, E. (1978) Effects of solution variables on the calcium sensitivity of the microtubule assembly system. J. Biochem., 84: 507-512. Minami, Y., Murofushi, H. and Sakai, H. (1982) Interaction of tubulin with neurofilaments: For- mation of networks by neurofilament-dependent tubulin polymerization. J. Biochem., 92: 889-898. ZOOLOGICAL SCIENCE 1: 205-213 (1984) Further Studies on the Physiological Role of Hexose-6-Phosphate Dehydrogenase: No Positive Correlation between Hexose-6- Phosphate Dehydrogenase and Enzymes other than NADPH-Cytochrome c Reductase TAKESHI KODAMA! Department of Zoology, Faculty of Science, Hokkaido University, Sapporo 060, Japan ABSTRACT — Intrarenal distributions of cytosolic NADP*-dependent dehydrogenases (6- phosphogluconate dehydrogenase, malic enzyme and isocitrate dehydrogenase), microsomal reductase (aromatic aldehyde-ketone reductase and NADH-cytochrome 6, reductase) and cytochrome b, and effects of phenobarbital, methylcholanthrene, fasting, castration and sex hormones on activities of these enzymes were examined, and the results were compared with the previous results on hexose- 6-phosphate dehydrogenase and the microsomal NADPH-cytochrome c reductase-cytochrome P—450 system. Asa result, no positive correlation in the distribution and responsiveness to the treatments was observed between the cytosolic dehydrogenases and microsomal NADPH-cytochrome c reductase, between hexose-6-phosphate dehydrogenase and the NADH-cytochrome 6, reductase-cytochrome b; system, and between hexose-6-phosphate dehydrogenase and aromatic aldehyde-ketone reductase. © 1984 Zoological Society of Japan INTRODUCTION Hexose -6- phosphate dehydrogenase (EC 1.1.1.47) is a microsomal enzyme which cat- alyzes the reduction of NAD(P)* through the oxidation of various hexose-6-phosphates, such as glucose-6-phosphate, galactose-6-phosphate and 2-deoxy-glucose-6-phosphate [1], and has proper- ties quite distinct from cytosolic glucose-6-phos- phate dehydrogenase (EC 1.1.1.49) [2-9]. In contrast to a well known function of glucose- 6-phosphate dehydrogenase, the physiological role of hexose-6-phosphate dehydrogenase has not yet been fully explored. It seems that this enzyme may play a role in supplying reduced equivalents to microsomal electron’ transport system. Consistent with this assumption § the folldwing findings are recognized: (1) hexose-6- Accepted September 24, 1983 Received June 4, 1983 ‘ Present address: Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-01, Japan phosphate dehydrogenase activity is high in the microsomes of hepatic cells, testicular inter- stitial cells, ovary, adrenal [7], the straight seg- ment of renal proximal convolution [10], striated duct and serous tubular cells of submandibular gland and plasma cells (Tanahashi and Hori, unpublished data), and (2) it can be induced by phenobarbital in the liver [5] and by human chorionic gonadotrophin in testicular interstitial cells [7]. Our data also demonstrates the inti- mate correlation between hexose-6-phosphate dehydrogenase, but not glucose-6-phosphate dehydrogenase, and NADPH-cytochrome c re- ductase (EC 1.6.2.3) with respect to the intrarenal distribution and the responsiveness to various treatments [11]. All these data are compatible with our notion that hexose-6-phosphate dehy- drogenase may be functioning as an NADPH generator in the microsomal electron transport system, thus involving in drug and steroid me- tabolism. In order to further test our notion, intrarenal distributions of and effects of various treatments 206 T. KODAMA on cytosolic NADPH-generating enzymes, mi- crosomal aromatic aldehyde-ketone reductase, microsomal NADH-cytochrome b; reductase (EC 1.6.2.2) and cytochrome 6; were examined in the present study. MATERIALS AND METHODS Reagents 6-Phosphogluconate, NADP’, NADPH and NADH were purchased from Oriental Yeast Co. Ltd.; 4-benzoylpyridine and 3-methylcholanthrene were from Wako Pure Chemical Industries Co.; cytochrome c (Type III) and isocitrate were from Sigma; Triton X- 100 and 17a-methyltestosterone were from Nakarai Chemicals Ltd.; 17f8-estradiol was from Teikoku Zoki Co.; sodium phenobarbital was from Sankyo Co. Ltd. All other chemicals were commercial products of the highest grade available. Treatments of animals Wistar rats were used throughout this study. Fasting was _ per- formed for 3 days with adult males (90-day-old; 270-300 g). Young males (50-day-old; 170-200 g) were given once daily intraperitoneal injections of phenobarbital (75 mg/kg body weight) dissolved in saline for 4 days following 2-day fasting or methylcholanthrene (40 mg/kg body weight) dis- solved in olive oil for 3 days. Orchidectomy was performed on day 30 and the castrated rats re- ceived daily subcutaneous injections of methyl- testosterone (5 mg/kg body weight) or estradiol- 178 (0.5 mg/kg body weight) dissolved in olive oil for 14 days from day 76 on. Control rats were injected with the vehicle only. Preparation of microsomes The cortex, outer and inner stripes of outer medulla and inner medulla in the kidney and testicular inter- Stitial tissues were separated by the methods of Kodama and Hori [11] and Takahashi er al. [7], respectively. The various organs and tissues were homogenized in 9 vols. of 0.25 M sucrose, and microsomal and cytosol fractions were isolated as described previously [11]. The microsomal pellets were washed once with 1.15°%% KCI and resedimented by centrifugation at 144,000 xg for 40min. The washed pellets were suspended in 1.157% KCl-25 mM Tris-HCl buffer (pH 7.5). Enzyme assays Enzyme activities were assayed spectrophotometrically at 37°C. The microsomal enzyme activities were assayed with the microsomes treated with 1% Triton X-—100. Hexose-6-phosphate dehydrogenase and NADPH- cytochrome c reductase activities were assayed as reported previously [11]. Aromatic aldehyde- ketone reductase activity was measured with NADPH and 4-benzoylpyridine in 80 mM _ phos- phate buffer (pH 6.3) by the method of Sawada et al. [12]. NADH-cytochrome b, reductase ac- tivity was measured with NADH and potassium ferricyanide as substrate by the method of Takesue and Omura [13]. The millimolar extinction differ- ence (cm™'*mM™') between ferricyanide and fer- rocyanide was assumed to be 1.02 at 420 nm [14]. Cytosolic 6-phosphogluconate dehydrogenase (EC 1.1.1.44) [8], malic enzyme (EC 1.1.1.40) [15] and isocitrate dehydrogenase (EC 1.1.1.42) [8] activi- ties were determined by the methods described in respective references with slight modifications. The activities are expressed by using “unit”. One unit of activity was defined as the amount of enzyme that reduced or oxidized 1 wmol of electron acceptor per min under the assay con- dition. The content of cytochrome b; in micro- somes was determined by measuring reduced minus oxidized difference spectrum of samples assuming the extinction difference of the cytochrome be- tween 424 nm and 409 nm to be 185 cm™*-mM * [16]. Protein was determined according to Lowry et al. [17] with bovine serum albumin as a standard. The significance of the differences between experimental means was assessed using Student’s t-test for paired variables. RESULTS Cytosolic dehydrogenases Intrarenal distributions of 6-phosphogluconate dehydrogenase, malic enzyme and isocitrate de- hydrogenase are shown in Table 1. 6-Phos- phogluconate dehydrogenase and malic enzyme are more active in the medulla than in the cor- tex. In the case of isocitrate dehydrogenase, the activity is evenly distributed in the three portions. Such distributional patterns of the Role of Hexose-6-Phosphate Dehydrogenase 207 TABLE 1. Intrarenal distributions of cytosolic NADP*-dependent dehydrogenases, microsomal reductases and cytochrome b, in adult males Enzymes Cortex 6-Phosphogluconate 55.8 +2.0 dehydrogenase (munits/mg protein) Malic enzyme 15.2 +0.9 (munits/mg protein) [socitrate 1.01 +0.06 dehydrogenase (units/mg protein) Aromatic aldehyde- 5.3 +0.5 ketone reductase (munits/mg protein) NADH-cytochrome b,; reductase (units/mg protein) 2.45+0.05 Cytochrome 6, 0.11 +0.009 (nmol/mg protein) The enzymes were assayed as described in the mean+S.E. of 4-6 animals. three enzymes are different from those of NADPH- cytochrome c reductase and cytochrome P-450 described previously [11]. These findings are unfavorable to a notion that one of the three cytosolic dehydrogenases might be related to the NADPH-dependent electron transport system. Effects of various treatments on the three enzymes are shown in Tables 2 and 3. As shown in Table 2, phenobarbital has no effect on the three enzymes, while methylcholanthrene causes slight increases of 6-phosphogluconate de- hydrogenase activity in the outer stripe and of malic enzyme activity in the cortex. Fasting reduced the malic enzyme activity in the outer stripe. These findings contrast to the previous results that all treatments are effective in stimu- lating the activities of hexose-6-phosphate dehy- drogenase and NADPH-cytochrome c reductase in the outer stripe and that such increases are also accompanied with increases in cytochrome P-450 and/or aryl hydrocarbon hydroxylase [11]. It is particularly noteworthy that fasting causes a decrease in malic enzyme, while it stimulates hexose-6-phosphate dehydrogenase, cytochrome c reductase and cytochrome P-450 [11]. Such in- dependent behavior of malic enzyme suggests that “Materials and Methods”’. Inner stripe of outer medulla + inner medulla Outer stripe of outer medulla 105.6 +2.8 106.6 +7.2 48.4 +1.7 26.0 +0.8 0.72 +0.03 0.93 +0.04 Be) -E Us) 4.9 (n=2) 2.45 +0.08 2.17+0.06 0.140.004 0.12 +0.007 Values represent it might not be functionally coupled to the mi- crosomal electron transport system. Activities of the three cytosolic dehydrogenases show sex difference in both the cortex and the outer stripe (Table 3): females have higher ac- tivities than males, except 6-phosphogluconate dehydrogenase activity in the outer stripe which is higher in males. The cause of such sex differ- ence might be explained in terms of either stimu- lative actions of estrogens or inhibitory actions of androgens. It could also be expected that castration causes an increase of activity if andro- gens are inhibitory. However, the data shown in Table 3 are rather complicated, and particu- larly puzzling in the case of 6-phosphogluconate dehydrogenase of the cortex. Nevertheless, con- clusion is at least possible that estradiol has an inhibitory action on 6-phosphogluconate dehy- drogenase and a stimulative action on malic enzyme and isocitrate dehydrogenase in both the cortex and the outer stripe. In comparing these data with the previous results that estrogen has a stimulative effect on hexose-6-phosphate de- hydrogenase, cytochrome c reductase, amino- pyrine demethylase and laurate hydroxylase [11], it becomes evident that 6-phosphogluconate de- T. KODAMA 208 "AJOAIOOdSaI ‘SfaAg] %{ TO pue %T “%¢ Je poyessed oy} WIOIJ JUSIOYIP APURDYIUSIC ,«,«, “AJOATIOAdSAI “S[OAST % ['O pue %T “% Ss 1 sojew Wnpe Wolf JUSIOPIP APJUCOYIUSIC 46g ‘e ‘¢LI-lolpens” :qa “QUOIOISOISOYAMIOW :.LIN ‘s[ewtue 9-p Jo “q's Uva oY) jUOSoIdeI sonjeA *,.SPOYISJ! 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JO SCLIS.I3}NG asejonpal LOOT C C00 + SPT Ol'0+ Ir’ 60°0 + Iv'7 LO'O+ 8t'7 80'0 + St°7 XMMOD —-*g BWO.1Y9OIKD- HAWN B]]Npow 191nNo (ulajo1d Stu/syunw) aS OF III SOF S'El 90OF L'El 90F El SOF 79 LOF 6 JO 9GLy8 30)NO asv}9Npad 9u0}o¥ g'0+ LP cOF VS cOn 19 Soa 1s OF P'E CO+F 67 x90 -apAyap]e snewoly poeol jomUuoy poieol | joUoOy pool jO.1UOD SsowWAZUq Buljse-f audi UR[OYS|AYIOW, jeugseqousyd ce AQupry ay} Ul §q SWOIYDOIAD puP sosPIONpas [PWOSOJIW UO Busey puL duoyUR[OYS[AYyIOW ‘yeyGseqousyd Jo sioaya p AAV 210 T. KODAMA hydrogenase could not be a candidate for the NADPH supplier to the microsomal electron transport systems involving drug metabolism and w-oxidation of fatty acids. In short, evidence has not been obtained that some of the cytosolic dehydrogenases examined are primarily responsible for the supply of NADPH to the microsomal ele- ctron transport system. NADH-cytochrome b; reductase and cytochrome bs The results shown in Table 1 indicate that NADH-cytochrome 5b; reductase and cytochrome bs are evenly distributed in the three renal por- tions. Effects of phenobarbital, methylcholan- threne and fasting are shown in Table 4. Pheno- barbital has no effect on these components at all, whereas methylcholanthrene causes a slight in- crease of cytochrome b; content in both the cortex and the outer stripe. Fasting results in a slight decrease of NADH-cytochrome 6; re- ductase activity in the outer stripe, in contrast to the increases of hexose-6-phosphate dehydroge- nase, NADPH-cytochrome c reductase and cyto- chrome P-—450 as reported previously [11]. Effects of castration and sex hormones are shown in Table 5. No sex difference is ob- served in the cytochrome 6; content, while the activity of NADH-cytochrome 5b; reductase is higher in males than in females. Neither me- thyltestosterone nor estradiol has a significant effect on this enzyme. Thus, no conclusive proof was obtained that hexose-6-phosphate dehydro- genase might be functionally related to the NADH-cytochrome 6; reductase-cytochrome )b; system. The decrease of NADH-cytochrome b, reductase activity in fasted rats is rather unfavor- able to such relationship. Aromatic aldehyde-ketone reductase Considerable activities of aromatic aldehyde- ketone reductase are present mostly in the outer stripe (Table 1). Its activity is decreased after phenobarbital treatment in both the cortex and the outer stripe, and upon fasting in the outer stripe only (Table 4). Sex difference is remarka- TABLE 6. Specific activities (units/g protein) of hexose-6-phosphate dehydrogenase, NADPH- cytochrome c reductase and aromatic aldehyde-ketone reductase in the microsomal fractions from various rat organs Hexose-6-phosphate NADPH-cytochrome c reductase Aromatic aldehyde- ketone reductase units/g protein Tissues dehydrogenase Liver 43.4+1.6 Adrenal 21.4+2.9 Testicular interstitial 18.9 cells (n=2) Outer stripe of renal 16.0-L0.2? outer medulla Ovary 12.4+0.3 Lung 7.8+0.4 Spleen 5.6+0.3 Lymph node 2ANSEOD Intestine 2.0-+0.1 Salivary gland 2.0+0.3 Brain 1.2+0.2 142.2+6.4 41.0+1.7 97.8+8.4 3.8+0.8 Sal) 24.6 42.5+1.9° 13.6+0.8 48.6+1.7 Del atau 44.0+4.8 Spl sell 21.2+0.5 0.2+0.2 15.3+0.4 Oe=0m 38.6+6.3 N. D. 12.5+0.4 N. D. 15.0+1.6 N. D. Organs of adult males and ovaries of adult females were examined. In the case of adrenal, testicular interstitial cells, ovary, lymph node and salivary gland, organs from 3-4 animals were pooled. The enzymes were assayed as described in ‘‘Materials and Methods’’. of 3 experiments. N. D., not detectable. * Cited from Kodama and Hori [11]. Values represent the mean-+tS.E. Role of Hexose-6-Phosphate Dehydrogenase 211 ble in the outer stripe: males are more active than females (Table 5). The activity is reduced by castration and restored to normal level by methyltestosterone. Treatment of castrated rats with estradiol results in a further decrease of the activity. Such an inhibitory action of estradiol on the reductase is in contrast te its stimulative action on hexose-6-phosphate dehydrogenase. Organ-specific distribution of this enzyme is shown in Table 6, in comparison with those of NADPH-cytochrome c reductase and hexose-6- phosphate dehydrogenase. The activity of aro- matic aldehyde-ketone reductase is low in the adrenal and ovary where the activities of hexose- 6-phosphate dehydrogenase and cytochrome c re- ductase are comparatively high. It is thus un- likely that the principal role of hexose-6-phosphate dehydrogenase is to provide aldehyde-ketone re- ductase with NADPH. DISCUSSION The previous paper [11] indicates that there is a similarity between hexose-6-phosphate dehy- drogenase and NADPH-cytochrome c reductase with respect to the intrarenal distribution and the responsiveness to various treatments, thus suggesting functional coupling of hexose-6-phos- phate dehydrogenase to microsomal NADPH- dependent electron transport system. Consistent with this suggestion, the present results show that the cytosolic dehydrogenases and the micro- somal reductases examined substantially differ from hexose-6-phosphate dehydrogenase and NADPH-cytochrome c reductase in the intrarenal distributions and responses to various treatments. This suggests that hexose-6-phosphate dehydroge- nase might not be functionally related to the microsomal NADH-dependent electron transport system and aromatic aldehyde-ketone reductase, and that NADPH-cytochrome c reductase might not be functionally coupled to the cytosolic NADPH generators. The supply of NADPH as well as activated substrates may be rate limiting in the microsomal mixed function oxidation [18-20]. Hence, it is important to know which enzyme is primarily responsible for the supply of NADPH. Kauf- fman ef al. [21, 22] emphasized the importance of the pentose shunt dehydrogenases in the sup- ply of NADPH to the microsomal mixed function oxidation system. In _ contrast, Shah and Pearson [23] suggested that elevation of the combined activities of malic enzyme and the pentose shunt dehydrogenases would be _ neces- sary for the provision of reducing equivalents in the face of extra drug stress. In accordance with these studies, we also found a significant increase of glucose-6-phosphate dehydrogenase activity in the livers of phenobarbital-treated rats [5, 11]. Howeyer, it was found in the previous and pres- ent studies that none of the cytosolic dehydro- genases examined respond to phenobarbital in the kidney, although activities of aryl hydro- carbon hydroxylase and aminopyrine_ de- methylase are increased. In the kidney of methylcholanthrene-treated rats, increases in the activities of glucose-6-phosphate dehydrogenase and malic enzyme in the cortex and that of 6- phosphogluconate dehydrogenase in the outer stripe were observed, whereas the activity of aryl hydrocarbon hydroxylase increased in both the cortex and the outer stripe. Thus, such independent behaviors between the cytosolic de- hydrogenase and the microsomal mixed function oxidation system suggest that the cytosolic de- hydrogenases might not be primarily responsible for the supply of NADPH to the microsomal system. There are two electron transport systems in microsomes; one is dependent on NADH and consists of NADH-cytochrome 46, _ reductase, cytochrome 4, and _ cyanide-sensitive factor, while the other is an NADPH-dependent system consisting of NADPH-cytochrome c_ reductase and cytochrome P-450. The former is_ re- sponsible for the desaturation of long chain fatty acids, while the latter is involved in the metabolism of various steroids and xenobiotics as well as in w-hydroxylation of fatty acids [24, 25]. It has also been known that there is pos- sible interactions between the two systems; elec- trons flow from NADH to cytochrome P-450 via NADH-cytochrome hb, reductase or cytochrome b;, or from NADPH to cytochrome bh; via cyto- chrome c_ reductase [26]. Hexose-6-phosphate pA ly. dehydrogenase has a high affinity to NAD* as well as to NADP*, and hence it is possible that the enzyme may be linked to the NADH-de- pendent system. However, the present study did not yield evidence favorable to this possi- bility. Sawada et al. [12, 27] reported an enzyme in liver microsomes of guinea pig which catalyzes the reduction of 3-ketosteroids, aromatic alde- hydes and aromatic ketones in the presence of NADPH (cytochrome P-—450 is not required). A most efficient NADPH generator for this reductase was found to be hexose-6-phosphate dehydroge- nase. There is no reason to believe that the microsomal electron transport system should have such a membrane-bound NADPH-generator as hexose-6-phosphate dehydrogenase, since the ac- tive site of NADPH-cytochrome c reductase is considered to be exposed on the cytoplasmic surface of microsomal membranes [28], thus being accessible to NADPH probably present in excess in the cytoplasm. Consequently, it is necessary to prove that cytoplasmic NADPH could not be used in vivo by NADPH-cytochrome c reductase for some reason in order to validate the hy- pothesis that hexose-6-phosphate dehydrogenase may be coupled to the microsomal electron transport system. On the other hand, aromatic aldehyde-ketone reductase is embedded in micro- somal membranes, thus being unable to react with cytoplasmic NADPH. It is plausible there- fore that hexose-6-phosphate dehydrogenase might be present in the membranes for such a reductase. Predominant location of this enzyme in the liver, testicular interstitial cells and the outer stripe of renal outer medulla as revealed in the present study appears to be favorable for the hypothesis of Sawada eft al. However, its low activity in the adrenal and ovary, and the phenobarbital-, fasting- and estradiol-induced decreases in the kidney are in contrast to the behaviors of hexose- 6-phosphate dehydrogenase. As this stage of knowledge, it seems to be a most plausible interpretation of the available data that hexose-6-phosphate dehydrogenase may be functionally coupled to the microsomal NADPH- dependent electron transport systems. T. KODAMA 10 11 12 REFERENCES Beutler, E. and Morrison, M. (1967) Localization and characteristics of hexose-6-phosphate dehydro- genase (glucose dehydrogenase). J. Biol. Chem., 242: 5289-5293. Hori, S.H. and Matsui, S. (1968) Intracellular distribution of electrophoretically distinct forms of hepatic glucose 6-phosphate dehydrogenase. J. Histochem. 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(1979) Intracellular distributions of glucose 6-phosphate dehydrogenase and hexose 6-phosphate dehydrogenase in rat testicular interstitial cells. J. Fac. Sci. Hokkaido Univ. Ser. VI, Zool., 22: 12-20. Kodama, T., Ohnishi, K. and Hori, S. H. (1981) Identity of cytosol and mitochondrial glucose 6-phosphate dehydrogenases. J. Fac. Sci. Hok- kaido Univ. Ser. VI, Zool., 22: 191-198. Oka, K., Takahashi, T. and Hori, S.H. (1981) Differential effects of the NADPH/NADP* ratio on the activities of hexose-6-phosphate dehydro- genase and glucose-6-phosphate dehydrogenase. Biochim. Biophys. Acta., 662: 318-325. Tanahashi, K. and Hori, S. H. (1980) Immuno- histochemical localization of hexose 6-phosphate dehydrogenase in various organs of the rat. J. Histochem. Cytochem., 28: 1175-1182. Kodama, T. and Hori, S.H. (1982) Possible functional coupling of hexose-6-phosphate de- hydrogenase to microsomal electron transport system in rat kidney and liver. Biochim. Biophys. Acta, 715: 151-161. Sawada, H., Hara, A., Hayashibara, M., Naka- yama, T., Usui, S. and Saeki, T. (1981) Micro- somal reductase for aromatic aldehydes and 13 14 a 17 18 19 20 21 Role of Hexose-6-Phosphate Dehydrogenase ketones in guinea pig liver. Purification, charac- terization, and functional relationship to hexose-6- phosphate dehydrogenase. J. Biochem., 90: 1077- 1085. Takesue, S. and Omura, T. (1970) Purification and properties of NADH-cytochrome 4, reductase solubilized by lysosomes from rat liver microsomes. J. Biochem., 67: 267-276. Schellenberg, K. A. and Hellerman, L. (1958) Oxidation of reduced diphosphopyridine nucleo- tide. J. Biol. Chem., 231: 547-556. Luine, V.N. and Kauffman, F.C. (1970) Tri- phosphopyridine nucleotide-dependent enzymes in the developing spinal cord of the rabbit. J. Neurochem., 18: 1113-1124. Omura, T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes: I. Evidence for its hemoprotein nature. J. Biol. Chem., 239: 2370-2378. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin pheno! reagent. J. Biol. Chem., 193: 265-275. Moldeus, P., Grundin, P., Vadi, H. and Orrenius, S. (1974) A study of drug metabolism linked to cytochrome P-450 in isolated rat-liver cells. Eur. J. Biochem., 46: 351-360. Thurman, R. G., Marazzo, D. P., Jones, L. S. and Kauffman, F.C. (1977) The continuous kinetic determination of p-nitroanisole O-demethylation in hemoglobin-free perfused rat liver. J. Pharmacol. Exp. Ther., 201: 498—S06. Weigl, K. and Sies, H. (1977) dependent on cytochrome /P-450 in_ isolated hepatocytes. The role of the tricarboxylates and the aminotransferases in NADPH supply. Eur. J. Biochem., 77: 401-408. Kauffman, F. C., Evans, R. K. and Thurman, R. Drug oxidations to nN 23 24 25 26 oH | 28 213 G. (1977) Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats. Biochem. J., 166: 583-592. Kauiiman, F.C... Evans, R.K., Remke; L. A., Berinsky, S. A., Ballow, C. and Thurman, R. G. (1980) Effects of 3-methylcholanthrene on oxi- dized_ nicotinamide-adenine dinucleotide pho- sphate-dependent dehydrogenases and_ selected metabolites in perfused rat liver. Biochem. Pharmacol., 29: 697—700. Shah, S. and Pearson, D. J. (1978) The effect of phenobarbitone on cytoplasmic NADP-linked dehydrogenase activities in rat liver. Biochim. Biophys. Acta, 539: 12-18. Lu, A. Y. H. and Coon, M.J. (1968) Role of hemoprotein P-450 in fatty acid omega-hydro- xylation in a soluble enzyme system from liver microsomes. J. Biol. Chem., 243: 1331-1332. Wada, F., Shibata, H., Goto, M. and Sakamoto, Y. (1968) Participation of the microsomal electron transport system involving cytochrome P-450 in w-oxidation of fatty acids. Biochim. Biophys. Acta, 162: 518-524. Williams, C. H. Jr (1976) Flavin-containing de- hydrogenases. In “The Enzymes’. Ed. by P. D. Boyer, Academic Press, New York, 3rd ed., Vol. XIII, pp. 89-173. Sawada, H., Hayashibara, M., Hara, A. and Naka- yama, T. (1980) A possible functional relation- ship between microsomal aromatic aldehyde-ketone reductase and hexose-6-phosphate dehydrogenase. J. Biochem., 87: 985-988. DePierre, L. W. and Ernster, L. (1977) topology of intracellular membranes. Biochem., 46: 201-262. Enzyme Ann. Rev. ' 7 | - = ‘ i x i | ' } ne ’ \ \ e i 4 ; ’ 3 j 7) x y - f E # i] { 4 ! “ ’ ~ q / 1 he OTD baad LOI, oy ee peri he RIoky iebesrey \ a rads 2 Lange 43 3 $34 Race: 7 4 * y : : edad } ule ce opi e | ae ee {SUR ey 4 f n sit I rs Nee Le nite re. Ties, ‘ts , | : peecateade) fran" rosie: a ZOOLOGICAL SCIENCE 1: 215-221 (1984) Pteridine-Metabolizing Enzymes in Rat Erythrocytes during Development SETSUKO KATOH and TERUMi SUBOKA Department of Biochemistry, Josai Dental University, Sakado, Saitama 350-02, Japan ABSTRACT — The developmental pattern of sepiapterin reductase and dihydrofolate reductase, which are both involved in the final two steps of the metabolic pathway of tetrahydrobiopterin, were studied in blood cells of fetuses and neonates of rats, and in erythrocytes of young and adult rats. Levels of dihydrofolate reductase activity 60-fold higher and sepiapterin reductase 20% lower than those found in erythrocytes of adult rats were observed in fetal blood cells at 20 days of gestation. Reticulocytes from adult rats injected with phenylhydrazine showed a relatively higher level of sepiapterin reductase and a 20-fold higher level of dihydrofolate reductase activity compared with those in erythrocytes of adult rats. In blood cells of the fetal rat at 20 days of gestation, a similar amount of reduced pteridine and 80% activity of dihydropteridine reductase were also found compared with those in erythocytes of adult rats. Based on the above observations, we propose that biopterin metabolism in rat erythrocytes may © 1984 Zoological Society of Japan be more stimulated during early development of the cell than in the mature period. INTRODUCTION Recently, the level of biopterin (L-erythro-6- dihydroxypropylpterin) content in blood or urine was determined to be diagnostic for a new type of human hyperphenylalanemia due to a deficiency of dihydropteridine reductase (DPR: EC 1.6.99.7) or a deficiency in the biopterin biosynthetic path- way [1, 2]. Reduced biopterin (5, 6, 7, 8-tetrahy- drobiopterin) is required for the enzymatic hydroxylation of phenylalanine, tyrosine and tryptophan as a cofactor of the H-donor with DPR, an enzyme which maintains the pteridine cofactor in tetrahydroform [3] (Fig. 1). The re- duced biopterin is essential for the conversion of phenylalanine to tyrosine or for the syntheses of biogenic monoamines such as _ norepinephrine, epinephrine, serotonin and melatonin. A _ postu- lated metabolic pathway of tetrahydrobiopterin in mammalian tissues described in many recent studies shows that tetrahydrobiopterin might originate Accepted September 24, 1983 Received June 22, 1983 from GTP by being catalyzed with some enzymes such as GTP cyclohydrolase, sepiapterin reductase (SPR: EC 1.1.1.153) and dihydrofolate reductase (DFR: EC 1.5.1.3) [4-8] (Fig. 1). Biopterin [9] and its metabolizing enzymes, SPR, DFR [10, 11] and DPR [12] and the catalyzing activity of dihydroneopterin triphosphate to sepiapterin or biopterin [8] have been found in erythrocytes of the adult rat, most of which are at levels far higher than those observed in humans. Thus, it is surmised that rat erythrocytes may synthesize biopterin, although the metabolism and the physiological role of biopterin in the blood of mammals are still unknown. In our previous study [10], we found that SPR is observed only in erythrocytes, not in leukocytes while DFR is detected in both erythrocytes and leukocytes in all the animal species tested. The activity of DFR in erythrocytes of the adult rat is quite low [10], however, this enzyme is expected to play a role in the biopterin metabolism in erythrocytes at some period during its development. In this study, we observed the developmental appearances of SPR and DFR, which are in- 216 S. KATOH and T. SUEOKA (a) GTP —— Dihydroneopterin —— —— Sepiapterin triphosphate | (b) Dihydrobiopterin-— (a) GTP cyclohydrolase (b) Sepiapterin reductase | (c) (c) Dihydrofolate reductase Tetrahydrobiopterin (f) (d) PAN OMS reauetase (d) | (le) (e) Aromatic amino acid hydroxylase Quinonoid-dihydrobiopterin— (f) non-enzymatic reaction Fic. 1. volved in the last two steps of the biopterin syn- thetic pathway, in erythrocytes of rats during fetal and neonatal stages. We also examined the occurrence of reduced pteridine and the activity of DPR in fetal blood cells at 20 days of gestation. MATERIALS AND METHODS Reagents and Animals Phenylhydrazine was purchased from Wako Pure Chemical Industries. NADH (grade IID), NADPH (Type I) and ferri-cytochrome c (Type VI) were from Sigma. Tetrahydro-6-methylpterin (A grade) was from Calbiochem. Dihydrofolate was prepared by chemical reduction of folic acid [13]. Sepiapterin was prepared from adult flies of the sepia mutant of Drosophila melanogaster [14]. Quinonoid-dihydro-6-methylpterin was pre- pared from tetrahydro-6-methylpterin by sponta- neous oxidation with ferri-cytochrome c [15]. Sprague-Dawley rats were used at stages rang- ing from 20 days of gestation to adult. The appearance of sperm in the vaginal cavity was examined after overnight mating of rats and the observation of semen was taken as the first day (1 day) of gestation. Preparation of reticulocytes Reticulocytosis was induced in male rats (250 g) by two injections of phenylhydrazine (2 mg/100 g in 0.2 ml of saline, injected sbcutaneously 48 hr apart). The animals were killed and blood fluids were collected 24 hr after the second injection [16]. Biosynthetic pathway of tetrahydrobiopterin. The total red cell count of the collected blood consisted more than 90° reticulocytes determined by staining with methylene blue. Collection of blood cells Fetal and newborn animals were decapitated, and young and adult animals were killed by cer- vical fracture. Blood was removed with 8.25 units of heparin per ml of whole blood. Blood cells were collected by centrifugation (400 X g, 10 min) and washed with 0.85% NaCl three times. Erythrocytes (or reticulocytes) of young and adult animals were obtained after removing the upper buffy coat from the cell precipitate of the first centrifugation of whole blood. Preparation of blood cells and tissue fractions for assays Fractions of blood cells were prepared as mixed homogenates from more than three fetuses or neonates. SPR and DFR in blood cells were extracted by homogenizing with 9 vol of 1mM potassium phosphate buffer, pH 7.5 by an NK- micronizer (Japan Precise Instrument Co.) (20,000 rev., 2min). The supernatant was obtained by centrifugation at 13,000 g for 30min. DPR in blood cells and enzymes in other tissues were ex- tracted with 3 vol of 0.15 M KCI by homogenizing (20,000 rev., 2min). For the determination of reduced pteridine, blood cells were added to 3 vol of distilled water and homogenized (25,000 rev., 2 min). The homogenate was boiled for 2 min and was centrifuged at 13,000xg for 30min after being chilled on ice. Pteridine Metabolism in Fetal Rat Blood 217 Assay of enzyme and reduced pteridine SPR and DFR: Activities were determined by methods described previously [10]. SPR was assayed in 100mM _ potassium phosphate buffer, DH 6.4, 50 uM sepiapterin, 0.1 mM NADPH, and enzyme solution in a final vol of 2m]. DFR was assayed in a reaction mixture containing 100 mM Tris-HCl buffer, pH 7.5, 504M _ dihydrofolate, 6.7 mM mercaptoethanol, 0.1mM NADPH and enzyme solution in a final vol of 2ml. A control for each assay was prepared by omitting NADPH for SPR assay, dihydrofolate or enzyme solution for DFR assay. DPR: The activity was determined by measur- ing the increase in absorbance at 550 nm of the reduced ferri-cytochrome c in a system containing 50mM_ Tris-HCl buffer, pH 7.5, 50M _ ferri- cytochrome c, 1M _ tetrahydro-6-methylpterin, 50 #~M NADH and enzyme solution in a final vol of 2 ml at 25°C [15]. A control for each experi- ment was prepared by omitting pteridine and enzyme solution. Reduced pteridine: The amount of reduced pteridine was determined by measuring the activity of the pteridine cofactor in the phenylalanine hydroxylase system [3]. The reaction mixture contained 100 mM Tris-HCI buffer, pH 7.3, 2 mM TABLE 1. L-phenylalanine, 10 mM dithiothreitol, 57 mUnits of phenylalanine hydroxylase (1 unit=1 pmol tyrosine formed from phenylalanine per min at 25°C with 0.1 mM tetrahydro-6-methylpterin), and an extract of blood cells in a final vol of 1 ml [17]. The eluate obtained from running an extract of fresh liver from 8 week-old male rats (3 vol of 0.15 M KCl) through a Sephadex G-—25 (fine) column was added as phenylalanine hydroxylase (no enzyme activity was detected in blood cells of the fetal rat or in erythrocytes of the adult rat described below). The amount of reduced pteri- dine in the extract of blood cells was assayed in the reaction mixture by incubation at 25°C for 20 min using authentic tetrahydro-6-methylpterin as a standard, and tyrosine formed was measured by the colorimetric method with a-nitroso-f-naphtol at 450 nm [18]. Protein determination Protein was determined by the method of Lowry et al. [19]. RESULTS Activities of sepiapterin reductase and dihydrofolate reductase in blood cells of fetal and neonatal rats Activities of sepiapterin reductase, dihydrofolate reductase and dihydropteridine reductase, and the amount of reduced pteridine in fetal blood cells Activity (per hr/ml cell pack) Samples Reduced pteridine (a)/(b)/(c) Sepiapterin Dihydrofolate A/B Dihydropteridine (per ml cell pack) reductase (A) reductase (B) reductase 6-MPH, equivalents pmol pmol 4Asso nmol Fetal blood cells (10)/(6)/(S) 8.044+2.25 5.334+2.48 ef | (44)/(22-33)/(13) 93.9+8.4 (1)/(69)/(3) 33.22 (1)/(36)/(3) 45.23 Adult erythrocytes (4)/(3)3 11.52+2.49 0.095+0.043 121 115.8+.9.9 (4)/(3)2 9.20+2.84 0.080+0 115 (6)/(3)3 24.82+1.36 Fetuses at 20 days of gestation and adults at 56 days after birth were used for the experiments. Enzymes and reduced pteridine were measured at 25°C by the methods described in the Materials and Methods. 6—-MPH,: tetrahydro-6-methylpterin. (c) Total number of mothers. The amount of reduced pteridine in fetal blood cells are shown as a mean value of 2 assays. (a) Number of test, (b) Number of animals pooled per test, 218 S. KATou and T. SUEOKA Fetal blood cells at 20 days of gestation showed a level of 80°% activity of SPR of adult erythrocytes (Table 1). On the other hand, they showed about a 60-fold higher level of DFR activity at this stage than that in erythrocytes from adults. The ratio between the activities of SPR and DFR in fetal blood cells (1.51) was far smaller than the value in erythrocytes of adult (115). A closing between the levels of SPR and DFR in the fetal blood cells is also seen in other tissues such as liver (0.47) [10], spleen (2.34) [10] and bone marrow (0.54) [20]. The developmental profile of the activities of SPR and DFR is shown in Figure 2 in blood cells of fetuses and neonates from 20 days of gestation to 7 days after birth, and in erythrocytes of young 2.9 2.0 Activity (umoi/h/ml extract) O05 20 21 22 i birth 2 3 Fic. 2. means are shown. The numbers of experiments are shown in parentheses. and adult rats at 28 and 56 days, respectively. DFR activity at 20 days of gestation decreased to about one-half of its level just before birth and this level was maintained during the newborn stage. DFR activity in the erythrocytes of young rats was as low as that observed in erythrocytes of adult rats. SPR activity increased 50% by birth and this level was also maintained during the new- born stage. SPR activity in erythrocytes of young rats gradually decreased until the adult stage. In blood cells of fetuses and neonates, and in erythro- cytes of young and adults, DFR was always present at lower levels than SPR whereas a reverse relation between the two enzyme levels was observed previously in rat liver development [21]. erythrocytes r4 28 56 AGE OF RAT, DAYS Activities of sepiapterin reductase and dihydrofolate reductase in blood cells of fetuses, neonates and in erythrocytes of young (28 days) and adult (56 days) rats. determined at 25°C as described in Materials and Methods. Activities were Means and standard error of the Each experiment with fetuses or neonates represents 3 to 6 pooled total blood cells from an animal. The values at 28 days were obtained by 4 experiments and the values at 56 days were by 4 male and 4 female experiments using 3 pooled erythrocytes from an animal. Sepiapterin reductase: open circle, dihydrofolate reductase: solid circle. Pteridine Metabolism in Fetal Rat Blood 219 Amount of reduced pteridine and the activity of dihydropteridine reductase in fetal blood cells The amount of reduced pteridine in fetal blood cells at 20 days of gestation was assayed using the phenylalanine hydroxylation system as the activity of the pteridine cofactor [3, 17] (Table 1). Cofactor activity was expressed as an equivalent amount of tetrahydro-6-methylpterin per ml of cell pack. Reduced pteridine in fetal blood cells was present in similar (or slightly higher) amounts as that of adult erythrocytes. However, both levels of reduced pteridine in fetal blood cells and erythrocytes of adults were too low to compare them with each other using this experimental method. The amount of reduced pteridine in adult rat liver was determined as 166.56-+0.01 nmol per g of wet weight from 9 male rats by the same method used in the blood cell assay. No phenylalanine hydroxylase activity was found in the extracts of either fetal blood cells or adult erythrocytes. Erythrocytes of adult rats contained two types of DPR, NADH-specific enzyme and NADPH- specific enzyme, upon examination by double immunodiffusion assay using antisera against these enzymes [22] courtesy of Dr. N. Nakanishi of our laboratory. In the extract of fetal blood cells, DPR was detected with 80% activity of that in adult erythrocytes using NADH as a coenzyme in the presence of tetrahydro-6-methylpterin (Table 1). As DPR activity was quite low, blood cells were extracted with smaller amounts of solution than used for SPR and DFR as described in the Materials and Methods. Also the DPR assay was performed immediately after the preparation of the enzyme solution since hemoglobin in the preparation precipitated easily by standing at 0°C. The two types of DPR in fetal blood cells were not examined in this study. From these results, rat blood cells (perhaps red cells) of early stage animals including fetuses and neonates seemed to have an active system of tetrahydrobiopterin biosynthesis. Activities of dihydrofolate reductase and sepiapterin reductase in reticulocytes Reticulocytosis was induced in the adult rat by treatment with phenylhydrazine. The activity of DFR measured about 20-fold higher in reticulo- cytes than in normal erythrocytes of adult rats (Table 2). DER activities in liver and spleen of animals treated with phenylhydrazine were scarcely different from the activities in tissues from un- treated animals. SPR activities, on the other hand, were similar in reticulocytes and erythrocytes, or in tissues of treated and untreated animals (Table 2). This shows that immature erythrocytes have TABLE 2. Effect of phenylhydrazine treatment on the activities of dihydrofolate reductase and sepiapterin reductase in red blood cells and tissues of adult rats Activity (“mol/hr/ml extract) Samples Phenylhydrazine ———---- —--— ——— treatment’ Dihydrofolate Sepiapterin reductase reductase Red blood cells (90% reticulocytes) + 0.330 2.94 (erythrocytes) _ 0.015 yd Liver + 11.40 5.71 - 9.00 5.37 Spleen 1.86 2.18 1.23 2.88 phenylhydrazine (+) or saline (—). The treatment was performed as described in Materials and Methods with injection of An equivalent weight of red blood cells or tissue from three adult rats (250g, male Sprague-Dawley) was collected and pooled in each treatment. The enzymes were measured at 37°C as described in Materials and Methods. 220 S. KATOH and T. SUEOKA high levels of both SPR and DFR activity. DISCUSSION In our recent study on the pteridine cofactor of the phenylalanine hydroxylation system of the fetal rat liver, we found that reduced pteridine, DPR, SPR and DFR occurred in the fetal liver at 20 days of gestation [21] when the activity of phenylalanine hydroxylase in the fetal liver was negligible [23]. These components involved in the formation of reduced biopterin were also detected in this study, in fetal rat blood cells at 20 days of gestation. Moreover, even at 18 days of gestation, reduced biopterin and GTP cyclohydrolase were found in fetal rat brain [24]. These observations show that the formation of reduced biopterin in the various tissues of rat begins at least 3-5 days before birth. Blood cells of the fetal rat during the 2nd half of the gestation stage originate from liver, spleen and bone marrow [25]. In our recent experiment on the liver of a fetal rat at 20 days of gestation [21], activity levels of DPR, DFR and SPR were 50, 50 and 80% respectively, of those found in livers of adult rats, while the amount of reduced pteridine was 0.6%. The values of these were converted to 1023 (4Asso), 7.90 (umol) and 5.98 (mol) per hr at 25°C per g of wet weight and 24.0 nmol tetrahydro-6-methylpterin equivalents per g of wet weight (this value was obtained in this study by the same method as in the blood cells), respectively. These values were all similar to the levels found in blood cells of fetal rats at the same stage of gesta- tion except for the level of DPR acitivity (Table 1). DFR activity in the fraction of fetal blood cells was far higher than the activity expected from the activity and the content of leukocytes in adult rat blood [10]. Immature leukocytes observed in the circulating blood of myelogenous leukemic rats [10] or in human patients with acute or chronic myelo- genous leukemia [26] do not show SPR activity. Thus, most of the activities of both SPR and DFR found in blood cells of fetal or neonatal rats seem to originate in the red cells. Therefore, we sug- gest that the formation of biopterin is more active during early development in red cells such as erythroblasts, reticulocytes or erythrocytes of fetuses and neonates than in the erythrocytes of the circulating blood of adult rats, and that biopterin formed in the cell remains inside thereafter. REFERENCES 1 Kaufman,S., Holtzman, N. A., Miéilstien, S., Butler, I.J. and Krumholz, A. (1975) Phe- nylketonuria due to a deficiency of dihydropteridine reductase. New England J. Med., 293: 785-790. 2 Rey, F., Harpey, J. P., Leeming, R. J., Aicardi, J. and Rey, J. (1977) Les hyperphenylalanemies avec active normal de la _ phenylalanine hydroxylase: Le deficit en tetrahydrobiopterin et le deficit en dihydropteridine reductase. Arch. Fr. Pediatr., 34: 109-120. 3 Kaufman, S. (1977) The phenylalanine hydro- xylating system from mammalian liver. In ‘““Advances in Enzymology”. Ed. by A. Meister, Vol. 35, Interscience, New York, pp. 245-319. 4 Fukushima, K., Eto, I., Saliba, D. and Shiota, T. (1975) The enzymatic synthesis of Crithidia active substance(s) and a _ phospholylated D- erythro-neopterin from GTP or GDP by liver preparations from Syrian golden hamsters. Bio- chem. Biophys. Res. Commun., 65: 644-651. 5 Etoh,I., Fukushima, K. and Shiota, T. (1976) Enzymatic synthesis of biopterin from D-erythro- dihydroneopterin triphosphate by extracts of kidneys from Syrian golden hamsters. J. Biol. Chem., 251: 6505-6512. 6 Matsubara, M., Katoh, S., Akino, M. Kaufman, S. (1966) Sepiapterin reductase. chim. Biophys. Acta, 122: 201-212. 7 Kapatos, G., Katoh, S. and Kaufman, S. (1982) Biosynthesis of biopterin by rat brain. J. Neurochem., 39: 1152-1162. 8 Yoshioka, S., Masada, M., Yoshida, T., Inoue, K.., Mizokami, T. and Akino, M. (1983) Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues. Biochim. Biophys. Acta, 756: 279-285. 9 Baker, H., Frank, O., Bacchi, C. J. and Hutner, S. H. (1974) Biopterin content of human and rat fluids and tissues determined protozoologically. Amer. J. Clin. Nutr., 27: 1247-1253. 10 Katoh, S., Arai, Y., Taketani, T. and Yamada, S. (1974) Sepiapterin reductase in blood of various animals and of leukemic rats. Biochim. Biophys. Acta, 370: 378-388. 11 Sueoka, T. and Katoh, S. (1982) Purification and characteristics of sepiapterin reductase from rat erythrocytes. Biochim. Biophys. Acta, 717: 265-— Dil 12 Katoh, S., Sueoka, T., Masuda, I., and Bio- Yamada, S. 13 14 15 16 17 18 19 20 Pteridine Metabolism in Fetal Rat Blood and Tawa, T. (1981) Studies on the pteridine metabolism of Tawa sarcoma (3) Occurrence of the enzyme and cofactor activities of phenylalanine hydroxylation system. Jpn. J. Oral Biol., 23: 419-426. Futterman, S. (1963) Preparation and properties of dihydrofolic acid. In ‘‘Methods in En- zymology”. Ed. by S.P.Colewick and N. D. Kaplan, Vol. 6, Academic Press, New York, pp. 801-802. Sugiura, K., Takikawa, S., Tsusue, M., Goto, M. (1973) Isolation and characterization of a yellow pteridine from Drosophila mel mutant se. Bull. Chem. Soc. Jpn., 46: 3312-3313. Hasegawa, H. (1977) Dihydropteridine reductase from bovine liver. J. Biochem., 81: 167-177. Kwiatkowska, J. (1973) Rabbit reticulocyte phosphofructokinase purification and some pro- perties. Biochim. Biophys. Acta, 321: 475-483. Bublitz, C. (1969) A direct assay for liver phe- nylalanine hydroxylase. Biochim. Biophys. Acta, 191: 249-256. Udenfriend, S. and Cooper, J.R. (1952) The chemical estimation of tyrosine and tyramine. J. Biol. Chem., 196: 227-233. 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: 265-275. Katoh, S., Sueoka, T., Takikita, U. and Yamada, 23 24 Ps) 26 221 S. (1978) Studies on the metabolism of pteridines in Tawa sarcome (2). Jpn. J. Oral Biol., 20: 571-580. Katoh, S., Sueoka, T. (1983) Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver. Int. J. Biochem., 15: 57-64. Nakanishi, N., Hasegawa, H. and _ Akino, M. (1979) Recent studies on NADPH-dihydro- pteridine reductase from bovine liver. In “Chemistry and Biology of Pteridines’”’. Ed. by R. L. Kisliuk and G. M. Brown, Elsevier North- Holland, New York, Amsterdam, Oxford, pp. 195-200. Murthy, L. 1., Berry, H. K. (1974) Development of phenylalanine hydroxylase activity in rat kidney. Arch. Biochem. Biophys., 163: 225—230. Kapatos, G., Kaufman, S., Weller, J. and Klein, D. (1983) The development of tetrahydro- biopterin and guanosine-5’-triphosphate cyclo- hydrolase: differential patterns in rat brain and pineal gland. Brain Res., 258: 351-355. Metcalf, D. and Moore, M.A.S. (1971) Haemopoietic cells. In “Frontiers of Biology’. Ed. by D. Metcalf, Vol. 24, Elsevier North- Holland, Amsterdam, London. Katoh, S., Sueoka, T., Hasegawa, H., Ueda, U., Takikita, U. and Yamada, S. (1975) Occurrence of sepiapterin reductase activity in the blood from rat and human leukemia. Bull. Josai Dent. Univ., 4: 49-55. _ < ; ik ~ ) j i" ’ ( f 1 ) , 4 ie be 3 Phin erties, | ie ae 2 Ty AS eer er: Porc i : lie Ae) i er wt aN te a ad ELFE. : Py P = aide. nif ve F i 4 t i ¢ oes iat “ + { I 4 i 4 13) a z uF ‘ as . = - - 's Niwa Tees Oe ma ZOOLOGICAL SCIENCE 1: 223-228 (1984) Hormonal Regulation of Biosynthesis of Major Plasma Proteins in Bombyx mori SUSUMU IZUMI, KENJI KIGUCHI' and SHIRO TOMINO? Department of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158, Japan and * Sericultural Experimental Station, Yatabe, Ibaraki 305, Japan ABSTRACT — A group of structurally related proteins, termed “30K Proteins’’, accumulates in the hemolymph of the last instar larvae of the silkworm, Bombyx mori. Hormonal regulation of biosynthesis of these plasma proteins was studied by means of allatectomy and in vitro culture of fat body. When corpora allata were exterpated from the day-l-old fourth instar larvae, 30K protein components appeared in the hemolymph 2 days after operation. The rate of the 30K protein synthesis in the fat body of the allatectomized larvae reached maximum 3 days after operation then declined. On the other hand, the fat body from the sham-operated larvae synthesized only a trace of 30K proteins throughout the fourth larval instar. Larvae that had been al- latectomized at the end of the fourth instar period normally molted. The induction of the 30K protein synthesis was also observed in the fat body of the allatectomized last instar larvae 2 days after feeding. From these results it is suggested that the biosynthesis of 30K proteins in the fat body is selectively induced by the disappearance of juvenile hormone from hemolymph under high nutritional condition. © 1984 Zoological Society of Japan INTRODUCTION In the silkworm, Bombyx mori, a group of structurally related proteins with molecular weights about 30K daltons comprise the major plasma proteins of the last instar larvae [1]. These proteins, referred to as “30K Proteins’, are particularly suitable as a model system for study- ing the developmental regulation of gene expres- sion in the holometabolous insects. 30K proteins are secreted from the fat body cells in a time dependent fashion and accumulate in the hemo- lymph of the late last instar larvae and of pupae [2]. Several components consisting of 30K pro- teins were purified to homogeneity and _ their structural and immunochemical characteristics have also been documented [2]. Furthermore, transla.ion of the fat body mRNA in a heterologous cell-free system revealed that the synthesis of 30K Accepted August 24, 1983 Received July 11, 1983 * To whom correspondence should be addressed. proteins is developmentally regulated at the pre- translational level in the fat body; i.e. the level of functional mRNA coding for 30K proteins is quite low in the fat body at the beginning of the fifth instar, which abruptly rises on day 3 followed by a massive secretion of 30K proteins in the hemolymph [2]. Since 30K proteins are the major biochemical characters specific for the metamorphic devel- opmental stage, it would be expected that both juvenile and molting hormones may participate in the regulation of synthesis of these plasma proteins in the fat body. Thus, the present experiments have been undertaken to study the effect of juvenile hormone on the synthesis of 30K proteins. Corpora allata, the source of juvenile hormone, were extirpated from the penultimate fourth instar larvae and the rate of 30K protein synthesis in the fat body of the allatectomized larvae was compared with that of the control larvae. In this communication, we present evidence that decline of juvenile hormone concentration in the 224 S. Izumi et al. hemolymph induces the synthesis of 30K protein in the fat body. Possible mechanism of the devel- opmental regulation of plasma protein synthesis in the B. mori silkworm is also discussed. MATERIALS AND METHODS Insect The F, hybrid strain of the Bombyx mori resulting from a cross between N. 124 and C. 124 was used. Larvae were reared on mulberry leaves at 25°C in a 16L: 8D photoperiod. Chemicals and culture medium L-[°°S]-Methionine (specific activity, 400 Ci/m mole) was obtained from New England Nuclear Co. Grace’s insect medium was purchased from Gibco Ltd. Other chemicals used were obtained from commercial sources. Allatectomy The female penultimate fourth instar larvae 24 hr after the third molt or shortly before the fourth molt were anesthetized with diethyl ether and corpora allata were extirpated with the sharpened forceps from the integumental space of the neck region. Sham-operation as control was performed as described above without extirpation of any tissues. Collection of hemolymph Hemolymph was collected from larvae by in- juring the abdominal legs. Hemolymph was bled from the wound into liquid nitrogen and stored as frozen beads at —80°C until use. Preparation of antibody Component 4 of 30K protein was purified from the larval hemolymph as described previously [2]. Antibody against Component 4 was prepared by subcutaneous injection at the back of a rabbit of Component 4 emulsified with Freund complete adjuvant as described previously [2]. Anti-Com- ponent 4 IgG was separated from serum by am- monium sulfate precipitation at 40% saturation and dialyzed against Tris-buffered saline (TBS; 20mM Tris-HCl containing 150mM NaCl, pH 7.5) (3, 4}. Culture of fat body in vitro Fat bodies were dissected out from larvae into the sterilized Grace’s insect medium and rinsed three times with the same medium. Approxi- mately each 200 vg of fat body was cultured in 50 wl of Grace’s insect medium containing 25 uCi [°°S]-methionine for defined time period in an Eppendorf centrifuge tube at 25°C under an atmosphere of 90% O, and 10% COQOz. Assay of protein synthesis At the end of culture time 1 ml of TBS was added to each culture and the mixture was homogenized. An aliquot (10 wl) of the homogenate was assayed for the acid-insoluble radioactivity as described previously [2]. The rest of the homogenate was centrifuged at 30,000xg for 5min. The pellet was dissolved in | ml of hot 1 N NaOH and protein concentration was determined by the method of Lowry [5]. The supernatant was mixed with 50 wl of anti-Component 4 IgG and incubated for 20 min at 37°C. Carrier Component 4 (20 yg) was then added to the mixture and incubation was continued for 30 min at 37°C and in the cold for 15 hr. Immunoprecipitate was collected by cen- trifugation, washed three times with TBS and dissolved in 100 pl of a buffer containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and 25 mM Tris-HCl, pH 6.8 by heating in boiling water for 2 min [6]. The radioactivity in the solution was determined by scintillation counting [2]. The rate of protein synthesis is expressed by the amount of [°’S]-methionine (cpm) incorporated/ mg TBS insoluble protein/hr. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in SDS was performed according to the method of Laemmli [6] using 10% acrylamide slab gel as described previously [2]. After electrophoresis, proteins were stained with Coomassie blue [7]. RESULTS Characterization of plasma proteins in the al- latectomized larvae Plasma Protein Synthesis in B. mori To study the effect of juvenile hormone on the expression of plasma proteins in Bombyx mori, change in the protein composition of the hemo- lymph of the allatectomized larvae was followed during development. Female penultimate fourth instar larvae were allatectomized 24 hr after the third molt. After allatectomy, hemolymph sam- ples were collected at 24 hr intervals and proteins were analyzed by SDS polyacrylamide gel electro- phoresis. As seen in Figure 1, the components of 30K proteins, which are barely detectable in the sham-operated controls (Fig. 1B) or in the normal fifth instar larvae [8], appear in the hemo- lymph of the allatectomized larvae at 2 days after operation and their concentration progressively Fic. 1. SDS polyacrylamide gel electrophoresis of plasma proteins of the allatectomized and the sham- operated larvae. Allatectomy or sham-operation was performed on the female fourth instar larvae 24 hr after the third molt as described in ‘‘Methods”. Hem- olymph samples from the allatectomized (A) and the sham-operated (B) larvae were collected at 24 hr intervals after operation. Each hemolymph sample was diluted five fold with saline and 10 yl of the diluted sample was electrophoresed in the Laemmli’s SDS gel system and stained for protein with Coomassie blue. Numbers under _ the electrophoreogram represent days after operation. 225 increases during development. By _ contrast, storage proteins, the other class of major plasma proteins (SP-1 and SP-2 in the figure), largely disappear from hemolymph 7 days after al- latectomy, since they are selectively taken up into fat body cells [9, 10]. Under our experimental conditions, the allatectomized larvae transformed to the precocious pupae 7 days after the operation. Subunit of vitellogenin (VITL-H, ref. 4) is dis- cernible in the hemolymph of the female precocious pupae. In general, developmental change in plasma proteins of the allatectomized fourth instar larvae is analogous to that observed in the normal fifth instar larvae [2]. Effect of allatectomy on protein synthesis in the fat body The rate of the 30K protein synthesis in the fat body of the allatectomized larvae was examined. Fat bodies were dissected from larvae at intervals after allatectomy. Each fat body preparation was cultured in a medium containing [°*S]-methionine and the rate of 30K protein synthesis was deter- mined by use of a specific antibody prepared against a 30K protein component (Component 4). Marked difference in the rate of 30K protein as well as total protein synthesis is observed between the allatectomized and control larvae as early as 24 hr after operation. The rate of 30K protein synthesis in the fat body of the allatectomized larvae increases and reaches maximum at 72 hr after operation and declines, while the sham- operated larvae synthesize only trace of 30K pro- teins throughout the fourth larval instar (Fig. 2). The results of experiments so far obtained strongly suggest that in the normal fourth instar larvae of B. mori, biosynthesis of 30K proteins in the fat body is suppressed by juvenile hormone in the hemolymph. Effect of allatectomy on the synthesis of 30K pro- teins in the last instar larvae During the normal development of B. mori, the level of mRNA coding for 30K proteins sharply rises in the fat body on day 3 of the fifth instar [2], at which time the hemolymph concentration of juvenile hormone is assumed to decline below threshold [11]. Accepting the notion that juve- 226 30 20 x10 cpm/mg/hr 5 6 days after operation Fic. 2. Developmental change in the rate of protein synthesis in the fat body. Allatectomy or sham-operation was performed as described in “‘Methods’” and under Fig. 1. Fat bodies were dissected from the allatectomized (O——O) or from the sham-operated (@——@) fourth instar larvae at 24 hr intervals after oper- ation. For each determination, fat bodies from three larvae were combined and incubated in a medium containing [°°S]-methionine as described in “Methods”. The rate of total protein synthesis (A) and that of 30K protein synthesis (B) were determined as described in the text. Each point represents determination on the combined fat bodies from three animals. nile hormone is solely responsible for suppressing the synthesis of 30K protein-mRNA, it was ex- pected that allatectomy of larvae any time before day 3 of the fifth instar should always bring about an elevation of the hemolymph concentration of 30K proteins. Thus, larvae were allatectomized shortly before the fourth molt and change in plasma protein pattern was followed during the last larval instar. Both the allatectomized and the sham-operated larvae normally molted at around 24 hr after the operation but the duration S. Izumi et al. of the fifth instar of the allatectomized individuals was shorter by 2 days than that of the sham- operated controls. As shown in Figure 3, the developmental change of plasma protein pattern of the allatecto- mized larvae is similar to that of the sham-op- erated controls, though increase in the concen- tration of 30K proteins is prominent in the hemolymph of the allatectomized larvae on day 3 of the fifth instar. However, in vitro culture of fat body clearly demonstrated that the synthesis of 30K protein in the allatectomized larvae is significantly stimulated after 2 days of operation (Fig. 4). The rate of 30K protein synthesis progressively increases, being maximal on the fourth day after allatectomy then declines. As expected, the developmental change of 30K pro- eianaaoae Ante ini ipl pie Sesh Corre seciesstianan ne iaccocesstancatl lc eccnsomsnasttttemecesuasantads Fic. 3. SDS polyacrylamide gel electrophoresis of © plasma proteins of the allatectomized and the sham-operated last instar larvae. Allatectomy or sham-operation was performed on the larvae shortly before the fourth molt. Hemolymph samples from the allatectomized (A) and the sham-operated (B) larvae were analyzed by SDS polyacrylamide gel electrophoresis as des- cribed under Fig. 1. Numbers under the elec- trophoreogram represent days after the fourth molt. Plasma Protein Synthesis in B. mori 227 tein synthesis in the allatectomized last instar larvae is analogous, though with some delay, to that observed with the allatectomized fourth instar larvae (Fig. 3). The result confirms that the induction of 30K protein synthesis in the fat body takes place in response to the disappearance of juvenile hormone from hemolymph. DISCUSSION The amount of the major plasma proteins (30K proteins) is negligible in the larval hemolymph of Bombyx mori during development until day 3 of the fifth instar [2]. When corpora allata are extirpated from larvae at the early fourth instar, the rate of 30K protein synthesis rises precociously in the fat body. It has been established from the experiment with the tobacco hornworm, Manduca sexta, that allatectomy results in the total disap- pearance of juvenile hormone from hemolymph within 24hr [11]. It seems, therefore, that the decrease of the hemolymph concentration of juvenile hormone below certain level provokes the synthesis of 30K protein-mRNA in the fat body of the allatectomized larvae. The low level of 30K protein in the larval hemolymph before the fifth instar could also be accounted for by assuming that the synthesis of these plasma proteins are suppressed by a high hemolymph concentration of juvenile hormone. Developmental change in the rate of 30K pro- tein synthesis in the allatectomized fourth instar larvae is analogous to, but somewhat different from, that observed in the normal fifth instar larvae. After allatectomy at the early fourth instar, the rate of synthesis of 30K proteins in- creases, being maximal on day 3 then declines. Thereafter, the activity stays at low level through- out development. During development of the normal fifth instar larvae, however, the second rise in MRNA activity for 30K proteins is observed at around a period of spinning [2]. This could possibly be brought about by the rise in the hemo- lymph concentration of ecdysteriod, since it has been known that the ecdysteroid titer of the hemo- lymph rises to the maximum at a time of spinning [12, 13]. Also, in our preliminary experiment, 20- hydroxyecdysone markedly stimulated the syn- thesis of 30K proteins in the cultured fat body [14]. We speculate that allatectomy at the early fourth instar not only clears juvenile hormone from hemolymph but may also advance the onset of ecdysone secretion from the prothoracic glands, resulting in the activation of 30K protein gene(s) by two processes in close succession in the fat body of the allatectomized larvae. This may be also true for the allatectomized last instar larvae (Fig. 4). Ww —5 x10 “cpm/mg/hr N ook 2 oo eS OO 7 days after 4th molt Fic. 4. Developmental change in the rate of the 30K protein synthesis in the fat body of the last instar larvae. Allatectomy or sham-operation was performed on the larvae shortly before the fourth molt. Protein syntheses in the fat body preparations were determined as described in ‘“‘Methods” and under Fig. 2. O—O: Rate of 30K protein synthesis in the allatectomized larvae. @——@®: Rate of 30K protein synthesis in the sham-operated larvae. Each point represents determination on the combined fat bodies from three animals. Considering from these observations, it is with little doubt that change in the hormonal environ- ment is a necessary requisite for regulation of 30K protein synthesis in the fat body cells of the B. However, some factor(s) in addition to the endocrine alterations must be taken into This has been indicated by the Figure 4. When the larvae were allatectomized at the late fourth instar period, the enhancement of 30K protein synthesis in the fat body is seen only after feeding. The mori larvae. consideration. experiment shown in result of our preliminary experiment showed that starvation of larvae after the fourth molt almost totally inhibits the biosynthesis of major plasma 228 S. Izumt et al. proteins (Izumi, unpublished observation). Tojo et al. [10] also reported the inhibitory effect of starvation on the biosynthesis of storage proteins in the B. mori larvae. Taking these facts together, it is suggested that the decrease of the hemolymph titer of juvenile hormone below the critical level high nutritional condition triggers the induction of 30K protein synthesis in the fat body. We do not have any evidence at present to explain the mechanism by which the level of 30K protein-mRNA in the fat body declines after full induction. This could be resulted from the selective cessation of transcription of gene(s) for 30K proteins or the inactivation (destruction) of gene(s) itself followed by degradation of the cytoplasmic mRNA. Studies on the mechanism of hormonal regulation of 30K protein gene transcription are in progress using an in vitro system with a cloned 30K protein DNA. un ler REFERENCES 1 Izumi, S., Fujie, J. and Tomino, S. (1979) Trans- lation of mRNA for major hemolymph protein in the silkworm, Bombyx mori. Zool. Mag., 88: 565. 2 Izumi, S., Fujie, J.. Yamada, S. and Tomino, S. (1981) Molecular properties and biosynthesis of major plasma proteins in Bombyx mori. Biochim. Biophys. Acta, 670: 222-229. 3 Osawa, S. and Tomino, S. (1977) Regulation by androgen of mRNA level for the major urinary protein complex in mouse liver. Biochem. Biophys. Res. Commun., 77: 628-633. 4 Izumi,S., Tomino,S. and Chino, H. (1980) Purification and molecular properties of vitellin from the silkworm, Bombyx mori. Insect Biochem., 10: 199-208. 5 Lowry, O. H., Rosebrough, N. J., Fan, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. 6 Laemmli, U.K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T,. Nature (London), 227: 680- 685. 7 Burgess, R.R. (1969) Separation and _ chara- cterization of the subunits of ribonucleic acid polymerase. J. Biol. Chem., 244: 6168-6176. 8 Mine, E., Izumi, S., Katsuki, M. and Tomino, S. (1983) Developmental and _ sex-dependent re- gulation of storage protein synthesis in the silkworm, Bombyx mori. Develop. Biol., 97: 329-337. 9 Tojo, S., Nagata, M. and Kobayashi, M. (1980) Storage protein in the silkworm, Bombyx mori. Insect Biochem., 10: 289-303. 10 Tojo, S., Kiguchi, K. and Kimura,S. (1981) Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm, Bombyx mori. Insect Biochem., 27: 491—497. 11 Fain, M. F. and Riddiford, L. M. (1975) Juvenile hormone titers in the hemolymph during late larval development of the tobacco hornworm, Manduca sexta (L). Biol. Bull., 149: 506-521. 12 Shaaya, E. and Karlson, P. (1965) Der Ecdyson- titer wahrend der Insektenentwicklung-IV. Die Entwicklung der Lepidopteran Bombyx mori L und Cerura vinula L. Develop. Biol., 11: 424-432. 13. Kiguchi, K. and Agui, N. (1981) Ecdysteroid levels and developmental events during the last larval instar in the silkworm, Bombyx mori. Abstract of Annual Meeting of Japanese Society of Sericultural Science. 14 Izumi, S., Tomino,S. and Kiguchi, K. (1980) Hormonal regulation of the biosynthesis of major hemolymph proteins in Bomyx mori. Zool. Mag., 89: 544. ZOOLOGICAL SCIENCE 1: 229-235 (1984) © 1984 Zoological Society of Japan Partial Biochemical Characterization of Humoral Factors Involved in the Nonfusion Reaction of a Botryllid Ascidian, Botrylloides simodensis YASUNORI SAITO and HIROSHI WATANABE Shimoda Marine Research Center, University of Tsukuba, 5—10-1 Shimoda, Shizuoka 415, Japan ABSTRACT — By means of an in vivo bioassay technique based on the micro-injection of blood plasma, it has been demonstrated that the blood plasma of Botrylloides simodensis has a specific activity corresponding to the colony specificity or allogeneic histoincompatibility. When intact blood plasma is exposed to various physico-chemical conditions, the specific activity is easily replaced by a non- specific activity. The specific and nonspecific activities have some characteristics in common, both being resistant to dialysis and heat-labile and dependent on bivalent cations. that both activities are attributable to the same factor in the blood plasma. These facts suggest Furthermore, the nonspecific activity appears in large molecule fractions either by ammonium sulfate fractionation or by Sephadex G75 gel filtration. INTRODUCTION In some compound ascidians, a phenomenon analogous to transplantation specificity has been known as “colony specificity’ [1-7]. Colony specificity manifests itself among others as fu- sion or nonfusion between colonies and is gov- erned genetically by multiple alleles at one locus. Two colonies having at least one allele in common are fusible, while two colonies having no allele in common are nonfusible [8-11]. Mukai [12] has reported that in Botryllus primigenus blood components of a colony are responsible for its fusibility. Later, Tanaka [13] has suggested in the same species that the al- logeneic rejection or nonfusion reaction (NFR) is induced by some humoral factor or factors in the blood. Recently, Taneda and Watanabe [14] have developed an in vivo bioassay technique which demonstrates that an NFR-inducing factor is Accepted September 29, 1983 Received June 30, 1983 Contributions from the Shimoda Marine Research Center, No. 422. The activity is not affected by trypsin, protease or neuraminidase. contained in the blood plasma of B. primigenus. This factor should be isolated and _ purified. Unfortunately, however, the colony of this spe- cies is so thin that it is difficult to collect a suf- ficient volume of blood plasma for use in bio- chemical approach. Botrylloides simodensis also has colony spe- cificity, and a remarkable NFR appears when two nonfusible colonies are brought into contact with each other by their artificial cut surfaces [4]. Moreover, the colony of this species is thicker than that of B. primigenus, facilitating the col- lection of blood plasma. Therefore, in this study we used B. simodensis and attempted par- tial characterization of the presumed NFR- inducing factor. MATERIALS AND METHODS Animals used A colonial ascidian Botryl- loides simodensis Saito and Watanabe [15] was used. In a colony, the individual blastozooids are grouped into a ladder system, and are con- nected with one another by a ramifying network of vascular vessels which terminate in ampullae 230 Y. Saito and H. WATANABE at the periphery of the colony. Many colonies collected at a rocky shore near the Shimoda Marine Research Center, the University of Tsukuba, were fastened to microscopical glass slides or glass plates (8cmx11lcm) by means of the method of Oka and Usui [16]. Then, they were cultured in cages immersed in seawater in the Nabeta Bay near the Center where the environment was perfectly natural. After cultur- ing for about two weeks, several colonies were selected and their fusibility was tested by ordi- nary fusion experiments. Preparation of blood plasma A colony was stripped off from the glass plate. After removing the debris and hydrozoans adhering to the colony surface, the colony was washed with filtrated sea- water (FSW). The seawater remaining on the colony surface and in the branchial sacs of the zooids was removed with filter paper. Then, the colony was cut into stripes about 3-5 mm wide and blood drops exuding through the cut surfaces of the stripes were collected in the cold (about 4°C). The collected blood was centrifuged at 12000 rpm at 4°C for 20 min to remove cellular debris. The resultant clear cell-free supernatant was used as intact blood plasma. The blood plasma thus prepared usually contained 2500— 3500 g/ml protein estimated with Lowry’s meth- od. The blood plasma was stored at 4°C and used within three days after preparation. Heat treatment In order to examine the heat stability of the NFR-inducing activity, blood plasma was heated in a water bath set at a desired temperature (45°, 50°, 55°, 56° or 60°C) for 30 min and then additionally at 100°C for 3min. Samples of heated blood plasma were used for bioassay. EDTA treatment This treatment was car- ried Out to examine the effects of bivalent cations on the NFR-inducing activity of blood plasma. Blood plasma was divided into five samples. One was stored at 4°C until use for assay; the Others were dialyzed at 4°C for 12hr against Tris-HCl buffer solution (TBS, 0.05 M Tris-HCl in 0.4M NaCl, pH 7.9) containing 10 mM ethyl- endiaminetetraacetic acid disodium salt (EDTA). After the EDTA treatment, three of the four samples were again dialyzed at 4°C for 12 hr; one against TBS with 10mM MgCl, another against TBS with 10mM CaCl, and the last against TBS with 10mM MgCl, and 10mM CaCl,. These five samples were used for bio- assay. Ammonium sulfate fractionation Four frac- tions were obtained from 1.5 ml blood plasma by salt fractionation with ammonium sulfate. The four fractions, Fl, F2, F3 and F4, were precipi- tated by ammonium sulfate of 0-20%, 20-40%, 40-60% and 60-100% saturation, respectively. Then, each fraction was dissolved in 0.5 ml FSW. This fractionation was carried out at 4°C. Gel filtration Sephadex G75 gel filtration was performed at 4°C on a column (43 cm~x 1.3cm ID) equilibrated with TBS. Two milli- liters of blood plasma was applied, and about 1.7 ml eluate volumes were collected. The opti- cal density of each tube was read at 280 nm for protein absorption. On the basis of the elution profile by scanning at 280nm, the tubes were divided into three groups and concentration by negative pressure dialysis was carried out for each group. The three samples thus concen- trated were used for assay. Enzymatic treatment Three enzymes, tryp- sin (Merck), protease (Type I, Sigma), and neur- aminidase (Type V, Sigma), were employed. So- lutions of respective enzymes at a concentration of 500 g/ml in FSW were prepared just before use. The following four samples were used as a set: intact blood plasma stored at 4°C (control); a mixture of 0.5ml blood plasma and 0.1 ml FSW; a mixture of 0.5 ml blood plasma and 0.1 ml enzyme solution; and a mixture of 0.5 ml FSW and 0.1 ml enzyme solution. For pro- teolytic enzymes, prior to injection, the latter three samples were incubated for 12 hr at the optimum temperature of the respective enzymes (35.5°C for trypsin and 37°C for protease). For neuraminidase, similarly the latter three samples were first dialyzed against 0.1 M acetate buffer solution, pH 5.0, for 12 hr at 4°C, incubated for 12 hr at 37°C and finally dialyzed against FSW Humoral Factors in Ascidian Nonfusion 231 for 12 hr at 4°C. Assay for NFR-inducing activity The in vivo bioassay system developed by Taneda and Watanabe [14] was employed. For each sample, about 7 wl was injected into each recipient through an ampulla with a micropipette (approximately 50 #m in tip diameter). Recipient colonies were about 5mm*xS5mm in size, each consisting of about 10 zooids. After the injection, the re- cipients were kept in the laboratory aquarium with continuously renewed running seawater. Four hours after injection, they were observed under a binocular stereomicroscope to assess the NFR-inducing activity of the injected samples. In the reactions of recipient colonies, the following four types were distinguished: (—): No harmful effect was induced except the ampulla was injured by injection. (+): Slightly harmful effects, such as weak contraction of ampullae and slight increase of Opacity in ampullae, were induced. (4+): Contraction of ampullae and increase of opacity in ampullae were distinctly recognized. (++): Withdrawal of ampullae from the fringe of the colony, amputation of vessels, disintegra- tion of ampullae, and/or degeneration of zooids were observed. In the text, the latter three types are referred to as NFR-like responses, or simply as NFRs. RESULTS Injection of blood plamsa Generally, in- jection of blood plasma of a fusible colony did not produce any harmful effect on the recipient. On the other hand, injection of blood plasma derived from a nonfusible colony induced an NFR-like response in the recipient. Both the proportion of recipients showing the NFR-like response and the intensity of the response gradu- ally increased until about 4 hr after injection. This is the reason why the assessment of the NFR-inducing activity was done 4 hr after injec- tion. The results of injection of freshly collected blood plasma in three different combinations of colonies TABLE |. Effects of injection of intact blood plasma Number of recipients Injected blood Total plasma t ++ — Auto BP” QO 3 24 70 97 F-Allo BP” CR al as MS 6 35 NF-Allo BP*? 50.70 wii 6 140 a) Blood plasma derived from syngeneic colonies. b) Blood plasma derived from fusible allogeneic colonies. c) Blood plasma derived from nonfusible allogeneic colonies. are shown in Table 1. When blood plasma de- rived either from a fusible syngeneic colony (Auto BP) or from a fusible allogeneic colony (F-Allo BP) was injected, most of the recipients showed no response. On the other hand, when blood plasma derived from a nonfusible allogeneic colony (NF-Allo BP) was injected, NFR-like responses were induced in the recipients. These results clearly show that blood plasma contains a factor or factors responsible for colony specificity. When intact blood plasma was frozen, or left at room temperature for a day, or stored at 4°C for more than three days after preparation, the above-mentioned colony specific activity became nonspecific. In other words, the blood plasma thus treated induced NFR-like responses not only in nonfusible colonies but also in originally fusible colonies. Therefore, until otherwise de- scribed, in the following experiments blood plasma stored at 4°C for less than three days was used. Effect of dialysis The pH range of blood plasma was from 7.6 to 7.8, slightly lower than that of seawater (pH 8.0-8.1). After dialysis of blood plasma against FSW for 12hr at 4°C, the NEFR-inducing activity was preserved as before. Injection of FSW alone produced no harmful effect. The nonspecific activity in long- stored blood plasma was also not affected by dialysis. Effect of heat treatment When _ blood plasma was heated, its transparency was re- duced due to the sedimentation of some blood 232 Y. Saito and H. WATANABE plasma components. The turbidity increased with elevation of temperature, and many particles were found in the sediment of blood plasma heated at 60°C or 100°C. These turbid samples of blood plasma were used for the injection. a= [oj + SSS ih Fic. 1. itioctine anne ns effect of heat treat- ment on the NFR-inducing activity of blood plasma in B. simodensis. The first histogram shows the activity of intact blood plasma stored at 4°C (control). Blood plasma was heated for 30 min at all temperatures except 100°C, where it was heated for 3 min. Recipient colonies used here were nonfusible to the colonies from which blood plasma was derived. As shown in Figurel, the NFR-inducing activity of blood plasma was nearly fully retained up to 50°C. However, the activity began to reduce distinctly at 55°C and was mostly lost by heating at 60°C for 30min. When long- stored blood plasma was heated, its nonspecific NFR-inducing activity was reduced in a similar fashion. Effect of EDTA treatment As shown in Table 2, the NFR-inducing activity of blood plasma was mostly removed by dialysis against TBS with 10mM EDTA. When the EDTA treated blood plasma was secondarily dialyzed against TBS containing both 10mM MgCl, and 10mM CaCl, the activity recovered to the original level. On the other hand, when the secondary dialysis was made against TBS con- taining neither Ca** nor Mg’*, the blood plasma failed to recover the activity (data not shown). The secondary dialysis against TBS with 10 mM TABLE 2. Effects of EDTA, calcium, and magnesium on the NFR-inducing activity Number of recipients” Injection —___—_+_—_—— Total # + = Intact blood plasma BZ 7 3 42 (control) Blood plasma 0 7 3i4/ 44 with 10 mM EDTA Blood plasma 9 13 2 44 with 10 mM MgCl, Blood plasma 21 13 13 47 with 10 mM CaCl, Blood plasma 30 12 5 47 with 10 mM MgCl, and 10 mM CaCl, a) Recipient colonies were nonfusible to the colonies from which blood plasma was derived. CaCl, was much more) effective in restoring the activity than against TBS with MgCl. The non- specific NFR-inducing activity of long-stored blood plasma was also dependent on bivalent cations. TABLE 3. NEFR-inducing activity of fractions obtained by ammonium sulfate fractionation Number of recipients”? Injection Total a Ste aa ela Fl 1 -9367ae 1 39 (0-20 % saturation) F2 15: 2k) 0 Gale (20-40 % saturation) F3 0 29410. . 2.4 (40-60 % saturation) F4 © »-.2 Ulee2Saat (60-100 % saturation) a) Recipient colonies were nonfusible to the colonies from which blood plasma was derived. Ammonium sulfate fractionation The NFR- inducing activity was found in three fractions, F1, F2 and F3, with the highest activity in F2 (Ta- ble 3). Importantly, however, the activity of these fractions had turned nonspecific. That is, these fractions induced NFR-like responses even in originally fusible colonies (not shown in Table 3). Humoral Factors in Ascidian Nonfusion kt G1i—i— G 2+ G3 —— 100 OD 280 NFR-INDUCING ACTIVITY FRACTION NUMBER Fic. 2. Elution profile by scanning at 280 nm of the B. simodensis blood plasma eluted from Sephadex G75 column. The first peak was eluted with void volume. The fractions were divided into three groups (Gl, G2 and G3) corresponding to the observed three peaks. Histograms show the NFR- inducing activities of these groups against non- fusible colonies. Trypsin treatment 100 % F - Recipient ee Ae Protease treatment 233 Gel filtration Elution profile by scanning at 280nm of the blood plasma eluted from Sephadex G75 column showed two large peaks and a small one (Fig. 2). The fractions obtained from this column were divided into three groups (Gl, G2 and G3) corresponding to these three peaks. Then, the NFR-inducing activity in each peak was examined. The activity was found only in G1, although it was again nonspecific. Effect of enzymatic treatment When blood plasma alone was incubated at an optimum tem- perature for enzyme activity (35.5°C or 37°C), the blood plasma induced nonspecifically NFR- like responses in the recipient colonies. Specific activity completely disappeared from the blood plasma within 2hr of incubation. In parallel with this change, filamentary sediment appeared in the blood plasma. Therefore, we could study Neuraminidase treatment Seer EJ + 1 FT E all AE mY ye fh Mi =A Samplea b c d a bed a bea plasma in B. simodensis. In all treatments (A, B and C), sample a is an intact blood plasma stored at 4°C; sample b is a blood plasma passed through the same procedure as enzymatic treatment but without enzyme; sample c is a blood plasma treated with enzyme; and sample d is an enzyme solution. The upper row shows the results when fusible colonies were used as recipients, and the lower row shows the results when nonfusible colonies were used as recipients. Tryspin treat- ment was performed for 12 hr at 35.5°C at pH 7.6~-7.8, protease treatment was performed for 12 hr at 37°C at pH 7.6~7.8, and neuraminidase treatment was performed for 12 hr at 37°C at pH 5.0. 234 Y. Saito and H. WATANABE the effects of enzymes only on this nonspecific activity. The trypsin solution did not induce any harmful effect in the recipients. Trypsin treatment did not reduce the nonspecific activity of blood plasma, which contained filamentary sediment (Fig. 3A). In another series of experiments, the incubated turbid samples were centrifuged at 12000 rpm for 20 min to remove the sediment, and the super- natants were injected into the recipients. They retained the same activity as before. When the protease solution was injected singly, a weak response very similar to the NFR oc- curred in the recipients. Protease did not reduce the nonspecific activity of blood plasma (Fig. 3B). The neuraminidase solution did not bring any harmful effect to the recipients. Neuraminidase treatment did not reduce the nonspecific NFR- inducing activity of blood plasma (Fig. 3C). Next, we prepared the supernatant of each sam- ple by removing the filamentary sediment through centrifugation, and tested its activity. The ac- tivity was unchanged. Further, when neurami- nidase treatment was carried out at pH 7.6-7.8 in a way similar to protease treatment, the non- specific activity of blood plasma was not affected. DISCUSSION This is the first approach to the biochemical characterization of humoral factors responsible for the colony specificity in ascidians. Blood plasma from a colony of Botrylloides simodensis induces, when injected, an NER-like response in a nonfusible colony; while it induces no response in a fusible colony. Thus, the blood plasma of this species has an activity correspond- ing to colony specificity. This specific activity is retained after dialysis of blood plasma against FSW, but is mostly lost by heating at temper- atures higher than 55°C. Further, the activity is dependent on bivalent cations, such as Ca?* and Mg’", especially the former. The specific activity in the blood plasma easily turns nonspecific by various gentle treatments, e.g., long-term storage at 4°C, freezing and thaw- ing, incubation at a moderate temperature, and other physico-chemical treatments. The blood plasma thus treated invariably induces NFR-like responses in fusible as well as nonfusible colonies. The above-mentioned nonspecific activity is rather stable, remaining unchanged for more than one year in the cold. Like the specific activity, the nonspecific activity is resistant against dialysis, heat-labile, and dependent on bivalent cations. Furthermore, the nonspecific activity is found in large molecule fractions ei- ther by ammonium sulfate fractionation or by Sephadex G75 gel filtration. The activity is not affected by trypsin, protease or neuraminidase. At present, it is not known whether the observed specific and nonspecific activities are carried by the same factor or by different factors in the blood pla- sma. The fact that both activities share some char- acteristics, e.g., stability to dialysis, lability to heat- ing, and dependency on bivalent cations, suggests that the former alternative is more probable. If this is the case, the factor might be a conjugate protein. To clarify these points, however, further study is needed. In some ascidians, naturally occurring hemag- glutinins have been reported; and such hemag- glutinins have often been considered to function as humoral recognition factors [17-19]. In fu- ture, our hypothetical factor may be related in some way or other to these hemagglutinins. However, little information is currently available. ACKNOWLEDGMENTS We are grateful to Professor Hideo Mukai of Gunma University for his helpful suggestion and critical reading of the manuscript and to the members of the Shimoda Marine Research Center for their kind assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and from Yamada _ Science Foundation to H. Watanabe. REFERENCES 1 Bancroft, F. W. (1903) Variation and fusion of colonies in compound ascidians. Proc. Calif. Acad. Sci., Ser. 3: 137-186. 2 Oka, H. and Watanabe, H. (1957) Colony- specificity in compound ascidians as tested by fusion experiments (A preliminary report). Proc. Japan Acad., 33: 657-659. 10 1] 12 Humoral Factors in Ascidian Nonfusion Karakashian, S. and Milkman, R. (1967) Colony fusion compatibility types in Borryllus schlosseri. Biol. Bull., 133: 473. Mukai, H. and Watanabe, H. (1974) On the occurrence of colony specificity in some compound ascidians. Biol. 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Tokyo Bunrika Daigaku, Sec. B, 7: 23-53. Fuke, M. T. and Sugai, T. (1972) Studies on the naturally occurring hemagglutinin in the coelomic fluid of ascidian. Biol. Bull., 143: 140-149. Anderson, R.S. and Good, R. A. (1975) Natu- rally-occurring hemagglutinin in a_ tunicate, Halocynthia pyriformis. Biol. Bull., 148: 357-359. Goombe; D. R.; . Schiuter, S..F.; Ey, P.L. and Jenkin C. R. (1982) Identification of the HA-2 agglutinin in the haemolymph of the ascidian Botrylloides leachii as the factor promoting adhesion of sheep erythrocytes to mouse macro- phages. Develop. Comp. Immunol., 6: 65-74. ' j a i ‘ te! ~ ~ i es i 5 ZOOLOGICAL SCIENCE 1: 237-244 (1984) Initiation of Sperm Motility in Ciona intestinalis by Calcium and Cyclic AMP MASAAKI MorRISAWA, SACHIKO MoRISAWA! and ROSARIA Dg SANTIS? Ocean Research Institute, University of Tokyo, 1-15—I Minamidai, Nakano, Tokyo 164, Japan, * Biological Laboratory, St. Marianna University, School of Medicine, 2905 Sugao, Miyamae, Kawasaki 213, Japan and *Stazione Zoologica di Napoli, Villa Comunale, 80121 Napoli, Italy ABSTRACT — The motility of Ciona spermatozoa, which had been prevented by calcium chelating agent EGTA (0.125-1.0 mM), was initiated by the subsequent addition of calcium (0.5-1.0 mM). In addition, when high concentrations of EGTA (up to 32 mM) were added to other sperm suspensions in which spermatozoa were already moving vigorously, the motility continued. These findings suggest that while calcium is not indispensable to maintain sperm motility, it is an effective external factor in the initiation of sperm motility. Spermatozoa demembranated with Triton X—100 were motile in the reactivating medium in which cAMP presumably synthesized from ATP in the medium by the contamination of endogenous adenylate cyclase: This was thought to be associated with incompletely removed or solubilized sperm membrane residue. On the contrary, when demembranated sperm were suspended in re- activating medium containing phosphodiesterase in which the auto-synthesized cAMP would be converted back to 5’-AMP, axonemes were completely quiescent even in the presence of an energy source, MgATP*-. Subsequent addition of a considerable amount of cAMP to the pho- sphodiesterase-immotile sperm induced axonemal movement instantly, suggesting that exposure of © 1984 Zoological Society of Japan axonemes to cAMP may be indispensable for the initiation of sperm motility in Ciona. INTRODUCTION Studies to clarify the initiation mechanism of sperm motility have been carried out for half a century and many factors have been suggested as external signals for the initiation of sperm motility in many animals [1]. Recent studies by the authors and their colleagues focussing on mono- valent cations and cAMP, resolved a sequential reaction which occurs during the initiation of sperm motility in natural spawning in salmonid fishes [2-8]. These studies showed that sperm motility in rainbow trout and chum salmon is suppressed by seminal potassium in the sperm duct and that decrease of potassium around sperm spawned in fresh water induces cAMP synthesis in the cells, Accepted September 29, 1983 Received July 20, 1983 followed by conversion of the axoneme from an immotile state to a motile one by cAMP, resulting in initiation of sperm motility. Changes of ex- ternal sodium have also been suggested as a sperm motility initiation factor in sea urchin [9] and rat [10] and regulation by cAMP-dependent protein kinase has been proposed as the main participant in the intracellular mechanism of initiation of sperm motility in echinoderms [11]. Concerning divalent cations, while evidence that calcium stimulates sperm motility has accumu- lated [12-14], few reports have suggested the initia- tion of sperm motility by calcium [15]. Since calcium is well known as an inducer of intracellular cAMP through the mediation of adenylate cyclase in the sperm [16-18], calcium and the cAMP system might be involved in the initiation of sperm motility. In order to clarify the role of calcium and cAMP 238 M. Morisawa ef al. in the initation of sperm motility, we developed experimental models in which any calcium con- tamination from seminal plasma would be re- moved by EGTA from sperm suspensions and any cAMP resulting from endogenous adenylate cyclase activity would be converted back to 5’- AMP by phosphodiesterase. It was showed that calcium and cAMP are respectively the external and internal signals indispensable for the initiation of sperm motility in protochordate, Ciona intes- tinalis. MATERIALS AND METHODS Ciona obtained in Naples Bay, Naples, Italy from May to September, 1982 were kept in a seawater tank and used within 3 days. Semen which was extruded from incision of an isolated sperm duct was collected by pipette and kept on ice. One wl of semen was diluted in appropriate volumes of artificial sea water (ASW) consisting of 30 g/l NaCl, 0.8 g/l] KCl, 1.3 g/l CaCl,, 6.6 g/I MgsO,, 0.18 g/l NaHCO; at pH 8.2 or the Ca’*- free ASW (CaCl, was replaced by NaCl) and placed on a glass slide without a cover slide at room temperature. Sperm motility was observed with a microscope at a magnification of x 100 and was evaluated in terms of grade (0-5): Grade 5-— all spermatozoa move vigorously forward; Grade 4— over 75% spermatozoa were motile; Grade 3— 51-75% were motile; Grade 2— 26-50% were motile; Grade 1— 1-25 °%% sperm were motile; Grade 0— all spermatozoa were immotile. The plasma membrane of the sperm was re- moved by mixing on ice 1 volume of semen with 20 volumes of extracting medium containing 0.15 M KCI, 0.5mM MgSO,, 2mM EGTA, 1 mM dithiothreitol (DTT), 2 mM Tris buffer pH 8.0 and 0.04% Triton X-100 for 30 sec. The demem- branated sperm (see Results) were resuspended in reactivating medium containing 0.15 M KCl, 2mM MgsSO,, 0.5 mM CaCl, 2 mM EGTA, 1 mM DTT, 20 mM Tris buffer pH 8.0 in the presence or ab- sence of 0.5 mM MgATP’ , phosphodiesterase or cAMP and were observed by dark-field microscopy with mercury illumination at a magnification of x 100. Movement of intact and demembranated sper- matozoa was recorded by videomicroscopy and percentages of moving sperm and speed of move- ment were measured by tracing the location of sperm heads on transparent sheet according to the method of Katz and Overstreet [19]. Seminal plasma or body fluid was obtained by centrifuging at 10,000 rpm the semen or body fluid extruded from sperm ducts or body surface by cutting the surface with scissors. Sodium, potas- sium, calcium and magnesium concentrations of the seminal plasma, body fluid and sea water of Naples were analyzed by an atomic absorption spectophotometer (Hitachi 180), and chloride con- centration by a Buchler digital chloridemeter. Osmolalities were measured by a melting point Knauer osmometer after thawing the freeze- stocked samples. All chemicals were reagent grade and water was deionized and glass distilled. RESULTS Effect of dilution on sperm motility in ASW in the presence or absence of Ca** As shown in Figure 1, when semen was diluted by ASW at a dilution ratio of 1: 200, Ciona sperm exhibited full motility (grade 5). Spermatozoa maintained full motility in ASW up to dilution ratios of 1: 4,000. Similarly, spermatozoa diluted in Ca**-free ASW at a dilution ratio of 1: 200 also exhibited full motility (grade 5). However, sperm motility decreased to grade 3, grade 2 and Fic. 2. Effects of EGTA and Ca**+ on sperm motility of Ciona intestinalis. Symbols in both a) and b) indicate averages obtained from three experiments at the beginning of May (©) and from three experiments at the end of May and beginning of June (@). a) one yl of semen was diluted with 200 yl of Ca?*-free ASW containing various concentrations of EGTA. b) one ul of semen was diluted with 200 1 Ca?*-free ASW containing 0.125 mM (@) or 1.0mM (0) EGTA and then appropriate volumes of CaCl, were added consecutively to increase Ca?+ concentrations of the sperm suspension and sperm motility for each Ca?* concentration was estimated. Initiation of Sperm Motility in Ciona 239 = 5 ® at Sd D > 3 3 ° 2 = E a4 rt) a ” 0 200 500 1000 2000 4000 Dilution rate Fic. 1. Effect of dilution on sperm motility of Ciona intestinalis. Symbols indicate sperm motility in ASW (@) or Ca’**-free ASW (0). Average of four experiments. (grades) Sperm motility Ww RO —— below grade | with increase of dilution ratios to 1: 500, 1: 1000 and 1: 2000 or 1: 4000 in Ca?’*-free ASW, respectively. Effects of EGTA and Ca** on sperm motility As shown in Figures | and 2, spermatozoa moved vigorously (grade 5) at a dilution ratio of 1: 200 in Ca’**-free ASW. When the semen was suspended in Ca**-free ASW _ containing 0.03—0.06 mM EGTA, the percentage of motile sperm decreased sharply in accordance with the increase of EGTA concentration. Almost all spermatozoa _ were quiescent in medium containing 0.125 mM EGTA but in a few spermatozoa twitches in the anterior part of a flagellum were recognized. Spermatozoa were completely quiescent at EGTA concentra- tions of over 0.125 and up to 1.0mM (Fig. 2a). When 0.06 mM of Ca’** was added to the immotile spermatozoa in Ca’**-free ASW containing 0.125 mM EGTA, some spermatozoa began to move (Fig. 2b). Numbers of motile spermatozoa in- creased with subsequent addition of Ca’** and almost 60°% of sperm became motile at a Ca’** concentration of 0.25mM. AIll spermatozoa be- 0 0.5 1.0 0 EGTA (mM) 0.5 1.0 15 Castitanmw ) 240 M. Morisawa et al. Tracks of RiGs 3: various conditions. Speed 159 + 7.4 198 + 9.2 215+ 10.6 m/sec (n=21) (n=16) (n= oe (n=29) AIG Tracks of sperm heads and shape of quiescent sperm of Ciona intestinalis under Note that motile sperm exhibit straight or curved tracks under all conditions, immotile sperm stand side by side and flagella were rigidly extended. The numbers of sperm for which speed was measured are indicated in parentheses. The dilution ratios of all experiments were 1: 1,000. came fully motile at Ca’* concentrations over 0.5 mM in experiments performed at the beginning of May. In identical experiments at the end of May or beginning of June (Fig. 2b), spermatozoa also moved vigorously in Ca’*-free ASW at a dilution ratio of 1: 200. Sperm motility gradually de- creased in Ca**-free ASW containing EGTA at EGTA concentrations over 0 to 0.5 mM and almost all spermatozoa were quiescent in medium con- taining 1mM EGTA. Subsequent addition of Ca** to the immotile spermatozoa in Ca’*-free ASW containing 1mM EGTA, induced restora- tion of sperm motility: almost 60°% of sperm moved at a Ca’** concentration of 0.5mM and all sperm exhibited full motility at Ca?* concen- trations over 1.0 mM. Tracks and speed of spermatozoa As shown in Figure 3, spermatozoa diluted at a ratio of 1: 1,000 moved in straight or curved lines in ASW at a speed of 159 wm sec’. Spermatozoa in Ca**-free ASW also moved in straight or curved lines but some spermatozoa were immotile. The immotile flagella were rigidly extended. The swimming speed of spermatozoa in Ca?*-free ASW (198 wm sec’) was significantly higher than that in ASW (p<0.01) and almost the same as that in Ca**-free ASW at a dilution ratio of 1: 200 (cf. Fig. 4). Spermatozoa suspended in Ca’*-free ASW containing 1mM EGTA were completely immotile at a dilution ratio of 1: 1,000 as well as 1: 200 (cf. Fig. 2) and the flagella were rigidly extended. Spermatozoa which immotile in the presence of |! mM EGTA became motile by addi- tion of 1mM Ca’*. Spermatozoa moved in straight or curved lines and the speed was 215 wm sec *, showing no significant difference to the speed in Ca**-free ASW. Effect of EGTA on the moving spermatozoa As shown in Figure 4, spermatozoa which were suspended in Ca’*-free ASW at a dilution ratio of 1: 200 exhibited full motility (grade 5) at a speed of 218-+-7.4 um sec * (n=23) (see also Figs. 1 and 2). When EGTA was subsequently added to suspensions of motile spermatozoa, the motility did not abate. All sperm remained motile with significant increase of speed (246+9.2 um sec’, n=21, p<0.05) at an EGTA concentration of 1mM. The speed of spermatozoa (219+6.2 wm sec’', n=30) returned to the level in Ca’*-free ASW without EGTA by subsequent addition of 1mM EGTA (final concentration of 2mM). Grade 5 sperm motility level was maintained through all EGTA concentrations from 4mM to 32mM (214-+-9.3 wm sec’, n=19 in 32mM EGTA). Initiation of Sperm Motility in Ciona 241 260 ° 240 \ E = wv © 220 oa a Ww 200 AN oO 1 2 4 8 16 32 EGTA(mM) Fic. 4. Effect of EGTA on motile spermatozoa of Ciona intestinalis. EGTA at a final concentration of 1 mM was added to motile sperm in Ca**-free ASW (dilution ratio of 1: 200) and recorded. After recording, EGTA was subsequently added to the sperm suspension and sperm motility was recorded at every EGTA concentration indicated in the abscissa. Sperm motility was grade 5 through all EGTA concentrations. Average data from five experiments, vertical bars represent the mean speed+S.E. Effects of phosphodiesterase and cAMP on the motility of demembranated spermatozoa When spermatozoa were treated with Triton X-100 in the extracting medium and then placed in MgATP”’ -deficient reactivating medium, axone- mal movement did not occur (Fig. 5). This sug- gests that removal of the sperm plasma membrane with the detergent caused the dispersal of the energy source for axonemal movement, resulting in immotility of the axoneme [20]. Thus, demem- branated sperm flagellae (axonemes) became motile by the supply of the energy source, MgATP’™ in the reactivating medium (Fig. 5): Axonemal movement did not occur within 30 sec after sus- pension, however, 7°% of axonemes became motile 1 min after suspension. The percentage of motile sperm increased with time; almost 50°% of sperm became motile after 3 min and all spermatozoa became motile with a speed of 116+11.3 um sec™’ 5 min after suspension. The speed of demem- 100 S ~ - 50 £ £ © roy wW Ot com ees | Senn ee a 0.05 1 eta h25 10 Time (min) Fic. 5. Motility of demembranated sperm of Ciona intestinalis in the presence or absence of ATP. Symbols indicate motility in the absence of ATP (@) or in the presence of ATP (O). Vertical bars represent means-+-S.E. of three experiments. 100 1} > ~ i250 ° £ £ © ro Ww +04@—Ohe—o en | Enno Ee ee oe 0. O05 1 2 J 10 15 Time (min) Fic. 6. Effects of phosphodiesterase and cyclic AMP on the motility of demembranated spermatozoa in Ciona intestinalis. Phosphodiesterase was present in the reactivating medium in the absence of cAMP for 10min and then 2mM, 1 mM and 1mM cAMP were added at 10, 11 and 12 min (indicated arrow) respectively in experiments (O, @ and A). Motility in the medium con- taining phosphodiesterase and 4 mM cAMP (()). branated sperm was almost two-thirds that of intact sperm swimming in ASW (cf. Fig. 3). 24? M. Morisawa et al. As shown in Figure 6, when demembranated sperm were suspended in reactivating medium containing MgATP? and 3.0 units/ml of phospho- diesterase from bovine heart (Sigma), spermatozoa were completely quiescent in two studies. In another experiment, 1-2% of sperm exhibited forward movement at 2-7 min and motile sperm increased to 2-4°% at 8-10 min after suspension. When cAMP was added to quiescent sperm 10 min after suspension at a final concentration of 2 mM, 5-80°% of sperm became motile within 1 min after cAMP addition. The percentage of motile sperm increased with further addition of cAMP and 20-90°% of sperm exhibited active movement with a speed of 67+3.8 wm sec’ at a final cAMP con- centration of 4mM. On the other hand, when demembranated sperm were suspended in reac- tivating medium containing 3.0 units/ml of phos- phodiesterase and then 4 mM cAMP was immedi- ately added, 85° of sperm became motile within slSee Bit & Goel OF Opak4l3 win gee, Wine motility was maintained for up to 5 min. Ion concentrations of seminal plasma and body fluid of Ciona As shown in Table 1, Na*, K*, Ca’* and Mg’* concentrations of the habitat sea water in Naples Bay (Mediterranean Sea) were higher than that of sea water around Japan (Kuroshio). Na* concen- tration and osmolality of body fluid from Ciona were almost the same as those of habitat sea water, and K*, Ca** and Mg’* concentrations of body fluid were slightly lower than those in the habitat sea water. In contrast, the Cl” concentration of body fluid was slightly higher. Na* concentra- tion in seminal plasma was almost the same as that of habitat sea water and body fluid. However, K* concentration of seminal plasma was 1.5—1.6 times higher than that of body fluid and sea water. On the contrary, Ca** and Mg** concentrations in seminal plasma were 80-90% of those in the body fluid and the habitat sea water. DISCUSSION There have been few attempts to clarify the relationship between calcium chelating agents and sperm motility. Young and Nelson [12] showed that EGTA suppressed sperm motility and calcium stimulated motility in sea urchin spermatozoa. This suggests that calcium is an important factor for sperm motility. However, they were unable to demonstrate the role of clacitum in the actual initiation of sperm motility because “attempts to restore motility to the immobilized spermatozoa by addition of calcium ions following treatment with EGTA were unsuccessful”’. In the present study, spermatozoa of Ciona exhibited full motility not only in ASW but also in calcium-free ASW at a dilution ratio up to 1: 200. Sperm motility was maintained with further dilu- tion by ASW, whereas increase of the dilution ratio in calcium-free ASW caused a decrease in sperm motility (Fig. 1). The decrease of motility follow- ing the increase of the dilution ratio in calcium- free ASW seems to be due to the decrease in the concentration of calcium which originates from the seminal plasma and surrounds spermatozoa (see Table 1). Furthermore, when spermatozoa were diluted by calcium-free ASW containing the calcium-specific chelating agent EGTA, sperm motility declined concomitantly with the decrease of calcium by increase of EGTA concentrations, finally halting completely at EGTA concentrations TABLE |. lon concentration of body fluid and seminal plasma of Ciona intestinalis Na* K* Catt Mg?* lig Osmolality (mM) (mM) (mM) (mM) (mM) (mosmole/Kg) Sea water *Kuroshio, Japan 458 eS 9.0 53.5 563 = Habitat, Naples 581 12.9 (1a 65.7 544 1140 Body fluid 584 11.9 2 58.1 596 1130 Seminal plasma 570 19.4 8.8 S208 ae ie SSS SSS SSS SSS SSS SSS SS * Quoted from ref. 25. Initiation of Sperm Motility in Ciona 243 of over 0.125 or 10mM. The motility of the quiescent sperm was restored by addition of cal- cium (Figs. 2 and 3), suggesting that calcium is an important factor for sperm motility. In addition, removal of external calcium from the suspension medium of spermatozoa whose motility had already been initiated had no effect on sperm motility (Fig. 4). These two phenomena strongly suggest that external calcium ions may not be necessary tO maintain sperm motility but that calcium ion are indispensable factor for the initia- tion of sperm motility in Ciona. As indicated in Figure 2, 0.125mM of EGTA and 0.5 mM calcium were enough to suppress and restore the sperm motility in Ciona collected at the beginning of May, whereas higher concentrations of EGTA and calcium were need for the same effects at the end of May or the beginning of June. Much higher concentrations of EGTA and calcium were necessary to obtain the same effects during the summer. Seasonal difference of sensitivity to EGTA and calcium may possibly be due to seasonal changes to calcium transport ability through the plasma membrane. Conflicting reports have been published con- cerning the effect of cAMP in the initiation of sperm motility. It has been reported that when the plasma membrane of spermatozoa has_ been removed by Triton X—100, movement is seen in the presence of only MgATP* without cAMP in sea urchin [20], bull [21] and hamster [22]. In contrast, sperm motility occurs only when axone- mes are exposed to cAMP before MgATP’ in salmonid fishes [5—8] and presumably in Ciona [23]. In the present study, when Ciona sperm were demembranated in the extracting medium and the axoneme transferred to reactivating medium con- taining MgATP* axonemal movement occurred as in sea urchin and mammals without any addi- tion of cAMP. However, there was a delay of 30 sec before movement occurred. The number of motile sperm increased and subsequently almost all sperm exhibited full motility (Fig. 5). Phosphodiesterase (PDE) from bovine heart (which rapidly hydrolyses cAMP [24]) was added to the reactivating medium to test whether cAMP is essential for the initiation of sperm motility (Fig. 6). No movement occurred for more than 10 min in the presence of PDE. This would sug- gest that when PDE was not added, cAMP was presumably synthesized from ATP in the reactivat- ing medium by adenylate cyclase in residual membrane material within 30 sec after suspending the demembranated sperm into reactivating me- dium and the auto-synthesized cAMP caused the gradual increase of motile axonemes. It also suggests that when PDE was added, the auto- synthesized cAMP converted to 5’-AMP, thus axOonemes were quiescent. To test the hypothesis that cAMP is need, cAMP was added to axonemes made quiescent by the previous addition of PDE 10 min after suspension. The motility induced suggested that cAMP is an indispensable factor in the initiation of sperm motility in Ciona. In salmonid fishes, decrease of potassium sur- rounding spermatozoa at spawning in fresh water induces intracellular cAMP and the cAMP induces initiation of axonemal movement and sperm motility [2-8]. In Ciona, the potassium concen- tration of the seminal plasma was 1.5 fold higher, while calcium concentration was on the contrary lower than that of the habitat sea water. Thus it is probable that potassium or calcium concen- trations surrounding spawned spermatozoa in sea water decrease or increase respectively. Although the exact effects of potassium as well as other factors such as gas tension etc. [1] on Ciona sperm remain unclear, it is possible that the decrease of potassium or changes in other factors at spawning induce calcium flux through the plasma membrane or that the increase of external calcium surround- ing spawned spermatozoa directly induce calcium influx. Since both calcium and cAMP can induce the initiation of sperm motility, it is probable that in Ciona calcium permeates the cell, induces in- tracellular cAMP synthesis and the endogenous cAMP then induces initiation of axonemal move- ment, i.e. sperm motility. ACKNOWLEDGMENTS The authors wish to thank Professor A. Monroy of the Stazione Zoologica di Napoli for encouragement, Drs. R. Pinto (S.Z.N) and F. Cotelli, Institute of Zoology, University of Mirano for critical advice during experiments, and the staff of the Stazione Zoologica di Napoli, Italy for providing materials. We also 244 wish to thank Associate Professor J. P. Barron, St. Marianna University, School of Medicine for his review of the manuscript. This work was supported by a grant-in-aid from the Japan Society for the promotion of Science to one of the author (M.M.). 10 REFERENCES Morisawa, M., Suzuki, K., Shimizu, H., Mori- sawa,S. and Yasuda, K. 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Borisy, Academic Press, New York, San _ Francisco, London, pp. 151-162. Morisawa, M., Okuno,M. and Morisawa, S. (1983) Direct evidence that cyclic AMP is an indispensable factor for initiation of sperm motility in salmonid fishes. In “The Sperm Cell’’. Ed. by J. André, Martinus Nijhoff publishers, The Nether- lands, pp. 369-371. Morisawa, M., Okuno, M., Suzuki, K. Morisawa, S. and Ishida, K. (1983) Initiation of sperm motility in teleosts. J. Submicrosc. Cytol., 15: 61-65. Nishioka, D. and Cross, N. (1978) The role of external sodium in sea urchin fertilization. In “Cell Reproduction”. Ed. by E.R. Dirksen, D. M. Prescott, and C. F. Fox, Academic Press, New York, San Francisco, London, pp. 403-413. Wong, Po YY" D:, Lee, W. Me and Tsang An Yuk: (1981) The effects of external sodium on acid release and motility initiation in rat caudal epididymal spermatozoa in vitro. Exp. Cell Res., 131: 97-104. Ishiguro, K., Murofushi, H. and Sakai, H. (1982) Evidence that cAMP- dependent protein kinase 13 14 16 Vy 18 19 20 21 Dupe 23 24 U5) M. MorisAwa et al. 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. Young, L.G. and Nelson, L. (1974) Calcium ions and control of the motility of sea urchin spermatozoa. J. Reprod. Fert., 41: 371-378. Morita, Z. and Chung, M. C. (1970) The motility and aerobic metabolism of spermatozoa in laboratory animals with special reference to the effects of cold shock and importance of calcium for the motility of hamster spermatozoa. Biol. Reprod., 3: 169-179. Morton, B., Harrigan-Lum, J., Albagli, L. and Jooss, T. (1974) The activation of motility in quiescent hamster sperm from the epididymis by calcium and_ cyclic nucleotides. Biochem. Biophys. Res. Comm., 56: 372-379. Morisawa, M., Ishida, K. and Suzuki, A. (1983) Roles of K* and Ca?* in initiation of sperm motility in rainbow trout. Develop. 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A study using demembranated sperm models. Exp. Cell Res., 127: 191-196. Brokaw, C. J. (1982) Activation and reactivation of Ciona spermatozoa. Cell Mot., Suppl. 1: 185-189. Butcher, R. W. (1974) Cyclic 3’, 5’-nucleotide phosphodiesterase from bovine heart. In “Methods in Enzymology, XXXVIII’’. Ed. by J. G. Hardman and W. O’Malley, Academic Press, New York, San Francisco, London, pp. 218-223. Ogasawara, T. (1982) Effects of prolactin and environmental calcium on water permeability of the eel gills. Dr. Thesis, Hokkaido University. ZOOLOGICAL SCIENCE 1: 245-254 (1984) Cross-Fertilization between Sea Urchin Eggs and Mussel Spermatozoa: Sperm Entry without Egg Activation KBNZI OSANAI and KBI-ICHIRO KYOZUKA Marine Biological Station, Tohoku University, Asamushi, Aomori 039-34, Japan ABSTRACT — Interphylum crossing was examined between sea urchin eggs (Hemicentrotus pulcherrimus, Strongylocentrotus intermedius and S. nudus) and mussel sperm (Mytilus edulis). Dejellied or denuded sea urchin eggs received to some extent mussel spermatozoa which underwent acrosome reaction by being exposed to excess calcium. The spermatozoa entered into the eggs without inducing visible egg changes and incorporated sperm organelles remained inactive. The “fertilized’’ eggs proceeded to cleavage and embryogenesis after being activated by the treatment with parthenogenetic chemical agents, urea and procaine hydrochloride. Urea-pretreated eggs were easily fertilized by heterologous spermatozoa. The present results suggest that (1) the egg plasma membrane is less species specific concerning sperm reception, (2) sperm-egg fusion does not cause egg activation and (3) organelles derived from mussel spermatozoa can function in sea urchin egg © 1984 Zoological Society of Japan cytoplasm. INTRODUCTION The crossing of sea urchin eggs with sperm obtained from an organism belonging to a dif- ferent phylum is useful to analyze sperm-egg interaction in fertilization process. Previous studies on interphylum crossing between sea urchin eggs and bivalve sperm show that sper- matozoa often penetrate into eggs without in- ducing fertilization envelope formation [1-3]. When Japanese sea urchin eggs were insemi- nated with mussel sperm, the eggs did not show any apparent signs of fertilization and remained unchanged in a light microscopic examination. Thus, we were led to ascertain whether the fail- ure of apparent fertilization was due to real unfertility or to egg inactivity despite sperm entry. In order to detect foreign sperm organelles incorporated in sea urchin eggs we employed following two methods: (1) direct detection of sperm nuclei in fixed specimens with a cytological method and (2) functioning of mussel sperm or- ganelles in sea urchin egg cytoplasm by activating Accepted September 8, 1983 Received August 8, 1983 the eggs with parthenogenetic agents. The present results revealed that mussel spermatozoa can enter into sea urchin eggs without egg activation. MATERIALS AND METHODS Gametes Sea urchins (Hemicentrotus pulcherrimus, Strongylocentrotus and S. and marine mussel (Mytilus edulis) were col- lected around the Marine Biological Station, Tohoku University, Asamushi, Aomori. Sea ur- chin eggs were discharged by injecting 0.5 M potassium chloride into the intracoelomic cavity and used after washing with sea water. Sea urchin sperm were obtained by dissecting testes in a small container. Extruded sperm (dry sperm) were stored in a refrigerator at about 4°C until use. Mussel sperm were obtained from mantles. The pieces of mantles (testes) dissected from the male were exposed to 0.5 M potassium chloride for a few minutes and then incubated in a small amount of sea water [4, 5]. Sperm were extruded to sea water. The sperm suspension was centri- fuged with a hand centrifuge to sediment tissue intermedius nudus) 246 K. OSANAI and K. KYOZUKA fragments. The supernatant containing sperma- tozoa was re-centrifuged at 3000 rpm for 3 min. The precipitate was resuspended in sea water and centrifuged again. The sedimented sperm was stored in a refrigerator. To remove jelly coat unfertilized sea urchin eggs were suspended in acid sea water (pH 5.2- 5.4) for 2 min or in Ca, Mg-free sea water con- taining 1mM _ ethyleneglycol-bis (S-amino ethyl ether) N, N’-tetraacetic acid (EGTA; Sigma Chemical Co.) for 1 min and then washed with fresh sea water. The vitelline layer was dis- rupted by suspending eggs in sea water contain- ing 0.1 mg/ml trypsin (Sigma Chemical Co.) for 2-10 min. To remove vitelline layer and corti- cal granules eggs were incubated in | M urea solution (pH 8, adjusted by the addition of sodi- um bicarbonate) for 10 min [6, 7]. The acrosome reaction of mussel spermatozoa was induced with excess calcium [8]. Diluted mussel sperm was added to sea urchin eggs in sea water containing one tenth volume of M/3 calcium chloride (ex- cess Ca-sea water). Final sperm concentration was 2-5 10° snermatozoa/ml, which was de- termined by counting with a hemocyte counter. Sea Water Natural sea water filtered through filter paper (Toyo Roshi No. 2) and Herbst’s artificial sea water prepared according to Motomura [9] were used. Ca, Mg-free sea water was prepared by substituting NaCl to CaCl, and Na,SO, to Mg SO. [10]. Activation of Inseminated Eggs The urea method [6] was employed to induce egg activation accompanied by cortical granule discharge. Sea urchin eggs were mixed with mussel sperm in excess Ca-sea water for 5 or 10 min and washed with sea water to remove free spermatozoa. The inseminated eggs were exposed to 1M urea for 5-10sec and washed several times with fresh sea water. To activate eggs without inducing cortical reaction the insemi- nated eggs were incubated in sea water containing 10mM procaine hydrochloride (Sigma Chemical Co). Procaine is known as a_ parthenogenetic agent which induces egg activation without corti- cal granules discharge [11, 12]. Microscopy To detect incorporated nuclei, eggs were fixed in Carnoy’s ethanol-acetic acid (3:1) mixture and stained with lactic acetic orcein overnight. After decoloration in 45-70% acetic acid the eggs were somewhat flattened with a cover slip and examined with a Nomarski differential interfer- ence microscope (Olympus BHB-333-N). For electron microscopy, dejellied or trypsin-pre- treated S. intermedius eggs were inseminated with Mytilus sperm in excess Ca-sea water and then fixed in Karnovsky’s fixative containing 8.5% sucrose for 45 min. The fixed specimens were postfixed in osmium tetroxide for 1 hr at 4°C, dehydrated through an ethanol series and embedded in Spurr’s resin. Thin sections were prepared with a Porter-Blum ultramicrotome, stained with uranyl acetate and lead citrate, and examined in a transmission electron microscope (JEM T-8). RESULTS Binding of Mussel Spermatozoa to Sea Urchin Eggs Mussel spermatozoa attached to the jelly coat surrounding sea urchin eggs, but slipped on the surface of the egg (Fig. la). To examine whether the failure of sperm binding was due to the ab- sence of acrosome reaction, mussel sperm were added to dejellied sea urchin eggs in sea water containing excess calcium. Thirty minutes after insemination, the eggs were sedimented with a hand centrifuge and washed with fresh sea water to remove free spermatozoa. Many spermatozoa remained adhered on the egg surface (Fig. 1b). Mussel spermatozoa bound also to the surface of denuded (trypsin-pretreated) sea urchin eggs in excess Ca-sea water. Electron microscopy re- vealed that all of the adhered spermatozoa under- went acrosome reaction and their acrosomal process contacted closely with the vitelline layer or with the protrusions which seemed to be derived from egg surface microvilli (Fig. Ic). Sea Urchin-Mussel Cross Fertilization 247 PRG: 1. inseminated in normal (a) or excess Ca-sea water (b). nation to remove free spermatozoa. micrograph showing a spermatozoon binding to the denuded egg surface. The bar indicates | ym. x 14000. Cytological Detection of Incorporated Sperm Nuclei In spite of the binding of many mussel sper- matozoa, the sea urchin eggs did not show appar- ent signs of fertilization, such as cortical granule discharge, sperm aster formation and cleavage. To examine whether the eggs incorporated sper- matozoa, cytological observation was carried out. The dejellied or denuded H. pulcherrimus eggs Attachment of Mytilus spermatozoa to Hemicentrotus eggs. Phase contrast micrographs. a, b: Dejellied eggs were The eggs were washed 30 min after insemi- x 300. c: An electron 5 min after insemination, were inseminated with M. edulis sperm in excess Ca-sea water. After various intervals the eggs were washed with fresh sea water and reincubated in sea water containing 0.1 mg/ml trypsin for 5 or 10min to remove spermatozoa remaining on the egg surface. After washing, the eggs were fixed and stained. Sperm nuclei were observed in some dejellied eggs (4 in 38 eggs, about 11%). The number of ‘‘fertilized’”> eggs increased to some extent after TABLE 1. Mytilus sperm incorporation in Hemicentrotus eggs Total number Number of sperm nuclei in an egg Sperm Pretreatment of eggs nuclei observed 0 | 2 3 4 6 10 per egg (1) Acid sea water 38 34 4 0.12 pH 5, 2min (2) Trypsin 117 76 33 4 3 l 0.46 0.1 mg/ml, 10 min (3) Urea 10 | 2 | 4 | | 4.00 1 M, 10 min (1) Dejellied eggs were mixed with M. edulis sperm in excess Ca-sea water for 20-30 min and then exposed to 0.1 mg/ml trypsin for 5 min. excess Ca-sea water for 10 min and re-exposed to trypsin for 5 min. Fixed 70—100 min after insemination. (2) Inseminated in Fixed 30 min after insemination. (3) Inseminated in normal sea water and fixed 20 min after insemination. 248 K. OsANAI and K. KYozZUKA the trypsin-pretreatment (35%). The majority of them (33 in 41 eggs) was monospermic (Table 1, Fig. 2a, b). The incorporated sperm nuclei re- mained condense in the egg periphery. This result shows that Mytilus spermatozoa can enter in sea urchin eggs without inducing egg activation. Sperm Entry into Vitelline Layer- and Cortical Granule-free Eggs Urea is known as a reagent which dissolves both the vitelline layer and the cortical granules of sea urchin eggs [6]. In isotonic urea solution the eggs undergo cortical granule exocytosis and the vitelline envelope separated from the egg surface is dissolved [7]. H. pulcherrimus eggs were exposed to 1 M urea for 10 min and then mixed with M. edulis sperm in excess Ca-sea water. The eggs were easily fertilized by heterologous spermatozoa and almost all eggs were polyspermic (Table 1). Incorporated sperm nuclei migrated to the inner part of the egg, where they decondensed to swollen pronuclei (Fig. 2d). Multiple asters developed near the sperm nu- clei. The eggs often underwent abnormal cleav- age. This result indicates that the urea treatment not only disrupts a barrier preventing the pen- Fic. 2. High magnification of a. 1630. : Sperm nuclei incorporated in Hemicentrotus eggs. a,b: Denuded eggs (0.1 mg/ml trypsin, 10 min) inseminated with Mytilus sperm in excess Ca-sea water. Fixed 70 min after insemination. x<340. b: c,d: Urea-pretreated eggs (1 M, 10min) inseminated with H. pulcherrimus sperm (c) or with M. edulis sperm (d) in excess Ca-sea water. 20 min after insemination. x 340. The bars indicate 10 ym. The arrows indicate incorported sperm nuclei. Sea Urchin-Mussel Cross Fertilization 249 TABLE 2. Embryogenesis induced with urea in dejellied Hemicentrotus eggs inseminated with Mytilus sperm Insemination in excess Ca-sea Urea-treatment Total number Developed water observed embryos (1) 20 min 5 sec 461 Ee sa ok (2) 20 7 619 6.5 (3) 20 7 329 12.8 (4) 30 10 541 3.3 (5) 35 5 332 18.1 Average 8.9 Dejellied eggs were inseminated in excess Ca-sea water. After 20-35 min they were washed with sea water and then exposed to | M urea solution for 5-10 sec. Cleavage or blastulation was observed as a criterion of development 2-3.5 hr (1, 5) or 17-25 hr (2, 3, 4) after insemination (7°-15°C). Fic. 3. Activation of Hemicentrotus eggs inseminated with Mytilus sperm by urea treatment. a-c: Dejellied eggs were mixed with M. edulis sperm in Ca-excess sea water for 20 min and then exposed to 1M urea for 5sec. a, b and c are 90 min, 2.5 hr and 17 hr after urea-treatment, respectively. Non-inseminated control. Unfertilized eggs were exposed to 1 M urea for 5 sec and placed in s water for 3 hr. 130. 250 K. OSANAI and K. KyYyozUuKA Fic. 4. Development of Hemicentrotus eggs fertilized with Mytilus sperm. Dejellied eggs were inseminated in excess Ca-sea water and then activated with urea. a: Swimming blastula, 20 hr after insemination. b-d: Early gastrulae, after about 2 days. Mesenchyme-like cells degenerate in the blastocoel. e: Pluteus after about 5 days. X280. TABLE 3. Embryogenesis induced with urea in trypsin-pretreated Hemicentrotus eggs inseminated with Mytilus sperm : Insemination Trypsin- : Total numbers Developed pretreatment 1” ee ape, Urea-treatment observed embryos (1) 10 min 20 min 7 sec 310 14.8% (2) 10 10 10 328 18.6 (3) 4 10 10 Dg 17.5 (4) 2 10 7 508 S75 (5) Z 10 if 33558) 10.5 Average 23.8 Trypsin-treated eggs were inseminated in excess Ca-sea water, exposed to 1 M urea and then placed in normal sea water. Cleavage or blastulation was observed as a criterion of development 3—4.5 hr (2, 3, 4) or 16-18 hr (1, 5) after insemination. TABLE 4. Induction of cleavage with procaine in Strongylocentrotus intermedius eggs inseminated with Mytilus sperm Incubation medium Cleavage (%) Average (%) (1) Sea water 0.4 0.3 0 0 = — — 0.2 (2) Procaine 13.3 14.7 4.9 30.2 Sad) 6.5 6.3 Hite 7/ Trypsin-pretreated eggs (0.1 mg/mi, 10 min) were inseminated in excess Ca-sea water for 10 min. After washin g, they were incubated in normal sea water (1) or sea water containing 10 mM procaine- HCI (2) (12°-15°C). Cleavage was observed 3-4.5 hr after insemination. Sea Urchin-Mussel Cross Fertilization Zo etration of mussel spermatozoa and a polyspermy block mechanism, but also induces parthenoge- netic egg activation, which leads the incorporated sperm organelles to function. Induction of Embryogenesis In order to induce embryogenesis in sea urchin eggs fertilized with mussel spermatozoa without egg activation, we made an attempt to activate the eggs with parthenogenetic chemical agents. Dejellied sea urchin eggs (H. pulcherrimus, S. intermedius and S. nudus) were inseminated with M. edulis sperm in excess Ca-sea water and then exposed to 1 M urea for 5-10 sec. The eggs Fic. 5. elevated the fertilization envelope and formed the monaster. Many of them did not cleave, but they cytolyzed after a night. Some eggs, about 9% of the urea-treated eggs, however, cleaved normal- ly and developed into blastulae (Fig. 3; Table 2). All of unfertilized eggs exposed to urea elevated the fertilization envelope (Fig. 3d), forming the monaster and then cytolized. In the sea urchin- mussel combination the cytolized eggs were con- sidered as unfertilized, while the cloven eggs were considered to be ‘“‘fertilized” by Mytilus sperma- tozoa. The extent of the fertilized eggs was comparable to that obtained in the cytological examination. The derived centrioles from S. intermedius eggs activated with procaine. a, b: Denuded unfertilized eggs were incubated in sea c,d: Denuded eggs were inseminated with M. edulis < 440, water containing 10mM _ procaine for 3.5 hr. sperm in excess Ca-sea water and then incubated in procaine for 2.5 hr. 252 K. OsAnal and K. KYyOZUKA Mytilus spermatozoa seem to become functionable after the activation of the cytoplasm. About 1 day after urea treatment, the “‘ferti- lized” eggs developed into swimming blastulae (Fig. 4a). In many of them the gut was scarcely invaginated and mesenchyme-like cells, various in size, liberated from the blastula wall, degen- erated in the blastocoel (Fig. 4b, c, d). However, some larvae underwent gastrulation and developed into pultei which were similar to normal sea urchin larvae in fundamental skeletal pattern (Fig. 4e). The ‘‘fertilized’’ eggs, which were indicated by embryogenesis after urea-treatment, increased to some extent after the disruption of the vitelline layer with trypsin. However, many of trypsin- treated eggs (about 76%) remained unfertilized. This result indicates that a factor preventing the penetration of heterologous spermatozoa remained still on the egg surface after the trypsin-treatment. Unfertilized sea urchin eggs formed a cytaster in sea water containing procaine, but they did not cleave. The failure of cleavage seems to be due to the defect of centrioles which are nor- mally brought by spermatozoa at fertilization. As sea urchin eggs incorporating a Mytilus sper- matozoon have the centrioles, the eggs are ex- pected to undergo cleavage under the presence of procaine. Trypsin-pretreated S. intermedius eggs were mixed with M. edulis sperm in excess Ca-sea water and then incubated in sea water contain- ing 10mM procaine hydrochloride. Amphiaster formation and cleavage proceeded in some eggs (about 12%) (Fig. 5c, d). As non-inseminated controls did not cleave (Fig. 5a, b), the divided eggs are considered to have been “‘fertilized” by Mytilus spermatozoa. At the two-cell stage the embryos were transferred to normal sea water and reinseminated with S. intermedius sperm. The blastomeres were refertilized, forming the hyaline layer. This finding suggests that the surface of the blastomeres retains a condition similar to unfertilized eggs concerning sperm reception and cortical change. DISCUSSION The present examination shows that Mytilus spermatozoa can penetrate into sea urchin eggs without inducing cortical granule discharge and egg activation. The previous electron micro- scopic observations on cross fertilization be- tween sea urchin eggs and bivalve spermatozoa demonstrated that the spermatozoa fuses with the eggs [2, 3]. This fact suggests that sperm- egg membrane fusion itself does not cause egg activation. The failure of egg activation is also shown in unfertilized sea urchin eggs fused arti- TABLE 5. Interaction between sea urchin eggs and bivalve spermatozoa Acrosome Sperm entry Fertilization reaction on Mono-or Egg Sperm vitelline normal excess envelope polyspermy layer sea water Cart formation Temnopleurus Mytilus —_ — 4- — mono hardwicki edulis Hemicentrotus — — +. — mono pulcherrimus Strongylocentrotus = = + — mono intermedius Strongylocentrotus = = + — mono nudus * Temnopleurus Cassostrea +-

. Sy : «+ - > La ~ &» - «hue — biel teas .* ™ dx Des s r 7 ya. i — > . - “ ve fn, wn 7 , fy - ’ “<4 - i Ve ° — 7 7 . i - ~ ? oy ~ . a > ee 4 "Ny . ; my 4 mF "47, We it ‘@ ~ ‘ _ - -_ & , = _ ras 2 4 260 N. YOSHIZAKI and CH. KATAGIRI reticulated appearance (Fig. 8). Electron micro- graphs of the granules isolated by density gradient centrifugation in Percoll (Fig. 9) revealed that the major constituent of the fraction used for rabbit immunization was the secretory granules found at the bottom of the cells. Behavior of antigen(s) specific to pars recta 2 portion of oviduct Indirect immunofluorescent staining was done on sections of the oviduct using the antiserum against secretory granules which had been isolated from the pars recta portion of the oviduct, as described in Materials and Methods. A specific stain oc- curred in the cells localizing preferentially in the bottom of epithelial folds in the pars recta 2 (Figs. 10 and 11). No specific stain was observed in any other types of cells either in the pars recta or the convoluta. Eggs from various portions of the reproductive tract were stained with this same absorbed anti- serum. The VCs of eggs from oviducal pars recta 2 and the uterus showed a strong fluorescence (Figs. 13 and 14), whereas those of eggs from the coelom and oviducal pars recta 1 did not (Fig. 12), indicating that the antigen(s) specific to the secre- tory granules of the cells in the bottom of oviducal epithelium is deposited on the VC during the passage of eggs through the pars recta 2 of the oviduct. On the other hand, the VC of activated uterine eggs showed no fluorescence at all (Fig. 15), although the F layer is naturally present in these eggs and would contain the material of the PF layer. We presume that in activated uterine eggs the antigenic determinant(s) reactive to the absorbed antiserum may be masked or undergo a conformational change under the influence of the cortical granule exudate. The jelly layer showed no fluorescence in any of the sections. It is highly probable that the fluorescence seen in non-acti- vated uterine eggs corresponds to the location of the PF layer detected by electron microscopic observation, and that the antigenic nature of this PF layer changes during its transition to an F layer upon egg activation. In agar diffusion analyses, the absorbed anti- serum employed in immunofluorescent studies formed a distinct precipitation band with the F- material and the VC of activated uterine eggs, but did not react at all with egg jelly, the VCs of coelomic and uterine eggs, or the cortical granule exudate (Table 1). The positive reaction ex- hibited by the F-material and the VC isolated from activated uterine eggs, as contrasted with their lack of immunofluorescence, may well be explained in terms of the reappearance of antigenic sites after EDTA-treatment. TABLE 1. Reactivity in immunodiffusion of preparations from egg or egg-envelope with antiserum against materials specific to oviducal pars recta 2 portion Preparation”) Reactivity”) VC of coelomic egg as VC of uterine egg 1 VC of activated uterine egg aL F-material ae jelly”? = cortical granule exudate = 1) 220-240 pg proteins/ml 2) 870 ug sugars/ml 3) + and —: presence and absence of a preci- pitin line, respectively. DISCUSSION The formation of the fertilization (F) layer in Xenopus laevis has been attributed to the associa- Fic. 6. idine blue. Fics. 7 and 8. Light micrograph showing sagittal section of anterior portion of oviduct stained with tolu- Note heavily stained cells in pars recta 2 (PR2) which are distinguishable from those unstained in pars recta 1 (PR1) and those weakly stained in pars convoluta (PC). x 290. Electron micrographs of epithelial cells in oviducal pars recta 2 portion, showing electron lucent secretory granules in cells on ciliated ridges (Fig. 7) and secretory granules with reticulated, electron dense core in cells in the bottom of the folds (Fig. 8). x 15,000. Fic. 9. Electron micrograph of the pars recta fraction obtained by the density gradient of Percoll and used for immunizing a rabbit. the bottom of epithelial folds. x 20,000. The fraction is mostly composed of secretory granules of cell in Fertilization Layer Formation in Xenopus 261 262 N. YOSHIZAKI and CH. KATAGIRI a 8 g . ;, ‘ eS - * ele a ‘ ¥ ty oy . J ' Pes ~ Fics. 10 and 11. recta 2 portion of oviduct. recta 2 (PR2) (Fig. 10), and between pars recta 2 and pars convoluta (PC) (Fig. 11). , PC 11 Immunofluorescent micrographs showing distribution of antigen(s) specific to pars Sagittal sections at the junction between pars recta 1 (PR1) and pars Note a preferential localization of antigen in cells in the bottom of epithelial folds in pars recta 2 and a secreted material in the lumen (double arrow). erythrocytes. x 100. tion of cortical granule exudates and the innermost jelly layer (J,) components in the presence of metals [7, 17]. The present study demands re- evaluation of this interpretation by demonstrating that pars recta material distinct from J, indeed preexists as a pre-fertilization (PF) layer on the outer surface of the vitelline coat (VC), and that it participates in the formation of the F layer by association with cortical granule exudates and possibly metals. in support of the preexistence of the PF layer as an entity distinct from J, is: (a) the occurrence in unfertilized eggs of a layer showing a distribution pattern similar to that of the F layer in fertilized eggs (Figs. 2 and 3); (b) the acquisition of a layer during the passage of eggs through the pars recta before they acquire J, (Fig. 4a); (c) the requirement that eggs sojourn in the pars recta for the formation of a mercaptan- Evidence Arrows indicate nonspecific fluorescence of resistant F layer (Fig. 5); (d) the localization in this PF layer of antigen(s) specific to the pars recta 2 portion but not J, (Figs. 10-14), and (e) the occurrence of PF materials in the F layer as evi- denced by immunodiffusion analyses. The present study further proposes that the antigenic nature of the PF layer changes during the transition from PF to F layer. Possible reasons why the PF layer in unfertilized eggs has been overlooked by previous researchers are several. First, the width of the PF layer is variable from egg to egg and is sometimes too thin to be distinguished from the jelly layer. Second, both PF and J, are solubilized by mercaptan treatment, so that J, preparations employed for previous chemical analyses [18] or reactivity studies with cortical granule exudates [17] could have been contaminated by a relatively minor Fertilization Layer Formation in Xenopus 263 Fics. 12-15. velopes of the antigen shown in Figs. 10 and 11. Yolk platelets show nonspecific fluorescence. fraction of PF materials. There is evidence that the PF-layer materials are glycoprotein in nature (unpublished observation). In a previous in vitro study [7], an F-like layer was produced at the site of F layer formation when unfertilized eggs were externally treated with cortical granule exudates (Fig. 1C in ref. 17). This have been duplicated in our laboratory, provided the eggs possessed a PF layer on the surface of the VC. Immunofluorescent studies combined with elec- results tron microscopic observations indicate that the materials of the PF layer are most probably derived from secretory granules in the special types of cells present at the bottom of the epithelium in the posterior one-fifth portion of the oviducal pars recta (pars recta 2). The necessity of the pars recta secretions for the formation of the F layer in Xenopus is unique compared with the situation in other anuran species, in which the transfor- mation of the VC to fertilization coat evidently occurs in coelomic eggs [19, 20]. In anurans such as Rana [8] and Bufo [9], the secretory products from the pars recta have recently been shown to be deposited on the VC as part of the fertilization processes. As discussed J, jelly layer. Immunofluorescent micrographs of sections of eggs from pars recta 1 (Fig. 12), pars recta 2 (Fig. 13), uterus (Fig. 14), and of uterine egg after activation (Fig. 15), showing localization in egg en- Arrows indicate a specific stain in the vitelline coat. x 400. elsewhere [9], the roles played by the molecules from the pars recta may be multiple, including the induction of acrosome reaction [10], sublytic effects on the VC [21], and possibly the enhance- ment of spermatozoan VC lysin as well. In Xenopus, the action of the first 1-cm segment of the reproductive tract has been implicated in the apparent dispersion of the filament bundles of VC during the passage of eggs through the oviduct [16]. oviduct, There is evidence that this portion of the the pars recta 1, contains antigen(s) distinct from the antigen(s) in the pars recta 2 demonstrated in the present study (unpublished observation). Such antigen(s) may indicate the presence of other materials necessary for fertili- zation in Xenopus. Also, the pars recta 1 cor- responds morphologically to the pars recta in other amphibian species. Hence, it is possible that the anterior four-fifths of the pars recta (pars recta 1) in Xenopus secretes materials which possess functions common to those documented in other anuran species. Efforts to analyze the role of the pars recta 1 portion along this line are in progress. 264 i) 10 REFERENCES Katagiri, Ch. 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ZOOLOGICAL SCIENCE 1: 265-271 (1984) Effect of Fibronectin on Cultured Cells Derived from Isolated Micromeres of the Sea Urchin, Hemicentrotus pulcherrimus Yus1 MIyAcui'!, MINEO IWATA?2, Hipgemi SATO! and Eizo NAKANO?:3 "Sugashima Marine Biological Laboratory, Nagoya University, Toba, Mie 517 and * Department of Biology, Nagoya University, Chikusa, Nagoya 464, Japan ABSTRACT — Effect of fibronectin on cultured cells derived from isolated micromeres of sea urchin embryos was examined. Fibronectin isolated from the sea urchin ovary (sea urchin fibronectin) accelerated the scattering of micromere-derived cells at the low concentration (0.01-0.1 mg/ml). The acceleration of the scattering of cultured cells also occurred on the fibronectin-coated surface. How- ever, at higher concentrations in a range of 0.2-0.8 mg/ml, sea urchin fibronectin enhanced the cell- to-cell and cell-to-substratum adhesion of micromere-derived cells. Sea urchin fibronectin also induced the formation of spicules in the serum-deficient culture medium. Similar results were obtained with the application of horse or calf plasma fibronectin but to a lesser extent. The present study suggests that fibronectin may have an important role in migration, adhesion and spicule for- © 1984 Zoological Society of Japan mation of cultured cells derived from isolated micromeres in vitro. INTRODUCTION Fibronectin is one of the major extracellular matrix proteins which is involved in cell-to-cell and cell-to-substratum interactions [1-2]. Fi- bronectin is also known to bind directly to a number of macromolecules, such as collagen, fibrin and glycosaminoglycans [1]. It seems fi- bronectin can mediate a variety of biological activities of cells by specific molecular inter- actions [3-5]. In sea urchins, fibronectin was first isolated from the ovary and characterized [6-7]. Im- munocytochemical studies also revealed that fibronectin is located in the sea urchin embryo at the gastrula stage [7-9]. Although these in situ experiments provided evidence for the dis- tribution of fibronectin, they gave little informa- tion on the functional expression or the molec- Accepted August 13, 1983 Received July 28, 1983 * To whom all correspondence should be addressed. ular involvement of fibronectin for cell adhesion and migration in sea urchin embryos. When the isolated micromeres of sea urchin embryos are cultured in sea water containing horse serum, they differentiate into the primary mesenchyme cells and form spicules in vitro [10— 12]. In sea water, the micromere-derived cells begin to scatter at the stage corresponding to the mesenchyme blastula stage of control em- bryos and turn into a mass of segregated cells [13]. These observations have led to the inves- tigations to determine whether fibronectin is involved in adhesion, migration and differenti- ation of micromere-derived cells. In the present report, we show that sea urchin fibronectin has effects on cell adhesion and mi- gration and also induces the formation of spicules in the serum-deficient culture medium. These data indicate that fibronectin may have a role for the dif- ferentiation of micromeres and primary mesen- chyme cells in the developing sea urchin embryo. 266 Y. MIYACHI et al. MATERIALS AND METHODS Culture and isolation of micromeres Japanese winter sea urchin, Hemicentrotus pulcherrimus, was used for the experiment. Gametes were obtained by intracoelomic in- jections of isotonic KCl. The eggs were ferti- lized in artificial sea water and transferred into Ca-Mg-free sea water. After fertilization, the fertilization membranes were removed by shak- ing and the denuded eggs were kept in Ca-free sea water at 18°C. At the 16-cell stage, the embryos were dissociated into single blastomeres by gentle shaking. The dissociated blastomeres were applied on a column of Ficoll (Pharmacia, Sweden) density gradient for the separation of micromeres. The gradient was made by mixing linearly 3 and 15% Ficoll in Ca-free sea water. This procedure was carried out at 4°C for 1.5-2hr. The micromeres were col- lected, washed twice with Ca-free sea water by gentle centrifugation and were suspended in Millipore filtered artificial sea water (buffered with 10mM EPPS*, pH 8.2) containing strep- tomycin (100 yg/ml), penicillin (100 U/ml) and fibronectin at various concentrations (0—0.8 mg/ml). The suspended cells were transferred into wells of 96-well plate (Coster, USA) and cultured at 18°C. Control embryos were sub- jected to the same culture temperature. In some of the experiments conducted, 4% horse serum (Gibco, not heat-inactivated, USA) was used as the control culture. Purification of fibronectin Fibronectin was purified from ovaries by a gelatin-agarose affinity column, followed by a heparin-agarose affinity column as_ described previously [6]. Fibronectin from the calf plas- ma was prepared according to the method of Engvall and Ruoslahti [14]. The horse plasma fibronectin used was a gift from Dr. M. Hayashi, Tsukuba University. * EPPS: N-2-hydroxyethyl piperazine-N ’-3-propane- sulfonic acid. Scattering Index Degree of scattering of the micromere-derived cells was estimated and quantified by measuring the scattering index according to the method of Sano [13]. The scattering index was determined by S/(A+S), where S is the number of single cells and A is the number of cell aggregates. Each value of the scattering index represented a mean of triplicates. Micrography Cells were observed under an inverted micro- scope with long working distance phase optics (Diaphoto, Nikon, Tokyo) and a polarizing microscope equipped with rectifier optics (Apo- photo, Nikon, Tokyo). Photographic records were made using Kodak Plus X film. RESULTS Figure 1 shows the micromere-derived cells maintained in sea water containing sea urchin fibronectin at different concentrations (0, 0.1, 0.5 and 0.8mg/ml) for 24hr after fertilization. These cells tended to scatter in the low con- centration (0.1 mg/ml) of sea urchin fibronectin, while no sign of scattering of the cells was seen at the high concentration (0.8 mg/ml). Degree of the scattering of micromere-derived cells was qualitatively determined by measuring the scat- tering index (Fig. 2). The maximum and plateau level of the scattering index was reduced as the concentration of sea urchin fibronectin increased. Furthermore, the period when the scattering index began to increase from zero level was delayed after the concentration-dependent manner. The micro- mere-derived cells started to scatter 10, 14, 16 and 20 hr after fertilization when the cells were main- tained in fibronectin-containing sea water at the concentration of 0, 0.2, 0.4 and 0.5 mg/ml, re- spectively. Even after 30hr from fertilization, the micromere-derived cells did not scatter at the high concentration of fibronectin (0.6—0.8 mg/ml). On the other hand, initial increase of scattering was accelerated by sea urchin fibronectin at the low concentration (0.01-0.1 mg/ml) 14hr after fertilization. The enhancement of scattering was Effect of Fibronectin on Micromeres 267 Fic. 1. Micromere-derived cells cultured in sea water containing sea urchin fibronectin. Cells were maintained in sea water (a) or in sea water containing fibronectin at the concentration of 0.1 (b), 0.5 (c) and 0.8 (d) mg/ml for 24 hr after fertilization. Bar indicates 50 um. % 100; (Fn| mg/ml x o - = o> 50- £ s 6 1S) Y) 0 - —— ———— i : = = = E aut 0°10 15 20 25 30 Time after Fertilization Fic. 2. Scattering of micromere-derived cells at different concentrations of sea urchin fibronectin. Isolated micromeres were maintained in sea water or sea water containing fibronectin (0.01— 0.8 mg/ml) for 30 hr after fertilization. The degree of scattering was estimated by the scattering index measured at 2-hr intervals. Open circle: in sea water. Closed circle: in sea water contain ing fibronectin. 268 Y. MIyACcui et al. Fic. 3. Spicules induced by fibronectin at the concentration of 0.5 mg/ml. The isolated micromeres were cultured in sea water containing sea urchin fibronectin (a), horse plasma fibronectin (b) and 4% horse serum (c). Bright contrast (a and c) and polarization microscopy (b). Arrows indicate spicules. Bar indicates 50 ym. 0 hh uy pri ies AGO xe) = = 59 C= ES ra) 0 0 0.2 0.4 0.6 0.8 Fic. 4. Formation of spicules by sea urchin fibronectin at different concentrations. Isolated micromeres were maintained in sea water containing sea urchin fibro- nectin at the concentrations of 0 to 0.8 mg/ml. Formation of spicules was shown by percent of cell aggregates producing spicules vs. total number of cell aggregates. Abscissa: concentration of sea urchin fibronectin (mg/ml). Ordinate: percent of spicule formation. Effect of Fibronectin on Micromeres also observed when the micromere-derived cells were cultured on the fibronectin-coated surface in artificial sea water. Fibronectin pretreated with either 2-mercaptoethanol or heat (100°C, 4 min) had no effect on the behavior of micro- mere-derived cells. When the micromere-derived cells were cultured at the high concentration of sea urchin fibronectin (0.5 mg/ml), the spicule formation was enhanced (Fig. 3a). Some spicules grew to triradiate forms, while others remained as granular or rod-like forms. The latter forms were also observed by mammalian plasma fibronectin isolated from either horse or calf plasma (Fig. 3b). In sea water containing horse serum, the elongation of spicules occurred (Fig. 3c). Figure 4 shows the formation of spicules as a function of the con- Fic. 5. Spicules induced by sea urchin fibronectin and horse serum. Cells were maintained in sea water containing fibronectin at the concentration of 0.5 mg/ml for 36hr after fertilization (a). The cells were maintained in sea water containing fibronectin for 24hr after fertilization, then fol- lowed by culturing in sea water containing 4% horse serum for 24hr (b). Arrows indicate spicules. Bars indicate 50 »m. 269 centration of sea urchin fibronectin in a culture medium. Spicules were not formed at a con- centration less than 0.1 mg/ml of sea urchin fibronectin, whereas they were formed in al- most all of the cell aggregates at the concentra- tion of 0.5—0.8 mg/ml. However, the spicules formed in the presence of fibronectin did not elongate and failed to grow into the skeleton. The successive elongation of spicules was observed only when the culture medium was changed to the sea water contain- ing horse serum and then cells were cultured for 24 hr at 18°C (Fig. 5). DISCUSSION Morphogenetic events during the early devel- opment of sea urchin embryos have long been a major interest in research for developmental bi- ologists [e.g., 10-12]. One of important phe- nomena is the _ differentiation of primary mesenchyme cells derived from the micromeres which are formed at the 16-cell stage. However, little is known of the molecular basis of differ- entiation of primary mesenchyme cells or the functional involvement of specific molecules. To overcome the difficulty of these experiments in situ, adequate model systems such as in vitro cultured cells were designed for the substituting experimentation. In sea urchin embryos, micromere-derived cells were shown to differentiate to primary mesen- chyme cells in vitro [10, 12]. Based on the recent observations, fibronectin could be a probable can- didate playing an important role in cell adhesion, migration and differentiation. In the present study, fibronectin promoted the scattering of micromere-derived cells at lower concentrations, while at higher concentrations it enhanced the cell-to-cell and cell-to-substratum adhesion. A minimal level of cell-to-substratum adhesion may be required to initiate cell scat- tering or cell migration but enforced adhesion may arrest the cell movement [15]. Present results support the hypothesis that fibronectin may be involved in embryonic ce!! adhesion and migration in sea urchins as well as mammals. Previous studies of fibronectin we re 270 Y. MIYACHI et al. focused on their involvement in migration and cellular differentiation of chicken embryonic cells [16-17] and amphibian primordial germ cells [18]. Cultured cells derived from the isolated micro- meres of sea urchin embryos provide us the opportunity to analyze the direct involvement of fibronectin for cell differentiation and the results will lead us to understand the interaction of embryonic cells with fibronectin and other ex- tracellular molecules. The initiation and successive elongation of spicules in micromere-derived cells occurred in vitro in the presence of horse serum [10], but no particular factor or specific molecule was found to be the initiator for the formation of spicules. The present report is the first case to show that fibronectin induced the spicules. Triradiate spi- cules as well as granular and rod-like spicules appeared when the micromere-derived cells were cultured in sea water containing sea urchin fi- bronectin. When primary mesenchyme cells that had begun the spicule formation in vivo were iso- lated and kept in a medium with the absence of the horse serum, the spicules ceased to grow into the skeleton [10]. The present study showed that the spicules induced by fibronectin also failed to elongate and grow into the skeleton. These ob- servations indicate that some factors other than fibronectin in serum may be involved in the process of elongation of spicules. Fibronectin is known to bind extracellular macromolecules such as collagen and _ glyco- saminoglycans. Probably, collagen-like proteins are prerequisites for the deposition of CaCO, and the successful skeleton formation in sea urchin embryos [19-20]. The blastocoelic fluid containing mucopolysaccharides may provide the primary mesenchyme cells with an appropriate molecular environment for the expression of their phenotype [10, 21, 22]. It is possible that sea urchin fibronectin interacts with these macro- molecules, regulating the differentiation of primary mesenchyme cells of sea urchin embryos. ACKNOWLEDGMENTS The authors are grateful to Dr. M. Hayashi for the gift of horse plasma fibronectin. Thanks are also due to Drs. M. K. Kojima, T. Kato and M. Masuda for the discussion of this problem. REFERENCES 1 Yamada, K.M. and Olden, K. (1978) Fibro- nectins — adhesive glycoproteins of cell surface and blood. Nature, 275: 179-184. 2 Ruoslahti, E., Engvall, E. and MHaymar, E. G. (1981) Fibronectin; current concepts of its structure and function. Coll. Res., 1: 85-128. 3. Sekiguchi, K. and Hakomori, S. (1980) Func- tional domain structure of fibronectin. Proc. Natl. Acad. Sci. USA, 77: 2661-2665. 4 Hayashi, M. and Yamada, K. M. (1983) Domain structure of the carboxyl-terminal half of human plasma fibronectin. J. Biol. Chem., 258: 3332- 3340. 5 Yamada, K. M., Kennedy, D. W., Kimata, K. and Pratt, R. M. (1980) Characterization of fibro- nectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J. Biol. Chem., 255: 6055-6063. 6 Iwata, M. and Nakano, E. (1981) Fibronectin from the ovary of the sea urchin, Pseudocentrotus depressus. Wilhelm Roux’s Arch., 190: 83-86. 7 Nakano, E. and Iwata, M. (1982) Collagen- binding proteins in sea urchin eggs and embryos. Cell Differ., 11: 339-340. 8 Spiegel, E., Burger, M. and Spiegel, M. (1980) Fibronectin in the developing sea urchin embryo. J. Cell Biol., 87: 308-313. 9 Katow, H., Yamada,K.M. and _ Solursh, M. (1982) Occurrence of fibronectin on the primary mesenchyme cell surface during migration in the sea urchin embryo. Differentiation, 22: 120-124. 10 Okazaki, K. (1975) Spicule formation by isolated micromeres of the sea urchin embryo. Amer. Zool., 15: 567-581. 11 Wolpert, L. and Gustafson, T. (1961) Studies on the cellular basis of morphogenesis of the sea urchin embryo. Development of the skeletal pattern. Exp. Cell Res., 25: 311-325. 12 Kitajima, T. and Okazaki, K. (1980) Spicule formation in vitro by the descendants of precocious micromere formed at the 8-cell stage of sea urchin embryo. Develop. Growth and Differ., 22: 265— 279. 13 Sano, K. (1977) Changes in cell surface charges during differentiation of isolated micromeres and mesomeres from sea urchin embryos. Dev. Biol., 60: 404-415. 14 Engvall, E. and Ruoslahti, E. (1977) Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int. J. Cancer, 20: 1-5. 16 18 Effect of Fibronectin on Micromeres Hynes, R. O. (1981) Fibronectin and its relation to cellular structure and behavior. In “Cell Biology of Extracellular Matrix”. Ed. by E. D. Hay, Plenum Press, New York, pp. 295-334. Ceitchley, D. R., England, M. A., Wakely, J. and Hynes, R. O. (1979) Distribution of fibronectin in the ectoderm of gastrulating chick embryos. Nature, 280: 498-500. Mayer, B. W., Hay, E. D. and Hynes, R. O. (1981) Immunocytochemical localization of fibronectin in embryonic chick trunk and area vasculosa. Dev. Biol., 82: 267-286. Heasman, J., Hynes, R. O., Swan, A. P., Thomas, V. and Wylie, C. C. (1981) Primordial germ cells of Xenopus embryos. Cell, 27: 437-447. 19 20 22 271 Nakano, E., K. Okazaki and lwamatsu, T. (1963) Accumulation of radioactive calcium in larvae of the sea urchin Pseudocentrotus depressus. Biol. Bull., 125: 125-132. Mintz, G. R., DeFrancesco, S. and Lennarz, W. J. (1981) Spicule formation by cultured embryonic cells from the sea urchin. J. Biol. Chem., 256: 13105-13111. Immers, J. (1956) Changes in acid mucopolysac- charides attending the fertilization and develop- ment of the sea urchin. Ark. Zool., 9: 367-375. Immers, J. (1961) Comparative study of the localization of incorporated '*C-labeled amino acids and *°SO, in the sea urchin ovary, egg and embryo. Exp. Cell Res., 24: 356-378. eo ripen, a TERI" Meme ah al Gilet ie ves. we hn : —= entity ee RET om JAY | 73 ised + ‘ fe od LA Wy a) a mas ie q i tty ' 7 é ta cf s oe i me z ‘ | / 1 ier pA N " j aa | ' a i = 4 i} { am . ey J + j 2 ) or) ZOOLOGICAL SCIENCE 1: 273-280 (1984) Early Genesis of Slow Action Potentials in Co-cultured Ectodermal Cells at Different Ages SHIZUO ITO and KAZUFUMI TAKAMUNB Department of Biology, Faculty of Science, Kumamoto University, Kumamoto 860, Japan ABSTRACT — Action potentials can be generated in the epidermis of newt (Cynops pyrrhogaster) embryos between Stages 24/25 to 35/36 following the injection of intracellular current pulses. These action potentials consist of two distinct components, a fast spike and a slow action potential. The slow component follows the fast spike and has been shown to be transmitted to other epidermal cells. Cultured presumptive epidermal cells taken from the pregastrula also generate two-step action potentials. The time course of the genesis of the action potential during epidermal differentiation was elucidated by plating the dissociated cells of the presumptive epidermal ectoderm of the pregastrula in close proximity to an older cell colony, known to possess both types of excitability. These preparations, composed of cells in cultures for 2 and 5 days, were used to examine cell to cell transmission of epi- dermal potentials via presumed junctions between the colonies. When the epidermis of an older cell was stimulated, the adjacent younger cell, unexcitable electrically, exhibited a slow depolarization under the influence of the older cell. Thus, it appears that the membrane responsible for the genesis of the slow potential may be present before the neurula stage. The mechanism of cell to cell trans- mission of the slow action potential is clearly not mediated by electrical transmission; we propose © 1984 Zoological Society of Japan instead that it has a chemical basis. INTRODUCTION By applying an intracellular current pulse, action potential, composed of a slow component preceded by a fast spike potential, can be recorded from any part (eye anlage, gill fold, balancer, somite and ventral region) of the epidermis of newt embryos ranging from the tail bud stage (Stage 24/25) to the hatching stage (Stage 35/36) [1]. The fast spike potential is a prerequisite for the production of the slow action potential of the stimulated cell but cannot by itself induce transmission of the slow action potential to other epidermal cells. The slow component evoked in one cell can be transmitted to other epidermal cells. Thus it is clear that a fast spike potential is elicited directly by an intracellular electrical stimulus, while a slow action potential can be induced by means of two Accepted August 17, 1983 Received June 23, 1983 pathways; one via a fast spike potential evoked by an intracellular current injection, another via cell to cell transmission of a slow action potential. In the former, a fast spike potential, which is usually followed by a slow action potential in normal saline, is elicited without the accompanying slow action potential (Na* channel) in Na*-free saline. When the fast spike, assumed to be attributed to a Ca** inward current, is blocked by application of Co** or verapamil saline, the slow action potential is consistently absent even in the medium where the Na* concentration is similar to normal saline [2]. These results suggest that slow action potentials are evoked by the genesis of fast spike action potentials, but are not induced directly by current injection. Evidence also indicates that the slow action potentials by themselves are capa- ble of eliciting slow action potentials in adjacent cells. Substitution of sodium bicarbonate-HCl saline for normal saline, which lowers the intracel- lular pH, results in an uncoupling of the fast com- 274 S. Iro and K. TAKAMUNE ponent. Under these conditions epidermal action potentials are transmitted from the stimulated cell to other cells. The epidermal potentials which are transmitted, however, consist solely of slow components. Fast spike action potentials in- duced electrically can still evoke slow action potentials in the stimulated cell, but the fast spikes are not transmitted to contiguous cells [2]. These results suggest that the fast spike potential of one cell does not trigger or induce fast action potentials in the membranes of contiguous cells. Therefore slow action potentials generated in contiguous cells are not dependent on the fast spike com- ponent. The cell to cell propagation of epidermal action potentials in the newt embryo must be attributed to some mechanism other than electrical transmission. Present experiments with use of co-cultured presumptive epidermal ectoderm cell colonies at different ages were performed in order to elucidate the onset of genesis and transmission of slow action potentials of the newt embryo. MATERIALS AND METHODS Collection of newt (Cynops pyrrhogaster) em- bryos and the culture conditions are similar to those reported previously [1, 3]. Co-culture of the Monolayered Presumptive Epi- dermal Ectoderm Cells at Different Ages Two pieces (1.01.0 mm) of presumptive epi- dermal ectoderm isolated from two pregastrula embryos were dissociated in Ca-Mg-free Steinberg’s saline for 60min. These dissociated cells were then cultured in a modified L-15 culture medium supplemented with 10% fetal calf serum (FCS) in a Falcon petri-dish (3002). At 3 days in culture, other freshly prepared dissociated cells, also taken from the pregastrula by a similar technique, were plated in close proximity to the older cell colony (Fig. 1). The dishes were left undisturbed until the younger cells adhered to the bottom of the dish. At 5 days in culture, two colonies of different ages corresponding to Stage 31/32 (referred to as mature cells) and to Stage 22/24 (referred to as immature cells), respectively, were brought into contact or overlapped. These preparations, composed of 2 and 5 day cultures, were used to examine cell to cell transmission of epidermal potentials via junctions between the colonies. Electrical Recording Electrical recordings were usually carried out Pas Ca:Mg-free L-15medium 3days stl Steinberg sol. (10%FCS) culture > tua sap Ca-Mg-free L-15medium 2days 5days Stl Steinberg sol. (10% FCS) culture culture Fic. 1. Schematic showing of co-culture technique of epidermal cell colonies at different ages. The dissociated cells, collected from 2 pieces of the presumptive epidermal ectoderm of the pregastrula, were cultured for 3 days. Freshly dissociated cells were treated by the same procedure and plated in close proximity to the older cell colony. Amphibian Epidermal Potentials 275 under in vitro conditions in a modified L—15 culture medium. All in vitro experiments were done at room temperature ranging from 20 to 25°C. Simultaneous recordings of the epidermal action potential were obtained from two separate cells (each impaled with a microelectrode filled with 3M KCl) under in vitro conditions. One micro- electrode was connected to a bridge-balance circuit which allowed simultaneous injection of a current pulse and recording of the resulting voltage (V;). The second microelectrode was used to impale the second cell and also to record the voltage (V;). Epidermal action potentials were generated in the first cell by passing outward or inward square current pulses (1.0-30nA through electrode V;; commonly 20 msec duration). In other experiments, measurements of inter- cellular electrical coupling in the cultured mono- layered cell colony were made by three micro- electrodes filled with 3 M KCl. Two electrodes recorded the electrotonic potentials (V, and V2) and the epidermal slow action potentials in two cells. The third electrode was used for intracel- lular current injection. RESULTS Sequential Changes in Genesis of Epidermal Action Potentials during in Vitro Cell Differentiation Epidermal cells begin to produce fast spike potentials in response to intracellular current pulses during a short critical period very early in the Stage 24/25 newt embryo. Although this critical period varies from specimen to specimen [3], by Stage 25 the epidermal cells produce an action potential containing both the fast and slow components. This compound potential persists until Stage 35/36 just before hatching. Dissociated cells of the presumptive epidermal ectoderm of the Stage 11 embryo (pregastrula) are inexcitable up to 2 days in culture (at 23°C). Intracellular anodal current injected into some cultured cells at 3 days (control stage 23/26) elicited only abortive or fast spike action potentials (Fig. 2A). Most cells in the colony, however, were still inexcitable in response to a current stimulus. At 4 days in culture, almost all cells examined A B \f af Fic. 2. Genesis of a two-step action potential in a monolayer cell colony taken from presumptive epidermal ectoderm of the pregastrula embryo. (A) Abortive action potential (f) evoked by inward current pulse, 3 days in culture. (B) A fast spike action potential (f ) early on the fourth day of culture. (C) A fast spike (f) with a slow action potential(s) late on the fourth day of culture. The potentials of B and C were recorded from the same colony in a continuous experiment over several hours. Calibration: 10 mV, 10 nA and 20 msec in A, B and 10 mV, 10 nA and S50 msec in C. Abbreviation in all figures: 0, zero potenital; _r, resting membrane potential; i, applied current pulse. produced either a fast spike alone or both a fast and slow action potential. Sequential changes of excitability were observed in cell colonies within the same culture dish at 4 days while recording continuously for several hours: Representative data showing such sequential changes in the action potential are shown in Figure 2B, C. In Figure 2B, at the beginning of experimentation, only a fast potential was recorded. After 6-8 hr, cells 276 S. Iro and K. TAKAMUNE in the same culture dish begin to produce typical fast spike responses followed by slow action potentials (Fig. 2C). These sequential changes of the action potential during development become more obvious in the cultured epidermal cells than in the intact embryonic cells. In Vitro Genesis of the Slow Potentials in Co- cultured Cell Colonies at Different Ages The boundary of each monolayered colony of co-cultured epidermal ectoderm cells can be recognized at different ages following inoculation; cells from 2 day cultures contain more yolk granules than do those from 5 day cultures. The presence of scattered ciliated cells produced in the 5 day colonies can also be used to distinguish Fic. 3. was about 800 pm. individual colonies. When epidermal two-step potentials (V, in Fig. 3B, C) were generated by intracellular current injection into a mature cell, a delayed slow depo- larization ranging from 20 to 40 mV was recorded in the immature cells. Typical results showing the epidermal action potential (V:) recorded from a mature cell and the concomitant slow depolariza- tion (V2) in an immature cell are presented in Figure 3B and C, where the recording site of the immature cell was about 800 um away from the mature cell (Fig. 3A, E, and E,). Immature cells, on the other hand, did not generate action potentials following intracellular current injection. This suggests that the slow depolarization evoked in the immature cell colony is not due to epidermal a re) 1 (A) Phase-contrast photomicrograph of co-cultured monolayer cell colonies at different ages (2 days and 5 days in culture). culture. **: The area of the cell colony at 2 days in culture. the retouched line shows overlapping parts of both cell colonies. electrode position of the experiment in Fig. 3B, C. *: The area of the cell colony at 5 days in *k*~ The area enclosed by E, and E, show the In both the interelectrode distance (B) Epidermal action potential (V,) of the mature cell induced by an outward current (3 nA, 17 msec) and transmitted slow depolarization (V2) in the immature cell (2 days in culture). (34 nA, 17 msec) injection. (C) Similar recording following an inward current Calibration: 10 mV, 10 nA and 50 msec. Amphibian Epidermal Potentials differentiation promoted by the mature cell colony. Slow potentials induced by the mature epidermal cell excitation appear to be transmitted to the immature cells which do not respond to current pulse injection. These experiments show that the early genesis of slow depolarizations in the im- mature cells takes place only via epidermal slow action potentials of the mature cells. As shown in Figure 3B, C, the magnitude of the slow de- polarization in the immature cells is considerably smaller than that of the mature cell stimulated by current injection; and the depolarization in V, characteristically has a gentle slope without the initial rapidly rising phase (fast action potential) Pa A | as occurs in V,. This suggests that the magnitude of the slow depolarization in the immature cells decreases as it is transmitted to the immature cells some distance from the current source. To test this possibility, the magnitudes of transmitted slow depolarizations (V, and V>) induced by slow depolarization of the mature cells were recorded simultaneously in two separate cells of an immature cell colony (2 days in culture) at various interelectrode distances. A current pulse for genesis of the slow potential was injected into a mature cell colony. The recording cell (V,) was always 200 ~m from the current source and was located within an immature cell colony some 2-3 A 1.0 B100 08 80 06 60 50 04 40 30 0.2 20 z 0.1 =a 10 S ='3 N > OE 0.05 Lt 4 2 0.01 1 0 0.2 04 0.6 08 0 01 0.2 0.3 04 0.5 Distance (mm ) Distance (mm ) Fic. 4. (A) The relative ratio (V,/V,) of both exitable slow action potentials at various interelectrode dis- tances within the immature cell colony (open circles) via an epidermal action potential generated by a current pulse applied to the mature cell colony, and decrease in electrical coupling (closed circles) within the monolayer cell colony at 2 days in culture. interelectrode distance (mm). Inset: Coupling is plotted with a logarithmic ordinate. Examples of electrotonic spread (a) interelectrode distance of Abscissa: 500 »m and of transmission (b) of slow action potentials interelectrode distance of 300 »m. Calibration: 10 mV, 40 nA and 50 msec in (a) and 10 mV, 4nA and 20 msec in (b). (mV) of electrotonic potentials at various interelectrode distances. was injected into cells within the epidermal cell colony at 2 days in culture. (B) Decrease in the amplitude Constant current (4.4 nA and 280 msec) Ordinate represents the amplitude of an electrotonic potential recorded in a cell (V,) which was impaled with a current injecting electrode, and of the potential following spread to adjacent cell (V,) at various interelectrode distances. respectively. 278 S. Iro and K. TAKAMUNE cells away from the boundary between the two colonies. As shown in Figure 4A (open circles), the values of the ratio (V2/V;) of the slow potentials recorded at the two sites declined in proportion to increasing intercellular distances. However, it is clear that the ratio (V2/V:) of the slow potentials with respect to intercellular distance is significantly larger than that of electrical coupling events (Fig. 4A, black dots) measured at the corresponding intercellular distance, as described later. Cell to Cell Electrical Coupling within 2 Day Cul- tured Cell Colonies A long (200-300 msec) depolarizing current pulse was injected into a 2 day cultured mono- layered cell in order to determine whether immature cells are capable of generating an excitable de- polarization, and also to determine whether the cells are electrically coupled (V./V.:) over various intercellular distances. Two separate micro- electrodes, one for current passing and one for voltage recording were visually inserted into one cell (V,) with an inverted phase-contrast micro- scope. By injecting a constant current (4.4 nA) we plotted the magnitudes of both electrotonic potentials (V, and V.) with respect to various intercellular distances, as shown in Figure 4B. The input resistance of the current injected cell (V,) ranged from 5 to 19 MQ. The electrotonic potential of V. declined exponentially to 5% at an interelectrode distance of 500“m and was nearly undetectable at 600 wm. At any rate, slow action potentials, as observed in the co-cultured cell colonies, were never recorded in V2. These data, obtained at various current intensities and at various intercellular distances, were converted to relative electrical coupling ratios (V2/V;) as shown in Figure 4A (black dots). Maximum coupling ratio as high as 0.6 at an interelectrode distance of 50 wm occurs between contiguous cells. From these data (Fig. 4A, black dots), it is suggested that electrotonic spread through intercellular pathways may terminate about 500um from V4. This distance corresponds to about 10-12 inter- posed cells. This electrical coupling arises primarily via low resistance junctional pathways between epidermal cells. However, time delays between both electrotonic potentials were never observed in the immature cell colonies. These results indicate that the slow action potential ob- served in the immature cell can not be attributed to either a voltage-dependent action potential or electrotonic spread of the original slow potential via cell to cell low resistance pathways, but rather Fic. 5S. (Stage 26/28). Transplantation resulting in early genesis of slow action potential in ectodermal cells. presumptive epidermal ectoderm of the Stage 11 (pregastrula) embryo was grafted onto the host embryo A piece of the The recipient embryo was kept undisturbed for 20-24 hr in modified Holtfreter’s solution at 23°C and allowed to develop to Stage 31 (host) with graft tissue (Stage 14/15). (A) Photomicrograph of the host embryo (Stage 31) impaled with electrode (V,), and graft tissue (T) impaled with electrode (V,), respectively. (B) Epidermal action potential (V,) of the host gill epidermis, evoked by an outward current pulse (2nA, and 20 msec), and propagated slow action potential (V,) in the graft. Distance between V, and V,: 1100 ym. The recording cell in the graft is located about 110 yum from the edge of the graft. (C) Slow action potential (V,) transmitted via the epidermal action potential (V,) of the host, evoked by an inward current pulse (20 nA, and 20 msec). Distance between V, and V,: 800 ym. The recording cell in the graft is located about 75 wm from the edge of the graft. Calibration: 10 mV, 10 nA and 100 msec in (B), 10 mV. 10nA and 50 msec in (C). Abbreviation in all figures: 0, zero potential; r, and r,, resting membrane potentials of both cells; i, applied current pulse. Amphibian Epidermal Potentials 279 is dependent on a mechanism which involves the slow action potential generated in the mature cell. These in vitro results have been corroborated by the similar in vivo experiments in which the pre- sumptive epidermal ectoderm of the Stage 11 embryo was transplanted onto the tail bud larva, known to possess both types of excitability; slow action potentials generated in host epidermal cells can be transmitted to graft cells which per se do not respond to current pulse injection (Fig. 5). DISCUSSION There are two separate problems in considering an epidermal action potential composed of a fast spike followed by a slow action potential. One problem concerns the mechanism underlying the sequential genesis of a two-step action potential during cell differentiation. A second problem concerns the kinds of cell membrane changes which are initiated by the slow component of the action potential and which underlie transmission to other cells. Questions regarding the first appearance of the epidermal action potential in the amphibian em- bryo and its development have been clarified previously [S—7]. However, Roberts and Stirling [6] and Spencer [7] did not distinguish between the fast and slow components of the action potential. By use of current injection into cultured epi- dermal cells during the course of differentiation, we observed first the appearance only of a fast spike action potential (3 days with a brief critical period). Subsequently the membranes generate a fast-slow action potential (at 4 days in culture). The fast spike has been shown to be dependent on Ca**, while the slow component on Na® [2]. Therefore, it is assumed that channel formation corresponding to these ions occurs in the presump- tive epidermal ectoderm cell membrane during differentiation into epidermal cells. The present experiments have revealed that immature cells in vitro do transmit a slow action potential to other immature cells when a slow action potential is generated in co-cultured mature epidermal cells by current injection. These cells by themselves, however, are incapable of generating either the fast or slow action potentials following current injection. From this observation, it is thought that Na* channels responsible for the genesis of the slow action potential occur prior to the appearance of Ca** channels. It was expected that the cells would generate two-step action potentials once the Ca** channels were established. However, so far we have been able to record only the fast spike action potential during the critical period. In order to explain this contradiction it is further postulated that some intermediate factor linking the Ca** and Na* channels may be pro- duced soon after the critical period. If the cell fires a fast action potential when the intermediate factor is sufficiently prepared for activation of the Na* channels, a slow action potential would be induced. Roberts and Stirling [6] have been reported that transmission of the epidermal action potential is the result of direct current flow between cells via low resistance functions. Another possibility is that some level of depolarization, generated by the first cell that fires, serves as a trigger to induce depolarization of the contiguous cell membrane, resulting in turn in the generation of a slow action potential in that cell (electrical transmission). The latter mechanism responsible for epithelial conduc- tion in various invertebrate animals is generally accepted by several investigators [7, 8]. At any rate, both cases suggest that propagation of the epidermal action potential is dependent on cell to cell electrical coupling and/or electrical transmis- sion via low resistance junctional membranes. Results presented in this report argue against the electrical transmission of epidermal potentials via low resistance junctions. We have shown that membrane depolarization by intracellular current injection into immature cells generates slow action potential nor induces trans- mission to other cells. Slow action potentials generated by mature cells, however, do propagate to immature cells presumably via junctions inter- conneting the cells. Accordingly, these results suggest that activation of the Na* channel respon- sible for the slow action potential is not due to a voltage dependency. Furthermore, any cell can transmit slow action potentials to other cells, even when the cells are electrically uncoupled by means of low intrace!- in vitro neither 280 S. Iro and K. TAKAMUNE lular pH [2]. Thus cell to cell transmission of the epidermal action potential, particularly the slow component, is assumed to occur at the cell mem- brane via a chemical mechanism. The question arises as to why a decline of the slow action potential propagated in an immature cell was recorded as it was transmitted to immature cell colonies more remote from the current source. Presumably the membrane responsible for genesis of the slow action potential is not suffic ently differentiated at these immature cell stages. ACKNOWLEDGMENT We are indebted to Dr. Darrell R. Stokes, Department of Biology, Emory University, for his critical reading, suggestions and help in preparing the manuscript. This work was supported in part by a grant 57540418 in aid to S. Ito from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 Sato, E., Adachi, S. and Ito, S. (1981) The genesis and transmission of epidermal potentials in an amphibian embryo. Devel. Biol., 88: 137-146. Sato, E.. Nakamura, O. and Ito, S. (1983) Ionic dependence and transmission of epidermal action potentials in a newt embryo. Devel. Biol., 97: 460-467. Ito, S., Inoue, H. and Adachi, S. (1982) The genesis and transmission of epidermal potentials of newt embryonic cells in vivo and in vitro. Develop. Growth and Differ., 24: 479-490. Roberts, A. (1969) Conducted impulses in the skin of young tadpoles. Nature (London), 222: 1265- 1266. Roberts, A. (1971) The role of propagated skin impulses in the sensory system of young tadpoles. Z. Vgl. Physiol., 75: 388-401. Roberts, A. and Stirling, C. (1971) The properties and propagation of a cardiac-like impulse in the skin of young tadpoles. Z. Vgl. Physiol., 71: 295-310. Spencer, A. N. (1974) Non-nervous conduction in invertebrates and embryos. Amer. Zool., 14: 917— 929. Anderson, P. A. V. (1980) Epithelial conduction: Its properties and functions. Progr. in Neurobiol., 15: 161-203. ZOOLOGICAL SCIENCE 1: 281-289 (1984) Regulation of Gonadal Differentiation in Frogs Derived from UV-irradiated Eggs TERUKIYO SHIRANE Department of Information and Behavioral Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan ABSTRACT — The gonada! differentiation in normal Rana nigromaculata was described and the process in animals derived from pressed and UV-irradiated eggs were examined and compared. The initial formation of medullae was basically the same (12 pairs of segments) in all primitive gonads of the normal (control) and UV-treated animals. The UV-treated animals, however, had fewer germ cells and medullary cells than the control. Ovarian development in normal larvae proceeded in cyclic rhythms, each cycle consisting of three steps: active proliferation of germ cells, growth of oocytes, and the swelling of secondary genital cavities (SGCs). In larvae derived from UV-irradiated eggs, Ovaries (with a few germ cells) did not show the initial two steps in the first cycle, but in the second cycle the first step was observed. Consequently, germ cells became surrounded individually or in groups by the wall-cells (medullary cells) of SGCs, hence reducing the SGC volume. These ovaries were eventually masculinized more or less completely, and functional (fertile upon artificial insemi- nation) sex-reversed frogs were produced. The results may suggest that the encounter of indifferent germ cells with medullary cells is necessary to induce the production of serologically detectable male (SDM) antigen as the underdeveloped ovaries kept the female characters until the stage when the © 1984 Zoological Society of Japan encounter occurred. INTRODUCTION Sex reversal from female to male occurs in Rana japonica and R. brevipoda if they derive from eggs whose vegetal hemisphere is irradiated at the 2-cell stage with UV [1]. UV-irradiated animals had poorly differentiated ovaries at metamorphosis with some degenerating oocytes in R. japonica and with few, if any, oocytes in R. brevipoda, and the two species showed each characteristic differentia- tion pattern. These ovaries apparently underwent transformation to the testis in later development. A possibility was thus pointed out that the inter- action between germ cells and surrounding somatic cells determines the primary sexual expression of gonads. The rate of sex reversal in these two species was not the same reflecting perhaps the difference in the differentiated state of ovaries at metamorphosis. Comparisons of the results for Accepted September 24, 1983 Received July 25, 1983 the two species were somewhat complicated be- cause the larvae grew at different seasons (thus at different water temperatures) and for different periods, and thus the possible effect of enviromental differences could not be completely eliminated. In the present study, the gonadal differentiation is examined in Rana nigromaculata, a species closely related to R. brevipoda. Both speceis grow under the same enviromental conditions. The outline of normal gonadal differentiation, although itself is similar to that of other Ranids, will be described first as this has not been done previously, and the gonadal differentiation in the UV-irra- diated group will be examined. Surprisingly, the female-to-male sex reversal, which takes place in UV-irradiated animals as in the two species re- ported in the previous paper, is already clear in this species in late larval stages. These results are discussed in relation to the role of interactio between germ cells and somatic elements or gonadal differentiation. 282 T. SHIRANE MATERIALS AND METHODS Rana nigromaculata Hallowell was used for experiments. To induce ovulation, hypodermic implantations of 2 to 3 dry frog pituitary glands each were given into the lower jaw of two mature females (batches I and II, respectively, in Table 1, see Results). Eggs were collected at 24 hr (at 20°C) after the implantation, and artificially in- seminated. Fertilized eggs were mechanically dejellied except for the innermost jelly layer. The eggs were pressed and UV-irradiated upon their vegetal hemisphere at the time when the first cleavage furrow reached three-fourths of the dis- tance from the animal pole. The apparatus for UV irradiation was described in a previous paper [1]. The pressure was regulated so as to give a 50 mg/egg weight. A total dose of 9300 erg/mm7 of UV (exposure time: 3 min) was administered. Larvae were reared at room temperature. The water temperature during the larval period varied from 13°C in May to 30°C in August. Most males matured sexually in the next breeding season after insemination. In contrast, most females were still quite immature at this time. Twenty tadpoles each were sacrificed at various developmental stages and their gonads examined TABLE 1. mental stages in Rana nigromaculata histologically. The gonads were fixed in 10% formalin solution, cut into 10 wm serial paraplast sections, and stained with haematoxylin and eosin. Some frogs were also sacrificed at intervals and their gonads were examined similarly. Develop- mental stages of larvae were determined following - those of Rana pipiens reported by Taylor and Kollros [2]. RESULTS In the experimental, only those eggs which had been irradiated at a right angle (see Materials and Methods) and began “normal” development were included. Some tadpoles (18/317 in the control and 24/330 in the experimental) and frogs (8/59 in the control and 8/66 in the experimental) died by accident or for some unknown reasons. Death ratios appear to be the same both in irradiated animals and in control animals. The gonads of sacrificed tadpoles and frogs were examined histo- logically (see below) and the sex of individuals determined (Table 1). The sex of accidentally died frogs was also determined but only by the gross morphology of gonads. The sex ratios of larvae and frogs in the control were essentially the normal 1: 1 at every stage examined, but those in Sex-ratios of tadpoles raised from pressed and UV-irradiated eggs at various develop- developmental stages batch sex Vv Vill xX XHI XV XVIII XX XXIII XxXV control J 2 1 10 9 9 11 11 (non-irradiated) ot 8 (5) 10(3) 11 11 9 9 II ge. 10 9 Y) So 10 11 11 M/F 0.67 1.00 1.00 1.22 1.22 1.22 0.81 0.81 1.22 experimental IA 2 10 10 (irradiated) FA 10 (5) 10 17 IB & 9 5 1 3S 11 (4) 15 19 IT 2 10 D, 1 SB 10 18 19 M/F 1.00 1.22 1.00 1.00 300 9.00 5.67 19.0 19.0 LL SSS Numbers in parentheses show the number of animals in which the migration of germ cells was not observed. For details in the experimental at stage XV and later, see text. Regulation of Gonadal Differentiation the experimental deviated considerably (mostly males) at stage XV and later. There was no indication that females died selectively. Rather, these abnormal sex ratios were the result of sex reversal (see below). Morphology and histology of gonadal differentiation in Rana nigromaculata Although the process of normal gonadal devel- opment in R. nigromaculata is essentially the same to that in other Ranids, it would be beneficial to give a brief account as this has not been made previously except for some scattered descriptions on various stages. When the operculum formation was completed, the larva had a pair of genital ridges about 0.6 mm in length, and the number of primordial germ cells (PGCs) per larva was 40-60. At stage III, the genital ridges grew slightly in length and size, and were transformed into the primitive gonads each of which was composed of 11-13 (mostly 12) segments with a migrating medulla. Each medulla consisted of 60-80 cells spread out front and rear. Over one-half of them held a cell-bridge between the gonad and the periphery of nephrotome. The number of germ cells (once primitive gonads are formed, the term ‘germ cell’ instead of PGC will be used.) became about three times as many, 152-201 per larva (average 175) and 82-108 per gonad. At this stage the gonads were still indifferent. At stage V, each medulla consisted of over 100 cells and most of them no longer held the cell- bridge. Twelve out of 20 animals examined had gonads of this type. Remaining 8 animals, how- ever, had gonads which looked as though they had 15-16 medullae, some still holding slender cell- bridges near and along the pathway of arteries. Although it remains to be established whether these slender cell-bridges represént newly formed medullae or the remnants of the original medullae, this is the initial morphological sign of testis- differentiation. Figure 1 shows the growth of gonads measured in plane and expressed in mm’. The growth of ovaries reflected the changes in the internal devel- opment: active proliferation of oogonia (at stages VIII, XV and XXIII), growth of oocytes (at stages 283 X, XVIII and XXV), and the swelling of secondary genital cavities (SGCs) in medullae (at stages XIII, XX and at one month after metamorphosis). The swollen medullae in developing ovaries maintained individuality until stage XX, when the second swelling occurred and their fusion took place except for the artery distributed portions, thus forming several (usually six) large ovarian cavities. In contrast, testes were initially characterized by the slender cell-bridges of medullary cells at the vicinity of artery-distributed portions. These artery-distributed portions apparently played a leading role in the conjugation of medullae. The migration of germ cells from the cortex into the medulla began at stage V at the anterior part of the testis and proceeded posteriorly. Only 4-6 anterior segments had migrated germ cells at stage VIII, but all had germ cells at stage XIII. Many medullary cells in these testes re-aggregated at some (usually 5—6) portions at stage XV which cor- responded generally to the artery-distributed portions. Some of these medullary cells consti- tuted the slender ducts (vasa efferentia) and others contributed to the partition of gonadal com- ponents (the formation of seminiferous tubules) at stage XX. Although two juvenile hermaphrodite seach with a normal-type testis and with an underdeveloped ovary (One at stage XV and one at stage XX, respectively) were observed, none of the total of 40 young frogs examined at two weeks and later after metamorphosis showed any gonadal abnor- malities either in gross morphology or in detailed histology. The results for 11 frogs examined at one year after insemination will be described later. Gonadal differentiation in R. nigromaculata raised from pressed and UV-irradiated eggs At the time when the operculum was completed, the genital ridges of experimental larvae were so slender and transparent that they could not be distinguished from other tissues even in fixed samples unless sectioned and observed under the microscope. Forty animals were examined at this stage, 10 in batch IA, 10 in batch IB, and 20 in batch II. The effect of irradiation upon the for- mation of PGCs did not vary much among these 284 T. SHIRANE er 1.0 o—o Ovary 4 e—e Testis b = = 0.8 3 G 0.6 qvensp Cc q Ee e) ©) C aie 2 0.4 Vv re 6 ve N yy 0-2 ee ete -¢——¢ 2 50 60 70 80 Days old XXL paul ay Vil X XM XY XV— XX XXV Developmental stage Fic. I; a, active proliferation of oogonia at genital cortex; Growth (in plane, mm?) of gonads in the control R. nigromaculata larvae. b, formation of oocytes; c, swelling of secondary genital cavities (SGCs). batches: most animals examined were PGC free. In batch JA and IB, which derived from the same parental female, only one out of 10 animals in each batch had some PGCs (18 and 20 cells, respec- tively). In batch II, two out of 20 animals had some PGCs (12 and 30 cells, respectively). Thus, even in these animals the numbers of PGCs were very much smaller than those in control animals (40-60 cells). Even at stage III, most of the primitive gonads were still transparent, and their segmented struc- tures could be observed through genital epithelium only in fixed samples. Germ cell-free animals were no longer present, though the number of germ cells per larva was still far less than that in control ones, 39-127 as opposed to 152-201. The appearance rate of germ cells per 10m cross section of gonads was 0.1-1.2 (0.4 on an average) in contrast to 1.5—2.0 (1.6) in the control. On the other hand, the pattern of medulla formation was essentially the same as in the control. In the gonads of animals with a few germ cells, however, the numbers of cells in each medulla were generally smaller, and those in some medullae at the posterior part were less than 40. Figure 2 shows the size of the left gonads (measured in plane, mm7’) in ex- perimental larvae in comparison with that of the control at various stages examined. Regulation of Gonadal Differentiation 285 At stage V, when the initial sign of testis-differ- entiation became obvious in the control animals, the characteristic movement of medullary cells in the primitive gonads as seen in the control was also easily distinguishable in most of the experimental larvae through the genital epithelium. Based on this morphological criterion, 10 animals out of 20 examined had testes. Five of them, however, did not yet show the migration of germ cells into medullae (Table 1). The testicular differentiation in sectioned preparations was basically the same as in the control, but the gonads had numerous vacant areas (instead of being filled with germ cells) which apparently gradually disappeared in later stages (XV—XVIII), thus producing the testes shorter in length and smaller in volume (Fig. 2). The ovarian differentiation was very poor in the experimental animals. Not only the first prolifera- tion of germ cells at the cortex (at stage VIII in the control, Fig. 1) but also the formation of oocytes (at stages X, XVIII and XXV in the con- trol, Fig. 1) was not as extensive as in the control. Formation of SGCs, however, proceeded in the normal manner and at a sheduled time (stage XIII, Fig. 1). Im these underdeveloped ovaries the swelling of SGCs resulted little in the increase in the ovarian volume (Fig. 2). After stage XV, most larvae had _ testis-like gonads in gross morphology. At this stage, nine larvae out of 20 had ovaries or ovary-like gonads in sectioned preparations. Of these, two had nearly normal ovaries with some grown oocytes on both sides, and 7 had smaller “‘ovaries’’ without grown oocytes. Among animals with such under- developed “‘ovaries’’, three had actively proliferat- ing oogonia in the cortex of one or both gonads. These smaller “‘ovaries” should have become larger if not for the fact that the reduction of SGC- volumes took place at the same time. The re- maining four larvae had extremely small “‘ovaries” X XV BE SSSA Rh BSS DSO dM» SAOQSAYN Control C] UV-irrad. NAASSANS 1 2 12 13 Size of gonads (x10' mm?) Stage I Vv Vit 20 Z ® ; o i =. 10 g v ; — VA > , 7 d e as. 1 2 ip ee Be ° 20 XX = w Q = testis ovary J mart Z 072345678 10 11 Fic. 2. (control) eggs of R. nigromaculata. Growth (in plane) of the left gonad in larvae raised from UV-irradiated eggs and from non-irradiated Twenty larvae each were examined at respective stages. 286 T. SHIRANE with only a few germ cells. They had individual medullae not fused together and two kinds of large vacant areas (primitive genital cavities with a few mesenchymal cells and blank SGCs). Both are ovarian characteristics and ordinarily these may be used to distinguish them from the proper testes with a few germ cells. However, in these ‘“‘ovaries’”” germ cells were often observed among the medullary cells that over-approached the genital cortex. Thus, these four larvae were con- sidered to be sex-transformed and were counted as males in addition to 11 larvae which had testes (Table 1). At stages XVIII-XX, such smaller “ovaries” with a very few germ cells can hardly be distin- guished from proper testes with a few germ cells even in the sectioned preparations, because these gonads were shortened and became to have a solid strand morphology as a whole due to the disap- pearance of various vacant areas. There were also smaller “‘ovaries’” (5/20 examined at stage XVIII and 3/20 at stage XX, respectively) which had some germ cells but no oocytes. The oogo- nium-like cells were also located in the interior of the gonad. The SGCs in these gonads were small in volume and were arranged irregularly. These latter “‘ovaries’’ were also considered as trans- formed into testes (Table 1) with their character- istic solid strand morphology, although they had only smaller number of medullary cells (about 100 per medulla). At stages XXIII-XXV, some animals among those counted as males still showed a plain indica- tion of transformation (five animals at stage XXIII and four at stage XXV, respectively). From the histological analysis of gonads in 19 males at stage XXV (Table 1), five males had testes normal in gross morphology and in histology, four had testes nearly normal in gross morphology but abnormal in sectioned preparations, their semi- niferous tubules being arranged irregularly on one or both sides (sex-reversed females), and the remaining 10 had cord-like testes with a few germ cells on both sides. In animals with cord-like testes on both sides their genetic sexes could not be distinguished. Of 46 frogs examined at two weeks and later after metamorphosis, 37 were males in gross morphology. Eleven out of these males were regarded as sex reversed females on the basis of their testicular structure in sectioned preparation: irregular arrangement of seminiferous tubules. Three males at two weeks could not be decided on their genetic sexes. The formation of tubular lumen (at two months in the controls) and the spermatogenesis in sex- reversed females were lagging far behind that in the proper males in the controls and in the experi- mentals. In addition, some large cells (25-30 wm in diameter) were noticed among spermatogonia and primary spermatocytes (most of which were less than 16 wm in diameter) in the testes of sex- reversed females. After then these cells seemed to grow slowly. They might grow further and eventually become oocyte-like cells as reported previously in sex-reversed females of R. brevipoda [1]. Sex ratios of progenies of males derived from UV- irradiated eggs At one year after insemination (9 month after metamorphosis), 11 frogs (6 females and 5 males) in the control and 12 frogs (3 females and 9 males) in the experimental were still available for examination. The right testis of each of these males was used to prepare sperm-suspension for artificial insemination, and the left testis was fixed for histological observations. The sperm-suspensions derived from five males in the control were fertile and each sample activated successfully more than 95% of the eggs obtained from wild-caught females. The progenies of two male parents were reared until the time they com- pleted metamorphosis and their sexes determined. Both gave a 1: 1 sex-ratio (Table 2). One of these parental males had the left testis which was nearly normal in histology but which had a few auxocytes without yolk granules (120-180 wm in diameter) (Fig. 3A). The other four males all had the left testis which was normal in histology (Fig. 3B). In the irradiated group, one male out of 9 had very small and immature testes in gross morphology on both sides which had only a few sperms both in the testis-suspensions and in the sectioned prepara- tion. Two males had nearly normal testes in morphology and histology. The other six males Regulation of Gonadal Differentiation 287 TABLE 2. Sex ratios of the progeny of males raised from pressed and irradiated eggs of R. nigromaculata No. of progeny* Parental males total females males M/F Control No. | 35 18 17 0.94 (non-irradiated) No. 2 40 21 19 0.90 Experimental No. 2 49 42 i 0.17 (irradiated) No. 5 43 42 1 0.02 9 males No. 7 47 47 0 0.00 6 others BSS 126 109 0.87 * Progenies were reared until the completion of metamorphosis, and then their sexes were determined. “ ~ a Mm a& «2 @ Fic. 3. Cross-sections of testes of R. nigromaculata frogs at one year after insemination. bar 25 pm. A: Testis of a control male (control No. 2, Table 2) showing some auxocytes, which suggests the juvenile hermaphrodite nature. B: Testis of a normal control male (control No. 1, Table 2). C: Testis of a male (experimental No. 2, Table 2) raised from UV-irradiated egg showing the presence of an auxocyte, which may suggest that this is a juvenile hermaphrodite. From the sex ratio of progeny, this frog was concluded to be a sex-reversed female. D: Testis of a male (experimntal No. 7, Table 2) raised from UV- irradiated egg. This frog was also concluded to be a sex-reversed female by the sex ratio of progeny) Some oocyte-like cells (arrows) are present at the periphery of tubules. 288 T. SHIRANE had slightly smaller testis on one or both sides. Of these six males, three had abnormal, sex- reversed testes in sectioned preparations. All these males were fertile when the testis-suspensions were examined for artificial insemination (Table 2). Three sex-reversed females (Nos. 2, 5 and 7 in the Table) produced progenies which were mostly or exclusively females. These results prove that they in fact sex-reversed females. The left testis of the male No. 2 had some large auxocytes with- out yolk granules and oocyte-like cells together with bundled-sperms in the seminiferous tubules themselves arranged abnormally (Fig. 3C). Males Nos. 5 and 7 (Table 2) also had in their left testis some oocyte-like cells as well as bundled- sperms (Fig. 3D). DISCUSSION The gonadal differentiation in R. nigromaculata derived from UV-irradiated eggs follows basically the similar pattern to that reported previously for R. japonica and R. brevipoda [1, 3, 4]. In R. nigromaculata, however, the underdeveloped Ovaries with a few germ cells were nearly com- pletely masculinized before the animals completed metamorphosis, contrary to the other two species in which sex reversal was still undetectable at this stage. It should be noted that the larval devel- opment took place under essentially the same enviromental conditions both in R. nigromaculata and R. brevipoda. We may thus disregard factors other than UV irradiation and conclude that the differences in the developmental stage at which sex reversal is detectable and in its frequency are due to the different genetic constitutions. The incidence of sex reversal by exogonadal treatments (i.e. hormones) varies among various species [5]. Details leading to the different re- sponses to the treatment, however, remain un- known. It may be worthwhile to examine the relationship between the developmental state of defective ovaries and the frequency of sex reversal in R. brevipoda and R. nigromaculata, since no functional sex reversal by exogonadal treatments has, to my knowledge, been reported, and since we can disregard environmental influences as noted above. We may thus concentrate on the endogenous regulatory processes for sex differen- tiation. In R. brevipoda, the defective ovaries in UV-irradiated animals became distinguishable in gross morphology from testes and normal ovaries in larvae at metamorphosis, inasmuch as they increased, though slightly, their volume by swelling of the SGCs (Shirane, unpublished). The animals with very small ovaries at metamorphosis had few oocytes, if any (in these ovaries no oocyte was formed at earlier stages) [1, 4]. Nevertheless, the mesenchyme between the medulla and the cortex developed to some degree as if it would prevent medullary cells (wall cells of SGCs) from invading into the cortex [4]. Sex reversal in these animals is probably induced at one or two months after metamorphosis by exogonadal stimulation for gonadal maturation. On the other hand, not only the formation of oocytes but also the development of mesenchymal tissue were inhibited in R. nigro- maculata ovaries with a few germ cells in the UV- irradiated animals. These defective ovaries, which became clearly distinguishable by stage XV in gross morphology, did not show the first and second steps in the first cycle of ovarian develop- ment. Nevertheless, an active proliferation of germ cells in some but not in all defective ovarian cortex did occur. This might have been regarded as an ovarian character corresponding to the first step of second cycle of normal ovarian develop- ment. Medullary cells migrated into the ovarian cortex apparently due to the poor development of mesenchyme between medullae and cortex. It might be that the gonadal differentiation dur- ing larval period is controlled such that the regula- tory system, reacting to the abnormality in gonadal components (shortage of information), keep off a wasteful differentiation. This system in R. brevipoda would work so as to stop the further ovarian differentiation when the primary differen- tiation is unsatisfactory, but it does not alter the direction of differentiation immediately. In R. nigromaculata, a new condition, the unexpected encounter of indifferent germ cells with medullary cells, immediately follows, and this may trigger the early sex reversal. In R. japonica, the formation of oocytes in ovaries of UV-irradiated animals was observed to occur somewhat later in development than in normal females. The number of oocytes Regulation of Gonadal Differentiation 289 was much smaller than the control, and many of these showed degenerating figures at metamor- phosis [3]. The deviation from normal sex ratio in the UV-irradiated animals due to female-to-male sex reversal was statistically insignificant in R. japonica contrary to that in R. brevipoda [1] and in R. nigromaculata (the present results). It has been known for R. japonica, however, that sex reversal can be easily induced by androgen [6]. The endogenous regulatory system in R. japonica, it appears, is considerably unstable or loose com- pared to that in R. brevipoda and R. nigromaculata. It appears certain that the sex reversal in the UV-irradiated frogs is caused by an unsatisfactory formation or growth of oocytes in ovaries with fewer germ cells. Is there any specific element(s) necessary for the formation and/or growth of oocytes which is lost from gonadal components by the UV irradiation? The formation of pri- mordial germ cells (PGCs) is more dependent on whether eggs are pressed or non-pressed upon UV irradiation than on the actual dosages adminis- tered. Sex reversal was scarcely observed in animals from non-pressed and irradiated (9300 erg/mm7’) eggs in R. japonica [1] and in R. nigro- maculata (unpublished). Some male frogs of R. nigromaculata from pressed and irradiated (7800 erg/mm’*) eggs were sex-reversed females: forty- seven out of 60 frogs just after metamorphosis were males, and 12 of these males were re- garded as sex-reversed females on the basis of histological observations (Shirane, unpublished). Whether the decreased number of germ cells in UV-irradiated animals is directly related to altered sex differentiation (insufficient production of a substance(s) by a smaller number of germ cells) or the UV irradiation directly causes the deficiency in some substances in some gonadal components remains to be elucidated. REFERENCES 1 Shirane, T. (1982) Gonadal differentiation in frogs, Rana japonica and R. brevipoda, raised from UV-irradiated eggs. J. Exp. Zool., 223: 165-173. 2 Taylor, A.C. and Kollros, J.J. (1946) Stages in the normal development of Rana pipiens larvae. Anat. Rec., 94: 7-24. 3. Shirane, T. (1970) On the formation of germ cells of the frogs larvae from eggs irradiated with ultraviolet rays. Mem. Fac. Gen. Ed. Hiroshima Univ. III, 4: 24-37. 4 Shirane, T. (1972) On the formation of primordial germ cells of Japanese pond frog, Rana brevipoda Ito. Mem. Fac. Gen. Ed. Hiroshima Univ. III, 5: 17—23. (in Japanese) 5 Witschi, E. (1967) Biochemistry of sex differenti- ation in vertebrate embryos. In “Biochemistry of Animal Development’. Ed. by R. Weber, Academic Press, New York, Vol. II, pp. 193-2235. 6 Kawamura, T. and Yokota, R. (1959) The off- spring of sex-reversed females of Rana japonica Guenther. J. Sci. Hiroshima Uniy. Ser. B, Div. 1, 18: 31-38. ; i ard pea dy ’ ae. ee Y > Sane Cet rs : a uw att: ag i r p , ; ; it . Le 'elluew’ cen rw inwy ne Boat PR WY tointba tials sige f : f i th j R A < ' j ‘ ‘ a i RUE “oe “5 ; i a ‘5 x j | eels i Se j \ fe j . ; \ ~~ ' A ra é \ | ted nary a : . f i a Ls oo Fi : Poy GT eh ied “ Ma, A ae ery AY LRH ~ wi 3 ~ : rr i be 7 P = ( : ‘ ZOOLOGICAL SCIENCE 1: 291-316 (1984) Hybridogenetic Reproduction of Allotriploids between Japanese and European Pond Frogs MIpDoRI NISHIOKA and HIROMI OHTANI Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Hiroshima 730, Japan ABSTRACT — A few male and female allotriploids were obtained from crossings between female Rana brevipoda from Japan and male Rana lessonae from Italy, together with abundant diploid hybrids. Many male and female allotriploids were also produced from the same crossings by refrigerating eggs after insemination. The allotriploids were constructed of two Rana brevipoda genomes and one Rana lessonae genome. The oocytes of mature female allotriploids contained 13 bivalents consisting of Rana brevipoda chromosomes, while the Rana lessonae chromosomes were eliminated from the oocytes. In the testes of mature male allotriploids, the seminiferous tubules were filled with bundles of normal spermatozoa as in the case of the testes of diploid male Rana brevipoda. The offspring of male allotriploids back- crossed with female Rana brevipoda were completely of Rana brevipoda type at the tadpole and adult stages. Almost all the offspring were diploids and females. They were the same as diploid Rana brevipoda in the electrophoretic patterns of serum proteins, hemoglobin and various kinds of enzymes extracted from skeletal muscles. All the above findings indicate that the male and female allotriploids made hybridogenetic reproduction in the sense that the two Rana brevipoda genomes form 13 biva- lents and produced haploid gametes by normal meiosis after the single Rana lessonae genome was eliminated. Albinic offspring appeared from backcrossings of some male allotriploids with albinic diploid female Rana brevipoda (B*B*). These male allotriploids were considered to have been raised from unreduced ova of female Rana brevipoda which were heterozygous for an albinic gene by fertilization with sperm © 1984 Zoological Society of Japan of male Rana lessonae and to be B*B*L and B*B*L in gene constitution. INTRODUCTION It is generally believed that Rana esculenta Linné distributed widely in Europe are hybrids between Rana lessonae and Rana ridibunda and that they keep their independence by hybrido- genesis, although many problems related to their reproduction still remain to be solved. Spontaneous occurrence of triploid Rana esculenta was reported by Hertwig and Hertwig [1] as the first discovery of polyploid animals in_verte- brates. These triploids were 18 tadpoles reared as the controls of pseudohybrids. Twenty-five years later, Wickbom [2] found that four of eight Rana esculenta collected on Bornhorn Island near the exit of the Baltic Sea were tri- Accepted September 29, 1983 Received August 16, 1983 ploids. Twenty-five years later, Giinther [3] discovered that seven of 24 Rana esculenta col- lected from East Germany were triploids. There- after, he [4] found that 39.6% of all the frogs collected from 19 places in the northern and eastern parts of East Germany were triploids. According to Uzzell and Berger [5], Uzzell et al. [6], Gunther and Hahnel [7], Giinther ef a/. [8] and Ginther [9], there are two classes of triploid Rana esculenta; allotriploids with two Rana lessonae genomes and one _ Rana_ ridibunda genome, and those with two Rana _ ridibunda genomes and one Rana lessonae genome. In the populations consisting of Rana _ esculenta alone or a mixture of Rana esculenta and Rana lessonae, allotriploids with two lessonae and one ridibunda genome dominate. Ginther et al. [8] and Giinther [9] have as- sumed that the presence of two genomes from 292 M. NIsHIOoKA and H. OHTANI ~ one or the other parental species in allotriploids permits synapsis to occur more readily and results in producing haploid gametes after the elimination of the other genome. The allo- triploids collected from East Germany usually produce haploid gametes with one Rana lessonae genome, as they generally consist of two Rana lessonae genomes and one Rana ridibunda genome. On the other hand, diploid female Rana esculenta collected from the same districts produce ova with one Rana ridibunda genome after the Rana lessonae genome was eliminated. In the populations con- sisting of Rana esculenta alone, these ova will produce Rana esculenta by inseminating with sperm of the male allotriploids, while in those consisting of a mixture of Rana esculenta and Rana _ lessonae, the ova will produce Rana esculenta by inseminating with sperm of the male Rana lessonae or allotriploids. In the present study, it was found that the allotriploids obtained from crossings between female Rana brevipoda from Japan and male Rana lessonae from Italy behave similarly to those between Rana lessonae and Rana ridibunda in the formation of gametes, although the diploid hybrids remarkably differ from diploid Rana esculenta in that they are completely sterile or that no elimination of one of the two genomes occurs in the ova at least. The production of male and female allotri- ploids from crossings between female Rana brevipoda and male Rana lessonae and some aspects of their hybridogenetic reproduction will be reported here. MATERIALS AND METHODS A total of 27 female and eight male Rana brevi- poda \to and a total of eight female and 13 male Rana lessonae Camerano were used in the present study. Thirteen of the female Rana brevipoda were crossed with the male Rana lessonae. Ten of these female Rana brevipoda were mated with four of the male Rana brevipoda as controls. The eight female Rana lessonae were also mated with eight of the male Rana lessonae in the control matings. Two of the female Rana brevipoda, (B)BB 79W8., Nos. 1 and 2, and three of the male Rana brevipoda, (B)BB 79W&, Nos. 1-3, were homozygous wild-type fifth-generation off- spring of a pair of frogs collected in 1970 from Konko, Okayama Prefecture. The remaining sin- gle male Rana brevipoda, (B)BB 82F6&, No. 4, used in the control matings was collected in 1982 from Konko. Nine other female Rana brevipoda, (B)BB 7T9H2., Nos. 3-8 and Nos. 11-13, used in cross- ings with male Rana lessonae were albino- heterozygous wild-type fifth-generation offspring which were obtained in 1979 and derived from the above-stated single pair of Rana brevipoda. The remaining two female Rana brevipoda, (B)BB 82F8., Nos. 9 and 10, used in crossings with male Rana lessonae were collected in 1982 from Konko. The other 14 of the 27 female and four of the eight male Rana brevipoda used in the present study were backcrossed with male or female hybrids or used in the control matings with male or female Rana brevipoda. Four of the female (B)BB 79W&., Nos. 3-6, were homozygous wild-type fifth-generation offspring of the above-stated single pair of Rana brevipoda. Seven others, (B)BB 79A2, Nos. 1-7, were albinic fifth-generation offspring obtained in 1979 from the same pair of Rana brevipoda. Two others, (B)BB 80W&, Nos. 1 and 2, were homozygous wild-type sixth-generation offspring produced in 1980 from the same pair. The remainder (B)BB 82W2, No. 1 was a homozygous wild-type seventh-generation offspring produced in 1982 from the same pair. On the other hand, three of the male Rana brevipoda, (B)BB 80W6, Nos. 1-3, were homozygous wild-type sixth-generation offspring produced in 1980 from the same pair. The remainder (B)BB 82W&, No. 1 was a homo- zygous wild-type seventh-generation offspring pro- duced in 1982 from the same pair. Rana lessonae used in the present study were the offspring of specimens collected in 1977 from Firenze, Italy. Although Uzzell and Hotz [10] have reported that the specimens collected from Valdarno, Toscana region belonged to the south- ern non-hybrid and hybrid forms of the Rana esculenta complex, it is evident that the present specimens belong to Rana lessonae, as they are the same as the Rana lessonae collected from Luxemburg in morphology, electrophoretic pat- Rana brevipoda, Hybridogenetic Reproduction of Allotriploids 293 terns of serum proteins, hemoglobin and 10 enzymes extracted from skeletal muscles and re- sults of crossing experiments made in various kinds of combinations. In 1977, the first-gen- eration offspring were produced from three matings between three females, (L)LL 77F&., Nos. 1-3, and three males, (L)LL 77F&, Nos. 1-3. All the offspring obtained from matings I and II were brown in dorsal ground color. About half of the offspring from mating III were green in dorsal ground color, while the others were brown. A total of eight females and 13 males were used in crossings and the control matings. Specifically, two females and two males, (L)LL 77 I-br2, Nos. 1 and 2 and (L)LL 77 I-brd, Nos. 1 and 2, obtained from mating I, three females and three males, (L)LL 77 Il-br2-, Nos. 3-5 and (L)LL 77 Il-br&d, Nos. 3-5, obtained from mating II, and three green females, (L)LL 77 Ill-gr2, Nos. 6-8, and six green males, (L)LL 77 Ill-gr&, Nos. 6-8 and 11-13, and two brown males, (L)LL 77 Wl-br&, Nos. 9 and 10, obtained from mating III were used in the crossing experi- ments with Rana brevipoda and the control matings. Ovulation was accelerated by injecting sus- pension of bullfrog pituitaries into the abdominal cavity. Hybridization was made during the breeding seasons of 1980 and 1982 by artificial fertilization. Triploid hybrids were produced by refrigerating eggs at 1-2°C for 2.5 hours, 20- 25 minutes after insemination to suppress the extrusion of the second polar body [11]. The chromosomes of tadpoles were observed in the tail-tips by the squash method with water- pretreatment without using colchicine solution [11]. The chromosomes of adult frogs were observed by the blood culture method [12, 13]. When analyzable mitotic figures were not obtained, the ploidy of frogs was determined by measuring the size of erythrocyte nuclei. The ploidy of pre- served frogs was determined by measuring the size of cell nuclei of uriniferous tubules in the kidney. The ploidy of frozen frogs was deter- mined by electrophoretic patterns of serum proteins, hemoglobin and various enzymes ex- tracted from skeletal muscles [14, 15]. Lamp- brush chromosomes were observed by the method of Gall [16] or Nishioka ef ail. [15]. The gonads of frogs were fixed in Navashin’s fluid, sectioned at 10-12% and stained with Heidenhain’s iron hematoxylin. Tadpoles were fed on boiled spinach or chard. After metamorphosis, nymphs at various devel- opmental stages and adults of the two-spotted cricket, Grvilus bimaculatus De Geer were given to the frogs. The following abbreviations are used for des- ignation of each kind of individuals. (B)BB —— Diploid Rana brevipoda (L)LL ——— Diploid Rana lessonae (B)BL ——— Diploid hybrids, Rana brevipoda &- < Rana lessonae & (B)BBL —— Triploid hybrids consisting of two Rana brevipoda genomes and one Rana lessonae genome F Field-caught frog W —— Homozygotic wild-type individual produced from a laboratory mating H —————— Wild-type individual heterozygous for an albino gene A ————— Allbinic individual homozygous for an albino gene OBSERVATION I. Developmental capacity of hybrids 1. Control series a. Rana brevipoda The following 10 control matings were made by using 10 female and four male Rana brevipoda. i) Two matings in 1980 between two I1- year-old females and one l-year-old male, (B)BB 7T9W 2., Nos. 1 and 2 x(B)BB 79W@, No. 1. ii) Six matings in 1982 between six 3-year-old females heterozygous for an albino gene and two 3-year-old normal males, (B)BB 79H&., Nos. 3— 8 x(B)BB 79W@%, Nos. 2 and 3. iii) Two matings in 1982 between two field- caught females and one field-caught male, (B)BB 82F2., Nos. 9 and 10 x(B)BB 82F@, No. 4. The results of these 10 matings showed that 74.8-94.4% of the respective number of eggs, 819 (85.5%) of 958 eggs in total, cleaved nor- mally, 70.9-87.0°%, 739 (77.1%) in total, hatched 294 M. NisHiokKA and H. OHTANI TABLE 1. Developmental capacity of the hybrids between Rana brevipoda and Rana lessonae No. of — No. of No. of No. of Farents NO. exe normal normally normally metamor- PMC ° leavases (tail-bud hatched feeding _phosed Female Male Cggs Cieavases embryos tadpoles tadpoles frogs (B)BB (B)BB Nos. 1-4 958 819 790 739 715 671 81.9 Nos. 1-10 (85:5 7%) (62.970) C7) 14-0) Ora) (L)LL (L)LL Nos. 1-8 536 469 473 421 404 399 85.1 Nos. 1-8 (87.57%) @64%) (857) “54 7)" C4475) (B)BB 79W, (L)LL 77 I-br, 922 238 SS 135 107 11 4.6 Nos. 1, 2 Nos. 1, 2 Q35:8%) d6:87) “A467, “ho, (ray (B)BB 79H, (L)LL 77 II-br, 834 688 595 467 355 190 27.6 Nos. 3-5 Nos. 3-5 (825%) GL.3Y)- C60,) 42.67) 72874) (B)BB 79H, QEL Ti Mike.) 971 258 233 184 83 49 19.0 Nos. 6-8 Nos. 6-8 Q6.67%) @407) (897) C85) @ 0,4) (B)BB 82F, (L)LL 77 Ill-br, 1124 803 632 531 273 184 22.9 Nos. 9, 10 Nos. 9, 10 G14) 6Ce2y) G27, G43," “der (B)BB (L)LL Nos. 1-10 3851 1987 1615 1317 818 434 21.8 Nos. 1-10 61.67) Gioy). GCG427) ) Cl2 A Gls) PMC, Percentage of metamorphosed frogs to normally, and 59.2-80.2%, 671 (70.0%) in total, attained completion of metamorphosis at the age of 52-99 days, 63.8 days on the average. This number of metamorphosed frogs corresponds to 81.9% of the normally cleaved eggs (Table 1). b. Rana lessonae Eight control matings in total were made by using eight female and eight male Rana lessonae. i) Two matings in 1980 between two 3-year- old, brown-type females and two 3-year-old, brown-type males, (L)LL 77 I-br2-, Nos. 1 and 2x(L)LL 77 I-br&, Nos. 1 and 2. ii) Three matings in 1982 between three 5- year-old, brown-type females and three 5-year-old, brown-type males, (L)LL 77 IIl-br&., Nos. 3-5 x (L)LL 77 Il-br&, Nos. 3-5. ili) Three matings in 1982 between three 5- year-old, green-type females and three 5-year-old, green-type males, (L)LL 77 III-gr9, Nos. 6-8 x (L)LL 77 IlI-gr&, Nos. 6-8. It was found that in the eight control matings, 70.4-96.7'%, of the respective number of eggs, 469 (87.5%) of 536 eggs in total, cleaved nor- mally, 70.1-89.0%, 421 (78.5°%) in total, hatched normally and 60.3-84.6%, 399 (74.4%) in total, attained completion of metamorphosis at the age of 46-50 days, 47.5 days on the average. This normally cleaved eggs. number of metamorphosed frogs corresponds to 85.1% of the normally cleaved eggs (Table 1). 2. Experimental series a. Crossings between female Rana brevipoda and male Rana lessonae The following 13 crossings were made between 10 female Rana brevipoda and 10 male Rana lessonae. i) Two crossings in 1980 between two 1- year-old females and two 3-year-old, brown-type males, (B)BB 79W&., Nos. 1 and 2x(L)LL 77 I-br&, Nos. 1 and 2. ii) Four crossings in 1982 between three 3- year-old females heterozygous for an albino gene and three 5-year-old, brown-type males, (B)BB 7T9HY, No. 3 x(L)LL 77 Il-brS, No. 3, (B)BB T9HY., No. 3x(L)LL 77 Il-brS, No. 4, (B)BB T9H2., No. 4x(L)LL 77 I-br&, No. 4 and (B)BB 7T9H8., No. 5x(L)LL 77 Il-br&, No. 5. iii) Three crossings in 1982 between three 3- year-old females heterozygous for an albino gene and three 5-year-old, green-type males, (B)BB T9H&., Nos. 6-8 x (L)LL 77 IlI-gr&, Nos. 6-8. iv) Four crossings in 1982 between two field- caught females and two 5-year-old, brown-type males, (B)BB 82F2., No. 9x(L)LL 77 IIl-bré, Hybridogenetic Reproduction of Allotriploids No. 9, (B)BB 82F2., No. 9x(L)LL III-br&, No. 10, (B)BB 82F2., No. 10 x(L)LL 77 III-br&, No. 9 and (B)BB 82F2., No. 10x(L)LL 77 Ill-bré, No. 10. The results indicated that in the 13 crossings, 14.1-93.8°% of respective number of eggs, 1987 (51.6%) of 3851 eggs in total, cleaved normally, 12.4-90.5°%, 1615 (41.9%) in total, became nor- mal tail-bud embryos and 11.5—79.8%, 1317 (34.2%) in total, hatched normally. Thereafter, 8.1-77.7%, 818 (21.2%) in total, became nor- mally feeding tadpoles and 1.2-42.6%, 434 (11.3%) in total, attained completion of meta- morphosis at the age of 44-162 days, 58.1 days on the average. The number of the metamor- phosed frogs corresponds to 21.8% of the normally cleaved eggs (Table 1). About one-third of the normally cleaved eggs died of incomplete in- Vagination at the gastrula stage, edema, under- development and other abnormalities before the hatching stage. Of the normally hatched tad- poles, about one-third died of edema or under- development without taking food. About the half of tadpoles which began to eat also died of underdevelopment, edema or some other abnor- 295 malities during the tadpole stage. b. Production of allotriploids by refrigerating eggs In 1982, three crossings were made between three 3-year-old female Rana brevipoda, (B)BB T9H&., Nos. 11-13, which were heterozygous for an albino gene and three 5-year-old, green-type male Rana lessonae, (L)LL 77 Ill-gr&, Nos. 11- 13. Most of the eggs of each female were re- frigerated at 1—2°C for 2.5 hours, 20-25 minutes after insemination. Of 2819 eggs in total, 544 (19.3%) cleaved normally, 232 (8.2%) became normal tail-bud embryos and 188 (6.7%) hatched normally. While 80 of the normally hatched tadpoles died of underdevelopment, edema or some other ab- normalities, the other 108 completed metamor- phosis at the age of 55-112 days, 65.4 days on the average. This number of metamorphosed frogs corresponds to 3.8% of the total number of eggs and to 19.9% of the normally cleaved eggs (Table 2). A part of the eggs of the three female Rana brevipoda, (B)BB 79H, Nos. 11-13, inseminated with sperm of the three male Rana lessonae, (L)LL TABLE 2. Developmental capacity of the hybrids between Rana brevipoda and Rana lessonae produced by refrigerating eggs after insemination Parents No. No.of No.of No.of No.of No. of Refrig- oF nGenal normal normally normally metamor- PMC eration pure oleaga ees tail-bud hatched feeding phosed Female Male 88 8S embryos tadpoles tadpoles _ frogs (B)BB 79H, (L)LL 77 Untreated 174 25 22 20 14 9 36.0 No. 11 IlI-gr, No. 11 (Ifa) (12:07,) (lo) nO) ¢o:.27,) Treated 1615 119 36 24 20 12 10.1 CW) oC 2275) Gee se) e.7 %) (B)BB 79H, (L)LL 77 Untreated 160 37 28 16 12 6 16.2 No. 12 III-gr, No. 12 RZoi 7a) Ie fo) Oa Zab CO Le yay CS) Treated 594 175 106 94 92 72 41.1 (937) Gis) USB fale) Ay. 602.172) (B)BB 79H, (L)LL 77 Untreated 113 18 16 7 5 5 27.8 No. 13 IlI-gr, No. 13 (i597) (14.2501 6.27,))4, 447.) (44%) Treated 610 250 90 70 54 24 9.6 (41:0 4) 4a). (lhe Za) GS 7) U.3.9 ZA) (B)BB 79H, (L)LL 77 Ill-gr, Untreated 447 80 66 43 31 20 25.0 Nos. 11-13 Nos. 11-13 (17.9%) (14.8%) ( 9.6%) (69%) ( 4.5%) Treated 2819 544 232 188 166 108 19.9 C9375) CBebyoh hl Zo) Ao vo). C38 Zo) PMC, Percentage of metamorphosed frogs to normally cleaved eggs. 296 77 Ill-gr&, Nos. 11-13, was reared as the control. It was found that 80 (17.9%) of 447 eggs in total cleaved normally, 43 (9.6%) hatched normally and 20 (4.5%) attained completion of metamorphosis at the age of 48-90 days, 59.1 days on the average, while the others died of edema, underdevelopment or some other abnormalities during the embryonic and tadpole stages. The number of metamor- phosed frogs corresponds to 25.0% of the nor- mally cleaved eggs (Table 2). II. Ploidy and gonads of mature hybrids 1. Control series a. Rana brevipoda Of 671 metamorphosed frogs produced from 10 control matings, 306 were living and sexually matured at the age of one year. Of these frogs, 152 were females and 154 (50.3%) were males in appearance. When the ploidy of 33 females and 37 males was examined, it was found that a single male was a triploid, and all the others were diploids (Fig. 1). The testes of the triploid male were very abnormal in inner structure and contained a few larger or smaller, abnormally shaped spermatozoa together with some pycnotic vd ‘e i% f T, ao Ww a | 2 3 4 5 XX O& KR kn oR 6 7 8 9 10 X® AK O&K 11 12 13 Fic. 1. Metaphase plate and the karyotype of a cultured blood cell from Rana brevipoda. x900. M. NisHIioKA and H. OHTANI nuclei, while those of the other diploid males were filled with bundles of normal spermatozoa. The ovaries of the 33 females were completely normal and filled with full-grown ova (Table 3). b. Rana lessonae Of 399 normally metamorphosed frogs pro- duced from eight control matings, 80 were con- tinuously reared. After one year, 66 of them were living and sexually matured. It was found in appearance that 32 were females and 34 (51.5 %) were males. When the gonads of 10 females and 10 males were examined, it was evident that all of these frogs were diploids (Fig. 2), as the ova- ries and testes were filled with full-grown ova and normal spermatozoa, respectively (Table 3). 2. Diploid hybrids and allotriploids obtained without refrigerating eggs Of 434 metamorphosed hybrids produced from 13 crossings between female Rana brevipoda and male Rana lessonae, 298 were living and sexually matured at the age of one year. When the sex was judged by appearance, 162 were females and 136 (45.6%) were males (Table 3). The polidy of these female and male hybrids were examined (4h \ oe : Q = =e am ' ” £P 1 2 3 4 5 AA AK AK OG) KK 6 7 8 9 10 3X BA AA 1 12 13 Fic. 2. Metaphase plate and the karyotype of a cultured blood cell from Rana lessonae. X900. Hybridogenetic Reproduction of Allotriploids 297 WG nw 2X AS KA 1 12 13 Metaphase plate and the karyotype of a from a diploid hybrid, x 900. Fic. 3. cultured blood cell (B)BB? x(L)LLS. by the blood culture method (Figs. 3 and 4) or measuring the nuclear size of erythrocytes and uriniferous tubules and confirmed by the elec- trophoretic patterns of blood proteins and various enzymes extracted from skeletal muscles. While the ploidy of 90 hybrids including 64 females and 26 males was not determined, 196 of the other 208 were diploids and 12 were tri- ploids. The triploid hybrids were considered to be (B)BBL in genome constitution, as it was evi- dent that they had been produced from unreduced eggs by fertilization with haploid spermatozoa. While 95 of the diploid hybrids were females and 101 (51.5%) were males, three of the triploid hybrids were females and nine were males (Table 3). a. Female diploid hybrids The structure of ovaries was examined in 86 of the 95 female diploid hybrids. There were no hybrids whose ovaries were the same in de- velopment as those of the control Rana brevipoda However, six (2,) of the fe- male hybrids had approximately normal ovaries. The ovaries of 29 others (2:2) were under- developed and semitransparent. These ovaries or Rana lessonae. y= % » oe my hse Ve Ce ee 5 ‘ y\ Mi Bah MH 3 Th rh wie Kaa AA XXK XXK AKA ARS 1 12 13 Fic. 4. Metaphase plate and the karyotype of a cultured blood cell from a triploid hybrid, (B)BBL 82 IIl-brS, No. 4. 800. contained some growing auxocytes being usually 0.1-0.2mm in diameter, together with many young oocytes and oogonia. Some of the growing auxocytes were often degenerating. The ovaries of the remaining 51 diploid females (3) had almost completely degenerated and were replaced by fat body (Fig. 5a, b, c). It was remarkable that five of six females with approximately normal ovaries were produced from a mating, (B)BB 82F2., No. 10 x(L)LL 77 III-br&, No. 10, and the other one was produced from the same male, (L)LL 77 IlI-brS, No. 10, by mating with another female, (B)BB 82F2., No. 9 (Table 3). b. Male diploid hybrids The structure of testes was examined in 101 male diploid hybrids. The testes of these male hybrids were all very abnormal in inner structure. In 30 males (&;), the testes were nearly normal in size and appearance but contained no normal spermatozoa. No or a few abnormally shaped spermatozoa and some pycnotic nuclei were found in the seminiferous tubules. However, som spermatocytes and spermatogonia were ! here and there. The testes of the other © 298 M. NisHIOKA and H. OHTANI TABLE 3. Sex ratio of one-year-old diploid hybrids between Rana brevipoda and Rana lessonae Parents NOOR No. of females No. of males Ploidy Series at Ana- Ana- Female Male irons Total lyzed 2, 2, "25 Votal lyzeducneeccece 8 frogs frogs (B)BB (B)BB 2n (B)BB _ 69 33 33 33 O O 36 36 3606 C(OllC*O Nos. 1-10 Nos. 1-4 3n (B)BBB 1 0 1 1 0 7 10 Unknown 236 119 0 iL 7/ 0 Total 306 52 33933) 0p OneelS4: 37 36), Bia. (L)LL (L)LL 2n (L)LL 20 10 10 10 O O 10 10 10 O O Nos. 1-8 Nos. 1-8 Unknown 46 Dy, 0 24 0 Total 66 32 10 10 O O 34 10 10 O O (B)BB (LLL, 7/7/ 3n (B)BBL 80 3 2D, Deed SOMO 1 1, AL OmerO 79W, I-br, I-br Noss te 2 Nos. 1,2 Unknown 2 DZ, De riQr ton ww 0 Total 5 4 AY 2D pal) ae 1 1, « LaeeO nO (B)BB 79H, (L)LL 77 2n (B)BL 82 96 44 44.0) -15 29 5 52 OD 14s Nos. 3-5 II-br, II-br Nos. 3—5 3n (B)BBL 82 6 0 6 6°67 OF 0 Il-br Unknown 34 2S) 0 9 0 Total 136 69 AV O) IS 2 67 58 6 1453s (B)BB 79H, (L)LL 77 2n (B)BL 82 35 19 10.510) O10 16 16 0 6 10 Nos. 6-8 III-gr, III-gr Nos. 6-8 (B)BB 82F, (L)LL 77 2n (B)BL 82 65 32 BG ila ID 33 33° 0) 10m 23 Nos. 9,10 _‘II-br, IlI-br Nos. 9,10 3n (B)BBL 82 3 1 Py PherOveo0 2} 2. 2°) OT III-br Unknown 54 37 sie WO) 1 5e 82 17 0 Total 12 70 TOY AMD ala 52 35° 22° 103 (B)BB (HEE 2n (B)BL 196 95 86 6 29 51 101 . 10 SOss0Ran Nos. 1-10 Nos. 1-10 3n (B)BBL 12 3 327830 On nO 9 9 9 0720 Unknown 90 64 39°" Ore. Oo 34 26 0 Total 298 162. 128 9 34 85 .136 110) (SesO0na 2,, Normal ovary, $,, Abnormal testis, °,, Underdeveloped ovary, 2,, Degenerated ovary, &,, Normal testis, $3, Extremely abnormal testis. hybrids (3) were extremely abnormal. They were small and semitransparent and contained neither normal nor abnormal spermatozoa. Even spermatogonia or pycnotic nuclei were scarce. Their testes had almost completely degenerated (Fig: Sd, e,.f). c. Female allotriploids Two of the three female allotriploids and one of the nine male allotriploids were produced from two matings, (B)BB 799W&., Nos. 1 and 2 x(L)LL 77 I-br&, Nos. 1 and 2, together with two females whose ploidy had not yet been determined. The Hybridogenetic Reproduction of Allotriploids 299 . ee | weed © mre Fic. 5. Rana lessonae. Normal ovary (2,) of a female hybrid, Degenerated ovary (2 Abnormal testis (3 ,) of a male hybrid, >“+OoaAang® ovaries of the two female allotriploids were nearly normal in appearance and contained numerous auxocytes which were 1.1-1.4mm in diameter. These two female allotriploids were numbered (B)BBL 80 I-br&., Nos. 1 and 2 (Table 3). Lampbrush chromosomes were examined in 11 and 33 oocytes obtained from the two female allotriploids. The former oocytes were 1.1 mm in diameter, while the latter were 1.3 or 1.4mm. All these oocytes contained 13 pairs of chromo- somes. On the basis of the landmarks of these ae ze AT: Cross-sections of the gonads of one-year-old diploid hybrids between Rana brevipoda and he ve x 130. Underdeveloped ovary (2) of a female hybrid, 3) of a female hybrid, Normal testis (3 ,) of a control diploid Rana brevipoda, Extremely abnormal testis (33) of a male hybrid. chromosomes, the two homologous chromosomes of each pair were of Rana brevipoda (Figs. 6, 7). They always formed a bivalent. The chromo- somes of Rana lessonae were eliminated from each oocyte. The chiasma frequency of bi- valents in the oocytes of the two allotriploid females was compared with that in 50 oocytes obtained from 11 control female diploid Rana The results indicated that the chiasma frequencies of the allotriploid and the control Rana brevipoda were 35.95 and 38.70 per oocyte, respec- brevipoda. 300 M. NisHIoKA and H. OHTANT Rana brevipoda Rana lessonae ] ] = lh ] ] ] ] ) é f F es B . P7 i 1 ; 1 1 3 4 5 6 i 8 9 10 11 12 13 1 3 #4 5 6 7 8 9 10 11 12 13 Fic. 6. Maps of the thirteen lampbrush chromosomes of Rana brevipoda and Rana lessonae. Marks drown with a solid and a dotted line represent a simple and a compound type of giant loops, respectively. A black spot indicates a sphere. A pair of short horizontal bars on each chromosome indicates the segment including the centromere. Fic. 7. Lampbrush chromosomes of a female allotriploid, (B)BBL 80 I-br2, No.1. 300 Hybridogenetic Reproduction of Allotriploids 301 tively, and that there was no large difference in this respect. The remaining female allotriploid, (B)BBL 82 Ill-br2, No. 3, was produced from a mating, (B)BB 82F2., No. 10x(L)LL 77 III-br&, No. 10, together with a male allotriploid, 33 diploids con- sisting of 14 females and 19 males and 13 frogs whose ploidy had not yet been determined. The ovaries of the single female allotriploid were nearly normal in appearance. On the surface of each ovary, about 20 gray or brown auxocytes being 0.6—0.8 mm in diameter were observable. Four of the 14 female diploid hybrids had nearly normal ovaries each of which had about 20 or more numerous gray or brown auxocytes on the surface. These auxocytes were 0.6-0.8mm in diameter in two females, (B)BL III-br2-, Nos. 3 and 4, and 1.1-1.4 mm in the other two, (B)BL 82 III-br&,, Nos. 5 and 6. The ovaries of the remaining 10 female diploid hybrids (2-. and &3) were very abnormal and degenerative (Table 3). Lampbrush chromosomes were observed in five oocytes of the female allotriploid, (B)BBL 82 III- br2, No. 3, and 41 oocytes of the above four female diploid hybrids, (B)BL 82 III-br2., Nos. 3-6, with nearly normal ovaries and of two more diploid females, (B)BL 82 III-br2, Nos. 1 and 2, produced from a mating, (B)BB 82F2, No.9 ) 4°) doe pee > 72 (=) dees @ ead (7) e— (=) & =a cain eluate ml Fic. 2. Sephadex LH~-20 elution pattern of pure steroids: '*C-progesterone ('4C-P), *H-aldosterone (°H-Aldo) and *H-corticosterone (7H-B)=6000 to 10000 dpm; 11-deoxycorticosterone (DOC), 11- dehydrocorticosterone (A) and cortisol (F)=100 to 600 wg. The radioactivity (continuous line) or the steroid amount (broken line; determined with optical density at 240 nm [21]) was indicated for each collected fraction. Plasma Corticosterone in Bufo 321 After extraction and chromatographic puri- fication of distilled water, less than 9 pg of cor- ticosterone-like material was assayed in this sample. When a known amount of pure corticosterone (from 1000 pg to 3000 pg) was treated like ordi- nary plasma samples, 104-+-4.5 % of the theoretical dose were found by radioimmunoassay. Figure 2 shows the elution profile of some steroids chromatographed on a Sephadex LH- 20 column. Progesterone, 11-deoxycorticosterone and cortisol were eluted in fractions different from the corticosterone ones. However, corticosterone, aldosterone and 11-dehydrocorticosterone were not separated. Corticosterone was assayed in the same plasma sample (collected in one female Bufo japonicus formosus captured in Tokyo in February) after methylene chloride extraction, and after chroma- tographic purification of the methylene chloride extract; the respective corticosterone plasma levels were 17.7 and 5.7 ng/ml. Dialysis method reliability Intra- and interassay variances were evaluated using the same Bufo plasma sample at the same dilution. The coefficient of variation of the percentage of corticosterone total binding was lower than 4%, in the same experiment and also between different experiments. When buffer was placed in the two compartments of the dialysis system, the percentage of binding was always lower than 5%. Corticosterone plasma levels In toads captured in Tokyo, corticosterone plasma levels were the highest in February during the breeding season when the animals were in the spawning ponds (0.01 sift y if ZOOLOGICAL SCIENCE 1: 327-331 (1984) © 1984 Zoological Society of Japan Two New Species of Cestodes Belonging to the Genus Vampirolepis (Cyclophyllidea : Hymenolepididae) from Cave Bats of Taiwan ISAMU SAWADA Biological Laboratory, Nara University of Education, Nara 630, Japan ABSTRACT — Vampirolepis taiwanensis sp. n. and Vampirolepis macrostrobiloides sp. n. are des- cribed from the bent-winged bat, Miniopterus schreibersii fuliginosus, and the leaf-nosed bat, Hip- posideros armiger terasensis, of Taiwan, respectively. V. taiwanensis sp. n. most closely resembles V. isensis Sawada, 1966 and V. ogaensis Sawada, 1974, but differs from V. isensis in smaller rostellar hooks and triangularly arranged testes and from V. ogaensis in fewer rostellar hooks and triangularly arranged testes. V. macrostrobiloides sp. n. is different from all other species in the genus so far recorded from bats in having macrostrobila, larger eggs, larger onchospheres and larger embryonic hooks. INTRODUCTION To date, the endoparasites fauna of cave bats on Taiwan has been unknown for the most part. The present study was carried out to clarify the systematic position of chiropteran cestodes on Taiwan. In December 1974 and November 1981, 59 bats were collected from several raceways, a stalactite grotto, a tunnel and a house, and ex- amined for cestodes. The bats were captured alive by hand and autopsied immediately at the collecting sites. Their alimentary canals were cut open as soon as possible, fixed in Carnoy’s fluid (three parts of 100° ethyl alcohol plus one part of acetic acid) and brought to Japan. After the alimentary canals were soaked in 45% acetic acid for 30 minutes for expanding, they were stored in 70°% alcohol. Cestodes, obtained from the alco- hol-preserved alimentary canals, were stained with Heidenhain’s iron hematoxylin, dehydrated in alcohol, cleared in xylene, and mounted in Canada balsam. Measurements are given in millimeters. Accepted September 24, 1983 Received July 21, 1983 RESULTS The data on the bats examined and the cestodes found are given in Tables 1 and 2, and the stations at which the bat collections were made are shown on the maps (Figs. 1 and 2). The bats, Rhinolophus monoceros, Myotis ad- versus taiwanensis and My. mystacinus latirostris, were infected with no cestodes. Vampirolepis taiwanensis sp. 0. (Figs. 3-6) Nine bats, Miniopterus schreibersii .fuliginosus, were obtained from two raceways in Taichung and Nantou Countries in December 1974, and were examined for cestodes. Three of them were found infected with three specimens of this cestode. In November 10, 1981, eleven bats, M. s. fuliginosus, were collected from a raceway in Nantou Country. Four of them were found infected with nine specimens of the cestode. Dezcription (based on three mature specimens): Mature worms 12-31 long by 0.8-1.4 wide. Meta- merism distinct, craspedote, margins slight|, serrat. All proglottides wider than long. Ge: pores unilateral, located a little posterior to n 328 I. SAWADA TABLE 1. Cestodes found in cave bats collected on Taiwan in December 1974 Serial Number of bats No. of Locality Date Bat species Cestode locality ex- infected % species in Fig. 1 amined 1 Raceway Shinchung-township, 8 Dec. Miniopterus 2 1 50 Vampirolepis Taichung Country schreibersii taiwanensis fuliginosus n. sp. ep Raceway Koushing-township, 12 Dec. M.s. V py 29 Vz Nantou Country fuliginosus taiwanensis Myotis 1 0 0 mystacinus latirostris My. adversus 1 0 0 taiwanensis 3 Raceway Puli-township, 12 Dec. Rhinolophus 3 0 0 Nantou Country monoceros 4 Stalactite Kenting, 20 Dec. M.s. 2D 0 0 grotto Pingtung Country fuliginosus R. monoceros 1 0 5 House Haulien-city, 23 Dec. Pipistrellus 5) 0 0 Haulien Country abramus TABLE 2. Cestodes found in cave bats collected on Taiwan in November 1981 Serial Number of bats No. of Locality Date Bat species Cestode species locality exe fal) |G in Fig. 2 amined fected 1 Raceway Kouhsing-township, 10 Nov. Miniopterus 11 4 36 Vampirolepis Nantou Country schreibersii taiwanensis fuliginosus 2 Raceway Puli-township, 10 Nov. Rhinolophus 12 0 0 Nantou Country monoceros Myotis adversus 2 0 0 taiwanensis My. mystacius 1 0 0 latirostris 3 Tunnel Puli-township, 11 Nov. Hipposideros 4 1 DIVE Nantou Country armiger teraensis macrostrobiloides Nn. sp. R. monoceros yy 0 0 M.s. fuliginosus 1 0 0 4 Raceway Kouhsing-township, 11 Nov. R. monoceros y) 0 0 Nantou Country M. mm. latirostris 2 0 0 of proglottid margin. Scolex 0.245-0.350 long by 0.245-0.301 wide, not sharply demarcated from strobila. Rostellum 0.084 long by 0.063-0.077 wide, armed with a single circle of 22-24 hooks. Hooks measuring 0.018-0.021 long; hook handle long; guard bluntly rounded at its end, slightly shorter than or equal to blade. Rostellar sac 0.098 by 0.063, muscular, pyriform, extending Suckers 0.070-0.084 long posterior to suckers. by 0.091—0.112, round to oval. Neck absent. Two New Species of Bat Cestodes on Taiwan 329 Fic. 2. Sketch map showing the collecting sites of the bats at Nantou Country in November 1981. For the locality numbers, see Table 2. Fic. 1. Sketch map showing the collecting sites of the bats on Taiwan in December 1974. For the locality numbers, see Table 1. Fics. 3-6. Vampirolepis taiwanensis sp. n. 3: Scolex. 4: Rostellar hooks. 5: Mature proglottid. 6: Egg. Scales in mm. 330 I. SAWADA Testes three per proglottid, ovoid, 0.055—0.069 by 0.065—-0.083, arranged in a form of triangle, one poral and two aporal. External seminal vesicle oval, 0.053-0.070 by 0.018, directly dorsal to seminal receptacle, situated in anterior half of proglottid. Internal seminal vesicle gradually enlarging to fill poximal portion of cirrus sac. Cirrus sac 0.140 by 0.014-0.018, extending ante- rolaterally beyond osmoregulatory canals. Vagina initially posterior to cirrus sac, passing hehind cirrus sac, gradually expanding into voluminous seminal receptacle measuring 0.028 by 0.014. Ovary 0.140-0.152 wide, medial, transversely elongated, in middle or in anterior half of pro- glottid. Vitelline gland, round, 0.021-0.028 by 0.021, just posterior to ovary. Uterus saccular, occupying area between osmoregulatory canals in gravid proglottides. Eggs spherical, 0.032 by 0.035—0.039, sorrounded by four envelopes; outer- most chorion’ thick, with rough — surface. Onchospheres spherical, 0.028 by 0.021-—0.025; embyonic hooks 0.014 long. Host: Miniopterus schreibersii fuliginosus. Habitat: Small intestine. Localities and dates: Shinchung-township, Taichung Country, December 8, 1974; and Kouhsing, Nantou Country, December 12, 1974 and November 10, 1981. Type depository: Biological Laboratory, Nara University of Education, Nara, Japan. Remarks: Vampirolepis taiwanensis sp. n. most closely resembles V. isensis Sawada, 1966 [1], in the number of rostellar hooks and V. ogaensis Sawada, 1974 [2], in the length of rostellar hooks. V. taiwanensis can be separated from V. isensis in that it possesses smaller rostellar hooks (0.018— 0.021 vs. 0.032) and in the arrangement of the testes (triangular distribution vs. trnasverse row). It differs from V. ogaensis in the number of rostellar hooks (22-24 vs. 30) and in the arrangement of the Fics. 7-10. 8: Rostellar hooks. 7: Scolex. 9: Mature proglottid. Vampirolepis macrostrobiloides sp. n. 10: Egg. Scales in mm. Two New Species of Bat Cestodes on Taiwan 331 testes (triangular position vs. transverse row). Vampirolepis macrostrobiloides sp. 0. (Figs. 7-10) Four bats, Hipposideros armiger terasensis, were collected in a tunnel at Puli-township, Nantou Country in November 11, 1981. One of them was found infected with a single large specimen of this cestode. Description (based on one mature specimen): Length of strobila 152, maximum width 3.8. Strobila margins serrate. All proglottides wider than long. Scolex somewhat round when the rostellum is invaginated, 0.245 long by 0.301 wide, not sharply demarcated from neck. Rostellum 0.077 in diameter, retractable into elongated rostellar sac measuring 0.161 by 0.119, armed with a single row of 18 hooks measuring 0.025 long. Hook handle long; guard bluntly round at its end, longer than blade. Unarmed suckers oval, 0.119 by 0.105. Neck region behind scolex 1.2 long by 0.025 wide. Genital pores unilateral, located a little posterior to middle. Testes three in number, subspherical, 0.166— 0.180 by 0.083, situated in posterior field of pro- glottid, arranged in a transverse row, one poral and two aporal. Aporal testis usually elongate, located between dorsal and ventral osmoregulatory canals. Cirrus sac 0.207 by 0.042, occupied by internal seminal vesicle measuring 0.124-0.138 by 0.042. External seminal vesicle oval, 0.207—0.290 by 0.124. Ovary transversely elongated and bilobed in mature proglottid, 0.415—0.443 by 0.069-0.083. Vitelline gland small, lobed or oval, 0.124 by 0.041-0.069, situated near midline in space between first and second testes in posterior field of proglottid. Vagina opening in genital atrium, extending along ventral surface of cirrus sac, increasing gradually in diameter, forming large seminal receptacle. Seminal receptacle more or less globular, 0.152- 0.208 by 0.055-0.083. Uterus arising directly from ovarian lobes as a lobe sac, gradually en- larging, filling entire whole part of proglottid. Eggs subspherical, 0.053-0.056 by 0.046, sur- rounded by four envelopes; outermost chorion thick, with smooth surface. Onchospheres spher- ical, 0.032—0.035 by 0.035—-0.039; embryonic hooks 0.018 long. Host: Hipposideros armiger terasensis. Habitat: Small intestine. Locality and date: Puli-township, Nantou Country; November 11, 1981. Type depository: Biological Laboratory, Nara University of Education, Nara, Japan. Remarks: The present new species is separated from all the known species of Vampirolepis which have so far been recorded from the bats [1-5] by the larger size of the strobila, eggs and oncho- spheres and by possessing longer embryonic hooks. ACKNOWLEDGMENTS The author is grateful to Mr. M. Harada, Laboratory of Experimental Animals, Osaka City University Medical School, and Mr. Ho-mu-Lin, Taiwan Pro- vincial Institute of Infectious Disease, for collecting bats on Taiwan. REFERENCES 1 Sawada,I. (1966) On a new tapeworm, Vam- pirolepis isensis, found in bats with the table of the morphological features of tapeworms in Vampiro- lepis. Jap. J. Med. Sci. Biol., 19: 51-57. 2 Sawada,I. (1974) Helminth fauna of bats in Japan XV. Annot. Zool. Japon., 49: 103-106. 3 Jensen, L. A. and Howell, K. M. (1983) Vampiro- lepis schmidtisp.n. (Cestoidea: Hymenolepididae) from Triaenops persicus (Hipposideridae) of Tanzania. Proc. Helminth. Soc. Wash., 50: 135-— 137. 4 Murai, E. (1979) Cestodes of bats in Hungary. Parasit. Hung., 9: 41-62. 5 Rausch, R. L. (1975) Cestodes of the genus Hy- menolepis Weinland 1858 (sensu lato) from bats in North America and Hawaii. Can. J. Zool., 53: 1537-1551. 1 hit)? Laeger) aaee oun wire TO athe Seeley ‘e vit? re Hie jae éoins . 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Ltd., rue BOTANICAL MAGAZINE toxyo An international journal for plant sciences published quarterly by the Botanical Society of Japan. For a century, the journal has continuously published outstanding papers by Japanese as well as foreign botanical scientists. Contributors to the journal are not limited to the members of the Society and their papers are ac- cepted by paying the page charge. Papers in a recent issue: ZHAO, Y.J., S. KAMISAKA AND Y. MASUDA: Osmoregulation in Hypocotyls of Etiolated Mung Bean Seedlings with or without Cotyledons in Response to Water-Deficient Stress JORDAN, J.L., L.S. JORDAN and C.M. JORDAN: Prominent Spermoderm Patterns of Poaceae Mor!, K., H. MATSUSHIMA AND M. TAKEUCHI: A Study of Cell Wall Regeneration of Protoplasts in Marchantia polymorpha L. HOSOKAWA, Y. AND K. KIYOSAWA: Diurnal Rb~ Transport in Phaseolus Pulvinus KIMATA, M.: Comparative Studies on the Reproductive Systems of Carvdamine flexuosa, C. impatiens, C. scutata, and C. lyrata, Cruciferae ST. JOHN, H.: Novelties in Hawaiian Pittosporum (Pittosporaceae) Hawaiian Plant Studies IG NISHINO, E.: Corolla Tube Formation in the Primulaceae and Ericales SUZUKI, K.: Breeding System and Crossability in Japanese Epimedium (Berberidaceae) TAKEDA, K. AND S. TOMINAGA: The Anthocyanin in Blue Flowers of Centaurea cyanus Hor!I, T., R.E. NORRIS AND M. CHIHARA : Studies on the Ultrastructure and Taxonomy of the Genus Tetvaselmis (Prasinophyceae) II. Subgenus Prasinocladia INVIDED TARA GAUTHERET, R.J.: Plant Tissue Culture: A History Order form Send to THE BOTANICAL SOCIETY OF JAPAN Toshin Building Hongo 2-27-2, Bunkyo-ku, Tokyo 113, Japan THE BOTANICAL MAGAZINE, TOKYO [1] Individuals: ¥ 5,000 p.a. [] Institutions: ¥ 12,500 p.a. Name (Please print): Address: Date: Signature: 2) SSSSSSSSSSsSFsMMseF TOMY MICRO HIGH SPEED REFRIGERATED CENTRIFUGE MIR-15A HIGH-SPEED, FURB@A are wis CENTRIFUGE FOF gi ferrite £e) Ro) 3 SAMPLES, WITH 'qaggho ae7 Wiel) TR] . ia ' START 11 Integrated with a Re- S Quick Start / Quick Stop frigerator High Quality | G Easy Operation #3 N - GN DRAIN TRAP S Maximizing Floor #7 Convenient Drain Space Utilization WESPECIFICATION | Max. Speed 15,000rpm Max. Centrifugal Force @l Safe Design 15,860 | Automatic with built-in Koki, a comprehensive centrifuge SCR20BA | 300 ~ 20,000 8 | microprocessor control, Digital version manufacturer, brings you a com- SS Ses] 2 | 2 | S| Avonatiocontrer, plete line of High Speed Refrig- 20PR-520| 300~ 20,000] =| & | = | Analog version erated Centrifuges, Table-Top SCR7B 8lel a | . Centrifuges and Miniature Cen- — Scavoa Tye coiled falar P90 ala trifuges with wide-ranging O5PR-22 300 ~ 5,000 g Compact type for biochemistry speeds, capacities and tubes/ ~gecs—taoe— a0 woe cee nes = For clinical Automatic control bottles versatility for your SCT4BD a : SCT4BE ae ES IDONENOINY ESE Automatic with microprocessor clinical laboratory routine as well SCT15B 2,000 ~ 15,000 2 Miniature control as basic research. MC-200 % ners & ; xclusive MC-201 xe} Hematoerit MC-202 ee Dual purpose f MC-300 430 ~ 2,800 Cell washing HITACHI Refrigerated/ Table-Top/Miniature Centrifuges Brit @ Nissei SangyoCo.Ltd. © Hitachi Koki Co.,Ltd. Instrument International Dept. Mori 17th Bldg., 26-5 Toranomon 1-chome, Minato-ku, Tokyo 105, Japan C.P.O. Box: 1316, Tokyo 100-91, Japan Phone: 03-504-7282 Telex: J 22412 NISSEICOA ZOOLOGICAL SCIENCE VOLUME 1 NUMBER 2 APRIL 1984 CONTENTS REVIEWS Dan, K.: The cause and consequence of unequal cleavage in sea urchins Kitamura, A. and K. Hiwatashi: Cell contact and the activation of conjugation in Para- THRO CTU. ioscan SoS EL 161 ORIGINAL PAPERS Physiology Tomioka, K. and T. Yamaguchi: Response modification of cricket sensory interneurons Cc (UTA EG Ya 01 | Bae en a nears anit Tae SOR A Re eR 169 Kawasaki, M. and K. Aoki: A directionally selective binocular unit in the midbrain tegmentum of Tribolodon hakonensis C ell Biology Hashimoto, M., E. Nishida and _ H. Sakai: Effects of betaine, taurine and isethionate on microtubule assembly Biochemistry Kodama, T.: Further studies on the phys- iological role of hexose-6-phosphate dehy- drogenase: No positive correlation between hexose-6-phosphate dehydrogenase and en- zymes other than NADPH-cytochrome c RCA CEASE » ais bass bronco 205 Katoh, S. and T.Sueoka: Pteridine-metab- olizing enzymes in rat erythrocytes during GPEVELO DINE Hs ooo: sn vedere eee ee 215 Izumi, S., K. Kiguchi and S. Tomino: Hor- monal regulation of biosynthesis of major plasma proteins in Bombyx mori .............+. 223 Saito, Y. and H.Watanabe: Partial bio- chemical characterization of humoral factors involved in the nonfusion reaction of a botryllid ascidian, Botrylloides simodensis ...229 Developmental Biology Morisawa, M., S. Morisawa and R. de Santis: Initiation of sperm motility in Ciona in- testinalis by calcium and cyclic AMP ......... 237 Osanai, K. and K. Kyozuka: Cross-fertili- zation between sea urchin eggs and mussel spermatozoa: Sperm entry without egg ACTIVATION 2.55000 0+ s1c00e00s 02 oo cee eee ee EEE 245 Yoshizaki, N. and Ch. Katagiri: Necessity of oviducal pars recta secretions for the forma- tion of the fertilization layer in Xenopus LQQVIS vccciiiecsthassctvatsantune se eet ee ee Eee 255 Miyachi, Y., M.Iwata, H.Sato and E. Nakano: Effect of fibronectin on cultured cells derived from isolated micromeres of the sea urchin, Hemicentrotus pulcherrimus...265 Ito, S. and K. Takamune: Early genesis of slow action potentials in co-cultured ecto- dermal cells at different ages .................... 273 Shirane, T.: Regulation of gonadal differ- entiation in frogs derived from UV-irradiated eggs Nishioka, M. and H. Ohtani: Hybridogenetic reproduction of allotriploids between Japa- nese and European pond frogs.................. 291 Endocrinology Jolivet-Jaudet, G., M. Inoue, K. Takada and S. Ishii: Circannual changes in corticos- terone plasma levels and binding of corti- costerone to plasma in Bufo japonicus JOVINOSUS © oocecscavcnscsdesscsuosecvios sont et epee 317 Taxonomy Sawada, I.: Two new species of cestodes belonging to the genus Vampirolepis (Cyclo- phyllidea: Hymenolepididae) from cave bats of Taiwan Printed by Daigaku Letterpress Co., Ltd. Hiroshima, Japan tinuation of the “Zoological Magazine” and the “Annotationes Zoologicae Japonenses ZOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Nobuo Egami (Tokyo) Toshin-building, Hongo 2—27-2, Bunkyo-ku, Associate Editors: Tokyo 113, Japan. (03)-814—5675 Susumu Ishii (Tokyo) . Officers: Seiichiro Kawashima (Hiroshima) President: Hideshi Kobayashi (Funabashi) Koscak Maruyama (Chiba) Secretary: Makoto Asashima (Yokohama) Assistant Editors: Treasurer: Kiyoaki Kuwasawa (Tokyo) Takeo Machida (Hiroshima) Librarian: Shun-Ichi Uéno (Tokyo) Kazuyoshi Tsutsui (Hiroshima) Editorial Board: Howard A. Bern (Berkeley, endocrinology) Aubrey Gorbman (Seattle, endocrinology) Robert B. Hill (Kingston, physiology) Yukio Hiramoto (Tokyo, cell physiology) Yukiaki Kuroda (Mishima, cell genetics) Hiromichi Morita (Fukuoka, sensory physiology) Tokindo S. Okada (Kyoto, developmental biology) Andreas Oksche (Giessen, neuroanatomy) Hidemi Sato (Nagoya, cell biology) Koichi Sekiguchi (Tsukuba, phylogeny) Mayumi Yamada (Sapporo, systematic zoology) Ryuzo Yanagimachi (Honolulu, reproductive biology) 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, Associate 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, Park Voorn 4, 3454 JR De Meern, P. O. Box 2073, 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 volumes. 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 1984, 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 1: 333-348 (1984) REVIEW Developmental Control of Cell Differentiation TUNBO YAMADA Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland ABSTRACT — In regulatory mechanisms involved in the developmental expression of cell types, one can distinguish between immediate and remote controls. In the immediate control, the gene activa- tion pattern characteristic for the cell type in question may be established in the cell lineage by the interaction of the genome with factors like nonhistone chromatin proteins or transcription complex proteins, by the level of methylation of specific sites of gnome DNA, or by the attachment pattern of genes with the nuclear matrix. The remote control is exercised by specific cellular events which lead the cell lineage to the condition where the immediate control can operate. In ascidian development, the remote control consists in the specific number of DNA replication cycles, which allows the “‘pre- existing’ cytoplasmic factors of the immediate control to interact with the genome. In vertebrate development where the interactions between different cell layers play a role in the remote control, available evidence seems to suggest that the progression through the cell cycle by the cell lineage is essential in the remote control. It is speculated that in normogenesis of vertebrate somatic tissues, the factors necessary for the immediate control becomes available within the cell lineage, when the remote © 1984 Zoological Society of Japan control is completed, and are not introduced from the outside by tissue interaction. 1. IMMEDIATE AND REMOTE CONTROL OF CELL DIFFERENTIATION Since the cell type of metazoan tissues can be characterized by the differential pattern of gene activation, which is relatively stable, the appear- ance of cell types during ontogenesis should be based on the establishment of such pattern in the cell lineage. The factors or conditions which have been considered to be responsible for the activation include non-histone chromatin proteins [1-4], transcription complex proteins [5, 6], the level of methylation at CCGG se- quences of genes [7, 8] and the contact of genes with the nuclear matrix [9]. Whether beside this type of regulation in which individual genes are controlled, a regulation at the cell type level exists remains an open question. There is a report which can be interpreted to suggest such a regulation [10]. Whatever the answer to this Received October 17, 1983 question, it appears probable that specific molec- ular mechanisms exist for the expression of cell types on the basis of the differential gene activa- tion. In the present discussion of developmental control of cell differentiation, we assume that such mechanisms operate as the immediate control of cell type differentiation. During ontogenesis the expression of cell type is preceded by a number of developmental events which prepare the cells for the expression, suggesting the existence of specific regulartory events as components of developmental process, which are geared with the immediate control. These events are called the remote control in this paper. Obviously the transition from the remote to the immediate control represents a critical phase in which the genetic mechanism of cell type differ- entiation becomes established. This phase may correspond to the quantal cell cycle proposed by Holtzer [11,12]. From the mode of its effect on a large number of developmenta! sys tems the thymidine analogue 5-bromodeoxyur dine 334 T. YAMADA (BrdU) inhibits this transition when incorporated into genome DNA [for review, 13, 14]. 2. CONTROL OF ENZYME SYNTHESIS IN ASCIDIAN DEVELOPMENT Since the time of Conklin [15] the appearance of the muscle cell lineage in ascidian embryos is known as the typical case of mosaic development, in which the partition of the specific cytoplasmic areas into blastomeres and subsequently into cell lineages determines the differentiation pathway. In one recent approach to this problem acetyl- cholinesterase was used as the marker of the muscle cell lineage. The enzyme appears in the presumptive muscle cells at the neurula stage, and subsequently the enzyme activity increases in the muscle cell lineage [16, 17]. Puromycin blocks the enzyme development when applied at the time of the appearance of the enzyme. The acti- nomycin D treatment suggests that the synthesis of mRNA for the enzyme begins during the gastrula stage. When embryos are cleavage- arrested with cytochalasin B or colchicine the development of the enzyme is not affected [16]. If, however, aphidicolin is used continuously from the 64-cell stage onward to inhibit DNA synthesis [18], the enzyme development is in- hibited [19, 20]. The aphidicolin treatment from the 76-cell stage onward is followed by the ap- pearance of a low level of the enzyme. By combining the cytochalasin B treatment with the aphidicolin treatment, 7 cycles of DNA rep- lication were found needed before obtaining the positive reaction for acetylcholinesterase. According to Conklin [15] the yellow crescent in the egg of Styela partita becomes localized later in the muscle cell lineage of the larva. Suggested by a Morgan’s experiment [21], Whit- taker [22] compressed the embryo during the third cleavage. The treatment alters the pattern of segregation, so that the yellow crescent is segregated into 4 cells instead of two cells as normally occurs at the 8-cell stage. Released from the compression and treated with cytochalasin B for preventing further cleavage, the embryo eventually develops acetylcholinester- ase in three or four cells instead of in two cells of the muscle cell lineage as found in the cleavage-arrested non-compressed §8-cell stage. This means that the two nuclei that normally enter into the ectodermal pathway without being associated with the yellow crescent, receive a part of the crescent and become involved in the synthesis of acetylcholinesterase. The results reviewed above allow to project a model according to which the essential factor(s) for the immediate control is “pre-existing” and localized in the egg cytoplasm, but its inter- action with genome DNA depends on the com- pletion of 7 DNA replication cycles [23]. Lat- er we discuss the significance of these cycles. That interactions with other cells are not essential for the localized appearance of acetylcholin- esterase was demonstrated by isolating two cells which contain the yellow crescent at the 8-cell stage [24]. The progeny of isolated cells showed the histochemical reaction for the enzyme at the time when it becomes positive in the control whole embryo. In another approach Whittaker [25] studied the appearance of tyrosinase as the marker of brain melanocytes, and alkaline phosphatase as the marker of endodermal cells. Obtained results were basically comparable to those found for acetylcholinesterase, showing the insensitivity of enzyme development toward cytochalasin B and localization of the enzyme in the specific blas- tomeres. Although the mosaic nature of developmental control of those enzymes is im- pressive, one should not forget that the mosaic nature of ascidian development has a limitation. Especially in formation of the brain an inductive interaction which has a restricted similarity with that found in vertebrate development occurs [26]. 3. CONVERSION OF NEWT IRIS EPITHELIAL CELLS Complete removal of lens from the adult newt eye is regularly followed by regeneration of the lens from the dorsal pupillary margin of iris epithelium [27-29]. The regenerated lens is almost indistinguishable from the original lens in morphology, but differs slightly from latter at the molecular level, indicating a higher percentage Developmental Control of Cell Differentiation 335 of y-crystallins, lower percentage of SH-crystallins, and different peptide maps of ;-crystallins [30, 31]. That this type of lens formation is dependent on neural retina was demonstrated by a number of surgical experiments [32, 33] and was put in analogy with lens induction by the optic cup in developing embryos [34]. Thus the system was regarded as one of the prominent examples of inductive control of tissue differentiation. The classical proposal [27, 35] based on histo- logical findings that the lens regenerate is formed by heavily pigmented iris epithelial cells (IECs) after the loss of pigment granules (melanosomes) was confirmed by organ culture experiments in which iris epithelium and iris stroma were sep- arately tested for the lens forming capacity [36] and by primary and clonal cultures of IECs (37, 38]. In the adult eye, all IECs are overtly differentiated and non-dividing. Lens removal is followed by the entrance of cells into cell cycles all over the epithelium [39-44]. The lens is formed by those cells which are located at the dorsal pupillary margin, have gone through a number of cell cycles and completed depigmen- tation (the allotypic pathway). The rest of proliferating IECs show partial depigmentation and revert to the fully pigmented condition after retreat from the cell cycles (the autotypic pathway). The mode of conversion of IECs into lens cells in cell culture differs from that in situ in the following points: (a) Whereas in situ, IECs located at the dorsal pupillary margin alone are converted, in cell culture IECs derived from the ventral half of the iris are able to convert as frequently as those derived from the dorsal iris half [37, 38,45]. (b) The time interval needed for the appearance of the lens phenotype in situ after lentectomy is about 15 days [46, 47], while in cell culture it is more than 28 days [37, 45, 48]. (c) The conversion in situ is dependent on a factor from neural retina. However, the conversion in cell culture occurs in the absence of specific factor from neural retina. It was pointed out that these deviations in the mode of conversion between two systems can be interpreted if one assumes that the completion of a specific number of cell cycles is required for the conversion, and those cells which do not complete the critical number of cell cycles revert to the original cell type [45]. The proposal was based on the following information available at that time: (a) Existence of differential pro- liferation among IECs in situ [49]. (b) In cell culture the same level of proliferation is observed in dorsal and ventral IECs [37, 38]. (c) The total cell cycle time of IECs in culture is about two times as long as that of the allotypic pathway in situ [50,51]. From these data it was argued that in situ only IECs in the allotypic pathway with the shorter cell cycle time can traverse the critical number of cell cycles during the limited proliferation period subsequent to lentectomy, while in cell culture the differential proliferation is lost so that all cells have the same probability of conversion regardless of their location within the epithelium. Since in cell culture, unlike in situ, the period of proliferation is not the limiting factor, cells are able to complete the critical number of cell cycles even with the relatively long cell cycle time. This is also the reason that the expression of the lens phenotype is delayed in cell culture compared with that in situ. Since the cell cycle time of IECs in the allotypic pathway in situ is significantly smaller than that in culture the suggestion was made that the role of neural retina in situ is growth promotion and not the induction of lens differentiation. This suggestion fits in with the finding that no specific factor of neural retina is needed to obtain the conversion in cell culture. In one of the tests of the hypothesis the cell cycle number traversed by IECs in both pathways in situ was estimated (Yamada and McDevitt, in preparation). For this study the fact was utilized that the progress through cell cycles by IECs is correlated with a decrease in the pigmentation level. The observation of the pigment level in the segregated IEC lineages’ in vitro showed that the number of cell cycles traversed by a cell lineage can be estimated by the pigment level. Assuming that the same holds true for IECs in situ, the pigmentation level of IECs at the dorsal pupillary margin (allotypic pathway) and at the ventral pupillary margin (autoty | ic pathway) in situ was estimated at selected stages of lens regeneration in serial histological section: 336 T. YAMADA and the number of traversed cell cycles before the expression of the lens phenotype in the allotypic pathway, and that before repigmentation in the autotypic pathway were estimated. The results indicated that the cell cycle number for the allo- typic pathway was 56, and that for the autotypic pathway was =4. In the remaining parts of the autotypic pathway, the final level of pigmentation was too high to be estimated by the method used, suggesting that the cell cycle number trav- ersed must have been =3. In an earlier work the estimation of the cell cycle number traversed before the expression of the lens phenotype by dorsal marginal IECs in situ (allotypic pathway) was made on the basis of the average total cell cycle time and the time elapsed after the first mitosis and before the appearance of crystallins [49]. The estimate was 6 cell cycles in agreement with the last cited result. Another test of the working hypothesis was made by estimating the cell cycle number traversed in cell culture by a lineage of IECs before the expression of the lens phenotype. The deter- mination of the critical cell cycle number in cell culture was made by using segregated cultures of IEC’. The maximum number of cell cycles traversed by a segregated culture was estimated by cell counts and the level of pigmentation. Since the progress through cell cycles by the progeny of an IEC is not uniform, the level of pigmentation was used to supplement cell counts in this estimation. After the completion of various cell cycle numbers the segregated cultures were fixed and tested for crystallins with indirect immunofluorescence. In this test using 231 segregated cultures, no culture was found posi- * Utilizing the fact that grooves produced on the surface of plastic culture substratum with a tip of surgical steel nife prevents the locomotion of IECs cultured in monolayer from one side to the other, single or small number of IECs seeded on a plastic plate are sur- rounded by the continuous deterrent zone. In this way the progeny of the segregated IEC can be studied separately from the rest of cell population. The deterrent effect of grooves diminished when culture becomes multilayered. Growth of segregated culture is improved when substratum is treated with fibro- nectin and a feeder layer of TVI cell line derived from newt iris cells is employed. tive until after completion of 4 cell cycles. After completion of 5 cell cycles, 25% of cultures became positive. The percentage increased after completion of 6 and 7 cell cycles and reached 100°% after completion of 8 cell cycles (Yamada and McDevitt, in preparation). No significant dif- ference was found between IECs from the dorsal iris half and those from the ventral half in this test. The result supports the working hypo- thesis in showing that also in cell cluture, ITECs go through about 6 cell cycles before expressing the lens phenotype, and that ventral IECs which in situ do not complete more than 4 cell cycles and do not become converted into lens cells can be induced to convert if they traverse more than 5 cell cycles in cell culture. According to the working hypothesis, the role of neural retinal in this system is the support of proliferation of IECs which is required in situ or in organ culture [52] but not required in cell culture. A direct test of this idea has not been done. However, a number of observations provide circumstantial evidence for the idea: (a) It was demonstrated that calf retina and vitreous body contain a growth factor, EDGF, a protein of 17,500 dalton with an isoelectric point of pH 4.5. [53]. (b) The role of neural retina in lens regen- eration from dorsal iris in organ culture [52] can be replaced by pituitary [54, 55]. A prep- aration of thyrotropin, one of pituitary hormones, which increases the mitotic index of IECs, supports lens regeneration in organ culture [56]. Also insulin was found to support lens regeneration in organ culture [57]. (c) When lens regeneration in situ is suppressed by removal of neural retina from the same eye, an inhibition of proliferation in IECs was observed [40]. While IECs are proceeding through cell cycles a number of changes occur in the cytoplasm and on the cell surface. The initiation of prolife- ration of IECs is associated with a steep drop in the cAMP level [58, 59], and enhancement of rRNA synthesis [60,61] coupled with ultra- structural alterations of nucleoli [62]. The first wave of DNA synthesis follows a rapid increase in the synthesis of proteins, and the increase of the glutamine synthetase activity [58, 63]. As stated, the progress through cell cycles is ac- Developmental Control of Cell Differentiation companied by the stepwise decrease of the level of melanosomes [64, 65]. This depigmentation process can be ascribed to dilution by cell division of melanosomes which are not synthesized, and exocytosis of melanosomes which occur both in situ and in culture. For the exocytosis, roles of lysosomes which become activated by the entrance in the cell cycle [66], the cellular level of cAMP [59, 67] and the cellular level of Ca** [68] have been suggested. The incorporation of °*H- DOPA into IECs generally correlates with the cellular level of melanosomes [69]. Thus _ it becomes indetectable in the allotypic pathway, while it is enhanced in the repigmentation phase of the autotypic pathway. A number of studies suggested that the stable condition of the cell surface of normal IECs is switched to a dynamic one, as cells are activated in proliferation: The enlargement of cell surface, shedding of cell surface material and formation of cell processes [67, 70, 71]. Evidence exists for a progressive decrease in the electrophoretic mobility of IECs while cells are progressing through cell cycles, and subsequent increase in the mobility during the redifferentiation phase [72]. The decrease is more pronounced in the allotypic pathway than in the autotypic pathway in situ. The factors involved in the cell mobility are sensitive to ribonuclease, chondroitinase and neuraminidase [73, 74]. It was speculated that when IEC progeny completes the critical cell cycle number, the alteration in cytoplasm and cell surface lead to the appearance of factors which react with the genome and determine the gene activation pat- tern characteristic for lens cells [75, Yamada and McDevitt, in preparation]. In the autotypic pathway, where the critical cell cycle number is not attained, the alterations in cytoplasm and cell surface are limited. Hence, when cells retreat from cell cycles, the autotypic gene acti- vation pattern may be established. In terms of the present scheme, the remote control of this system is provided by the number of cell cycles traversed by the cell lineage, while the nature of the immediate control] has not been identified. 337 4. EPITHELIO-MESENCHYMAL INTERACTIONS IN MAMMALIAN DEVELOPMENT The epithelio-mesenchymal interaction in rat pancreatic development was selected as a model of embryonic induction and submitted to analysis with the transfilter method [76, 77]. Subsequently the system was extensively studied at cellular and molecular levels [for review, 78, 79]. After terminal differentiation the pancreatic epithelium gives rise to acinar cells which produce digestive enzymes, duct cells which function in transport of enzymes, and three types of endocrine cells producing glucagon, insulin and somatostatin respectively. The differentiation of the epithelial component of pancreatic rudiment depends on the presence of the mesenchymal component. This effect of mesenchyme can be exerted through a Millipore filter in the transfilter arrangement. Later studies showed that the mesenchyme has a factor which is required for DNA synthesis and proliferation of epithelial cells [80]. In the absence of mesenchyme or the mesenchymal factor, the proliferation of the pancreatic epi- thelium remains at the minimum level and is followed by the differentiation of endocrine cells producing glucagon. In the presence of mesen- chyme or the mesenchymal factor the epithelium proliferates extensively and the differentiation of acinar cells predominates. This effect of mesen- chyme on proliferation and differentiation of the epithelium is not specific to pancreatic mesen- chyme, but is shared by measenchyme of other sources. That the mesenchymal factor acts on the surface of target cells was indicated by the ability of the partially purified factor after covalent binding to Sepharose beads to stimulate DNA synthesis in pancreatic epithelium. These results make probable that in development of pancreas the appearance of various cell types is controlled by the extent of proliferation which is dependent on the growth factor produced by the mesenchyme. There may be different requirements for proliferation for the precursors of different cell types, or multipotential stem cells prod different cell lineages according to the leve! o proliferation. In any case the remote co 338 T. YAMADA seems to be exerted by the extent of proliferation. Another prominent example of the epithelio- mesenchymal interaction which was submitted to the transfilter analysis, and whose mechanism was closely examined is the mouse metanephric system [81]. In this system, the metanephrogenic mesenchyme differentiates secretory tubules nor- mally under the influence of the ureter bud, and fails to form tubules in the absence of the inducing tissue. or most experimental studies the ureter bud was replaced by embryonic spinal cord which has the equivalent morphogenetic effect. The mesenchyme of other sources does not respond to form metanephric tubules when faced to ureter bud or spinal cord in the transfilter arrangement. These situations were interpreted to suggest that the matanephrogenic mesenchyme is already ‘‘predetermined’’ when tested [82]. Hence the induction is permissive rather than instructive. Under the condition of the transfilter arrangement, the cell processes penetrate through filter pores, and the surface contact between inducing and target cells does occur [83]. Ac- cording to a recent report [84], when the meta- nephrogenic mesenchyme and spinal cord are put in the transfilter arrangement, the incor- poration of thymidine into DNA of target cells is enhanced compared with that of the non- induced control mesenchyme cells. The period of the enhancement coincides with the period during which the inductive influence is exerted. The authors conclude that the induction of tubule formation is accompanied by a contact mediated stimulation of DNA synthesis and that during the induction period, the target cells should pass one cell cycle during which they become irre- versibly committed to tubule formation. It appears, however, not fully excluded that the stimulation of DNA synthesis has to be accom- panied by other unknown event to produce the morphogenetic response. 5. THE VEGETALIZING FACTOR AND THE DIFFERENTIATION OF MESODERMAL TISSUES IN MPHIBIAN EMBRYOS The generally accepted view that in amphibian development, the three germ layers exist from the start was challenged on the basis of a number of surgical experiments mainly conducted by two groups of workers, one led by Nieuwkoop and the other led by Nakamura [85, 86; for review, 87, 88]. These experiments supported the idea that the definite pattern of mesodermal develop- mental tendency becomes established between the cleavage and_ gastrula_ stages, while the ectodermal and endodermal tendencies are present from the start of ontogenesis, localized in the animal and vegetal hemispheres respec- tively. The establishment of mesodermal tend- ency in the marginal zone of the animal hemisphere occurs by interaction of corresponding cells with the vegetal hemisphere which possesses the endodermal tendency. The general aspect of this interaction can be called vegetalization as proposed by Nakamura [88], because the develop- mental pattern is shifted toward that of a more vegetal level, although the substantial part of the interaction can be defined as induction [87]. Independent from this series of studies, it was found that some tissue proteins have the ability to induce mesodermal and endodermal tissues in amphibian presumptive ectoderm [89, 90; for review, 91-93]. The Tiedemann’s group succeeded to purify and characterize the factor from chick embryos as a protein with the molecu- lar weight of 30,000—32,000 [94,95]. Since the morphogenetic effect of the factor is a shift of development from the animal pattern to a more vegetal pattern, it was called the vegetalizing factor. It appears that the mechanism by which this factor works on ectoderm cells may give us a clue for the mechanism of the establishment of the mesodermal tendency in normogenesis dis- cussed above. Recently Grunz [96] treated Xenopus presumptive ectoderm with the Tiedemann’s factor at different concentrations and for different time intervals. The weakest treatment led to the formation of blood and heart (ventral mesodermal tissues) from ectoderm. The increasing intensity of treatment shifted the differentiation gradually to pronephros (lateral mesodermal tissue), somites and to notochord (dorsal mesodermal tissues). The most intensive treatment was followed by formation of endoderm Developmental Control of Cell Differentiation 339 alone. Some neural and mesectodermal tissues were also produced. This experiment together with a number of related experiments imply that in normogenesis, the establishment of the mesodermal tendency occurs on the basis of a single factor similar to the Tiedemann’s factor transmitted from the vegetal hemisphere to the animal hemisphere, and that the dorso-ventral organization of the mesoderm thus emerges reflects a gradient of the intensity of the inter- action of the factor with the cells. Since the Tiedemann’s factor has the affinity with DNA [91] and its morphogenetic effect disappears when bound to BrCN-Sepharose [97], it was suggested that the factor does not work at the cell surface, but becomes internalized and involved in the activation of genes in the responding cells. The fact, however, that the factor gives rise to a multitude of cell types and its specific effects are concentration dependent argues that the set of genes it may activate does not directly specify the individual cell types of induced tissues, but functions in the remote control of cell type dif- ferentiation. An earlier work on the morphogenetic effect of the dorsal mesoderm on lateral and ventral mesoderm of TJriturus neurula indicated that the effect is the shifting of the differentiation pattern of responding mesodermal region toward the more dorsal level, and not the direct specification of cell types or tissues [98,99]. This suggests a hierarchical organization in the mesodermal differentiation corresponding to the topographi- cal position of the prospective significance along the dorso-ventral axis within the trunk mesoderm. The proposal was made that the mesodermal cells at various dorso-ventral levels can be char- acterized by levels of a hypothetical physiological activity which decides the future developmental pathways, called “morphogenetic potential”, diminishing from the dorsal level toward the ventral level, and the effect of the dorsal meso- derm consists in the elevation of the potential in the responding cells [99]. Corresponding to the level of the potential attained at a critical stage, specific tissues can be expressed. Recent ex- periments of Slack and Forman [100] made on the presumptive mesoderm of Xenopus gastrulae showed that the model can be expanded to the gastrula stage. Now this speculative model conforms with the above cited results of Grunz [96] which show that a single factor interacting with ectodermal cells gives rise to various types of mesodermal differentiation, and the dorso- ventral sequence of these differentiations cor- responds to the intensity of the reaction. The fascinating aspect of the system, presumptive ectoderm treated with the vegetalizing factor, is that all representative tissues belonging to three germ layers of amphibian larvae can be generated by ectodermal cells under the influence of a single molecule, while without the factor these cells produce only ciliated epidermal cells and secretory sucker cells. The appearance of neural and mesectodermal tissues can be the indirect effects of the factor via induced mesodermal tissues. The appearance of mesodermal tissues may be the direct effect of the factor on presumptive ectoderm as will be discussed in the following paragraph. The endoderm is certainly induced directly by the factor, but the expression of tissues of the digestive system probably depends on an interaction of induced endoderm with the mesodermal tissues produced in the system [101-104]. That a network of specific interactions leading to differentiation of tissues and cell types which make up the whole normogenesis can be unleashed by a single molecule working on a developmentally homogeneous cell population presents a challenge of the first order. Some comments are necessary for the statement made in the last paragraph that the mesodermal tissues are directly produced by the vegetalizing factor. Earlier the suggestion was made that these mesodermal tissues are secondarily induced by an interaction between induced endoderm and the remaining ectoderm [105, 106]. The idea was defended by the experiment in which presumptive ectoderm treated with the Tiedemann’s factor was disaggregated into single cells for 20 hr and then reaggregated. The result indicated an inhibition of the formation of dorsal mesodermal tissues [106]. The authors interpreted that the inhibition of mesodermal differentiation was caused by sup- pression of the cell contact between prim induced endoderm and ectoderm. However. | details of reported results fit better with the | 340 T. YAMADA bility that the disaggregation suppressed the expression of dorsal mesodermal tissues which depends on contact among mesodermal cells, but it is neutral for the expression of ventral mesodermal tissue which is less dependent on such contact. Furthermore, the assumption of the secondary induction of mesodermal tissues con- tradicts with the results of series HI of Grunz’s experiments [96] in which a high percentage of dorsal mesodermal tissues appear in the absence of endoderm or differentiated endodermal tissues after the treatment of gastrula ectoderm with the Tiedemann’s factor. Guinea pig bone marrow which contains a factor similar to the Tiedemann’s factor in its morphogenetic effects [90] when applied to presumptive ectoderm gives rise to mesodermal tissue with (68%) or without (32%) accompanied endoderm or endodermal tissues [104]. The observation that the treatment of ectoderm with purified sample of the vegetalizing factor leads only to the production of endoderm [105] was originally interpreted to mean that the mesodermal induction obtained with the crude samples was due to a contamination or secondary induction. However, the new results of Grunz [96] indicate that meso- dermal tissues can be obtained with the factor, if the intensity of the treatment is adequately reduced. These two considerations seem to justify the discussion made in the last paragraph. Lithium ion is known to cause vegetalization in amphibian presumptive ectoderm [107-110] which is very similar to that produced by the Tiedemann’s factor. Flickinger et al. [111] showed that a lithium treatment inhibits DNA synthesis in ectoderm-+mesoderm of frog embryos. Ac- cording to Togashi and Asashima [112] the electrophoretic mobility of presumptive ectoderm cells is elevated by a treatment with lithium or with fish swimbladder in a comparable manner. It was shown that the latter tissue contains a strong vegetalizing factor [113]. Since in newt IECs the cell electrophoretic mobility is inversely correlated with the proliferative activity [72, 73], the data of Togashi and Asashima may suggest a reduction of proliferative activity by the lithium treatment. In summary the possibility is open that vegetalization of ectodermal! cells is associated with a reduction of their proliferative activity as already proposed by Flickinger [114]. In this respect it is interesting to note that lithium has significant effects, positive or negative, on the proliferation of mammalian cells, which are not shared by other ions [115, 116]. 6. GENERAL DISCUSSION The systems discussed above are selected on the basis of the insight they provide into the nature of the developmental control of cell differentiation. It is rather impressive to find that in most of those systems the cell cycle progression is proposed to play a dominant role in the remote control. Ex- amples of developmental control through the cell cycle progression are increasing in the literature. The control of head regeneration in hydra by the head activator seems to be another case [117, 118]. The head activator, one of the factors which modulate the regeneration pattern of hydra, is involved in the formation of the head. It is present in aconcentration gradient along the body axis with the maximum at the head. The factor has been partially purified as an acidic peptide of ca. 1,000 molecular weight. It has the property of growth factor. The interstitial cells which function as stem cells in this system are activated to enter into the cell cycle after cutting. In the presence of the head activator the stem cells produce nerve cells. Mainly on the basis of cell labelling studies it was proposed that the length of the G2 phase is decisive in the option of differentiating into nerve cells or remaining as stem cells. The head activator shortens the G2 phase of activated stem cells and channels them into the pathway of nerve cells. In hydra the G1 phase is insignificant. It has been reported that by applying different concentrations of retinoic acid to a teratocarcinoma cell line, expression of different cell types can be obtained [119]. The authors suggest that during nor- mogenesis the commitment of pluripotential cells to specific developmental pathways may be con- trolled by a gradient of retinoic acid or a similar factor. According to a number of studies, retinoids affect the transcription of messenger RNA for ornithine decarboxylase which is required for the G1 progression [120]. Hence it is conceivable that in the above experiment, a differential in the suppression of the cell cycle progression is the key Developmental Control of Cell Differentiation 341 for the appearance of different cell types whether operating on a restricted number of distinct cell lineages, or on single pluripotential cell lineage. Recently extensive sequence similarity has been found between the transforming protein derived from the Simian sarcoma virus onc gene p-sis and a human platelet-derived growth factor [121]. This finding implies that the basic event in carcinogenesis is enhanced proliferation in agreement with some of earlier opinions. The coincidence in the basic feature of the control mechanism indicated for carcinogenesis and that discussed here for deve- loping systems, especially for cell-type conversion appears meaningful and suggests a common fundamental principle. There exists one approach in reprogramming of cell type differentiation in mammalian cell lines, which seems to have some impact on the theme of the present paper. When C3H/10T3 CL8 mouse cells are treated with the nucleoside analog, 5 azacytidine or some other cytidine analogs, striated muscle cells and chondrocytes are formed, which do not appear in the untreated culture [122-124]. After incorporation of the analog, DNA strands are found undermethylated. The expression of muscle differentiation requires a latent period of 10-16 days during which active proliferation occurs. At least two mechanisms can be envi- sioned: (a) Some genes involved in the prolifera- tive activity are activated by undermethylation of DNA. A mechanism analogous to the model of IEC conversion (section 3) operates and the allotypic cell types are expressed. (b) The allo- typic differentiated state is directly determined by the undermethylation of DNA _ bases and perpetuated through proliferation by maintenance methylation. Certain conditions of cell density, contact or fusion should be required for the final expression of the allotypic cell types. Since DNA methylation does not occur in Drosophila and some other arthropods [125] and not all vertebrate genes seem to be controlled by methylation as originally proposed [126], the value of DNA methylation as a means of controlling the gene activity might be somewhat limited. How- ever, in many systems of developmental control of cell differentiation, DNA methylation may be playing a basic role in conjunction with the cell cycle progression. It is an attractive hypothesis that the requirement of DNA replication cycles for the expression of enzymes in ascidian development is associated with stepwise demethylation of specific DNA bases [23]. According to Eguchi and Watanabe [127] a treatment of lentectomized newt eye with N-methyl-N ’-nitro-N-nitrosoguanidine (MNNG) leads to formation of supernumerary lenses. This implies that some IECs in the autotypic pathway are shifted into the allotypic pathway by the treatment. Since MNNG is now shown to be an inhibitor of DNA methylation [128, 129], it is possible that the reprogramming effect of MNNG is caused by the undermethylation of certain DNA bases, like in the last cited experiment. Since, however, the supernumerary lenses were found to be produced by depigmented cells, it is likely that undermethylation of DNA bases alleged to occur by the treatment, activated some genes involved in proliferation, and the enhanced proliferation shifted the cells into the allotypic pathway, ac- cording to the model discussed in section 3. Concerning the organizer action in amphibian embryos, no general discussion will be attempted in this review. Only some of aspects related to the main theme of this paper will be briefly discussed. The facts that presumptive ectodermal cells of early gastrula can produce neural tissue after a brief treatment with extreme pH [130], and mesodermal and endodermal tissues after a treatment with lithium chloride [108, 109] argue for the assumption that these cells are able to produce by themselves all factors necessary for the im- mediate control of a large number of cell types belonging to three germ layers, if adequate con- ditions are given. This makes rather probable that also in normogenesis, inductive formation of those cell types does not require the introduction of any factors of the immediate control from outside. This means that the organizer action in normogenesis relies On a remote control. Con- cerning the properties of the remote control operating in neuralization (dorsalization) of pre- sumptive ectoderm nothing is definitely known. The role of cell surface is emphasized by a num’ of observations: Macromolecular neura factors retain their inductive effects when bo: 342 T. YAMADA Sepharose [92, 97]. The neural differentiation of ectodermal cells responding to dorsal mesoderm can be specifically inhibited by treating cells with lectins SBA and PSA [131]. On the other hand Concanavalin A elicits neuralization of competent ectodermal cells [132-134], and the effect can be cancelled by a-methyl-D-mannose. A study of incorporation of *H-thymidine into ectodermal DNA indicates that the treatment with this lectin temporarily reduces the incorporation rate, but subsequently keeps the rate higher than in the control. This result is in general agreement with the data on the mitotic index of ectodermal ex- plants cultured with or without a neural inductor [135]. These two results seem to suggest two possibilities: (1) The epidermal pathway is as- sociated with an early retreat from the cell cycle, and the shift into neural pathway (dorsalization) is possible by keeping the cells in the cell cycle during this phase. (2) The suppression of DNA replication during the period of neural competence reprogrammes the cells into the neural pathway. In connection with the second possibility it is important that the neural competence of pre- sumptive ectoderm has been found to be a function of the cell cycle progression [136]. The epidermal differentiation may depend on interaction of the epidermis-specific factors of immediate control with replicating DNA of the cell generation of neural competence. If after the competence period these epidermis-specific factors disappear, one should be able to avoid epidermal differentiation by delaying DNA replication. This would shift the cells into the neural pathway. Such pre- liminary discussion serves to show that a remote control of neuralization by the cell cycle progres- sion is within the realm of possibility. According to Flickinger [114] the role of cell division in determination in amphibian embryos is mediated by the control of the transcriptional pattern. Synthesis and accumulation of two classes of D-RNA with different developmental implications are controlled by cell division. One class of D-RNA is transcribed on redundant genes and thought to code for proteins involved in cel- lular maintenance. The other class is transcribed on less redundant genes which are involved in differentiation. The treatment with lithium chloride of prospective ectoderm causes a reduction in number and rate of cell division and allows transcription of more redundant genes, and accumulation of the corresponding class of D- RNA. This leads to formation of mesoderm and endoderm. On the contrary, when prospective ectoderm is neuralized by sodium bicarbonate, an increase in the number and rate of cell division occurs. This condition leads to transcription of less redundant genes and accumulation of the second class of D-RNA. This scheme is in general agreement with the discussion made in this review and suggests that the solution of the deve- lopmental control of cell differentiation may be found in the correlation between cell cycle progres- sion and transcriptional pattern. The role of proliferation in the remote control of cell type differentiation discussed in this review implies that proliferation affects the subsequent mode of differentiation by altering the available population of the factors of immediate control. Such effect of proliferation should not be, how- ever, generalized. There exist many cases where the differentiated state of cells can be maintained unexpressed through a number of cell generations and expressed with great fidelity, when adequate conditions are given. As one example of such perpetuation of the differentiative potential through repeated cell replications, Holtzer ef al. [12] studied reversible blocking of the expression of the differentiated state in myogenesis, chondrogenesis, and melanogenesis caused by treatments with TPA, RSV and BrdU. On the basis of their results, they propose that each specific cell lineage has a relatively stable structural arrangement of a set of specific genes related to the differentiated state, which can be transmitted through prolifer- ation without being expressed. If this is the case, in reprogramming of the differentiation pathway, such structural arrangement of genes should be the target of alteration. The arrangement should be modified or converted during the passage of reprogramming cell cycles. Such _ speculative model of maintenance and reprogramming of the differentiated state has a strong affinity with the idea that the gene activation pattern is controlled by the attachment of genes with the nuclear matrix [9]. This idea implies that the topological aspect Developmental Control of Cell Differentiation of chromatin plays a dominant role in the potenti- ality of gene expression. 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(1974) Mitotic activity and cell proliferation in primary induc- tion of newt embryo. Develop. Growth Differ., 16: 29-40. Suzuki, A. and Ikeda, K. (1979) Neural com- petence and cell lineage of gastrula ectoderm of newt embryo. Develop. Growth Differ., 21: 175-188. ZOOLOGICAL SCIENCE 1: 349-364 (1984) © 1984 Zoological Society of Japan REVIEW Experimental Studies of Mammalian Fertilization* M. C. CHANG Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, USA INTRODUCTION I started my research work on ram sperm under the direction of the late Dr. John Hammond and Dr. Arthur Walton in 1939 at the School of Agri- culture, Cambridge University. My work was mainly concerned with the oxygen uptake of ram sperm and the preservation of ram sperm for artificial insemination and I had a paper on the effects of low temperature and acclimatization on the respiration and survival of ram sperm pub- lished with A. Walton in the Proceedings of the Royal Society [1]. After I received my Ph. D. degree, in December 1941, due to the Second World War I was unable to leave England. Dr. A. Walton arranged a grant for me from the British Agriculture Research Council and I did some studies of sperm production of a pony stallion [2] and rams [3], fertilizing capacity of spermatozoa following cold treatment of the scrotal testes of rabbits [4] and artificial production of monstrosities in the rabbit [5]. During this period I realized that the study of fertilizing capac- ity of sperm was more important than that of the morphology, physiology and metabolism of sper- matozoa and planned to do more work on the fertilizing capacity of mammalian sperm. In order to do this we have to know more about the physiology of mammalian fertilization. At that Received October 24, 1983 * This paper was presented at the annual meeting of the Zoological Society of Japan, Ehime University, October 4, 1983 when the writer was invited as a Guest Scholar by the Faculty of Agriculture, Kyoto University. time what we knew about physiology of fertiliza- tion was mainly from the study of sea-urchin whose fertilization takes place outside of the body. Then I realized again that if we would like to know more about the physiology of fertilization in mammals, we ought to have a good procedure to fertilize mammalian eggs in vitro. In 1944, we knew that there were only a few scientists able to fertilize rabbit eggs in vitro, for instance Dr. G. Pincus [6] in America and Dr. J. Yamane [7] in Japan. Be- cause my work on the monstrosity of rabbit was close to a study by Pincus, I wrote to him in 1944 and asked him casually whether or not I could work in his laboratory for a year so that I could learn the techniques to fertilize rabbit eggs in a culture dish. It happened that he got a grant to work on egg transfer in cattle. He thought that I could be useful for this project and offered me a Fellowship in his newly established Worcester Foundation for Experimental Biology, at that time associated with Clark University, Worcester, Mass. From 1945 to 1980, besides my work on transplantation of rabbit eggs [8], oral contra- ceptives [9] and other studies, I was more con- cerned with studies of in vitro fertilization. This paper, without reference to my review papers, attempts to summarize the results of our studies of fertilizing capacity of sperm, fertili- zability of eggs, the in vitro fertilization of eggs in various mammals conducted especially by my Japanese associates in my laboratory, and other related observations. I must say at this occasio’ that by their hard work we have achieved a deal towards the understanding of mamr fertilization by simple biological experime: 350 FERTILIZING CAPACITY OF SPERMATOZOA I started my study of fertilization in vitro when I got to the Worcester Foundation for Experimen- tal Biology in the spring of 1945. I followed the procedures reported by Pincus, told by Pincus, including any ideas that came to my mind, and worked with Pincus on a few occasions, but I was unable to obtain rabbit eggs fertilized in vitro. Only on one occasion, in August 1945, did I find a few rabbit eggs cleaved in egg clots after incubation for one day with washed ejaculated sperm in a Carrel flask. Then I went to Washington D. C. for a holiday. When I came back, I could not repeat the same experiment. But from 1945 to 1950 I did many experiments to study the fertilizing capacity of rabbit spermatozoa as follows: Inspired by a study of the relation between ‘density’ of sperm suspension and fertility after artificial insemination of rabbits by my supervisor, A. Walton [10], and following the observation that the epididymal sperm disintegrated by application of ice to the scrotal testis of rabbits [11], the impor- tance of sperm number and the functional integrity of spermatozoa, rather than the morphology of spermatozoa, in relation to the probability of fertilization, were reported [12]. The effect of dilution of sperm [13], lack of effect of hyaluroni- dase and seminal plasma on the fertilizing capacity of rabbit spermatozoa [14, 15], the spermicidal effect of various sera on sperm of different species [16], the effects of heterologous seminal plasma and sperm cells on fertilizing capacity of rabbit sper- matozoa [17] and the harmful effect of blood sera from some species [18] and that of seminal plasma of different species on the fertilized rabbit eggs [19] were described. The fertility and sterility as revealed in a study of fertilization and development of rabbit eggs [20] and no effect of phosphorylated hesperidin on fertilizing capacity of rabbit sperm [21] were reported. In a study of effects of radiocobalt irradiation of rabbit sperm on fertilization and early develop- ment, it was found that the motility of sperm was damaged at very high doses of irradiation, 32,000- 65,000 r, with little damage to their fertilizing capacity but the development of fertilized eggs was M. C. CHANG impaired after irradiation of sperm at 800r [22]. In a study of frozen rabbit sperm to —79°C, it was found that the motility and fertilizing capacity were better protected in the presence of dimethyl sulfoxide (DMSO) than glycerol during freezing and thawing [23]. The motility and metabolism of sperm in the presence of accessory secretions and calcium were reported by Morita and Chang [24-26]. Although the fertilizing life of sperm in the female tract is about 20 hr in many mammalian species, it was found that by uterine insemination of ferrets before ovulation, the fertilizing life of ferret sperm could last for 126 hr [27]. By the same method, it was reported that the fertilizing life of mouse sperm in the female tract lasted about 12 hr [28] rather than 6 hr as reported by other investigators and that of golden hamster sperm lasted about 10 hr [29]. The fertilizing life of rat sperm in culture, in the mature and immature female tract lasted respectively for 10, 18 to 20 and 10 to 14hr while the fertile life of rat egg lasted about 9 hr after ovulation [30, 31]. The fertilizing capacity of rabbit epididymal sperm was not increased by suspension of sperm in whole rabbit seminal plasma as compared with those suspended in Ringer or Tyrode solution [32] and the treatment of hamster sperm with deoxyri- bonuclease I significantly depressed the fertilizing capacity of hamster sperm and caused a delayed fertilization [33]. The fertilizing capacity of hamster sperm, however, was inhibited by treat- ment with synthetic protease inhibitors but not by the natural trypsin inhibitors [34]. In a series of studies of fertilizing capacity of hamster spermatozoa [35-41], it was respectively reported that: (1) Hamster epididymal sperm lost their fertilizing capacity 12 days after castration and their fertilizing capacity could be maintained for 12 days by daily injections of 10 yg of testos- terone or 1 mg of dehydroepiandrosterone but not by 1 mg progesterone. (2) Injection of 66 mg/kg a-chlorohydrin to the male hamsters induced sterility within 4 days as shown by the insemination of epididymal sperm of treated animals and the infertility was not related to deficiency of andro- gen. (3) Daily injections of 37mg AY-—22,352 induced sterility in the male hamster within 4 days Experimental Studies of Mammalian Fertilization 351 and a marked loss of fertilizing capacity occurred by 24hr after the second injection. (4) The fertilizing life of mouse and hamster sperm in the cauda epididymis lasted for 25 days and the number of sperm decreased sooner in the hamster than in the mouse but the initial decrease of fertilizing capacity occurred much earlier in the mouse than in the hamster. (5) Treatment of hamsters with prostaglandin however did not affect the fertilizing ability of spermatozoa. (6) When silastic tubes containing estradiol were implanted into the seminal vesicles or implanted subcutaneously in the male hamsters, fertilizing capacity of sperm was lost 12 days later but was regained 10 weeks after the removal of estrogen. (7) Sperm in the vas deferens of hamsters lost their fertilizing capacity 3 days after ligation of the initial part of the duct and 2 days if the testes were removed at the time of ligation; unilateral castration for 3 days had no effect on the fertilizing capacity of sperm from the ipsilateral unobstructed duct, whereas sperm from the contralateral ligated duct associated with the remaining testis were unable to fertilize eggs. Moreover it was found that daily injections of estriol for 12-14 days also caused sterility of male rats and mice [42]. The fertilizing capacity of sperm from rats and mice treated with antifertility drugs was examined for in vivo and in vitro fertilization [43, 44]. It was found that injection of a-chlorohydrin 4.4 mg/kg/day for 4 days could inhibit fertilizing capacity of rat sperm as tested in vitro and in vivo while it required 44.2 mg/kg/day for 4 days to inhibit the fertilizing capacity of mouse spermatozoa; injection of two related com- pounds, CL-88,236 or AY-22,352 again revealed the insensitivity of mice to these drugs but 10 mg/ kg/day of AY—22,352 could reduce significantly the fertilizing capacity of mouse sperm in vivo and in vitro. Finally it was reported that oral administra- tion of Gossypol could induce sterility of male rats and hamster at a dose level of 5 to 10 mg/kg daily for 12 days but had no effect on rabbits at the same dose level for 5 to 14 weeks [45]. FERTILIZABILITY OF MAMMALIAN EGGS By artificial insemination of rabbit at various times after ovulation it was found by my supervisor Sir John Hammond [46] that the fertile life of rabbit eggs lasted about 6 hr after ovulation. By transferring rabbit eggs recovered at various times after ovulation into the oviducts of mated rabbits I [47] found that rabbit eggs were capable of fertilization up to 8hr after ovulation but their fertilizability dropped sharply 4 hr after ovula- tion. When the newly ovulated rabbit eggs were treated in vitro at 38°C for 6-12 hr, 24°C for 12- 24hr, 10°C for 48-72 hr and at 0°C for 24-48 hr, some of them were still fertilizable. When unfer- tilized eggs kept at 0°-10°C for 24hr and trans- ferred into the oviducts of unmated rabbits, cleavage up to 8-32 cells was obtained. The fertilization of rabbit eggs after storage at O° or 10°C for one day, as well as the probability of subsequent development [48] and the development of parthenogenetic rabbit blastocysts with deploid chromosomes induced by low temperature storage of unfertilized rabbit eggs [49] were reported. In a study of the maturation of ovarian oocytes in culture and their maturation, activation, fertiliza- tion and subsequent development in the oviducts I [50] found that the maturation of oocytes as a whole is more important than nuclear maturation. The fertilization and normal development of follicular oocytes after transfer into the oviducts of mated rabbits [51] and the fertilization and degen- eration of eggs after transfer to the uterus of mated rabbits [52] were described. Later study of transferring eggs into the uterus of mated animals or injection of hamster eggs with hamster sperm into the uterus showed either no fertilization [53] or a few fertilized and degenerated eggs [54]. The mammalian fertilization and the possibility of its control were reported at the Sth International Congress of Planned Parenthood in Tokyo [55]. Subsequently by uterine insemination of golden hamster at various times before or after ovulation it was found that the fertilizable life of hamster eggs after ovulation lasted about 9hr. The cortical granules were significantly decreased at 6 hr after ovulation and after losing fertilizability, initial activation of eggs, such as the extrusion of the second polar body and the formation of one or two pronuclei, was observed [56]. Following delayed insemination, the fertil life of mouse eggs after ovulation was foun: 352 M. C. CHANG about 15 hr [57], longer than previously reported. By in vitro fertilization of mouse eggs recovered at various times after ovulation, it was found that the fertile life of mouse eggs was about 10-12 hr after ovulation [58]. This indicates that the manipulation of eggs in vitro may cause their loss of fertilizability sooner. The similar size of the male and female pronu- cleus in newly fertilized sheep eggs [59], the presence of succinic dehydrogenase in the hamster and rabbit eggs [60], the morphology and timing of fertilization in the Mongolian gerbil and deer mouse [61] and the fertilization and early devel- opment of Chinese hamsters [62] were reported. Attempt to induce polyspermy in the rabbit by delayed insemination and treatment of females with progesterone was unsuccessful because the presence of three pronuclei in the egg was most likely due to the formation of a pronucleus from the second polar body or to the fragmentation of the female pronucleus [63]. The effects of radiocobalt irradiation of unfer- tilized rabbit eggs in vitro on their fertilization and subsequent development in vivo were investigated. It was found that the proportion of fertilized eggs decreased to 48% after irradiation at 32,000r, but the proportion of normal blastocysts and the embryonic development decreased to 3% and to 0% respectively after irradiation at 800r [64]. The effects of irradiation of gametes and zygotes on the embryonic development of rabbits and hamsters were summarized [65] and the morphological changes of sperm head in the ooplasm of mouse, rat, hamster and rabbit were described [66]. After the removal of the corona radiata from rabbit eggs and transferring them into the oviducts of mated rabbit, it was found that cumulus oophorus does not play a part in fertilization but that corona radiata plays an important role in protecting the fertilizability of eggs [67]. CAPACITATION OF RABBIT SPERMATOZOA IN THE FEMALE TRACT AND FERTILIZATION IN VITRO OF RABBIT EGGS In an article entitled ‘Fertilizing capacity of rabbit spermatozoa deposited into the follopian tube’, I [68] concluded that “‘it is quite clear that fertilization occurs when the spermatozoa have been in the tube for six hours before ovulation, which is perhaps the time required for a physio- logical change in the spermatozoa enabling them to attain fertilizing capacity.’ Dr. C. R. Austin [69] also had similar observations published in an Australian journal. Based upon these two reports and his observations on the penetration of rat eggs examined at various times after mating, Austin [70] introduced the term ‘capacitation’ in the literature of reproductive biology and concluded that “the sperm must undergo some form of physiological change or capacitation before it is capable of penetrating the egg”. The capacitation of rabbit sperm in the uterus [71], the ““decapacita- tion” of rabbit uterine sperm treated with seminal plasma of rabbit, human and bull and the “‘reca- pacitation”’ of treated rabbit sperm deposited into the oviducts [72] and the removal of decapacitation factor from seminal plasma by high-speed centrifu- gation [73] were described. Capacitation of rabbit sperm in the oviduct and in the uterus [74], the increase of oxygen uptake and glycolytic activity of rabbit sperm after uterine incubation [75], the disturbance of egg transport and sperm capacita- tion in the pseudopregnant and progestin-treated rabbits [76, 77], and finally the fertilization of denuded rabbit eggs in vitro by sperm recovered from the uterus or the vagina [78] were reported. The fertilization of rabbit eggs by tubal insemina- tion of uterine capacitated sperm was shown to depend on the Ca** level which was not implicated in decapacitation [79]. Based on the discovery of sperm capacitation in the female tract, Thibault et al. [80] of France reported the cytological evidences of fertilization of rabbit in vitro. Finally the production of young, genetically true to their parents, following the transfer of eggs fertilized in vitro by sperm re- covered from the uterus [81, 82] was described. Although the possibility of capacitation of rabbit sperm in vitro was reported by many investigators and appeared to be difficult, finally it was achieved according to a procedure of washing sperm twice and incubating for 10 hr by my former associate Niwa and his associates [83]. Experimental Studies of Mammalian Fertilization 353 FERTILIZATION OF HAMSTER EGGS IN VITRO Following the fertilization of rabbit eggs in vitro, the first successful in vitro fertilization of golden hamster eggs was conducted in my laboratory by Yanagimachi [84, 85]. By semination of newly ovulated eggs in a tissue culture medium 199 containing glycine covered with mineral oil, it was observed that about 60% of eggs were fertilized by sperm recovered from the uterus 4-5 hr after mating, 30-50 °%% by uterine sperm recovered 0.5 hr after mating and 17% by epididymal sperm. A\l- though the rate of fertilization was low by epi- didymal sperm, the possibility of capacitation of epididymal sperm in culture without the partici- pation of the female tract and biological secretions was well demonstrated. The process and time of sperm penetration in vivo and in vitro were de- scribed by Yanagimachi [86] who observed that the sperm from uterus penetrated eggs earlier than those from the epididymis; the acrosome was absent before the penetration of the zona pellucida; the time required for sperm head to traverse the zona pellucida and the perivitelline space was 3-4 min and 1-2 sec, respectively. Using a movie camera to examine tubal eggs after delayed mating Yang ef al. [87] observed that it required about 7 min for the hamster sperm to pass through the zona pellucida. The capacitation of hamster sperm treated with rabbit tubal fluid and bovine tubal cystic fluids was described by Iwamatsu and Chang [88]. Subsequent studies by Yanagimachi and other investigators have shown that capacitation of hamster sperm and fertiliza- tion in vitro of hamster eggs can be achieved in media containing follicular, tubal fluids, or blood sera and finally only in the presence of bovine serum albumin. In a modified Krebs-Ringer bicarbonate solution supplemented with bovine serum albumin for mouse gametes and a modified Tyrode’s solution with heated hamster serum for hamster gametes, it was found that the optimal osmolarity was between 308 and 372 mOsm for the fertilization of mouse eggs, between 292 and 392 mOsm for hamster eggs [89]. Obviously hamster gametes could withstand the variation of osmolality better than the mouse gametes. In similar media for mouse and hamster gametes and in a modified Krebs-Ringer bicarbonate solution with heated rat blood serum, the optimal pH value for in vitro fertilization of mouse, hamster and rat eggs was, respectively, 7.3-7.7, 6.8-8.2 and 7.8 [90]. It seems that in vitro fertilization of rat eggs requires a narrow range of pH higher than 7, while hamster and mouse eggs can withstand a relatively wide range of pH. In a study of in vitro fertilization of hamster eggs [91], it was found that the proportions of eggs fertilized were sig- nificantly different in different media and the fertilizing capacity of hamster spermatozoa was maintained for 12 hr during incubation with frozen guinea pig spermatozoa. In vitro fer- tilization of hamster eggs by ejaculated or epididy- mal sperm in the presence of seminal plasma or male accessory gland secretions was examined [92]. It was observed that there was no difference in the penetration rates, the time of sperm penetration and the optimal sperm concentration between the ejaculated and epididymal spermatozoa; the swelling of zona pellucida, a high incidence of polyspermy and degeneration of eggs were observed in the presence of accessory gland secretions; fertilization by epididymal sperm was inhibited in the presence of seminal vesicle and ventral prostate secretions but not in the presence of coagulating gland and dorsal prostate secretions. Fertilization of Chinese hamster (Cricetulus griseus) eggs in vitro was described by Pickworth and Chang [93] who pre-incubated sperm from the vas deferens of Chinese hamster in a medium containing 1% bovine serum for 2—3 hr and then added Syrian and Chinese hamster eggs in clots. Chinese hamster eggs recovered from oviducts and follicles were also added to the pre-incubated sperm suspensions and incubated for 5 to 8hr at 37-38°C under mineral oil equilibrated with 5% CO: in air. She found that 22% and 10% of Chinese hamster oviductal eggs were respectively fertilized in the medium with and without bovine serum but no sperm tail got into the egg; no follicular eggs were fertilized and no Syrian hamster eggs were attached and penetrated by Chinese hamster spermatozoa. It should be pointed here that this was the first experiment to intro crystalline bovine serum albumin in the m 354 M. C. CHANG for in vitro fertilization at a time when workers in this field were using animal fluid, such as follicular fluid and sera to facilitate fertilization in vitro. STUDIES OF FERTILIZATION OF MOUSE EGGS IN VIVO AND IN VITRO From the in vivo study of fertilization, the meiosis of mouse eggs before and after sperm penetration, the inhibition of fertilization and the production of heteroploidy in eggs of mice treated with colchicine were described [94, 95]. Soon after the first successful in vitro fertilization of mouse eggs by capacitated sperm recovered from the uterus of mated mice [96], in vitro fer- tilization of mouse eggs by epididymal sperm in the presence of heated bovine follicular fluid was reported by Iwamatsu and Chang [97] who ob- served that after incubation of egg clots with epididymal sperm at 37°C in an atmosphere of 5% carbon dioxide and 95% oxygen for 1-6hr the pattern of sperm motility changed and the acro- some on the sperm head disappeared; while the penetrated eggs was increased to 37% 4hr after semination and 2 hr was required for the capacita- tion of mouse sperm in vitro. Further investiga- tion of in vitro fertilization of mouse eggs by Iwamatsu and Chang [58, 98, 99] revealed respec- tively that: (1) The proportion of penetrated eggs increased from 13% at 2hr to 67% at 8hr after semination with fresh epididymal sperm in the presence of bovine follicular fluid but the propor- tion of penetrated eggs increased from 17% at 20 min to 90% at 2hr after semination with sperm pre-incubated for 3-4 hr, showing the capacitation of mouse sperm in vitro. (2) The fertile life of mouse eggs lasted about 10-12 hr after ovulation and the failure of transformation of sperm head and activation of eggs occurred in the aged eggs; although many sperm could pass through the zona pellucida, a vitelline block pre- vented no more than 4 sperm to enter the vitellus. (3) The mouse oocytes recovered from the follicles 4hr before ovulation could be fertilized, but their zona reaction to block further sperm entry was weaker than those recovered 2 hr before or at various times after ovulation; sperm capacitation and penetration in vitro could be influenced by the sperm and eggs from different strains of mouse and by the concentration of calcium ions and the pH value of media; in vitro sperm penetration through the zona pellucida in oocytes at any stage of maturation was observed but the proportion of penetrated oocytes increased from the breakdown of germinal vesicle stage to telophase I, showing the acquisition of fertilizability just before, or at the time of, completion of the first maturation division. Before 1971, animal fluids, such as follicular fluid or heated sera, were introduced into the media for the capacitation of sperm and fertiliza- tion of eggs. After he left my laboratory and returned to Japan, Toyoda and his associates [100] reported that mouse eggs could be fertilized in vitro in a medium containing bovine serum albu- min and sodium pyruvate without any biological fluid. Using a medium containing bovine serum albumin, sodium pyruvate and sodium lactate, it was found that after the removal of follicular cells attached to the eggs by hyaluronidase, the propor- tion of penetrated eggs was decreased in the mouse eggs but not in the hamster eggs [101]. This may indicate that after the removal of follicular cells and corona radiata, the mouse eggs lose their fertilizability faster than do hamster eggs. Further study showed that about 10% eggs either fertilized in vitro or in vivo could develop into blastocysts in culture but only 13% and 16% of two-cell eggs fertilized respectively in vitro and in vivo developed into normal young after transferring them to the oviducts of pseudopregnant mice [102]. The importance of sodium pyruvate, sodium lactate and serum albumin for the capacitation of mouse sperm and fertilization of mouse eggs in a modified Krebs-Ringer solution containing glucose was examined [103]. It was found that without any one of these compounds in the medium, about 8-10% motile spermatozoa lost their acrosome but no egg was penetrated after incubation for 5—7 hr. Addition of sodium pyruvate increased the pro- portion of motile spermatozoa without acrosome (19-34%) and the proportion of fertilized eggs (3.2-25.5%). Incubation of spermatozoa and eggs in the medium containing serum albumin of various species caused a further increase of the proportion of motile spermatozoa without acro- Experimental Studies of Mammalian Fertilization 355 some (50-65%) and that of fertilized eggs (60— 86%). The best medium for sperm capacitation and fertilization was the basic medium containing bovine serum albumin, sodium lactate and sodium pyruvate (97.6% fertilization). In a study of fertilization of mouse eggs in vitro [104-106], it was respectively concluded that: (1) Treatment of eggs with or without wheat germ agglutinin or deposition of wheat germ agglutinin into the ovarian bursa affected the vitellus and in- hibited fertilization. (2) There were no differences in the proportion of eggs fertilized or in their stages of fertilization between the young and aged ham- sters or mice. (3) Sperm from CD-1 and C3H males were capable to fertilize large numbers of eggs (73-85%) from CD-1, C3H and CS7BL/6 females but sperm from C57BL/6 males were able to fertilize only small numbers of eggs (14-30%) from C57BL/6, CD-—1 and C3H females. In a study of in vitro fertilization of mouse eggs [107] it was found that the optimal sperm concen- tration was about 10 to 63 x 10* sperm/ml and the minima! number required to fertilize a majority of mouse eggs was about 100 to 840 spermatozoa, which was much lower than that required for the fertilization of rat eggs, 3,000 to 6,000 [108]. IN VITRO FERTILIZATION OF RAT EGGS Early studies of in vitro fertilization of rat eggs revealed that in vitro penetration of rat eggs by either uterine or epididymal sperm failed, but after the dissolution of zona pellucida by treatment of eggs with 0.02°% chymotrypsin for 15 to 30 min in vitro, penetration of rat eggs was possible either by uterine or epididymal sperm [109]. The successful in vitro fertilization of rat eggs by uterine sperm in a Krebs-Ringer bicarbonate solution containing crystalline bovine serum albu- min was reported by Miyamoto and Chang [110, 111] who found that the presence of bovine serum albumin or rat serum in the medium appeared to be very important for in vitro fertilization of rat eggs by uterine sperm but no eggs were fertilized by pre-incubated epididymal sperm. The pro- portions of fertilized eggs were higher when sperm were recovered 4-Shr after mating (85-95%) than those recovered either 0.5-1 hr (4-7%) or 10-11 hr after mating (32-55%), showing that capacitation of rat sperm in the uterus required about 5 hr and the deterioration of uterine sperm 10 hr after mating. Finally the fertilization of rat eggs in vitro by epididymal sperm in a modified Krebs-Ringer bicarbonate solution supplemented with glucose, pyruvate, lactate, bovine serum albumin and antibiotics incubated in 0.4 ml medium under oil at 37°C in a gas phase of 5% CO, in air was achieved by Toyoda and Chang [112, 113]. They reported that sperm penetration through the zona pellucida started 5hr after semination and the transformation of sperm head into the male pronucleus occurred about 2 hr later; the percentage of eggs undergoing fertiliza- tion was 88% 7-20hr after semination and cleavage of fertilized eggs occurred 30 hr after semination; following the transfer of fertilized eggs to recipient pseudopregnant rats, 43 normal young were obtained. The capacitation of epididymal sperm in a medium with high K/Na ratio and cyclic AMP for in vitro fertilization of rat eggs was also described. In a series of investigations of in vitro fertilization of rat eggs [108, 114-116] it was stated respectively that: (1) The superovulated eggs from immature rats were much easier to fertilize than those from mature rats but the optimal concentration of epididymal sperm (0.7-1.5 10° sperm/ml) from mature rats also played an important role. (2) When epididymal sperm were pre-incubated in a diluted form, capacitation of sperm was achieved in 3—5 hr, the penetration rates were increased and the interval between semination and sperm penetra- tion was reduced; the rate of fertilization was higher when incubated with 5% CO, in air (77%) than in air alone (24%); in a smaller than a large droplet, in the presence of (21%) than in the absence of follicular cells (6%). (3) The effect of low sperm concentration on the capacitation of rat sperm in vitro was described. (4) The optimal sperm concentration for in vitro fertilization of rat eggs was 0.16 to 0.51 x 10°/ml and the minimal number of sperm was about 3,000 to 6,000. In another series of studies of in vitro fertilizatio: rat eggs [117-119] it was found respectively tha (1) Follicular oocytes could be penetrated on) 356 M. C. CHANG at a stage from the condensed germinal vesicle to metaphase I, but the failure of formation of male pronucleus and the abnormality of eggs were ob- served in the penetrated young oocytes after culture and in the aged eggs recovered from oviducts 14 to 20hr after ovulation. (2) The penetration of the zona-free rat eggs also required the capacita- tion of spermatozoa. (3) The pretreatment of rat sperm with high concentration of NaCl in the medium did not facilitate the capacitation of rat sperm and the fertilization of rat eggs. In another study of in vitro fertilization of rat and mouse egg by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs [120], it was concluded that the penetration rate by ejaculated sperm was lower in the rat (0 to 8 %) than in mouse eggs (11 to 41%); although the optimal sperm concentration for in vitro fertilization was similar by ejaculated or epididymal spermatozoa, the time of sperm penetration in mouse eggs was delayed by semination with ejaculated sperm; although lac- tate, pyruvate and glucose were all important for the in vitro fertilization of rat eggs, pyruvate could be supplied by the follicular cells surrounding the egg. REMARKS ON IN VITRO FERTILI- ZATION OF HUMAN EGGS Our biological studies of mammalian fertilization in vitro was aimed at a better understanding of various aspects of fertilization and the improvement of animal breeding. Since the delivery of a normal girl in England in 1978 after the transplantation of a human egg fertilized in vitro for a sterility case, the in vitro fertilization of human eggs suddenly became fashionable and was conducted throughout the world. It was thought appropriate on this occasion to pass a few remarks on in vitro fertiliza- tion of human eggs. With reference to our work [85] and that of Barros and Austin [121] on in vitro fertilization of hamster eggs, the first authentic in vitro fertilization of human eggs was reported by Edwards et al. [122] who stated that ‘‘Our impres- sion is that this pre-incubation (of sperm) led to the attachment of more spermatozoa to the zona pellucida, and to a higher incidence of penetrated and pronucleate eggs.””> My good friends Austin and his associates [123] estimated that the time required for capacitation of human sperm in vitro was about 7 hr, which was much more than the present estimation. Based on our experience and described in this article, it seems that it requires about 1 to 2 hr for the hamster and mouse sperm to be capacitated in the uterus and in vitro, thus in vitro fertilization of hamster and mouse egg is relatively easier. The capacitation of rat sperm in the uterus and in vitro took about 5 to 6hr, while that of rabbit sperm in the uterus took about 6hr and in vitro took about 10hr. Thus the in vitro fertilization of rat and rabbit eggs is more difficult. I do not have much experience with human eggs, except that in 1945 with my good friend Dr. E. Finkle, we put one or two human oocytes with human sperm into the rabbit oviduct in the laboratory of Dr. John Rock of Harvard University. Dr. Yanagimachi [124], who worked on human eggs in Australia, had informed me that human eggs are the easiest to fertilize in vitro; the problem is perhaps the procedure to transfer the fertilized human eggs. Then it was not nice of Edwards [125] to call me a respected Chinaman, but I will not call him a swashbuckling Briton. CROSS FERTILIZATION IN VIVO AND IN VITRO Cross fertilization between different mammalian species was first conducted by artificial insemina- tion of domestic rabbits (Oryctolagus cuniculus) with cottontail rabbit (Sylvilagus floridanus) sperm. It was found that about 39% of domestic rabbit eggs could be fertilized by sperm from cottontail rabbits, but the fertilized eggs degenerated before or at blastocyst formation [126, 127]. When domestic rabbits were inseminated with hare (Lepus timidus or L. europaeus) sperm, all eggs were fertilized but they failed to cleave beyond the early blastocyst stage [128]. Reciprocal fertili- zation between the domestic rabbit (Oryctolagus cuniculus) and snowshoe hare (Lepus americanus) by artificial insemination was conducted. It was found that 96% of rabbit eggs could be fertilized by snowshoe hare sperm, and the fertilized eggs degenerated during cleavage and blastocyst forma- tion, but only one of nine eggs from 10 snowshoe Experimental Studies of Mammalian Fertilization 357 hares might have been fertilized by rabbit sperm. After insemination of 12 rabbits with a mixture of equal numbers of rabbit and snowshoe hare sperm, 66 % of eggs were fertilized by hare sperm and 34% fertilized by rabbit sperm [129]. The ferret eggs could be fertilized by mink sperm by uterine in- semination, but the hybrid embryos, implanted without delay, degenerated within 22 days [130]. The mink eggs, however, could not be fertilized by ferret sperm; although ferret blastocysts could develop in the mink uterus for a few days without implantation, while mink blastocysts could implant without delay in the ferret uterus but degenerated later [131]. By uterine insemination the reciprocal fertilization between ferret (Mustela furo) and weasel (M. erminea) was possible and the ferret eggs fertilized by weasel sperm implanted without delay, but only one living hybrid young was ob- tained 42 days after insemination [132]. The reciprocal fertilization following mating between two species of deermice (P. maniculatus and P. polionotus) was possible, but failure of implantation was observed [133]. Reciprocal fertilization in vitro between these two species of deermice was possible, and their respective sperm could pene- trate the zona pellucida, but they were unable to fertilize domestic mouse eggs in vitro [134]. FERTILIZATION OF ZONA-FREE EGGS IN VITRO In vitro penetration of zona-free eggs by sperm of different species was investigated [135-137]. It was observed that very few mouse eggs were penetrated by rat or hamster sperm, rat eggs by hamster sperm, hamster eggs by rat sperm and the transformation of sperm head into pronucleus as well as the formation of female pronucleus failed in these cases; but very high proportions of zona- free rat and hamster eggs were penetrated by mouse sperm and the formation of male and female pro- nuclei occurred normally. The penetration of hamster and rabbit zona-free eggs by capacitated rat and mouse spermatozoa was shown by the enlargement of the sperm head but activation of eggs failed. The zona-free hamster eggs could be penetrated by sperm of six species, including guinea-pig, deermouse and rabbit spermatozoa, but zona-free rat, mouse and rabbit eggs could not be penetrated by guinea-pig sperm and zona-free rat and mouse eggs could not be penetrated by sperm from two species of deermice and Mongolian gerbil; in vitro fertilization of intact prairie deer- mouse (P. maniculatus) eggs was possible but not possible for the white-footed mouse (P. leucopus) eggs. In a study of relationship between sperm con- centration and polyspermy in intact and zona-free mouse eggs [138] it was observed that high pro- portions of eggs examined | or 4hr after incuba- tion with pre-incubated sperm were undergoing fertilization in both the intact and zona-free eggs; the average number of sperm attached to the zona pellucida or vitellus was slightly higher in the presence of a higher concentration of sperm; polyspermy was increased from 25 to 200 10° sperm/ml, but no clear correlation between the incidence of polyspermy and further increase of sperm concentration in both the intact and zona- free eggs; there were a functional zona reaction and a vitelline block formed in a short time to prevent further entry of sperm. The time of sperm attach- ment and cortical granule breakdown in intact and zona-free mouse and hamster eggs were examined at various times after semination with capacitated sperm by a vital staining technique [139]. It was observed that the breakdown of cortical granules after attachment of homologous sperm occurred sooner in the hamster eggs than in the mouse eggs but occurred much slower in hamster eggs exposed to mouse sperm, although more mouse sperm attached to and penetrate the zona-free hamster eggs. FERTILIZATION OF FROZEN- THAWED EGGS I have long been interested in the storage of eggs in low temperature for transplantation [140] and tried to freeze fertilized rabbit eggs suspended in serum containing fructose slowly or rapidly frozen to —65°C without success [141]. After the dis- covery of deep freezing sperm in the presence of glycerol by my good friends Audry Smith Chris Polge [142], the freezing of sperm an tilized eggs now became a common practice 358 M. C. CHANG In vitro fertilization of mouse and hamster eggs after freezing and thawing was first conducted in our laboratory [143]. It was observed that about 10% of the mouse eggs and 80% hamster eggs appeared to be normal after having been stored at —70°C and —196°C in the presence of dimethyl sulfoxide (DMSO), while about 90% normal hamster eggs and 10% normal mouse eggs could be fertilized in vitro. By improvement of the freezing procedures it was found that unfertilized mouse eggs suspended in a medium containing 1.5M DMSO could be cooled at 0.33°C/min to —45°C, then at 1°C/min to —75°C with good recovery and the normal eggs could be fertilized in vitro and developed into blastocysts in culture or into normal young after transferred to recipient mothers [144]. The unfertilized eggs of mouse, rat, golden hamster and Mongolian gerbil were cooled to —75°C at different rates of cooling [145]. It was observed that the best cooling rate for the mouse and rat eggs was from —4°C to —45°C at 0.33°C/min followed by 1°C/min from —45°C to —75°C; hamster eggs could withstand a wide variation in the rate of cooling; Mongolian gerbil eggs withstood cooling poorly and the eggs from mature mice and rat withstood freezing better than those from immature females. Finally the fer- tilization in vitro of rat ovarian oocytes after freez- ing and thawing was reported by Kasai et al. [146]. IMMUNOLOGICAL STUDY OF FERTILIZATION Immunological studies of fertilization was started when Tsunoda came to my laboratory in 1975. In a series of studies by Tsunoda and Chang [147-154], it was reported respectively that: (1) Without the effect of ovulation and the number of eggs released, the fertilization rates following mating were significantly reduced in the female mice after injection of homologous sper- matozoa, testis with epididymis and ovary but in the female rat only after injection of ovarian homogenate. When the mouse eggs were in- seminated in vitro, the fertilization rate was very low after injection of egg donors with ovarian homogenate, better with testis and epididymis and best with sperm suspension. (2) Unabsorbed anti-hamster ovarian serum from rabbits showed 8-9 precipitate bands with hamster ovary, 3 bands with rat and one band with mouse ovarian extracts. Treatment of hamster and mouse eggs with anti- ovarian serum blocked in vitro fertilization of these eggs due to the formation of precipitate on the zona pellucida. A single injection of anti- hamster ovarian serum inhibits in vivo fertilization in mice but not in rats. (3) Anti-rat ovarian serum from rabbits significantly inhibited fer- tilization of rat, mouse and hamster eggs in vitro as compared with normal serum, and fertilization also failed in many rat eggs without a precipitate on their zona pellucida. The fertilization rate was significantly depressed following uterine in- semination of rat sperm exposed to anti-rat Ovarian serum. A _ single injection of anti-rat ovarian serum to females inhibited fertilization in vivo of rat and mouse eggs more severely than hamster eggs. (4) Fertilization of mouse, rat and hamster eggs in vivo could be inhibited after a single intraperitoneal injection of respective anti- ovarian serum from rabbits. These anti-ovarian sera absorbed with ovary did not prevent fertiliza- tion, but absorbed with the liver and kidney in- hibited fertilization in mice and hamsters but not in rats. Fertilization in rats and mice however was not inhibited by a single injection of anti- Ovarian serum obtained from isoimmunized rats and mice respectively, but in vitro fertilization of mouse eggs was inhibited in the presence of isoimmune anti-mouse ovarian serum. (5) In vitro fertilization of mouse eggs was inhibited in the presence of unabsorbed and absorbed anti- mouse ovarian serum from rabbits with liver and kidney but not in the presence of such antiserum absorbed with the ovary. A single injection of females with such antiserum inhibited fertilization in mice but not in rats and hamsters. Uterine insemination with sperm exposed to anti-rat or anti-hamster sperm serum inhibited fertilization in rats, mice and hamsters, while anti-mouse testis and epididymis serum only inhibited fertilization in mice. Following an intraperitoneal injection of anti-ovarian serum from rabbit, the fertilizability of intact mouse eggs was suppressed in vitro but the penetration of the zona-free mouse eggs was not affected. The fertilizing capacity of mouse Experimental Studies of Mammalian Fertilization 359 sperm recovered from the uterus of injected females was affected more by injection of anti-testis and epididymis than by injection of anti-ovarian serum. (6) Treatment of denuded eggs with either anti- mouse egg or anti-zona serum caused a more pronounced precipitate on the zona pellucida of the mouse eggs than on that of rat or hamster eggs; the fertilization of mouse eggs in vitro was com- pletely inhibited by unabsorbed anti-egg or anti- zona serum absorbed with liver and kidney but not by anti-egg serum absorbed with ovary. A complete inhibition of in vivo fertilization in different strains of mice was observed after a single injection of anti-zona or anti-egg serum, and such treatment also inhibits fertilization in rats but not in hamsters. (7) A single intraperitoneal injection of female mice with rabbit anti-sera against denuded mouse eggs and isolated zona pellucida inhibited fertilization in vivo for 20-25 days. The fertilizing capacity of mouse sperm was suppressed by treatment of sperm with anti- egg but not with anti-zona serum. Treatment of mouse eggs with these antisera did not affect their development in culture but inhibited the shedding of the zona pellucida, (8) Treatment of unfer- tilized and fertilized mouse eggs with sera from old females significantly reduced the rate of fer- tilization and the potential development in culture; some sera from old mice induced a faint precipitate on the zona pellucida by immunofluorescence test, indicating the presence of an antibody against the zona pellucida from the sera of old females. SUMMARY AND CONCLUSION The results of experimental studies of various aspects of fertilizing capacity of sperm, fertiliza- bility of eggs and fertilization in vivo and in vitro conducted by me during the past 40 years and conducted by my associates working in my labo- ratory were summarized in this article. The possibility of fertilization in vivo and in vitro in relation to the number of sperm, the fertile life of sperm in the female tract, the fertilizability of eggs before and after ovulation, the possibility of fer- tilization following various treatments of sperm, eggs, and other treatment of males or females were also presented. The capacitation of sperm and the successful in vitro fertilization of eggs of rabbits, golden hamsters, Chinese hamsters, mice, deermice and rats were described. Finally our experimental results concerning the cross fertilization between different species in vivo as well as in vitro, the fertilization of zona-free eggs in vitro, the fertili- zation of frozen and thawed eggs and the immu- nological studies of fertilization were presented. During these 40 years when the study of mam- malian fertilization was in an early stage of development, my associates, especially my Japanese associates, have done a great deal of work and they are all becoming leading scientists in this field of research. I am very much looking forward to seeing their future achievements in the study of mammalian fertilization in a more physiological and biochemical basis. These studies were supported mainly by grants from the National Institute of Child Health and Human Development and The Ford Foundation. Thanks are due to Ms. L. Hager for typing this manuscript. REFERENCES 1 Chang, M. C. and Walton, A. (1940) The effects of low temperature and acclimatization on the respiratory activity and survival of ram _ sper- matozoa. Proc. Royal Soc., London, 129: 517-527. 2 Chang, M.C. (1943) Sperm production of a pony stallion and treatment of spermatozoa in vitro with special reference to artificial insemin- ation of mares. J. Agr. 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Ae W24NG a : malindises CUE PRN RAL ep =) vhe> tang srdas ay Tae ae yen nda, ove: We Tae [ a oriwet - bez. ey Taal o a \ 2 1a, 4 f £ a 3 j ah h é: a . ¥ on ery, 4B hip OL ae tak) ata CHS HRD ON SR. 19 iO tratoiA ihe’ ZOOLOGICAL SCIENCE 1: 367-374 (1984) Freeze-Fracture and Histofluorescence Studies on Photoreceptive Membranes of Medusan Ocelli NosBuo TAKASU and MASAO YOSHIDA!:2 Department of Anatomy, School of Medicine, Osaka University, Nakanoshima, Osaka 530, and ‘Ushimado Marine Laboratory, Okayama University, 130-17 Kashino, Ushimado, Okayama 70 1-43, Japan ABSTRACT — Intramembranous particles (IMPs) in various membranous components of medusan ocelli were studied using four different types in basic organisation; flat (Aurelia aurita), cupulate with everted (Spirocodon saltatrix) and inverted (A. aurita) sensory cells and lens-bearing (Charybdea rastonii). In addition, a histofluorescence technique specific for retinal-based proteins was applied to the lens eye. Apart from the flat ocellus of A. aurita, average diameters and densities of IMPs on the P-faces of the microvillar and ciliary membranes of the sensory cells in the other three types ranged 8-9 nm and 5,000—6,000/m7, respectively, and the densities were significantly higher than those in non-photo- receptive constituents of the ocelli. In the flat ocellus of A. aurita, P-face particles in the microvillar and the ciliary membranes were much less in densities and larger in diameters. In the lens eye of C. rastonii, the specific fluorescence was restricted to the ocular cavity where putative sensory micro- villi were densely packed. From these results, we suggest that with the exception for the flat ocellus of A. aurita, the microvilli © 1984 Zoological Society of Japan and the cilia in the three types of ocelli are in fact involved in primary photoreception. INTRODUCTION Medusae have various types of ocelli, from a simple flat type to a well-developed lens-bearing one[1]. Fine structural studies have revealed that, except for the flat type as in Leuckartiara octona [5] and in Aurelia aurita [6], ciliary membranes projecting from putative photosensory cells into the ocellar cavity are also differentiated to varying degrees [5-11]. For some of the medusan ocelli, the photoreceptive function has been physiolog- ically inferred [2-5] and Yoshida and Yoshino [12] have reported in developing ephyrae of A. aurita that light-dependent swimming pattern changes with the differentiation of cupulate ocelli. For those ocelli, however, there is as yet no convincing evidence that the presumed receptive mem- branes bear in fact some kind of photosensitive pigment(s). In well-differentiated photoreceptors, Accepted October 24, 1983 Received September 29, 1983 2 To whom reprint requests should be addressed. intramembranous particles (IMPs) revealed by freeze fracture techniques are significantly denser in membranes of the photoreceptive site [13-15] and such particles are assumed to embody mostly rhodopsin molecules [16-19]. On the other hand, Ozaki et al. [20] succeeded in demonstrating the presence of retinal-related substances by a method of histofluorescence in photoreceptors of four taxonomically remote species. Ina previous paper [21], we have shown that putative photoreceptive membranes in seastars not only contain IMPs significantly more than any other membranous components in the ocelli but also become to fluoresce by the method of Ozaki et a/. [20]. In this paper, we will show that a similar line of approach is applicable to medusan ocelli. MATERIALS AND METHODS Materials used were Spirocodon saltatrix Tilesius (anthomedusa), Aurelia aurita Lam (semaeostomea) and Charybdea rastonii H: (cubomedusa). The former two were co!! 368 N. TAKASU AND M. YOSHIDA near the Ushimado Marine Laboratory and the last one, at Konamihama, Shimane Prefecture. Details for preparation of replicas of freeze- fractured tissues, morphometry of IMPs and induction of retinal-related fluorescence have been described elsewhere [21]. RESULTS Gross appearance The ocelli used in the present experiment are all different in the basic structural organization, ranging from a simple flat type to a well-differen- tiated lens eye [1, 5]. a) Flat type (Fig. 1); The large ocellus of Aurelia aurita is composed of a layer of pigmented sensory cells and each sensory cell bears a cilium which is surrounded by a tuft of microvilli arising from the apical surface of the same cell [6]. b) Cupulate type with everted sensory endings (Fig. 2); In Spirocodon saltatrix, pigmented cells and deeper-lying sensory cells form a cup shape and numerous microvilli in the cup lumen are derived from the sheath membrane of a single cilium arising from the apical end of each sensory cell. The pigmented cells also project few and short microvilli [6, 10]. c) Cupulate type with inverted sensory endings (Fig. 3); In the samll ocellus of Aurelia aurita, cilium-bearing free ends of ectodermal sensory cells face, via a thin mesogleal layer, the endodermal pigmented cells which align into a cup shape [6]. d) lLens-bearing eye (Fig. 4); The cubomedusan eye has a lens, an ocular cavity and a cup of pig- mented sensory cells [8]. The ocular cavity is filled with numerous microvilli arising from ciliary sheath membrane of the sensory cells and a few thick and long pigmented processes occupy the whole depth of the cavity. The microvilli often arrange in a regular array (Fig. 4b). Fia. 1. surrounding microvilli (Mv). corresponding to arrow in a. Fracture surfaces of cilium and microvilli in the flat ocellus of Aurelia aurita. x 4,600. b: Longitudinally fractured microvilli (Mv). x 66,000. SC, pigmented sensory cell. a: Low magnification. c: Cross-fractured cilium (Ci) and b and c were obtained at sites x 66,000. Medusan Photoreceptive Membranes 369 Mt ae 03 >? ‘ ‘ re 21 tb ™“* s by an arrow in inset. 66,000. Inset: Low magnification, 2,900. MvI and MvII, P-faces of micro- villi which are assumed to be derived from sensory cells and pigmented cells, respectively. OC, ocellar cavity; CB, cell body layer. Intramembranous particles Electron microscopic profiles of fractured surfaces prepared from the four different types of ocelli as described above are shown in Figures 14. All the membranous components examined revealed richly particulate P-faces and nearly smooth E-faces. In most cases, the fractured membranes could be identified topographically by referring to profiles of low magnification. Mea- surements of the size and the density of intramem- branous particles (IMPs) were limited to the P-faces of microvilli and plasma membranes of the sensory and the pigmented cells as well as to those of the sheath membranes of cilia arising from putative sensory cells. The IMP densities in various membranous components of different types of ocelli are sum- marized in Table 1. It is noteworthy that the IMPs in microvilli and cilia of the cupulate ocelli of A. aurita and the lens eye of C. rastonii are significantly denser than those in the plasma membranes of the sensory and the pigmented cells (P<0.001). The mean diameters of IMPs in these membranous components ranged from 8.16 1.05 nm in the plasma membrane of the sesory cell body in A. aurita to 9.21+1.04 nm in the plasma membrane of the long pigmented cell process in C. rastonii. In the case of Spirocodon ocelli, however, it was difficult to distinguish between the microvilli of the sensory cells and those of the pigmented cells. When all the data on the mean diameters and the densities of IMPs measured at 496 sites of P-faces of microvilli are assembled into an X—Y coordinate (Fig. 5), two separate groups become apparent; dense aggregation of small particles (Group I; mean diamter 8.92+0.93 nm, n=148) and wider distribution of large particles (Group II; mean di- ameter 9.83 + 1.03 nm, n=184). By analogy t~ densities in other known photoreceptive micr we assume that the group I represents par’ 370 N. TAKASU AND M. YOSHIDA es Oe Me Medusan Photoreceptive Membranes 371 8 9 10 11 Average diameter (nm) Fic. 5. Relationship between average diameters and densities of IMPs exposed in P-faces of microvillar membranes in the ocellar lumen of Spirocodon saltatrix. Each point represents datum obtained at a site that appeared fairly flat. In 3 replica samples 496 sites were explored. Points are segregated into two groups, I and II, which are assumed to correspond to micro- villi from sensory cells and pigmented cells, respectively. the microvilli of the sensory cells. Note that the density of GroupI (6,491+595/ym?) is now comparable to those in sensory microvilli of A. aurita (5,352 +1,336/um’*) and C. rastonii (6,405 + 582/ m7’). The flat ocellus of A. aurita is exceptional so that the IMPs are significantly less dense in the micro- villi than in the sheath membranes of cilia and the plasma membrane of the cell body (P<0.001). It must be added that mean diameters of IMPs in what have been called sensory endings in the flat ocellus of A. aurita are larger (11.65-+1.59 nm in microvilli and 11.10+1.35 nm in ciliary sheath) than those in other types of ocelli. Histofluorescence Each sensory club of C. rastonii bears six photo- sensory organs, viz., a large and a small lens eyes and two pairs of simple ocelli, so that frozen sections aimed to cut the large lens eye often contained parts of the simple ocelli. An example is shown in Figure 6a. Before reduction with sodi- um borohydride, weak non-specific fluorescence was induced by UV irradiation in non-ocullar and ocullar regions of the sensory club (Fig. 6b). When reduced with sodium borohydride after a light denaturation by cold formaldehyde solution (18-20%), a brilliant yellow-green fluorescence was induced by UV irradiation not only in the ocular cavity of the lens eye but also in the cup lumen of the small ocellus (Fig. 6c). The specific fluore- scence lasted for about 90 sec under UV irradiation. This observation clearly indicates that a substance that becomes fluorescent upon reduction is spatially restricted to the ocullar cavity where the microvilli are densely packed. DISCUSSION In recent years, many freeze-fracture studies on well developed eyes in invertebrates, mainly in arthropods [13-17], as well as in vertebrates [22, 23] have shown that IMPs are densely present in the P-faces of the photoreceptive membranes. The Fic. 3. inset. and mesoglea. 66,000. cup of endodermal pigmented cells (PC). Ci, Basal part of cross-fractured cilium. Fic. 4. Fracture surface of the large lens eye of Charybdea rastonii. Fracture surface of the cupulate ocellus of Aurelia aurita obtained at the site shown by an arrow in Irregularly arranged microvilli (Mv) of sensory cells are found in the layer between sensory cells Inset: Low magnification. Sensory cells (SC) face, via mesoglea (Me), the x 2,200. S, P-face of plasma membrane of sensory cell body; a: Low magnification. The ocullar cavity is filled with numerous microvilli (Mv) which arise from the ciliary shafts (Ci) of sensory cells (SC) The process of long pigmented cell (Pr) projects into the ocullar cavity. «2,800. b: Regularly arrar microvilli. x 66,000. c: Ciliary shaft of sensory cell and irregularly arranged microvilli. x 66,000 and c were obtained at sites corresponding to the arrow in a. 372 N. TAKASU AND M. YOSHIDA TABLE 1. medusan ocelli (Mean density +SD/pm7) The densities of P-face particles in various membranous components in four types of S. saltatrix small ocellus A. aurita C. rastonii large ocellus Sensory cell microvillus 6,491 +595 (148) cilium cell body Pigmented cell microvillus 1,903 +496 (184) cell body 5,352+1,336 (164) S385se 723 (7) 1,189+ 170 (15) L3o5ae 4's (s)) 1,201 +482 (54) 2,441 +727 (15) 2,592-+277 (15) 6,405 +582 (125) 5,902+295 (7) 2,795+490 (25) 1,554+290* (8) Number of places examined are shown in brackets. In the case of S. saltatrix, these values in microvillus of sensory and pigmented cell correspond, respectively, to Group I and II in Fig. 5. * Process of long pigmented cell. Fic. 6. Histofluorescence in the eyes of Charybdea rastonii. photographed with ordinary light microscopic optics. b: Autofluorescence prior to the reduction treatment. ocullar cavity of the large lens eye; Le, lens. c: Specific fluorescence induced by reduction with sodium borohydride. restricted to the ocullar cavity and the cup lumen of the simple ocellus. generally accepted view is that those IMPs re- present mostly the visual protein, rhodopsin. With an exception of the flat ocellus of A. aurita, average diameters and densities of P-face particles in the microvillar membranes of the sensory cells in the medusan ocelli ranged 8-9nm and 5,000- 6,000/m’, respectively, both of which are within the ranges of published data of IMPs in well- differentiated photoreceptive membranes. P-face particles were also present at high densities in the ciliary membranes of the sensory cells in the cupulate ocellus of A. aurita and in the lens eye of C. rastonii, suggesting that those two membranous components (microvilli and cilia) contain the same substance. In the eyes of C. rastonii, the specific fluorescence _ ocelli of seastars [21]. a: Cryosectioned and unstained sensory club Arrow indicates one of the simple ocelli. OC, Note that fluorescence is x 100. ascribable to retinal-based proteins [20] was restricted to the ocular cavity, where numerous IMP-rich microvilli derived from ciliary membranes are densely packed. We may assume that the microvillar IMPs embody retinal-related proteins. Since the specific fluorescence was not detected upon reduction without preceding denaturation, most of those P-face particles may be related to the presence of not a retinochrome- but a rhodopsin- like substance. The topographical correspondence of the histofluorescence and the high density of IMPs has also been shown recently in primitive Microvilli from putative photosensory cells in the cupulate ocelli of S. saltatrix and A. aurita are too sparse for successful application of the histofluorescence method. Medusan Photoreceptive Membranes However, the presence of IMPs at a comparable density suggests that those putative microvilli embody a similar photopigment. Indeed, Yoshida [24] has biochemically detected the presence of a retinol-like substance in the ocelli of S. sa/tatrix. The above argument makes it dubious whether the so-called flat ocelli of A. aurita are involved in photoreception, as terminologically inferred. P- face particles in the microvillar and the ciliary membranes of the flat ocellus were much less in densities and larger in average diameters than in any other medusan_ putative photosensory membranes. The difference in structure probably reflects a difference in function. The flat ocellus of A. aurita may be a pigment spot associated with a function, if any, other than photoreception. ACKNOWLEDGMENT We thank Mr. Isozaki and Mr. Godo of Ushimado Marine Laboratory for their technical help. The work was partly supported by a Grant-in-Aid from the Ministry of Education to M. Y. REFERENCES 1 Yoshida, M. (1973) Photoreception in medusae. Mar. Sci., 5: 12-17. 2 Hisada, M. (1956) A study on the photoreceptor of a medusa, Spirocodon saltatrix. J. Fac. Sci. Hokkaido Univ. Ser. VI, Zool., 12: 529-533. 3 Tamasige, M. and Yamaguchi, T. (1967) Equili- brium orientation controlled by ocelli in an anthomedusa. Zool. Mag. (Tokyo), 76: 35-36. 4 Yoshida, M. (1969) The ocellar pigment of the anthomedusa Spirocodon saltatrix: Does its photoreduction bear any physiological significance? Bull. Mar. Biol. St. Asamushi, 13: 215-219. 5 Singla, C.L. (1974) Ocelli of hydromedusae. Cell Tiss. Res., 149: 413-429. 6 Yamasu, T. and Yoshida, M. (1973) Electron microscopy on the photoreceptors of an antho- medusa and a scyphomedusa. Publ. Seto Mar. Biol. Lab., 20: 757-778. 7 Eakin, R.M. and Westfall, J. A. (1962) Fine structure of photoreceptors in the hydromedusan, Polyorchis penicillatus. Proc. Nat. Acad. Sci. (Wash.), 48: 826-833. 8 Yamasu, T. and Yoshida, M. (1976) Fine stru- cture of complex ocelli of a cubomedusan, Tamoya bursaria Haeckel. Cell Tiss. Res., 170: 325-339. 9 Singla, C. L. and Weber, C. (1982) Fine structure 10 11 14 16 17 20 21 22 373 studies of the ocelli of Polyorchis penicillatus (hydrozoa, anthomedusae) and their connection with the nerve ring. Zoomorphology, 99: 117-129. Toh, Y., Yoshida, M. and Tateda, H.(1979) Fine structure of the ocellus of the hydromedusan, Spirocodon saltatrix. 1. Receptor cells. J. Ultra- struct. Res., 68: 341-352. Yamamoto, M. and Yoshida, M. (1980) Fine structure of ocelli of an anthomedusan, Nemopsis dofleini, with special reference to synaptic organi- zation. Zoomorphology, 96: 169-181. Yoshida, M. and Yoshino, Y. (1980) Differenti- ation of ocelli in ephyrae of Aurelia aurita. In ““Developmental and Cellular Biology of Coelen- terates”’. Ed. by P.Tardent and R. Tardent, Elsevier North-Holland, Amsterdam, pp. 343-346. Eguchi, E. and Waterman, T. H. (1976) Freeze- etch and histochemical evidence for cycling in crayfish photoreceptor membranes. Cell Tiss. Res., 169: 419-434. Fernandez, H. R. and Nickel, E. E. (1976) Ultra- structural and molecular characteristics of crayfish photoreceptor membranes. J. Cell Biol., 69: 721-732. Chi, C. and Carlson, S. D. (1979) Ordered mem- brane particles in rhabdomeric microvilli of the housefly (Musca domestica L.). J. Morph., 161: 309-322. Boschek, C. B. and Hamdorf, K. (1976) Rhodo- psin particles in the photoreceptor membrane of an insect. Z. Naturforsch., 31c: 763. Harris, W. A., Ready, D. F., Lipson, E. D., Hudspeth, A. J. and Stark, W. S. (1977) Vitamin A deprivation and Drosophila photopigments. Nature, 266: 648-650. Larrivee, D. C., Conrad, S. K., Stephenson, R. S. and Pak, W. L. (1981) Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster. J. Gen. Physiol., 78: 521-545. Schinz, R. H., Lo, M-V. C., Larrivee, D.C. and Pak, W. L. (1982) Freeze-fracture study of the Drosophila photoreceptor membrane: Mutations affecting membrane particle density. J. Cell Biol., 93: 961-969. Ozaki; iata,m. and Hara, T. (1982) Location of retinochrome and rhodopsin in the retina as revealed by fluorescence microscopy. Jap. J. Ophthalmol., 26: 103. Takasu, N. and Yoshida, M. (1983) Photic effects on photosensory microvilli in a seaster, Asterias amurensis (echinodermata: asteroidea). Zoomor- phology, in press. Jan, L. Y. and Revel, J-P. (1974) Ultrastri localization of rhodopsin in the vertebrate J. Cell Biol., 62: 257-273. 374 N. TAKASU AND M. YOSHIDA 23 Corless, J. M., Cobbs, W. H., Costello, M.J. and 24 Yoshida, M. (1972) Detection of retinol-like Robertson, J.D. (1976) On the asymmetry of substance and the relative abundance of frog retinal rod outer segment disk membranes. carotenoids in different tissues of the anthomedusa, Exp. Eye Res., 23: 295-324. Spirocodon saltatrix. Vision Res., 12: 169-182. ZOOLOGICAL SCIENCE 1: 375-382 (1984) Effects of Nymphal Stage Optic Nerve Severance or Optic Lobe Removal on the Circadian Locomotor Rhythm of the Cricket, Gryllus bimaculatus KBNJI TOMIOKA and YOSHIHIKO CHIBA Environmental Biology Laboratory, Biological Institute, Faculty of Science, Yamaguchi University, Yoshida, Yamaguchi 753, Japan ABSTRACT — Bilateral optic nerve severance and optic lobe removal were performed on the 8th- instar nymphs of the cricket Gryllus bimaculatus DeGeer. Effects of the operations on circadian activity under LD12: 12 were investigated. Six optic nerve-severed animals revealed free-running rhythms in both the nymphal (diurnal) and adult (nocturnal) stages, indicating that the compound eye is the photoreceptor necessary for entrainment. In the majority, the adult rhythm was ultimately roughly synchronized to light-off, probably due to partial regeneration of the optic nerves. Activity in nine of 16 optic-lobeless nymphs was too low to evaluate the effect of the operation, but in the remaining seven, no significant rhythm could be detected. Four to five days after the imaginal moult, these animals showed the normal sexual-maturation-related increase of activity, but this was still arrhythmic. This arrhythmicity of lobeless nymphs and adults indicates that the optic lobe is indispensable to both nymphal and adult rhythmicity. Some lobeless animals as adults exhibited an atypical rhythm peaking in the light phase; this may be attributable to nerve regeneration between © 1984 Zoological Society of Japan the retina and the cut end of optic tract. INTRODUCTION A central purpose in circadian rhythm study is to localize the driving oscillator(s). In adult insects such as the cricket, Teleogryllus commodus, and the cockroaches, Leucophaea maderae and Periplaneta americana, destruction or transplantation of candidate tissues has yielded results suggesting that the optic lobe contains crucial elements for the circadian rhythms of locomotion [1-5], stridulation [6], and spermatophore formation [7]. During the course of post-embryonic develop- ment, male Gryllus bimaculatus shows remarkable reversal in the phasing of their circadian locomotor pattern: the activity peak which is diurnal in the nymphs becomes nocturnal 4 to 5 days after the imaginal moult, when signs of sexual maturation are first manifest [8]. The nymphal (diurnal) and adult (nocturnal) rhythms both free run in constant dim light (8—12 lux) or darkness, but differ not only Accepted October 12, 1983 Received August 31, 1983 in phase but also in wave-form and free-running period. The question arises as to whether the ‘optic lobe theory’ based only on adult activity can apply to such post-embryonic changes in this circadian system. This paper reports results of experiments carried out to explore this question. MATERIALS AND METHODS Nymphs (8th instar) of male Gryllus bimaculatus DeGeer were obtained from a laboratory culture kept under 26°C and LD 12: 12 (light intensity: about 100 lux; light-off 18:00 hr local time), the ‘standard’ environmental conditions. Locomotor activity of individuals was recorded as described by Tomioka and Chiba [8]. Statistical evaluation of the activity rhythms was based on the Chi-square periodogram [9]. Operation and autopsy Intact animals, whose locomotor activi‘: been recorded under the standard condition 376 K. TOMIOKA AND Y. CHIBA taken from their activity chamber between 09: 30 and 12: OOhr for operation. An incision was made along the margin of the compound eye, the eye capsule was pried open so that the optic lobe and optic nerves were exposed, and then either optic nerves were cut with a pair of fine scissors or the optic lobe (including the lamina and medulla, Fig. 1) was removed after cutting both the optic tract and the optic nerves. The eye capsule was then put back into place. The whole procedure took about 5 min, and was performed without anesthesia. The post-operative animal was re- placed immediately in its activity chamber. G. bimaculatus zB ea Y. >: C Fic. 1. Schematic drawings of cricket (upper) and cockroach (lower) brain illustrating optic nerves (on), optic tract (ot) and the region of neuropiles, lamina (la), medulla (me), lobula (lo). As soon as the experiment was over, the head capsule was carefully opened to see whether the severed or removed parts had regenerated. In addition, the relevant parts were extracted for more precise histological examination using Mallory’s triple staining process. RESULTS Optic nerve severance In six 8th-instar crickets, optic nerves were severed bilaterally. Despite this operation, the locomotor rhythm reversed from the nymphal (diurnal) to the adult (nocturnal) in five animals, with both forms of the free-running just as in the intact animals held in DD [8]; the daily amount of activity was also similar to that of intact animals. Figure 2A depicts the event recorder trace of an individual yielding a typical result. This animal was held in the actograph from the 8th instar and was operated on at 12: 00 hr on day 6 (3 days before the imaginal moult). After the operation the nymphal rhythm which had been synchronized with the LD cycle started to free run with a period apparently longer than 24 hr. Five days after the imaginal moult (on day 14), intense activity suddenly appeared, more or less as expected, in the trough of the previous nymphal rhythm, and then free ran. For-several days the nymphal and the adult rhythms seemed to coexist. These tendencies were found in 5 of the 6 animals. Another noticeable fact in these five animals is that the adult rhythm did not free run for long, but in due course became entrained roughly to the onset of darkness, in Figure 2A by around day 29. In the sixth animal, the adult rhythm started with two peaks per circadian cycle, but with the two peaks free-running at different periods and eventu- ally fused into a single peak (Fig. 2B). This complicated activity pattern made it hard to judge in this animal whether the normal nymphal-adult rhythm reversal had occurred; it is reminiscent of the ‘rhythm splitting’. reported in Hemideina by Christensen and Lewis [10]. Autopsy revealed the optic merve to be regenerated partly in all six animals, recoupling the optic lobe with compound eye but in a deficient form. Histological examination confirmed that some neural coupling had occurred, however. Removal of optic lobes In 16 animals, both optic lobes were removed in the 8th instar. Nine of these animals had to be excluded from analysis because their activity was seriously decreased by the operation (Fig. 3A). The remaining seven which were as active as typical intact nymphs in constant darkness [8], revealed no detectable rhythmicity at all (Fig. 3B, days 1-7). Four to five days after the imaginal moult, their activity started gradually to increase. At the same time, signs of sexual maturation, such as the Cricket Circadian Rhythms Sid = = = ——— re yaa —~-————s ee a et — oe - - Vr ——r — - a ne ee ———————————————————— TN — Te as eee oe im—_—> —___——— Bee SS See ——— Sane Sonal eine Tr eS OO ee S—S a rr rm rs wn OO eee EE ar nr es en Oe en ie een ee eee ee ee a a ee 8 ee ee Os ee ee ers 12 24 12 24 12 Time of day Fic. 2. Event recorder traces of locomotor activity of two crickets receiving bilateral optic nerve severance. Crickets were 8th instar on day 1. Operation (arrow) was carried out at 12: 0O0hr on day 5 (A) and at 11: 30 hr on day 8 (B). Most such crickets revealed free-running rhythms and rhythm reversal from nymphal to adult patterns shown in A, whereas the adult activity shown in B, with two peaks eventually fusing into one peak was seen only once. Records are double-plotted to facilitate visual evaluation of the flow of activity peaks. im: imaginal moult. White and black bars indicate lighting regime. 378 K. TOMIOKA AND Y. CHIBA RTT Tr Tr Tr TIT ON 8 FST TT 40— Se ee Swe a Tr an ST TT re TT Ts 18 rt 8 EB rr ys ST er es I er eT TU BNR ers Ce Tae Ua Oe en ee ee nn En eon er rm ne 6 28) eee a nn Ce ee ee ee ae ee ey Ul ee oo SP eh EY eth NAP oe AN peat pr Pe pre OS yg SE py pry pr TY free — — | | 1 B | | \= EE Ee pS a ee ce ce ee ee Se Te ee a Eee, VA i te ld ll a se it re sk a e= i ml i —— — — i _ rr rr rr lm Se SS ee ee sss 3:50 DS ee et nT SEE EERE ene im I, See eT) ee Cs AW 1 1 Se CE) | 12 24 Time of day Fic. 3. ets were 8th instar on day 1. Locomotor activity of two crickets receiving bilateral optic lobe removal. Crick- Operation (arrow) was carried out at 09: 30 hr (A) and 11: 30 hr (B) on day 4. For further explanations see text and Fig. 2. performance of calling songs, were manifested. As with intact or optic-nerve-severed animals, this is the time of rhythm reversal. The increased activity was still arrhythmic in ten of the 16 animals as they became adult (Figs. 3A and 4A), but the remaining six showed a faint but significant rhythm having an atypical pattern (Figs. 3B and 4B). The activity was relatively high in the light phase, but appeared to be less well entrained than in intact animals. The operation resulted in partial hyperactivity in seven animals and hypoactivity in one, the other eight being normal. By partial hyperactivity we mean that hyperactivity continued for several consecutive days alternating with several con- secutive days of normal activity. Table 1 sum- Cricket Circadian Rhythms 150 100 amplitude 100 10 15 379 2 e - x oe? .005 e oo e e e "S ee oe" wae. 6. anne 0 ‘cee cece ‘eoce oo 8 20 25 trial period Fic. 4. Periodograms of adult locomotor activity data of two crickets receiving bilateral optic lobe removal. A and B correspond to animals A and B in Fig. 3, respectively. Lines correspond to critical values of x? for a=0.005. Sig- nificant components are given as points lying above the line (B) but could not be detected in A. TABLE |. Summary of optic lobe removal results Regeneration of No. of adults No. with activity level neural connection No. —_— Rhythmic Arrhythmic Normal Hyper* Hypo Complete medulla present 2 2 0 2 0 0 medulla absent 2 2 0 0 2 0) Incomplete 10 0 10 (3*) 5 4 (2*) if Indefinable 2 2 0 1 1 0 * Eyeless individuals. are also included. marizes these results in relation to neural regeneration. Observation by naked eye showed that 14 of the 16 animals had nerves regenerating, variably in extent, from the cut end of the optic tract towards the compound eye. Excluding three animals whose compound eyes had been completely re- moved with the lobes, eleven had regenerated nerves apparently reaching the frontal margin of the compound eye, but histological examination * Animals which showed hyperactivity for only several consecutive days revealed that neural connections were actually made in only four animals; in two of these, how- ever, some medulla tissue had been left in situ by the operation. Connection was judged as incom- plete in eight, with connective tissue between the regenerating nerve and the compound eye. In the remaining two animals, no certain conclusions could be drawn. The six rhythmic adults con of four completely regenerated and two indefin ones. 380 K. TOMIOKA AND Y. CHIBA Chi-square-test showed that the optic lobe removal was associated with the rhythm’s disap- pearance, and that neural reconnection was significantly related to the occurrence of the atypical rhythm, no matter whether the two ‘indefinable’ animals were regarded as ‘incomplete’ (y?=8.88, p<0.005) or ‘complete’ (y*=16, p< 0.005). There was no significant relation between neural reconnection and activity level. DISCUSSION The anti-phase relationship and the differences in wave-form and free-running period that develop between the late nymphal (diurnal) and adult (nocturnal) forms of the rhythm in male Gryllus bimaculatus were seen also in animals that had undergone optic nerve severance in the nymphal stage and then recorded in LD. This indicates that the operation is equivalent to holding animals in constant darkness, and also that the compound eyes are the photoreceptors involved in normal entrainment to light cycles, in both the adult (as is well known for the other insects: Leucophaea maderae [11], Teleogryllus commodus [4]) and in the nymph. However, five optic-nerve-severed animals (i.e. excluding the diphasic one) all eventually re- synchronized with the LD cycle, with their activity peaks as adults occurring around the onset of darkness — though not as punctually as in intact animals. This reentrainment would be attribu- table to regeneration of their optic nerves. An interesting question arising here is why the activity peak continued to free run almost straight without any obvious sign of a phase-shift, until reaching the light-dark transition. The typical observation in such studies is that a phase-shift occurs in response to a light cycle placed in an unusual phase relation to the rhythm, but here it did not. The simplest explanation is that the nerve regenerated enough to function again as a pathway for light information at about the same time as the activity peak reached the LD transition. But it is hard to imagine that such a coincidence occurred in all five animals when their free-running periods all differed so that their activity peaks reached the LD transition on different days. It seems more likely that regeneration was not sufficiently complete to convey strong enough information about the zeitgeber to force the rhythm to resynchronize by phase-shifting; in other words, light was felt subjectively too weak. This pos- sibility is suggested by the fact that in the intact cricket, the weaker the light intensity, the longer reentrainment takes (Tomioka and Chiba, unpubli- shed data), as is known also for the other animals [12]. In general, when evaluating the effect of Operations in this kind of study, activity level should be given careful consideration. Operations often result in hyper- or hypo-activity which makes it hard to judge effects on any oscillation underlying the overt activity [13]. One extreme is when activity disappears altogether, in which case the state of the oscillation cannot even be guessed at. This problem was not encountered in the optic nerve severance experiment, since that operation seldom affected activity level, but it did arise in the optic-lobe-ectomized nymphs. Over half of the 16 nymphs receiving this operation became almost completely inactive and, therefore, could not have their rhythms evaluated. Since inactive animals appeared at this high rate, immediate conclusions as to the rhythmicity of operated nymphs may be inappropriate. Nevertheless, no significant rhythms were detected in the remaining seven nymphs that retained normal activity levels, which suggests that optic lobe removal did cause them to lose rhythmicity. The daily activity level of all the optic-lobe- ectomized animals increased simultaneously with sexual maturation to the level shown by intact animals. Thus, for adults at least, there was little problem related to activity level and the effects of the operation were comparatively clear- cut, again suggesting the optic lobes to be indis- pensable for the normal rhythmicity. However, even in the adults some confusion remained, since six animals showed the atypical activity peaking in the light phase. Even though the relation between this rhythm and nerve re- generation was Statistically significant, unequivocal evidence was provided by only two animals, with their optic lobes completely removed and their nervous connections clearly remade. Cricket Circadian Rhythms 381 A few normal crickets show a higher activity in the light phase than in the dark, even after their rhythm should have reversed to produce a noctur- nal peak. When such a cricket receives optic lobe removal or optic tract severance, its activity does not immediately become arrhythmic, but rather tends to be higher in the light phase for several days (Tomioka and Chiba, in preparation). In view of this, the explanation that the atypical post- operative activity is attributable to nerve regen- eration may be premature. Cricket ( Teleogryllus commodus) stridulation and cockroach locomotion are reported to show rhythmicity under a temperature cycle in darkness after optic lobe removal [14, 15]. This rhythm is explained as essentially exogenous, and the tem- perature cycle is thought to be acting through a strongly damped oscillator [15]. But, the present atypical rhythm occurred in constant temperature, and so does not correspond to the temperature- sensitive rhythm. This cricket has one median and two lateral ocelli. These ocelli, however, seem not to be involved both in normal entrainment to light cycles and in the expression of the atypical rhythm. This is suggested by the facts that all the optic- nerve-severed crickets free ran in LD and that the atypical rhythm appeared in only 6 of 16 lobeless animals measured. Both nymphal and adult rhythms were made to free run by optic nerve severance, and made to disappear by optic lobe removal. These facts indicate that the optic lobes play a crucial role for both forms of the rhythm, but are both controlled by the same oscillator or by separate oscillators in the lobes? No essential progress to answer this question has been made since the previous paper [8], though the differing nymphal and adult free-running periods argue for it. In cockroaches, cells in the ventral portion of the lobe near the lobula are thought to be crucial to the maintenance of rhythmicity [3, 16]. Page [16] also states that only one intact lobula region is sufficient for persistence of rhythmicity. How- ever, what we refer to as the optic lobe in the cricket does not contain the lobula but the lamina and medulla only (Fig. 1). In our operation, the tract was cut at its distal end about 700 ~m from the lobula region, which was, therefore, left intact. Nevertheless, the locomotor rhythm disappeared, proving that the crucial mechanism resides in the lamina plus medulla region but not in the lobula region as reported for cockroaches. It is still uncertain, however, whether the optic lobe includes a self-sustained oscillator independent of the lobula and the cerebral lobe. Page [5] elegantly demonstrated that transplantation of the optic lobes to a lobeless cockroach led to a restora- tion of the circadian activity rhythm in the recipient with the free-running period of the donor. But this does not prove conclusively that the optic lobe has a self-sustaining ability. In our cricket, the circadian ERG (electro-retinogram) rhythm per- sists in the neurally isolated ‘optic-lobe-compound- eye’ system, suggesting that this system does include the driving oscillator of the ERG rhythm [17], a finding supported by Fleissner’s [18] work with the ground beetle Anthia sexguttata. The next problem is whether this oscillator drives the locomotor rhythm as well. ACKNOWLEDGMENT This study was supported by grants to Y. C. from the Ministry of Education, Science and Culture (Grant-in- Aid for Special Project Research on Mechanisms of Animal Behaviour) and the Chugai Pharmaceutical Company. REFERENCES 1 Nishiitsutsuji-Uwo, J. and _ Pittendrigh, C. S. (1968) Central nervous system control of circadian rhythmicity in the cockroach. III. The optic lobes, locus of the driving oscillation? Z. Vergl. Physiol., 58: 1446. 2 Roberts, S. K. (1974) Circadian rhythms in cock- roaches: Effects of optic lobe lesions. J. Comp. Physiol., 88: 21-30. 3 Sokolove, P. G. (1975) Localization of the cock- roach optic lobe circadian pacemaker with microlesions. Brain Res., 87: 13-21. 4 Sokolove, P.G. and Loher, W. (1975) Role of eyes, optic lobes, and pars intercerebralis in locomotory and stridulatory circadian rhythms of Teleogryllus commodus. J. Insect Physiol., 7’ 785-799. 5 Page, T. L. (1982) Transplantation of the cock- roach circadian pacemaker. Science, 216: © 6 Loher, W. (1972) 10 11 382 Circadian control of stri- dulation in the circket Teleogryllus commodus Walker. J. Comp. Physiol., 79: 173-190. Loher, W. (1974) Circadian control of sper- matophore formation in the cricket Teleogryllus commodus Walker. J. Insect Physiol., 20: 1155- 1172: Tomioka, K. and Chiba, Y. (1982) Post- embryonic development of circadian rhythm in the cricket, Gryllus bimaculatus: a rhythm reversal. J. Comp. Physiol., 147: 299-305. Sokolove, P. G. and Bushell, W. N. (1978) The chi-square periodogram: its utility for analysis of circadian rhythms. J. Theor. Biol., 72: 131-160. 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. Nishiitsutsuji-Uwo, J. and Pittendrigh, C. S. (1968) Central nervous system control of cir- cadian rhythmicity in the cockroach. II. The pathway of light signals that entrain the rhythm. Z. 12 13 14 15 16 17 18 K. TOMIOKA AND Y. CHIBA Vergl. Physiol., 58: 1-13. Aschoff, J., Hoffmann, K., Pohl, H. and Wever, R. (1975) Re-entrainment of circadian rhythms after phase-shifts of the zeitgeber. Chronobiologia, 2: 23-78. Brady, J. (1969) How are insect circadian rhythms controlled? Nature, 223: 781-784. Rence, B. and Loher, W. (1975) Arrhythmically singing crickets: thermoperiodic reentrainment after bilobectomy. Science, 190: 385-387. Page, T. L. (1981) Neural and endocrine control of circadian rhythmicity in invertebrates. In “‘Handbook of Behavioral Neurobiology. vol. 4 Biological Rhythms’. Ed. by J. Aschoff, Plenum Press, New York, London, pp. 145-172. Page, T. L. (1978) Interactions between bilater- ally paired components of the cockroach circadian system. J. Comp. Physiol., 124: 225-236. Tomioka, K. and Chiba, Y. (1982) Persistence of circadian ERG rhythm in the cricket with optic tract severed. Naturwissenshaften, 69: 395-396. Fleissner, G. (1982) Isolation of an _ insect circadian clock. J. Comp. Physiol., 149: 311-316. ZOOLOGICAL SCIENCE 1: 383-388 (1984) © 1984 Zoological Society of Japan Innervation of the Heart of a Prosobranch Mollusc, Rapana thomasiana MAKOTO KOBAYASHI, MIB NAKAMURA and TomMog HASIMOTO Physiological Laboratory, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan ABSTRACT — The heart of a prosobranch mollusc, Rapana thomasiana, is innervated by four cardiac nerves arising from the right visceral ganglion, and three from the left. These nerves con- tribute in regulating the heart activity. Right cardiac nerve (RCN) 3a, RCN 4, left cardiac nerve (LCN) 1, and LCN 2 are atrial nerves, and stimulation of these nerves enhanced the amplitude of heart beat. RCN 1 and RCN 3b are ventricular nerves which caused inhibition of heart activity. Results indicating a relationship between heart beat and whole body movements were obtained, sug- gesting neurohormonal effects on heart activity. INTRODUCTION Innervation of the molluscan heart has been described in a number of species since the pio- neering work of Carlson [1]. In gastropods, in addition to innervation of the cardiac muscu- lature, the structural and functional organization of the neural networks concerned with regulating heart activity has recently been investigated in a few species of opisthobranchs [2, 3], prosobranchs [4] and pulmonates [5, 6]. However, still little is known about the neural control of visceral motor function compared with the wealth of knowledge concerning somatic motor systems. In a prosobranch, Rapana thomasiana, the heart activity is controlled by cells in the visceral ganglia, and several cardiac nerves innervate the myocar- dium. In this species, however, it is very difficult to identify the central neurons taking part in the regulation of heart beat. In the present study, innervation of the heart was examined anatomically, and the physiological role of the cardiac nerves on heart beat was investigated in order to understand the control system of heart activity. Accepted October 18, 1983 Received August 30, 1983 MATERIALS AND METHODS Specimens of a prosobranch, Rapana thomasiana, were used. After removing the shell, the dorsal side of the viscera was cut open and dissected under a binocular microscope. The visceral ganglia and cardiac nerves were exposed by carefully removing the connective tissues and white glial cells covering them. Although the ganglia and large nerves could easily be identified by their orange-yellowish color, the fine inner- vations were examined by vital staining with methylene blue. Physiological experiments were carried out on preparations including the heart, the visceral ganglia and the cardiac nerves all interconnected and mounted on the surrounding tissues. For the recording of heart beat two types of prep- arations were employed. In one preparation, the aorta was cut, and a cannula was inserted into the ventricle through the cut end of the aorta; this is referred to as preparation A. In the other, the atrial end was cut, and a cannula was inserted into the atrium; this is referred to as preparation B. In both preparations, the cut end of the heart together with the cannula v tied with a thread which was connected ‘o strain gauge for tension recording. 384 M. KosayAsHi et al. Artificial sea water (ASW) consisted of (mM): NaCl, 461.9; KCl, 9.4; CaCl., 9.0; MgCl, 53.8; and Tris buffer 20.0 (pH adjusted to 7.8 by ti- tration with HCl). Since the heart did not beat spontaneously unless perfused, the heart was constantly perfused with ASW _ through the cannula. The frequency of heart beat positively correlated with perfusion rate, and slower rates frequently produced irregular beat- ing rhythm. The perfusion rate was, therefore, maintained at a level of about 1.0 ml/min, suffi- cient to keep the heart rate at 30—40 beats/min, a little faster than that observed in intact animals. Electrical stimulations were applied to visceral ganglia or cardiac nerves by using a pair of silver- silver chloride wires of about 100 “m in diameter. In some experiments, a preparation of the whole organism was employed. Jn this preparation, the entire central nervous system was exposed, in- cluding the circumesophageal ganglia, the connec- tives between them and the visceral ganglia, the visceral ganglia, and the cardiac merves (see Kobayashi [7] for the anatomy of the circum- esophageal portion of the central nervous system). In this case also, two types of preparations, A and B, were prepared depending on the recording methods. Since the tissues surrounding the heart were pinned to a Sylgard floor of a chamber, the recording of heart beat was not mechanically disturbed by the movement of the whole body. By using the whole organism preparations, the effects of stimulating some of the head ganglia (i.e., supra-oesophageal or sub-oesophageal) on heart beat and on the movement of the whole body could be observed, and the relationship between these effects could be investigated. All experiments were performed at room temperature of 20-24°C. RESULTS A dorsal view of a pair of visceral ganglia and the related nerves is shown in Figure 1. Ana- tomical examination has revealed that four cardiac nerves arise from the right visceral gan- glion (RVG), and three nerves arise from the left visceral ganglion(LVG). They are designated, from right to left, as right cardiac nerve (RCN) 1- was to SPOG imm Fic. 1. A schematic drawing of a pair of visceral ganglia and their related nerves (dorsal view). LCN, left cardiac nerve; LVG, left visceral ganglion; RCN, right cardiac nerve; RVG, right visceral ganglion; SBOG, sub-oesophageal ganglion; SPOG, supra-oesophageal ganglion. 4 and left cardiac nerve (LCN) 1-3, respectively. RCN 3 was the largest nerve and divided into two branches, 3a and 3b. RCN 3a, RCN 4, LCN 1 and LCN 2 were observed to innervate the atrium through the ventral wall of the peri- cardium, and some of their branches traveled through the atrium to the ventricle. RCN 1, RCN 3b and LCN 3 were found to come to the ventricle from along the outside wall of the aorta. Figure 2 presents schematic drawings of the two methods for perfusing hearts and recording their tension. The typical responses of these two preparations to stimulation of RVG is also shown, and a striking contrast can been seen. In preparation A, a train of electrical pulses (1 msec, 5 V, at 10/sec) applied to RVG for only 1 sec produced a remarkable enhancement of the amplitude of heart beat, and the effect remained for a few minutes. In preparation B, on the con- trary, a conspicuous inhibition of heart beat was produced by the same electrical stimulation. The following experiments were performed using the intact atrium preparation A, and the effects on heart beat of stimulating RVG and LVG were compared (Fig. 3). Stimulation of LVG yielded essentially the same results as stimulation of RVG; that is, the amplitude of heart beat was strongly enhanced but the frequen- cy was unchanged. After the commissure between the left and right ganglia was cut, similar enhancing Innervation of Molluscan Heart 385 transducer Ey A cannula | @® Suman Fic. 2. Schematic drawings illustrating methods for perfusing hearts and recording their tension in two kinds of preparations, A and B, and typical responses to the stimulation of RVG in both preparations. In each record, electrical pulses of stimulation of 1 msec, 5V, 10/sec were given for 1 sec at arrow. A before commissure cut Fic. 3. Comparison of responses to stimulation of LVG and RVG in preparation A. A and B show records before and after cutting the ganglionic commissure, respectively. Al and BI, responses to LVG stimulation; A2 and B2, those to RVG stimulation. In each record, stimulations (1 msec, 5 V, 5/sec) were given for 1 sec at arrow. effects were obtained by stimulating each gangli- on. These results indicate that excitatory nerves, which primarily enhance the force of heart beat, arise from both ganglia. In the next experiments, therefore, the effects on heart beat of stimulating each cardiac nerve were examined. The procedure was to cut each nerve and to stimulate the distal stump. Of eight nerves or nerve branches tested, stimulation of the following four nerves enhanced the heart beat: RCNesau RCN 4. °LEN*t and CN ‘2: Stim- ulation of the other nerves produced no appreciable change in heart beat. These physiological results imply that the above four nerves contain predom- inantly excitatory fibers and, consistent with our anatomical findings, innervate the atrium. The experimental results are illustrated in Figure 4. Figure 4A shows the effects of stimulating LVG and RVG after cutting the ganglionic commissure, but with the cardiac nerves still intact. Figure 4B illustrates the enhancing effects obtained by stimu- lating the cut end of the four atrial nerves. In Figure 4C it is shown that stimulation of each ganglion has no effect on heart beat after all the cardiac nerves were cut. Figure 5 illustrates the effects of varying the frequency of the stimulus applied to one of the excitatory atrial merves. RCN 3a, which was the most effective in enhancing the amplitude of heart beat, was stimulated by a train of elec- trical pulses of 1 msec, 5 V, at different frequencies of 1/sec—-10/sec. As the stimulus frequency raised, both the amplitude of beat and the dura'i: of the beat enhancement were gradually incre: 386 M. KosBAyYASHI et al. 30 sec Fic. 4. Responses to stimulation of visceral ganglion and cardiac nerve in preparation A. Gangli- onic commissure was cut before recording. A, responses to stimulation of visceral ganglion before cutting cardiac nerves; BI-B4, _ re- sponses to stimulation of the cut end of the cardiac nerve shown at the upper right of each record; C, responses to stimulation of visceral ganglion after cutting all cardiac nerves. In each record, stimulations (1 msec, 5 V, at 5/sec) were given for 1 sec at arrow. Similar results were also observed when the pulse duration or intensity was increased at a constant frequency. The experiments shown in Figures 6 and 7 were carried out with preparation B, in which the atrial end was cut while the ventricle and aorta remained intact. Stimulating either LVG or RVG led to a remarkable inhibition of heart beat, and the inhibition was usually followed by a transient increase of contraction height (Fig. 6). After the ganglionic commissure was cut, stimu- lation of RVG yielded inhibition in the same way as before the cutting, whereas LVG stimulation produced only small fluctuations in the amplitude of heart beat. These results imply that inhibitory fibers are contained in nerves arising from RVG. 1 iin 1/sec i | a ae a ; ' ; | OTL BERET IEEE EE 2 2/sec HT HANI PALTV ga et VEEP HET WN He yy TYAN FTA ’ t : 6 a | 3 5/sec if Vf! VATE WW i REE eee ee 4 IL. 4. veiniche 10/sec jhe colds ola I5se Fic. 5. Effects of varying stimulus frequency on response to stimulation of RCN 3a in prepa- ration A. Stimulations of 1 msec and 5 V at a frequency of 1/sec-10/sec were given to the cut end of the nerve for 1 sec at arrow. Then, the action of each cardiac nerve on heart beat was examined by stimulating the cut end of each nerve, as had been done with preparation A. Figure 7 illustrates a part of the results of such experiments. As shown in this figure, RCN 1 and RCN 3b were found to have conspicuous inhibi- tory effects on heart beat. Inhibition by stimu- lation of RCN 1 was duration dependent; that is, it continued during the period of stimulation and was followed by a transient increase of con- traction height (Fig. 7A). Stimulation of RCN 3b, on the other hand, produced tonic inhibition which persisted for a long period (Fig. 7B). Stimulation of LCN 3 yielded a slight increase Innervation of Molluscan Heart 387 A before commissure cut wa al lt B after oie ia cut wa tl 15 sec 1 mt Fic. 6. Comparison of responses to stimulation of LVG and RVG in preparation B. A and B show records before and after cutting the ganglionic commissure, respectively. Al and Bl, responses to LVG stimulation; A2 and B2, those to RVG stimulation. In each record, stimulations (1 msec, 5 V, at 5/sec) were given for 1 sec at arrow. of contraction height in some specimens, but no significant change in the others. The other nerves had no appreciable effects on heart beat. These results indicate that inhibitory nerves in RCN 1 and 3b innervate the ventricle through the aortic end. In experiments using the preparation of the whole organism, the effects of stimulating the supra-oesophageal ganglion (SPOG) or the sub- oesophageal ganglion (SBOG) on heart beat were examined. In these experiments, the stimulation was applied after all the cardiac nerves had been cut off. The effects were not constant but quite variable in different specimens (Fig. 8). In prep- aration A, stimulation of SPOG or SBOG usually produced enhancement of heart beat to- gether with body movement (Fig. 8A). In preparation B, on the other hand, stimulation of SPOG or SBOG caused a slow gradual in- crease of beat amplitude in a few specimens (Fig. 8B), tonic inhibition in some (Fig. 8C), and no appreciable effects in others. The inhibition of heart beat usually followed the movement of the whole body. These successive phenomena were Observed to occur, not only by electrical stimulation, but also spontaneously. i preparation @® SPOG SBOG B preparation ® SPOG SBOG a to + preparation ® SPOG SBOG 30 sec Fic. 8. Effects of stimulation of SPOG and SBOG on heart beat in the preparation of the whole organism. Stimulations (1 msec, 5 V, at 10/sec) were given to the ganglion shown at upper right of each record for 1 sec at arrow. A, B and C are records from different specimens. ui i a Fic. 7. ‘ Responses to stimulation of RCN I (A) and RCN 3b (B) in prepara- a o 15 sec tion B. Stimulations (1 msec, 5 V, at 10/sec) were given to the cut end of the nerve during the period shown by the underline beginning at arrow. Stimulus duration was | sec (Al, Bl and B2), 2 sec (A2) and 5 sec (A3). 388 M. KosayAsHIi et al. DISCUSSION The present study has revealed that four and three cardiac nerves arise from RVG and LVG, respectively, and these nerves contribute to the regulation of heart beat. The experiments have shown that RCN 3a, RCN 4, LCN 1 and LCN 2 are excitatory atrial nerves, whereas RCN 1 and RCN 3b are ventricular nerves and predominantly inhibitory. The significance of the contibution of many nerves to the regulation of heart activity is not clear at present, but the mechanisms of action of each nerve on the myocardium appear to be different. Stimulation of RCN 1 caused a phasic inhibition which is duration dependent, while RCN 3b produced a prolonged, tonic inhibition. The different effects could reflect differences in the transmitters or in the active sites on the myo- cardium. In Aplysia and Helix cardioregulatory meurons are scattered throughout the central ganglia, and their functional mechanisms are dif- ferent from one another [2, 5, 6, 8, 9]. In the present experiments the chronotropic effect on heart beat was scarcely elicited. by stimulation of excitatory nerves. The constant perfusion rate,employed in the experiment might be one reason for the lack of chronotropic effect. However, the application of some putative neuro- transmitters caused variable chronotropic effects under constant perfusion rate [10] and, therefore, the exclusive inotropic action of the excitatory nerves appears to be the direct effects of nerve stimulation. Although the gastropod hearts are myogenic, they do not usually beat spontaneously after dissection unless they are perfused [2, 11]. Mayeri et al. [2] have suggested that this may be due, either to the lack of some unidentified component normally found in hemolymph, or to the disruption of normal hemodynamics caused by dissection. The present experiments with the whole organism preparation have suggested a relationship between the evoked slow movements of the whole body and heart beat. We suppose that stimulation of the central ganglia may release into the hemolymph neurohormones that act on both somatic ‘and cardiac muscles. Thus, the regulation of heart activity may be carried out both by multifunc- tional neural networks: and by circulating neuro- hormones. REFERENCES 1 Carlson, A.J. (1905) Comparative physiology of the invertebrate heart. I. The innervation of the heart. Biol. Bull., 8: 123-159. 2 Mayeri, E., Koester, J.. Kupfermann, I., Liebe- swar, G. and Kandel, E. R. (1974) Neural control of circulation in Aplysia. I. Motoneurons. J. Neurophysiol., 37: 458-475. 3 S.-Rozsa, K., Salanki, J., Véro, M., Kovaéevic, N. and Konjevi¢é, D. (1980) Neural network re- gulating heart activity in Aplysia depilans and its comparison with other gastropod species. Comp. Biochem. Physiol., 65A: 61-68. 4 Kuwasawa, K. and Hill, R. B. (1973) Regulation of ventricular rhythmicity in the hearts of pro- sobranch gastropods. In “Neurobiology of Invertebrates”. Ed. by J. Salanki, Akadémiai Kiado, Budapest, pp. 143-165. 5 S.-Rozsa, K. (1979) Analysis of the neural network regulating the cardio-renal system in the central nervous system of Helix pomatiaL. Amer. Zool., 19: 117-128. 6 S.-Rozsa, K. (1979) Heart regulatory neural network in the central nervous system of Achatina fulica (Férussac) (Gastropoda: Pulmonata). Comp. Biochem. Physiol., 63A: 435-445. 7 Kobayashi, M. (1981) A preliminary report on the regulatory mechanisms of the antagonistic radular muscles. In “Advances in Physiological Science, Vol. 23. Neurobiology of Invertebrates’. Ed. by J. Salanki, Akadémiai Kiado, Budapest, pp. 83-96. 8 Koester, J., Mayeri, E., Liebeswar, G. and Kandel, E.R. (1974) Neural control of circulation in Aplysia. II. Interneurons. J. Neurophysiol., 37: 476-496. 9 S.-Rozsa, K. (1976) Neuronal network under- lying the regulation of heart beat in Helix pomatia L. In: ““Neurobiology of Invertebrates’. Gastro- poda Brain. Ed. by J. Salanki, Akadémiai Kiado, Budapest, pp. 597-613. 10 Kawakami, H. and Kobayashi, M. (1984) Phar- macological approach to the analysis of regulation of molluscan heart activity. Zool. Sci., 1: 387-395. 11 Hill, R. B. (14958) The effects of certain neuro- humors and of other drugs on the ventricle and radula protractor of Busycon canaliculatum and on the ventricle of Strombus gigas. Biol. Bull., 115: 471-482. ZOOLOGICAL SCIENCE 1: 389-397 (1984) Pharmacological Approach to the Analysis of Regulation of Molluscan Heart Activity HARUMI KAWAKAMI and MAKOTO KOBAYASHI! Physiological Laboratory, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan ABSTRACT — The effects of a few putative neurotransmitters and their antagonists on the cardiac activity of a prosobranch gastropod, Rapana thomasiana, were investigated. Acetylcholine (ACh) had strong inhibitory effects on the heart, and the amplitude and frequency of heart beat decreased as the concentration of ACh was increased. ACh antagonists, propantheline and benzoquinonium, depressed or blocked the inhibitory action of ACh, as well as that by stimulation of cardiac nerves, RCN 1 and RCN 3b. Serotonin and FMRFamide (Phe-Met-Arg-Phe-NH,), on the other hand, showed excitatory effects on the heart, with FMRFamide having greater enhancing effects than © 1984 Zoological Society of Japan serotonin. methysergide. prevented by methysergide. The effects of serotonin, and none of those of FMRFamide, were blocked by The excitation caused by stimulation of nerves, RCN 3a, RCN 4 and LCN 1, was not The possible physiological role of these putative neurotransmitters, especially that of FMRFamide, in the heart of Rapana is discussed. INTRODUCTION It is well known that the molluscan heart can beat spontaneously and that the rhythm of the heart beat is controlled by the action of neurotrans- mitters. In general, acetylcholine (ACh) is an inhibitory substance for the molluscan heart, while serotonin is an excitatory substance which enhances cardiac activity [1]. However, this generality cannot be applied to all molluscan hearts, for the mode of action of these substances is quite variable. In experiments using two related bivalve species, Shigeto [2] has shown that ACh _ inhibits spontaneous beating and _ hyperpolarizes_ the membrane in oyster myocardium, but increases the beat frequency and depolarizes the membrane in mussel myocardium. Serotonin excites most bivalve hearts, but depression has also been ob- served [3]. FMRFamide (phenylalanyl-methionyl-arginyl- phenylalanine amide), extracted and purified from clam ganglia, is a potent cardioexcitatory neuro- peptide [4, 5]. FMRFamide usually enhances the Accepted October 18, 1983 Received August 30, 1983 ’ To whom requests for reprints should be addressed. molluscan heart beat at concentrations as low as 10°°-10°* M, but it is strongly inhibitory to some bivalve hearts [3]. In a prosobranch gastropod, Rapana thomasiana, cardiac activity is regulated by the action of several cardiac nerves arising from a pair of visceral ganglia [6]. However, the cardioactive substances released from these nerves as neurotransmitters are not yet known. In the present study, the myocardial effects of ACh, serotonin and FMRFamide were compared with the inhibitory and excitatory actions elicited by cardiac nerve stimulation. In addition, known antagonists were tested for their ability to block both the pharmacological actions of the putative transmitters and the effects of nerve stimulation. Thus, we meant to examine the physiological role of these substances in the regulation of the heart activity in Rapana. MATERIALS AND METHODS Preparations of the heart, with the viscera! ganglia and cardiac nerves mounted on the rounding tissues, were made from Rapana asiana. The methods of dissecting the tiss: 390 H. KAWAKAMI AND M. KOBAYASHI of making the preparations were the same as those described. in the accompanying paper [6], and the same two types of preparations, A and B, were used in the present experiments. The techniques for recording heart beat and stimulating the cardiac nerves electrically were also the same as those described previously [6]. The heart was constantly perfused with artificial sea water (ASW) through a cannula at a rate of about 1.0 ml/min. Drugs were applied by trans- ferring a circulation tube from the normal ASW to the drug-containing ASW, and back. By using this simple method, no change in perfusion rate occurred when solutions were changed, and a small bubble inserted in between two different solutions in the tube easily showed the starting time of drug application or washing. The following drugs were used; acetylcholine chloride (ACh, Daiichi Seiyaku), atropinium sulfate (atropine, Merck), d-tubocurarine chloride (curare, Tokyo Kasei), hexamethonium bromide (hexamethonium, Nakarai Chemicals), propantheline bromide (propantheline, Sigma), benzoquinonium chloride (benzoquinonium, Sterling-Winthrop Res. Inst.), 5-hydroxytryptamine creatinine sulfate (serotonin, Sigma), FIMRFamide (Peninsula Lab.), and methysergide-hydrogenmaleinate (methysergide, Sandoz). Dose-response curves of the putative neuro- transmitters, ACh, serotonin and FMRFamide, were obtained by perfusing successive doses of the drug, from lower to higher concentrations for 1 to 2 min each, interruped by 15 min washing with ASW. To examine blocking effects, the antagonist for a putative transmitter was applied at a relatively high concentration, unless nonspecific effects of the drug itself were produced, whereas the transmitter was given at a relatively low concentration, but sufficient to elicit its effects. All experiments were performed at room tem- perature of 20—24°C. RESULTS Inhibitory effects The inhibitory effects of ACh and nerve sti- mulation were examined by using preparation B, although ACh produced essentially the same effects on both preparations A and B. Figure | shows the effects of ACh on heart beat at six different goncentrations. The threshold concentration was around 10°g/ml. As the concentration was increased, both the amplitude and frequency of heart beat were reduced, and finally the beat stopped in diastole. These re- lationships are graphically illustrated in Figure 2. In this graph, the amplitude and frequency of heart beat were expressed as a percentage of the control, and they decreased in parallel as ACh concentration was increased. The duration of the arrest of best was also dependent on the ACh concentration, but it was quite variable in diffe- rent specimens. To examine the possibility that ACh acts as an Effect of ACh 1 fh sunitiil i ist a I —— 10°9 g/mi 2 Aen Ta ———— SS 2x1079 me | [{—— 5x 1079 \. Hi | 29] 1 min Pineal 107! Fic. 1. Effects of various concentrations of ACh on heart beat. ACh was applied during the period shown by the horizontal line under each record. Regulation of Molluscan Heart Activity 391 100r* RS Cc E eel ois = o eo < ee) 5 +e amplitude = o—o frequency 1 £ +—+ arrest AY 7 o — bo ) 0 © 10710 10°79 10°8 1077 ACh concentration (g/ml) Fic. 2. Dose-response curves of ACh effects. Amplitude and frequency of heart beat and duration of arrest of beat are plotted against the concentration of ACh. Each point is expressed as percent of the control, and it shows mean and SE (vertical line). inhibitory neurotransmitter in the heart, effects of several known blockers of ACh receptors were investigated. Of five drugs tested, atropine, curare and hexamethonium, up to 10°* M, had no signi- ficant effects on the action of ACh (10° g/ml). Sometimes, agonistic effects on heart beat were produced by these blockers. In contrast, propantheline (10°°M) partially blocked the effects of ACh (Fig. 3). In the presence of propantheline, the inhibition of ampli- 1 Control 4 Prop. 10°M 5 Wash after 30 min 2 Prop. 10M 2 Prop.10°M after 10 min | T 1 Control Bi ——————— ACh 10°8 g mi after 25 min after 10 min after 20 min } jb! a | ACh 1 min Fic. 3. Effects of propantheline (Prop.) on the inhibitory action of ACh. ACh was applied during the period shown by the horizontal line under each record. Treatment with 10°°M propantheline reduced the inhibition of heart beat by ACh (tracing 2). The concentration of ACh was unchanged throughout. In this and subsequent figures, designation of a drug without its concentration means that the dose is the same as the preceding one. 3 Prop.10°M after 20 min 6 Wash after 20 min } ie id UVB iii SSS ee 1 min Fic. 4. Effects of propantheline (Prop.) on the inhibitory action by stimulation of RCN 3b. In each record, stimulations (1 msec, 5 V at 10/sec) were given to RCN 3b for 1 sec at a triangle. 392 H. KAWAKAMI AND M. KOBAYASHI tude and frequency of heart beat by ACh were 68-+7.6% (n=5) and 624+7.1% (n=8) of the control, respectively; i.e., 30 to 40% of the ACh inhibition was blocked by propantheline. The recovery from the blockade was accomplished within 10 min by washing with normal ASW. Electrical stimulation of the right cardiac nerves, RCN 1 and RCN 3b, caused an extreme inhibition of heart beat [6]. Experiments shown in Figure 4 were conducted to observe the effects of pro- pantheline on the inhibition caused by stimulating RCN 3b. When 10°°M propantheline was ap- plied, the inhibition produced by nerve stimulation gradually declined and was completely blocked after 30 min. However, the amplitude of heart beat was enhanced by nerve stimulation as the blocking proceeded, and the enhancement re- mained for a while after washing with ASW. These results suggest that the electrically stimulated bundle of nerve fibers (1.e., RCN 3b) may contain some excitatory nerves as well as the dominant inhibitory nerves. Thus, as the effect of inhibitory nerves was blocked by the antagonist, the excitatory effect was unmasked. This excitation was not prevented by the serotonin antagonist, methy- sergide, applied for 30 min. A 1 Contro! B11 Control ll (ll “RCN3b 2 BQ3x105m 2 BQ3x10°m ll a “RCN3b -——_1 ACh 10° 8g —_____j ACh The inhibition of heart beat caused by stimulating RCN 1 was also blocked by pro- pantheline. Moreover, the gradual blocking of inhibition and the subsequent enhancement of contraction height were again observed and were similar to those processes in the case of RCN 3b stimulation. Figure 5 illustrates the effects of another ACh blocker, benzoquinonium, on the inhibition of heart beat, by RCN 3b stimulation and by ACh application, in the same specimen. This experi- ment shows that a longer time was needed to block the inhibitory effect by nerve stimulation than that by ACh application. An extreme inhibition by ACh was completely blocked by 3x10°M benzoquinonium within only 1 min, while the blockage of the inhibition by nerve stimulation was attained 20 min later. This difference may be due to the distribution of ACh receptors on the extra- synaptic membrane as well as on the subsynaptic membrane. Thus, perfused drug could reach the extra-synaptic receptors rapidly, but would take a longer time to reach the subsynaptic ones. The blocking effects of benzoquinonium were washed out within 10 min. Ween Fic. 5. Comparison of effects of benzoquinonium (BQ) on the inhibition of heart beat by RCN 3b stimulation (A) and that by ACh application (B) in the same preparation. sec at a triangle in each record of A. Stimulations (2 msec, 6 V, at 20/sec) were given to RCN 3b for 1 In tracing A2, stimulations were applied 20 min after the treatment with benzoquinonium, and in A3 they were applied 20 min after washing with ASW. ACh was applied during the period shown by the horizontal line under each record of B. In tracing B2, ACh was applied 1 min after the treatment with benzoquinonium, and in B3 ACh was applied 22 min after washing with ASW. Regulation of Molluscan Heart Activity 393 Excitatory effects those of FMRFamide on the heart beat of the same The excitatory effects of drugs and of nerve specimen. Both drugs enhanced heart beat, but stimulation were studied with preparation A. Figure 6 compares the effects of serotonin and es0 od 5HT xe *—e FMRFamide A 5HT B) FMRFamide ~ . 5 200 10 °™M i v Tv =] ‘a 150 = < 10°'M 3 q 100‘. ee Tico eee Dee oe 10s: Concentration (M) 10°M _ 1min Fic. 7. Dose-response curves of the effects of Fic. 6. Effects of various concentrations of serotonin (S HT) and FMRFamide. Amplitude serotonin (5 HT) and FMRFamide on the of heart beat is plotted against the concentration heart beat. Serotonin (A) and FMRFamide of the drug. Each point is expressed as percent (B) were applied during the period shown by of the control, and it shows mean and SE the horizontal line under each record. (vertical line). B) 200 < o— 5HT x «—e FMRFamide « oe 2 °° Pal (S) c b =] Cc a Ae i TT } a. am i (i ot iii ae Te Concentration (M) Fic. 8. Effects of FMRFamide and serotonin (5 HT) on the frequency of heart beat. In A, opposite effects of FMRFamide in different specimens are illus- trated. Tracing | is from one specimen and tracings 2 and 3 are from another. FMRFamide was applied during the period shown by the horizontal line under each record. In B, the frequency of heart beat is plotted against the concentra- tions of serotonin and FMRFamide; the results of two separate groups of speci- mens are shown for FMRFamide. 394 H. KAWAKAMI AND M. KOBAYASHI the contraction height and the duration of enhance- ment produced by FMRFamide were greater than those by serotonin at each concentration tested. Dose-response curves, obtained from the inotropic effects of both drugs, demonstrate that the maximum effect of FMRFamide is about three times greater than that of serotonin and two curves are not in parallel (Fig. 7). This graph also shows that the threshold concentration for FMRFamide (10°? M) is slightly lower than that for serotonin (3x10? M). These results indicate that FMRFamide has greater enhancing effects on heart beat than serotonin and that the mechanisms of action of these two drugs may be different. The frequency of heart beat was slightly increased by the application of serotonin at each concen- tration tested, as shown in Figure 6A and Figure 8B. FMRFamide, on the other hand, showed diverse chronotropic effects on different specimens (Fig. 8A). In specimens with a relatively slow beat, FMRFamide produced positive chronotropic and inotropic effects, whereas in relatively rapidly beating hearts, the beat frequency was either not chang2d_ significantly or was reduced by FMRFamide. The reduction in frequency was especially prominent when a higher dose of FMRFamide was applied which caused a greater enhancement of the amplitude of beat (Fig. 8A—3). The inotropic effects of FMRFamide may domi- nate its chronotropic effects, a condition similar to the effects of excitatory cardiac nerve stimulation [6]. In Figure 8B, the relationship between the concentration of FMRFamide and the frequency of beat is illustrated in two separate groups of speci- mens, one showing a positive chronotropic effect and the other showing a negative effect. Methysergide, a potent blocker of serotonin receptors in bivalve hearts [7], was recently found to act as a strong serotonin antagonist in the buccal muscles of Rapana [8]. In experiments illustrated in Figure 9, the effects of methysergide on the excitatory actions of serotonin and FMRFamide are compared. The enhancing effects of serotonin (10°’ M) were easily blocked by 10°°M methy- sergide only 1 min after the application (Fig. 9A). Although this blocking effect was hardly washable, serotonin of a higher concentration (3 x 10°’ M) A1 Control wqunnaate nig Ht Fee aH Wi! | Wy Pt | 5HT 10°-'M 2 UML 10°°M after 1 min HH HHUA A : WE | yl HU AUP Yi T es ail (QO 5HT 3° Wash after 60 min | MH | ) 1a| A eee ee ee en i —_—— 1 min [ae 5 HT B1 Control FMRFamide 10-°M 2 UML 10-°M after25imin | me FMRFamide 1 min Fic. 9. Effects of methysergide (UML) on the enhancing action of serotonin (5 HT) and FMRFamide. Serotonin (A) and FMRFamide (B) were applied during the period shown by the horizontal line under each record. produced enhancing effects as usual (not shown). The action of FMRFamide, on the contrary, was not affected by methysergide at the same concen- tration. Application of a higher concentration of methysergide (10 * M) for 60 min was also inef- fective at blocking the action of FMRFamide. These results clearly show that the receptors for serotonin and for FMRFamide are different. An enhanced amplitude of heart beat was also elicited by electrically stimulating the cardiac nerves, RCN 3a, RCN4 and LCN 1 [6]. In experiments shown in Figure 10, the effects of methysergide on the enhancing action of nerve Regulation of Molluscan Heart Activity 395 A 1° Control 2 UML 107M after 60 min B1> Control 5HT 10°mM 2 UML 10°mM after 1 min Whe i \ i TH EPA e | Nf hn 1g Hy HANA ii 5HT Vive est $i 11min Fic. 10. Comparison of the effects of methysergide (UML) on the enhancing action of nerve stimulation and of serotonin in the same specimen. In A, stimulations (2 msec, 8 V, at 5/sec) were given to RCN 3a, RCN 4 and LCN 1 for 1 sec ata triangle. line under each record. stimulation and on the action of serotonin were compared in the same specimen. The excitatory effects of nerve stimulation were not affected by the application of 10°° M methysergide for 60 min or more, and there were no significant differences in the results obtained by stimulating the three different nerves, RCN 3a, RCN 4 and LCN 1 (Fig. 1OA). The enhancing effects of serotonin, on the contrary, were blocked by the same con- centration of methysergide only 1 min after its application (Fig. 10B), as usual (cf. Fig. 9A). Serotonin is probably not involved in the nervously induced excitation of heart. DISCUSSION In most molluscan hearts, the remarkable inhibitory effects of ACh have led it to be considered as an inhibitory neurotransmitter [9, 10]. Now we have found that the inhibition of heart beat by stimulating the cardiac nerves, RCN 1 and RCN 3b, and by applying ACh directly, is pre- vented by the same antagonists, propantheline and benzoquinonium. These results indicate that ACh is the inhibitory neurotransmitter at the neuro- muscular junction in the heart of Rapana. The mode of inhibitory action of the two cardiac nerves, RCN 1 and RCN 3b, was different; the In B, serotonin was applied during the period shown by the horizontal former caused a phasic inhibition, while the latter produced a tonic inhibition [6]. Nevertheless, both inhibitory effects were blocked in the same manner qualitatively by the same antagonist. Therefore, the same transmitter, ACh, is probably acting at neuromuscular junctions of RCN 1 and RCN 3b. But the active sites of ACh at the myocardial synapses of the two cardiac nerves may be different. Both serotonin and FMRFamide had excitatory effects on the heart of Rapana. However, FMRFamide showed greater positive inotropic effects, longer maintenance of the effects and a lower threshold concentration than serotonin. Dose-response curves obtained from the inotropic effects of these two substances were not parallel. Moreover, the effects of serotonin were blocked by methysergide, whereas the responses to FMRFamide were unaffected. These results in- dicate that the two substances are acting on separate, specific receptors and that their mech- anisms of action may also be different. Similar differences in the effects of serotonin and FMRFamide on the ventricle of a bivalve, Mercenaria mercenaria, were observed by Higgins et al. [11] and by Greenberg and Price [12]. 7 have considered that serotonin is a fast-: transmitter, whereas the role of FMRFamide 396 H. KAWAKAMI AND M. KOBAYASHI be a long-term maintenance of muscular rhythmi- city and tone, evocative of an earlier proposal by Welsh [13]. In Rapana, the excitatory effects of stimulating the cardiac nerves, RCN 3a, RCN 4 and LCN 1, were not affected by a dose of methysergide which could block the action of serotonin within 1 min. Of course, a longer time is usually needed for an antagonist to block the action of nerve stimulation than to block the action of applied transmitter; such was the case with the ACh antagonists. Yet propantheline and benzoquinonium prevented the response to nerve stimulation 20-30 min after their application. Therefore, methysergide must have fully reached the receptors on the subsynaptic membrane after a treatment of more than 60 min; yet it had no effect on the action of the excitatory nerves. Stimulation of the inhibitory nerves, RCN 1 and RCN 3b, following treatment of the heart with propantheline or benzoquinonium caused excita- tion. Similar phenomena have been observed in the hearts of the bivalves, Mercenaria [14] and Tapes [15]. In these cases, the unmasked exciation was blocked by methysergide, and thus serotonin was considered to be involved in the cardioexcitation by nerve stimulation. In Rapana, on the contrary, methysergide did not prevent the excitation in the propantheline- or benzo- quinonium-treated preparations. From these several results we assume that serotonin is not a mediator involved in nervously induced excitation in the heart of Rapana. If this assumption is true, Rapana would become a very rare case in the regulation of molluscan heart, since serotonin excites most molluscan hearts and is accepted as an excitatory transmitter in those cases [3]. In a preceding paper a possible con- tribution of neurohormones to the control of cardiac activity was suggested [6]. Serotonin could be a candidate substance, contributing to the movement of the whole body, as well as to the regulation of the heart beat. FMRFamide is a neuropeptide extracted from the ganglia of a clam, Macrocallista nimbosa and has strong excitatory effects on bivalve hearts [4, 5]. FMRFamide also shows a potent ex- citatory action on the prosobranch buccal muscles [16], and a FMRFamide-like substance has been extracted from the pulmonate Helix neuron [17]. Although the presence of FMRFamide in Rapana neurons has not yet been demonstrated, the peptide may act as a neurotransmitter at the heart of this animal. This idea is supported by the finding that FMRFamide produced positive ino- tropic effects similar to those of nerve stimulation. However, the involvement of FMRFamide in the excitation by nerve stimulation could not be demonstrated in the present investigation, since no specific blocker for FMRFamide is known [18]. To clarify the physiological role of FMRFamide in the heart of Rapana, many problems, including its distribution within the organism and its pharma- cological analysis, have yet to be examined. ACKNOWLEDGMENT The authors are grateful to Sandoz Ltd. and to Sterling-Winthrop Res. Inst. for supplying a sample of methysergide and benzoquinonium, respectively. REFERENCES 1 Leake, L.D. and Walker, R. J. (1980) Inver- tebrate Neuropharmacology. Blackie & Son, Glasgow. 2 Shigeto, N. (1970) Excitatory and _ inhibitory actions of acetylcholine on hearts of oyster and mussel. Amer. J. Physiol., 218: 1773-1779. 3 Painter,S.D. and Greenberg, M.J. (1982) A survey of the responses of bivalve hearts to the molluscan neuropeptide FMRFamide and to 5- hydroxytryptamine. Biol. Bull., 162: 311-332. 4 Price, D. A. and Greenberg, M. J. (1977) Struc- ture of a molluscan cardioexcitatory neuropeptide. Science, 197: 670-671. 5 Price, D. A. and Greenberg, M. J. (1977) Puri- fication and characterization of a cardioexcitatory neuropeptide from the central ganglia of a bivalve mollusc. Preparative Biochem., 7: 261-281. 6 Kobayashi, M., Nakamura, M. and Hasimoto, T. (1984) Innervation of the heart of a prosobranch mollusc, Rapana thomasiana. Zool. Sci., 1: 383- 388. 7 Wright, A. M., Moorhead, M. and Welsh, J. H. (1962) Actions of derivatives of lysergic acid on the heart of Venus mercenaria. Brit. J. Pharmacol., 18: 440-450. 8 Fujiwara, M. and Kobayashi, M. (1983) Modu- lation of neuromuscular transmission by serotonin in the molluscan radular muscles: Involvement 10 11 12 13 Regulation of Molluscan Heart Activity of cyclic nucleotides. Comp. Biochem. Physiol., 75C: 239-246. Kuwasawa, K. and Hill, R. B. (1973) Regulation of ventricular rhythmicity in the hearts of pro- sobranch gastropods. In “Neurobiology of Inver- tebrates”. Ed. by J. Salanki, Akadémiai Kiado, Budapest, pp. 143-165. Hill, R. B. and Yantorno, R. E. (1979) Inotro- pism and contracture of aplysiid ventricles as related to the action of neurohumors on resting and action potentials of molluscan hearts. Amer. Zool., 19: 145-162. Higgins, W. J., Price, D. A. and Greenberg, M. J. (1978) FMRFamide increases the adenylate cyclase activity and cyclic AMP level of mol- luscan heart. Europ. J. Pharmacol., 48: 425-430. Greenberg, M. J. and Price, D. A. (1979) FMRFamide, a cardioexcitatory neuropeptide of molluscs: An agent in search of a mission. Amer. Zool., 19: 163-174. Welsh, J.H. (1955) Neurohormones. In “The 16 17 18 397 Hormones”’. Ed. by G. Pincus and K. V. Thimann, Academic Press, New York, Vol. 3, pp. 98-151. Loveland, R. E. (1963) 5-Hydroxytryptamine, the probable mediator of excitation in the heart of Mercenaria (Venus) mercenaria. Comp. Biochem. Physiol., 9: 95-104. Phillis, J. W. (1966) Innervation and control of a molluscan (Tapes) heart. Comp. Biochem. Physiol., 17: 719-739. Price, D. A. and Greenberg, M. J. (1980) Phar- macology of the molluscan cardioexcitatory neuropeptide FMRFamide. Gen. Pharmacol., 11: 237-241. Cottrell, G. A., Price, D. A. and Greenberg, M. J. (1981) FMRFamide-like activity in the ganglia and in a single identified neurone of Helix aspersa. Comp. Biochem. Physiol., 70C: 103-107. Nagle, G. T. and Greenberg, M. J. (1982) Effects of biogenic amines, FMRFamide and acetyl- choline on the radula protractor muscle of whelk. Comp. Biochem. Physiol., 73C: 17-21. + he a eae ahd Bp ee ee ee ote ei mi WE Tt, OC ees mew vibesiyly. ao 7 ‘7? } ; tt i) 2 | P lOO. i fej innSs> Wo 191610 sit eae iaivek (ual) wer mito ieniin sterhtind » Hehe Mepis Caer ize 7 $ ¢ ho 7 ear itch ° atypplaltmeny. Tatkaas rs eg) Miele? © ts 4 i bh ' é a »; aa su Laet STS re —— a alt 7 Pee TG MATA. | ae 1 | , . fit c's ei i = a ul b os 4 Phe t OM ese 2 7 all : — cue NS oe PS at Pom. te a ‘ 2 = * Oa ‘ ‘ ei i) etd & } wl ee thee . . 2 i. " (ha ey im ) i." ie Osa eaS 4m , . muh! r Peta re he ia. ! a 7 0 gs 7 Ta. : i cw | be’ 3 { i" geen Ase aio gt io. Gia Mise elt eIOLeniSy : A aes < mae ¥ hi Mees Stat ( rm WtD (Migs aes > is as mt 7 J STiirtonoI tae CY] Do aa eS a nC) a 2 é = ( hee aint. iy ihe as ( ea . - Pee + 7m d _ 5 iC a = 2 : _ & S 0) a | il f zie | a" % J ; : F Hie, \ = - J 4 a et P : Le Tein i Sei A _ Lo, Le eee ,, 1 1 f y ‘ c i ; , , \ a me “ \ ZOOLOGICAL SCIENCE 1: 399-403 (1984) © 1984 Zoological Society of Japan Frustule Movement of Fresh-Water Polyp, Microhydra sowerbii (Lankester) HIROSHI HASHIMOTO Department of Biology, Faculty of Education, Shizuoka University, 836 Ohya, Shizuoka 422, Japan ABSTRACT — The frustule produced asexually from the polyp of the fresh-water hydroid, Micro- hydra sowerbii (Lankester), migrates incessantly with very slow gliding motion on various substrates. The velocity of locomotion of the frustule is sensitively affected by temperature, but at a constant temperature, the velocity varies with body size. speed as compared with shorter specimens. As a rule, longer specimens show higher values of When migrating, the body length of this animal is slightly but distinctly fluctuated with a constant rhythm. The typical form of this rhythmical move- ment is distinctly influenced by temperature while its amplitude remains unchanged. INTRODUCTION The frustule of the fresh-water hydroid Micro- hydra is a planula like organism budded off on the parent polyp (Fig. 1). In spite of absence of cilia or special locomotory organs, the frustule moves on until it transforms into the polyp [1]. The dispersal of this hydroid which is permanently sessile is mostly due to the migration of the frus- tule. The locomotion of the frustule is, however, quite beyond recognition because of its very slow motion with the average velocity of 1 mm per hour at 25°C. Since the first reports on Microhydra [2, 3], many investigators noticed and referred the migration of the frustule, but no detailed obser- vation has been made. Kuhl [4] observed and analysed the movement of this animal by means of low speed photography and first referred the fact of the contraction and extension of the body in the migrating frustule. This paper deals with the velocity and the peri- odical body contraction in the migrating frustule of Microhydra, with a discussion on the similarity of the mechanism of locomotion between the frustule and the pseudoplasmodium of the cellular slime mold. Accepted November 11, 1983 Received September 2, 1983 MATERIALS AND METHODS The material, Microhydra sowerbii (Lankester) [5], was taken in a reservoir at Nagashima, Shimada City, Shizuoka Prefecture, Japan. For the obser- vations, the hydroids were settled on the frosted glass boards, 2.5 x 8.3 cm, and kept in Petri dishes, 2cm in depth and 8.5cm in diameter. A fresh- water Oligochaeta, Aulophorus furcatus (Miiller), cultured in the laboratory was used as the prey of the polyp. Young and active frustules, not more than two days after their release from the parent polyp, were used. The measurement by ocular micrometer was normally made under 150 magni- fications. The experiments were made in winter (January-February), 1974-1977, and the tempera- ture of the medium containing the frustule was controlled by changing the degree of room heating. RESULTS Velocity of locomotion The track of the migrating frustule is not always straight, but often forms irregular arches or loops [4]. However, because the velocity of locomotion was so slow, the track within one minute vertically straight. In the present study, i' confirmed that the velocity of an individual fr 400 H. HASHIMOTO Fic. 1. Frustule of Microhydra. Arrow indicates direction of locomotion. Bar=0.2 mm. 1.4 EZ 120) 0.8 VELOCITY (mm/hr) 0.6 0.4 15 20 DS ad TEMPERATURE Fic. 2. Effect of temperature on the velocity of migration. Each point with vertical bar indi- cates the average+S.D. of twenty individuals of moderate size, 0.6-0.7 mm. at a constant temperature remains unchanged for many minutes [4]. The speed of the frustule was determined by measuring the distance of progress of its caudal (mm /hr) VELOCITY 0.2 0.3 0.4 0.5 0.6 0.7 0.8 mm BODY LENGTH Fic. 3. Correlation between velocity and body length of frustule. Measurement was made at 25°C. edge, which is physiologically cephalic, in ten minutes, and these values were converted into the velocity per hour. As is shown in Figure 2, the values at 15, 20, 25, and 30°C were 0.38+0.05, 0.69-+0.08, 1.00+0.07, and 1.31+0.07 mm/hr. Each value is an average of twenty individuals of moderate size, 0.6-0.7 mm long. The velocity of the migrating frustule thus increased in proportion to the temperature. The speed of the frustule is very variable among individuals, especially according to the body size. The correlation between body length and velocity was examined at 25°C. The values of 63 specimens were shown in Figure 3. As is seen, longer speci- mens show higher values of speed as compared with shorter specimens. Periodical change of body length In the forward movement, the frustule body length changes periodically. Because of their extreme slowness, the body length changes are readily traced by measuring the body length every 15 seconds. At a constant temperature, the frequency and amplitude of the body length changes are kept relatively stable. A cycle of this movement is divided into two periods, the extension period and the contraction period. In the extension period, the frustule distinctly elon- gates to become narrower anteriorly. When body length reaches a maximum, the extension stops, Frustule Movement of Microhydra 401 and the body suddenly slackens and gradual shortening begins. This is the contraction period, in which fore end increases its width and therefore the middle portion becomes somewhat narrower. The amplitude of this movement, i.e., the difference between the minimum and the maximum body length is variable with individuals, but is usually within 10-12% of the maximum body length. The extension and contraction of the migrating frustule are shown by means of the comparison of traces of the locomotion of two points, the fore and rear ends of the body (Fig. 4A, B). The fore end regularly and rhythmically retracts corre- sponding to the contraction period, whereas the rear end always progresses with almost uniform velocity. This mode of the movement differs from usual vermicular movement, creeping motion, 0.3 0.2 0.1 A 0 5 10 15 min mm 0.3 0.2 0.1 0 5 10 15 min Fic. 4. Pattern of advance of fore (A) and rear (B) end of migrating frustule. A and B are based on different specimens. ~ = & a > 1S) Zz ~ = 10 ce 5 15 20 25 30 oC TEMPERATURE Fic. 5. Frequency of rhythmical movement, con- traction and extension of body, in migrating frustules showing effect of temperature. Each point with vertical bar indicates the average+ S.D. of twenty individuals of moderate size, 0.6- 0.7 mm. mm S D Go BODY LENGTH 0.70 0.66 0.68 0.64 5 10 iba «itee 25 min Fic. 6. Typical forms of rhythmical moveme: migrating frustules at different temper* conditions, 30 (A), 25 (B), 20 (C), and 15°C ()) 402 in which fore end never retracts and both fore and rear ends alternately repeat a progress and a passing stop [6, 7]. The period of a cycle from the beginning of extension to the end of contrac- tion is apparently prolonged at low temperatures and shortened at high temperatures. The lengths of the cycles at 15, 20, 25, and 30°C were calculated in term of frequency, that is, 5.0-+0.7, 9.2+1.0, 13.5+0.9, and 18.0+1.3 times per hour and those were graphed in Figure 5. Each value is the average of 20 individuals of 0.6-0.7 mm long. The frequency of the periodical movement is thus finely correlated to the temperature. The examples of typical waves during 28 minutes at each grade of temperature were illustrated in Figure 6. Those waves show that the amplitude of this movement is not affected by temperature. Lower limits of temperature to induce the regular waves of the periodical movement seems to be about 12°C. An incomplete and irregular wave was still observed in some specimens at 10 to 12°C, but no wave was found below 10°C. DISCUSSION Hanaoka [6] states that the frustule of a marine stalked medusa, Thaumatoscyphus distinctus, moves forwards by means of the creeping as in the manner of an earthworm, in which alternation of the extending of the anterior portion and the drawing of the posterior portion is repeated. Otto [7] also reported a similar planula movement of Haliclystus. The mode of locomotion in those creeping planulae is rather universal in the gener- alized vermicular organisms. As stated above, in the locomotion of the Microhydra frustule, the anterior part regularly retracts backwards after the extension periods, but the posterior part always advances keeping almost uniform velocity as shown in Figure 4. The mode of locomotion of the frustule of Microhydra thus can be distin- guished from that of the other vermiform animals. Dejidar [1] considered that the forward movement of Microhydra frustule may be induced by the contraction of the epidermal cells or possibly the germina! longitudinal muscles, and he used the term gliding instead of creeping for the locomotion of this animal. Kuhl [4] stated that the locomotion H. HASHIMOTO of the frustule results from the cooperation of the epidermal muscles of two germinal layers. The motive power of locomotion occurs in the anterior half of the body. He took special notice of the fact that the posterior end is continuously dragged during the extension and contraction of the body. In Microhydra, the body of the frustule is almost cylindrical in shape, and its contact with the base for stability is accomplished by the stickiness of the mucous substance which is secreted from the ectodermal cells and left as a thin slime sheath like a collapsed sausage casing on the base behind the migrating frustule. The sheath itself does not move, but the frustule moves within it. In fact, the frustule is capable of locomotion even if the slime sheath completely loses touch with the outer base, substrates. These facts are suggestive of the locomotion of the pseudoplasmodium of a cellular slime mold Dyctyostelium discoideum, in which the traction occurs between the sheath and the pseudo- plasmodium rather than between the sheath and the environment and the sheath lies on the base as a long track behind the advancing body of the pseudoplasmodium [8]. In the cellular slime mold, pseudoplasmodium, the slug-like cell mass, moves with a slow gliding motion, not the creeping motion [8]. The velocity of this organism is said to be 0.3 to 2.0mm per hour at 20°C, and in Dictyostelium mucoroides, it roughly corresponds to the speed of the individual amoeboid cells which compose the pseudoplas- modium [9], and in D. discoideum, the larger the migrating slug the faster it moves [10]. The speed of the frustule of Microhydra is 0.1 to 1.1 mm per hour at 25°C and is correlated to the body size too (Fig. 2). The parallelism between these quite different types of organism is of interest. It may be owing to the similarity of the mechanism of locomotion. REFERENCES 1 Dejdar,T. (1934) Die Siisswassermeduse Craspedacusta sowerbii Lankester in monographi- scher Darstellung. Z. Morph. Okol. Tiere, 28: 595-691. 2 Potts, E. (1885) Microhydra ryderi. Sci. Bull., 5: 5. 3 Ryder, T. (1885) The development and structure Frustule Movement of Microhydra of Microhydra ryderi. Amer. Nat., 19: 1232. Kuhl, G. (1947) Zeitrafferfilm-Untersuchungen iiber den Polypen von Craspedacusta sowerbii (Ungeshlechtliche Fortpflanzung, Okologie und Regeneration). Abh. senckenberg. naturf. Ges., 473: 1-72. Naumov, D. V. (1960) Hydroid and Hydro- medusae of USSR. Akademii Nauk, Moskva- Leningrad, pp. 509-512. Hanaoka, K. (1934) Notes on the early develop- ment of a stalked medusa. Tokyo Imp. Acad. Proc., 10: 117-120. Otto, J. J. (1976) Early development and planula movement in AHaliclystus (Scyphozoa: Stauro- 403 medusae). In ‘“Coelenterate Ecology and Behavior’. Ed. by G. O. Mackie, Plenum Press, New York and London, pp. 319-329. Bonner, J. T. (1967) The Cellular Slime Molds. Princeton University Press, pp. 92-103. Samuel, E. W. (1961) Orientation and rate of locomotion of individual amoebas in the life cycle of the cellular slime mold Dictyostelium mucoroides. Develop. Biol., 3: 317-335. Bonner, J., Koontz, P. G., Jr. and Paton, D. (1953) Size in relation to the rate of migration in the slime mold Dictyostelium discoideum. Mycologia, 45: 235-240. > Fieg* A Mist voy . (iui oii ~~ es ' in A sh wt . 3 “poh 9 tall tied [Pip 208 ary. : ; my 'itidely 1 ( wie a. eo | ul cane be ney Lye Vien we ately “= o oboe (je otic ~ ‘ c - a ; en ONS 4 ” 7 os “ ee” é me, ee > “ " ‘a , r : ee a a Fic. 9. A transverse section of a portion of the retracted axoneme (arrow) in the cytoplasm of a prophase cell in 7. toreumaticus. Note the large chromatin fibers (CH) and their absence in the region near the nuclear membrane (NM). Xx 22,000. already mentioned by Harris [37]. In telophase, the diplosomes were positioned somewhat more inward than in anaphase, but still remained within the sub-cortical region of the cells. Two centrioles in a diplosome retained the ‘L-shaped’ arrangment during prometaphase to anaphase (Figs. 10 and 11) but not in telophase (Fig. 12). In early telophase, the longitudinal axes of mother and daughter centrioles no longer lay on the same plane starting the ‘skewed’ ori- entation. This indicated that the two centrioles of a diplosome become disoriented or split apart at early telophase. The ‘skewed’ orientation was sustained as the general arrangement of the basal body and the proximal centriole (Figs. 2, 3 and 5). It was recognized that the daughter centrioles had elongated and attained almost maximum length of 0.35 wm in telophase. The regeneration of: the accessory structures associated with the future basal body seemed to begin on the mother centriole in metaphase (Fig. 11), and was prominent in early telophase (Fig. 12). The basal foot, transitional fibers and a short rootlet were observed on the mother centriole of the early telophase cell (Fig. 12). However, the mother centriole had neither as- sembled the axoneme nor attached to the cell surface, and the basal foot and the rootlet hac not been in contact with microtubules. 7 results revealed that the mother centriole at each spindle pole during division wa: transformed into the basal body in eac' M. MASUDA AND H. SATO oF ase % a od Fic. 10. Transverse serial sections of a prometaphase cell in 7. toreumaticus. The nuclear membrane is fragmenting, and astral and spindle microtubules are assembling around two diplosomes. The former basal body has lost its accessory structures and is not distinguishable from the former proximal centriole. Mother (MC, and MC,) and daughter (DC, and DC,) centrioles still maintain the ‘L-shaped’ orientation of the diplosome. x 27,000. cell. Soon after the division, the ‘skewed’ pair of centrioles in each daughter cell started to migrate towards the apical cell surface and initiate the In this study, the behavior of cilia, basal bodies assembly of axoneme of the regenerating cilium. and centrioles was surveyed throughout the mitotic DISCUSSION Centriole Cycle in Ciliated Cells 457 wer, « ¢ ‘ « 2 Fic. 11. Oblique sections of a metaphase cell in 7. toreumaticus blastula. Spindle microtubules are cleary shown with remnants of astral microtubules, and a diplosome is located in vicinity close to the cell surface. The mother centriole (MC) shown in (a) seems to start the regeneration of the acces- sory structures characterizing the basal body. (a) and (b) 33,000; (c) x 85,000. cycle of ciliated cells of sea urchin larvae both in prophase 2 min before the nuclear me vivo and from an EM approach. We observed breakdown, and regenerated the cilia in that the ciliated cells resorbed their cilia in cells during early interphase 3 min 458 M. MASUDA AND H. SATO Fic. 12. Serial sections of the spindle pole region in a early telophase cell in 7. toreumaticus. rootlet (SR), while they are not directly in contact with microtubules around the diplosome. centriole (DC) has elongated to almost maximum size of the mother centriole. The mother centriole (MC) of the diplosome has regenerated transitional fibers (TF), a basal foot (BF) and a short The daughter Note the ‘skewed’ orien- tation of these two centrioles and the reformation of the nuclear membrane around the chromosomes. x 26,000. completion of mitcsis. We also confirmed that the basal bodies of the cilia in interphase cells were reversibly converted to the centrioles at the spindle poles in dividing cells. These present findings are believed to show the first evidence that demonstrates the occurrence of mitosis as well as the cyclic changes of centrioles in met- azoan cells which bear cilia of the ‘9+2’ axonemal pattern. Overall features of the morphological changes in cilia, basal bodies and centrioles coincide with the results obtained from cultured mammalian cells with the ‘9+0’ cilia in mitosis [18, 21] and from flagellated spermatocytes of the sea urchin, Hemicentrotus pulcherrimus, in meiosis [28]. However, questions such as these arise. How is the resorption of cilia performed? How long does it take for basal bodies to be transformed into mitotic centrioles, and vice versa? The answers to these questions would be difficult to obtain through only the effort of the EM study; however, Nomarski microscopic observations of living cells allowed us to obtain accurate answers. The events on ciliary resorption can be divided into three phases: The phase one is the quick reduction in ciliary length, which takes only a few sec. The phase two, which takes a few min to complete, is the process of slow resorption of the ciliary remnants. The phase three is the disintegration of the retracted axonemes in the cytoplasm, which takes also a few min. The EM study reveals that naked axonemes exist Centriole Cycle in Ciliated Cells 459 within the cytoplasm in prophase, suggesting that the shortening of the cilia is performed by the rapid retraction of whole axonemes into the cytoplasm, while the ciliary remnants are the axoneme-free ciliary membranes left after the re- traction of axonemes. Bloodgood [13] has clas- sified the mode of the flagellar and ciliary loss of algae, fungi and protozoans into five categories, notifying that the loss caused by the retraction of axonemes into the cytoplasm (category 3) takes only a matter of sect to occur while the disinte- gration of the retracted axonemes takes 10 to 60 min. The mode of the ciliary loss of sea urchin larval cells is consistent but has slight difference in detail with that of the third category described by Bloodgood [13]. For example, the ciliary remnants are left after the resorption of cilia, and the disintegration of the retracted axonemes required only a few min to complete. The accessory structures characterizing the basal bodies are also reversibly lost during mitosis. They are disintegrated in prophase but regenerated again from metaphase. These structures can act as MTOCs in interphase and are likely to play a role in organizing cytoplasmic microtubules. Tilney and Goddard [35] suggested that the satellite of the basal body, the identical struc- ture with the spherical cap of the basal foot, initiated in vivo assembly of microtubules in ciliated larval cells of the sea urchin, Arbacia punctulata. In mitotic cells, spindle and astral microtubules are assembled around the diplosome, however, they are not in direct contact with either the spherical cap of the basal foot or the striated rootlet during their disintegration in prophase or in their regeneration during metaphase to telo- phase. Therefore, it can be assumed that the ultrastructural changes of the accessory structures Fic. 13. initiation of procentriolar formation in early interphase. (c) The ciliary resorption and the disintegration of (d) The initiation of the regeneration of acces- (e) The disorientation of mother and daughter cen- The daughter centriole reaches the maximum size until telo- diplosomes in late interphase. the accessory structures in prophase. sory structures in metaphase. trioles in telophase. phase. Scheme of the centriole cycle in ciliated cells of sea urchin blastulae. (a) The (b) The separation of two 460 M. MASUDA AND H. SATO represents the morphological process of the interconversion between the basal body and the centriole in mitosis. Then we can estimate the times required for the transformation of basal bodies into mitotic centrioles and vice versa. It may take 2 min for the basal bodies to be con- verted into mitotic centrioles, and less than 16 min to complete the reverse process. Many studies have been reported dealing with the behavior of centrioles in the cell cycle and the concept of the centriole cycle has been well es- tablished [20, 38-41]. In brief, the centriole cycle consists of four morphological events which are coordinated with other events in the cell cycle: the disorientation or splitting of two centrioles in a diplosome; the initiation of pro- centriolar formation; the elongation of pro- centrioles; and the physical separation of two diplosomes. We can recognize these four cyclic events in the mitotic cycle of the ciliated larval cells of sea urchins (Fig. 13). Two distinct dif- ferences are noted between the centriole cycle of the larval cells and that of the mammalian cells. One is in the mode of the disorientation. and the other is of the separation. In mammalian cells, the ‘L-shaped’ arrangement of a diplosome breaks up during G, period and the mother and daughter centrioles begin to show more or less a random orientation. However, in sea urchin larval cells, the disorientation of centrioles is performed by an orderly change from the ‘L-shaped’ to the ‘skewed’ orientation in early telophase, then the latter state is retained until the separation of two diplosomes begins. The separation of two diplosomes in mammalian cells, including the cells possessed primary cilia [21], begins in prophase and proceeds in association with the process of spindle formation. However, in the ciliated larval cells of sea urchins, the separation of diplosomes occurs during interphase which is performed by the translocation of the proximal centriolar pair from the ciliary base to the nuclear periphery, leaving the basal body pair at the ciliary base. Furthermore, the centriole cycle of the ciliated larval cells is somewhat different from the be- havior of “mitotic centers’ in non-ciliated cells of sea urchin embryos during the early development. In the latter case, the centrioles should be splitted, replicated and begin to be separated at about the same time during telophase [42-44]. One of the striking features of the centriole cycle found in this study is the fact that a newly formed centriole requires two cycles to be func- tionally matured but needs only one cycle to attain the full size of centrioles. A procentriole in a generation of the ciliated cell cycle will act aS a proximal centriole in the next generation, and then it will change into a basal body in the third generation. Although the proximal centriole acts as a mother centriole and can form its daughter, only the basal body can assemble the ciliary axoneme and also organizes cytoplasmic micro- tubules via the ciliary rootlet complex and the basal foot. In this respect not the length of centrioles but rather the existence of the accessory structures on centrioles seems to be a reliable marker for the centriolar maturation. Conse- quently, each ciliated cell possessed only one fully matured centriole (basal body) while it contains two mother centrioles. The same situation has recently been demonstrated by Vorobjev and Chentsov [20] in the centriole cycle of cultured pig kidney embryo cells. The fact that only one of the two mother centrioles in the interphase cell is activated and retains the capacity for organizing microtubules may explain why there is only one cilium in each cell throughout inter- phase. ACKNOWLEDGMENTS We would like to thank Dr. Koichi H. Kato, Institute of Biology, College of General Education, Nagoya City University, for his generous assistance with the electron microscopy. REFERENCES 1 Gibbins, J.R., Tilney, L.G. and Porter, K. R. (1969) Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. 1. The distribution of micro- tubules. J. Cell Biol., 41: 201-226. 2 Amemiya, S., Akasaka, K. and Terayama, H. 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(1971) Cell pro- liferation in the neural tube: An _ electron microscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch., 115: 226-264. Tachi, S., Tachi, C. and Lindner, H.R. (1974) Influence of ovarian hormones on formation of solitary cilia and behavior of centrioles in uterine epithelial cells of the rat. Biol. Reprod., 10: 391-403. Archer, F. L. and Wheatley, D. N. (1971) Cilia in cell-cultured fibroblasts. II. Incidence in mitotic and post-mitotic BHK 21/C13 fibroblasts. J. Anat., 109: 277-292. Tucker, R. W., Pardee, A.B. and Fujiwara, K. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 461 (1979) Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell, 17: 527-535. Vorobjev, I. A. and Chentsov, Y. S. (1982) Centrioles in the cell cycle. I. Epithelial cells. J. Cell Biol., 93: 938-949. Rieder, C. L., Jensen, C. G. and Jensen, L. C. W. (1979) The resorption of primary cilia during mitosis in a vertebrate (PtK,) cell line. J. Ultrastruct. 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(1982) Flagellum formation and centriolar behavior during sperma- togenesis of the sea urchin, Hemicentrotus pulcher- rimus. Acta Embryol. Morph. Exper., n.s., 3: 49-66. Tyler, A. and Spiegel, M. (1956) Elevation and retraction of the fertilization membrane of echino- derm eggs fertilized in papain solutions. Biol. Bull., 110: 196-200. Kushida, H. (1975) Hardness control of the Quetol 651 cured block. 24: 299. Iwaikawa, Y. (1967) Regeneration of cilia in the sea urchin embryo. Embryologia, 9: 287-294. Burns, R. G. (1973) Kinetics of the regeneration of sea urchin cilia. J. Cell Sci., 13: 55-67. Immers, J. and Lundgren, B. (1972) Aspects of differentiation and function of cilia and adjacent structures of the sea urchin larva. Acta Embryol. Exper., 1972: 177-197. Pickett-Heaps, J.D. (1969) The _ evolutic the mitotic apparatus: an attempt at comp ultrastructural cytology in dividing plar Cytobios, 3: 257-280. J. Electron Microscopy, 462 35 36 37 38 39 40 Tilney, L.G. and Goddard, J. 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ZOOLOGICAL SCIENCE 1: 463-470 (1984) Acid Mucopolysaccharide in Embryos of the Horseshoe Crab, Tachypleus tridentatus (Chelicerata, Arthropoda)' Tomio ITow and KoIcHI SEKIGUCHI?*> Department of Biology, Faculty of Education, Shizuoka University, Shizuoka 422, and *Institute of Biological Sciences, University of Tsukuba, Niihari-gun, Sakuramura, Ibaraki 305, Japan ABSTRACT — The distribution, type, quantity and biosynthesis of acid mucopolysaccharide (AMPS) in embryos of the horseshoe crab (Chelicerata, Arthropoda) were examined. In embryos at early developmental stages, most of the AMPS is sulfated type and fills in the perivitelline space between the chorion and the blastoderm. It is already found in unfertilized eggs and it is rarely synthesized after fertilization. ‘When the germ disc appears (stage 7), it is separated from the blastoderm by the secretion of amembrane. It disperses into sea water after the rupture of the chorion (stage 18 or 19). The other type of AMPS is synthesized in the embryonic body and is non-sulfated. At the hatching stage (stage 21), the non-sulfated AMPS decreases and sulfated AMPS is found in the endoskeleton, the intestine and in the articulation of the appendages. © 1984 Zoological Society of Japan INTRODUCTION In the cortical regions of the embryos of several marine animals belonging to the Annelida, Echi- nodermata and Vertebrata, there is a layer of acid mucopolysaccharide (AMPS) [1-4]. AMPS also exists in the intercellular matrix and it is related to many biological functions such as the construction of cells, the toughness and flexibility of tissues, calcification, the control of electrolytes and water, wound healing, lubrication, and the maintenance of stable transports [5]. In embryos of the horseshoe crab, a marine arthropod, the existence of AMPS in the cortical layer has been reported by Sekiguchi ef a/. [6] and Bennet [7], and the cortical reaction was examined in detail by Brown and Clapper [8], Bannon and Brown [9], and Brown and Barnum [10]. AMPS in the cartilage of adults has been described [11- 13], as well as the effect of inhibitors of the syn- thesis of AMPS on the embryonic development Accepted December 6, 1983 Received July 13, 1983 Contribution No. 421 from the Shimoda Marine Research Center. Present address: Ushiju 354, Kisai-cho, Kitasaitama- gun, Saitama, Japan. [14, 15]. However, the exact distribution and type of AMPS present in the embryos at different developmental stages have not been elucidated thus far. This paper deals with the characterization of AMPS in embryos of Japanese horseshoe crab, Tachypleus tridentatus. MATERIALS AND METHODS Male and female horseshoe crabs, Tachypleus tridentatus, were collected in Saga and Fukuoka Prefectures in Japan. They were transferred to laboratories at the Shimoda Marine Research Center and Shizuoka University where the present studies were conducted. The mature eggs were obtained from the body cavity of females by dissection. The eggs were fertilized by artificial insemination of sperm obtained from male indi- viduals. The fertilized eggs were reared in filtered sea water in a plastic tray. The stages of embry- onic development were identified according to Sekiguchi [16]. For light microscopic studies, the embryos were fixed in Bouin’s, Carnoy’s, or FAA solv! embedded in paraffin, and sectioned from ym in thickness. The sections were stai Mayer’s hematoxylin and eosin. The © 464 T. Itow AND K. SEKIGUCHI of AMPS was determined by histochemical methods using 1 °% alcian blue (pH 1 or 2.5), 0.05- 0.1°% toluidine blue (pH 0.1 or 4.0) and Hale’s reaction. The type of AMPS was determined by methylation and saponification. If a tissue con- tained non-sulfated AMPS, it was stained again after saponification. A digestion method using testicular hyaluronidase was also used for the determination of type of AMPS. Periodic-acid Schiff reaction (PAS) and digestion by amylase were used for the determination of distribution and type of neutral polysaccharide. For electron microscopy, specimens were pre- fixed in 5% glutaraldehyde and 4% paraformal- dehyde in cacodylate buffer (pH 7.4) and then post-fixed in 1% osmium tetroxide solution. These specimens were embedded in Epon 812 or Spurr low embedding resin (Polyscience Inc., USA), and sectioned with a Poter-Blum ultra- microtome. The sections were stained with saturated uranyl acetate and Reynold’s solution. The type, quantity and biosynthesis of AMPS were determined by the following biochemical methods. The AMPS was extracted from em- bryos at various developmental stages using the method of Aoki and Koshihara [17], and measured quantitatively by the method of Scott [18] using chondroitin sulfate as an internal standard. For qualitative analysis, the extracted AMPS was fractioned by Dowex 1-column chromatog- raphy. Hyaluronic acid, chondroitin sulfate and heparin were used as marker for the chromatog- raphy. Non-sulfated AMPS is mainly eluted by 0.25 M and 0.5 M NaCl, heparan sulfate by 1.25 M Stage 21 Fic. 1. The distribution of AMPS at different developmental stages. A: sulfated AMPS, A: non- sulfated AMPS. The AMPS which was observed in the embryos at stage 21 was mainly sulfated AMPS. The bar represents 1mm. A: articulation of appendages, skeleton, EX: exuvium of embryonic molting, inner egg membrane, IN: intestine, Pre. 2. c. Stage 13 N: nucleus, The cortical region of horseshoe crab embryos. The bar represents 1 ym. CH: chorion, PS: perivitelline space (layer of AMPS). CH: chorion, EN: endo- I: intermediate membrane (I-membrane), IE: PF: perivitelline fluid, PS: perivitelline space (layer of AMPS), T: general tissue of embryonic body, Y: yolk. a. early blastula stage (stage 4), b. stage 9, I: I-membrane, JE: inner egg membrane, 465 Mucopolysaccharide in Horseshoe Crab 466 T. Irow AND K. SEKIGUCHI NaCl, chondroitin sulfate by 1.5 M NaCl, and heparin by 2.0 M NaCl. Electophoresis was also carried out using the methods of Seno ef al. [19]. Digestion by hyaluronidase was also used. For the examination of biosynthesis of sulfated AMPS, embryos at different stages were reared for 72 hr in sea water containing 5 wCi *°*S—Na,SO,. AMPS was extracted from the treated embryos, and the radioactivity was measured with a liquid scintillation counter. In addition, the extracted AMPS was fractioned on a Dowex column to examine the type of synthesized AMPS. RESULTS AND DISCUSSION Sulfated and non-sulfated AMPS were found in several portions of embryos at different develop- mental stages. The distribution of AMPS is 1d O > yy 2 10 aL {e > w V3 eae Fic. 3. different developmental stages. embryos at stage 21. chorion number is more than 3 at each stage. Oy ali 20 21 The quantity of AMPS at different stages of horseshoe crab embryos. summarized in Figure 1. There was a great deal of AMPS in the peri- vitelline space between the chorion (the first egg membrane, the vitelline envelope or the outer egg membrane) and the surface of the periplasm at the stages between the unfertilized egg and the blastula stage (Fig. 2-a). After stage 7, when the germ disc appeared, the AMPS was separated from the blastoderm by a membrane which was secreted from the blastodermal cells. Hereafter, the authors call this membrane the intermediate mem- brane (I-membrane). It corresponds to the thin membrane which was reported by Sekiguchi ef ai. [6] (Fig. 2-b). The I-membrane became thicker and was pushed up by an other membrane which began to be secreted at stage 11. The latter mem- brane is called the inner egg membrane (the second- ary egg membrane or deutovum) [20] (Fig. 2-c). 1st instar early late larva The abscissa shows the In the horseshoe crab, the Ist instar larva has the same form as the *: the quantity of AMPS extracted from the embryos after the rupture of the The AMPS which was attached to the ruptured chorion was only 0.3 »g/embryo. Bars show standard deviation. Sample Mucopolysaccharide in Horseshoe Crab 467 TABLE 1. Histochemical reaction of embryos of the horseshoe crab ay eiion, General Inner egg exuvium, tissues of Chorion Yolk (the layer intestine, embryos membrane of AMPS) articulation Alcian blue (pH 2.5) a 4 4 om = et Alcian blue (pH 1.0) tt tt ++ Toluidin blue (pH 4.0) Ht an ie oa = Toluidin blue (pH 0.1) +t SI = a = = Hale’s reaction tt tt ++ = - = Alcian blue (pH 2.5) = nf . la 7 % after Methylation Alcian blue (pH 2.5) after < 2s 4h * s at Methylation +Saponification Bee ectirenidiace, divest tt tt an ae vi ¥ Peridoic-acid Schiff (PAS) = = ++ = aa tt PAS after Amylase digest _ — = = a tH +=Weak reaction; ++=Moderate reaction; The AMPS remained in the perivitelline space until stage 18. The first embryonic molting occurs at stage 18. At stage 18 or 19, the chorion is ruptured by the enlargement of the inner egg membrane. Then, the inner egg membrane wraps the embryo instead of the chorion. The AMPS in the perivitelline space was discharged following the rupture of the chorion. As the total quantity of AMPS in eggs decreased greatly after the rupture of the chorion (Fig. 3), we were able to determine that almost all AMPS in the eggs before the rupture of the chorion existed in the perivi- telline space. This AMPS was sulfated AMPS and mainly eluted in the fraction of heparan sulfate (Table 1, Fig. 4). Nevertheless, sulfated AMPS was rarely synthesized during the period imme- diately after fertilization (stage 1) to the develop- ment of appendage rudiments (stage 16) (Fig. 5). The AMPS may be synthesized during oogenesis. A small quantity of sulfated AMPS was found Fic. 4. The rate of each fraction of AMPS by column-chromatography. AMPS was extracted from horseshoe crab embryos at each stage shown in the ordinate. The AMPS was frac- tioned by NaCl. In the case of the extraction from the embryos at stage 18, their chorions were not ruptured. ( ): the days after the insemination. + Strong reaction; —=No reaction. in an other place. It existed on the outer surface of the chorion at stage 1 and was gradually dis- NaCl 0.25 M & 1-25 M 15M 2.0M 0.5 M un-fertilized egg Stage 10 (11 day.) Stage 18 (23 day.) suse 20 oa) [iS Stage 21 (55day,) Ist instar larva (70 day.) Perivitelline fluid at Stage 20 Endoskeleton in adults Hyaluronic acid Chondroitin sulfate Heparin 468 T. Irow AND K, SEKIGUCHI 150 3 7 13 16 Developmental Fic. 5. The biosynthesis of sulfated AMPS in horseshoe crab embryos. late 21 20 20 18 VS stage early Incorporation of *°S- Na,SO, into AMPS which was extracted from the embryos at the different stages shown in the abscissa. persed into the sea water (Fig. 1, Table 1). The synthesis of sulfated AMPS was recognized after the first embryonic molting (stage 18) (Figs. 4 and 5). In the period from stage 18 to 20, the sulfated AMPS was found on the outer surface of the embryonic body, the exuvium of the embryonic molting, and the internal surface of the intestine. In the perivitelline fluid which exists between the inner egg membrane and the embryonic body, sulfated AMPS was also found at these stages. The sulfated AMPS which was synthesized at these << = OM 0.25M 0.5 M NaC! fraction of heparan sulfate. ef 1.25 M NaCl Fic. 6. The column-chromatography of AMPS incorporating **S—Na,SO,. racted from the embryos at late stage 20, and was fractioned by NaCl. raction was counted. The ordinate shows CPM/embryo. The radioactivity was mainly found in the AMPS eluted by 1.25 M NaCl, that is, the The ordinate shows the counts per minute (CPM)/total AMPS in an embryo. stages was mainly eluted into the heparan sulfate fraction (Figs. 4 and 6). However, the synthesized sulfated AMPS seemed to be small in quantity. As mentioned later, biochemical and histochemical observations showed that a major part of AMPS at stage 19 and 20 was non-sulfated AMPS. In the embryonic body at stage 21 (the stage after the 4th embryonic molting, or the stage of hatching), sulfated AMPS was found in the endoskeleton, the epithelium of the intestine and in the articulation of appendages (Fig. 1, Table 1). as 3-0 —<—_—_—_ — — | 1.5 M 2:0 M M The AMPS was ex- The radioactivity of each The abscissa shows each fraction of Mucopolysaccharide in Horseshoe Crab 469 In addition, it was found in the perivitelline fluid at stage 21 as well as the period from stage 18 to 20. As sulfated AMPS has an active polyanionic nature in controlling electrolytes and water in extracellular fluid and has a role in lubrication [21], the sulfated AMPS in the perivitelline space may protect the horseshoe crab embryos from the high salinity of sea water and the mechanical force. Embryos of many other marine animals have a great deal of sulfated AMPS in the perivitelline space, too. The cartilage of marine animals con- tains a great deal of sulfated AMPS as compared with animals living on land or in fresh water [22- 26]. These facts support the above-mentioned supposition. Non-sulfated AMPS was found in the tissues of the embryonic body at the stages before 20. It was uniformly distributed in the embryonic body and was thought to be mainly hyaluronic acid, judging from histochemical and biochemical examinations (Table 1 and Figs. 4 and 7). When Stage 20 (Hyaluronidase digest) Stage 20 Stage 19 Hyaluronic acid Chondoroitin sulfate AMPS was extracted from embryos at stage 19 or 20 and examined by electrophoresis, only one band was Observed. The band was identified with that of hyaluronic acid and was digested by hy- aluronidase (Fig. 7). Histochemical observations also showed that the AMPS of embryonic bodies at stages before 20 was mostly non-sulfated AMPS (Table 1). The non-sulfated AMPS decreased greatly at stage 21 (Figs. 3 and 4). According to Meyer [27], non-sulfated AMPS controls water and protects animals against bac- terial attack. The non-sulfated AMPS in horse- shoe crab embryos may have these roles and may also play a role in cell construction during mor- phogenesis and in Vertebrata, as well [28]. The tissues of the embryonic body also con- tained neutral polysaccharide. As it was digested by amylase, it was probably glycogen. In the yolk, there was no AMPS, just neutral mucopoly- saccharide. In the chorion and inner egg membrane, AMPS and neutral polysaccharide a Start © Fic. 7. The electrophoresis (cellulose acetate strips) of the extracted AMPS. 470 were not found (Fig. 1, Table 1). ACKNOWLEDGMENTS The authors thank Prof. Hiroshi Watanabe, the director, Prof. Arinobu Ebara, the former director, and Dr. Toshiki Makioka of the Shimoda Marine Research Center for their hospitality. The authors also thank Prof. Hidetoshi Koshihara, Dr. Yasuko Koshihara, Prof. Nobuko Seno (Ochanomizu Univer- sity) and Prof. Yoshihiko Ohta (Shizuoka University) for their valuable advice and hospitality. 10 11 72 REFERENCES Pasteels, J.J. (1965) Etude au _ microscope électronique de la réaction cortical. I. La réaction corticale de fécondation chez Paracentrotus et sa chronologie. II. La réaction corticale de loeuf vierge de Sabellaria alveolata. J. Embryol. Exp. Morph., 13: 327-339. Pasteels, J.J. (1966) La réaction corticale de fécondation de loeuf de Nereis diversicolor, étudiée au microscope électronique. Acta Embryol. Morph. Exp., 6: 155-163. Crrkowitz, E. (1971) The hyaline layer; its isolation and role in Echinoderm development. Dev. Biol., 24: 348-362. Reverberi, G. (1971) Experimental embryology of marine and fresh-water invertebrates. North- Holland, Amsterdam. Brimacombe, J.S. and Webber, J. M. Mucopolysaccharides. Amsterdam. Sekiguchi, K., Itow, T. and Takasawa, T. 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SEKIGUCHI Bull., 125: 355-356. Person, P. and Philpott, D. E. (1969) The biology of cartilage I. Invertebrate cartilages: Limulus gill cartilage. J. Morphol., 128: 67-93. Itow, T. (1980) Teratogenesis of the horseshoe crab by a-lipoic amide (6, 8-thioctic amide) in marketed vitamin tablets. J. Nutr. Sci. Vitaminol., 26: 237-245. Itow, T. (1982) Effect of the glutamine-analogue “azaserine’” on embryonic development of the horseshoe crab. Develop. Growth and Differ., 24: 295-303. Sekiguchi, K. (1973) A normal plate of the development of the horse-shoe crab, Tachypleus tridentatus. Sci. Rep. Tokyo Kyoiku Daigaku B, 15: 153-162. Aoki, Y. and Koshihara, H. (1972) Inhibitory effects of acid polysaccharides from sea urchin embryos in RNA synthesis in vitro. Exp. Cell Res., 70: 431-436. Scott, J. E. (1960) Methods of biochemical analy- sis, Vol. 8. Interscience, New York. Seno, N., Anno, K., Kondo, K., Nagase, S. and Saito, S. (1970) Improved method for electro- phoretic separation and rapid quantitation of isomeric chondroithin sulfates on cellulose acetate strips. Anal. Biochem., 37: 197-202. Sekiguchi, K., Makioka, T. and Sato, T. (1976) On the formation of the inner egg-membrane in the Japanese horse-shoe crab, Tachypleus tridentatus. Zool. Mag., 85: 57-64. Dorfman, A. (1985) Studies on the biochemistry of connective tissue. Pediatrics, 22: 576-589. Person, P. and Mathews, M.B. (1967) Endo- skeletal cartilage in a marine polychaete, Eudistylia polymorpha. Biol. Bull., 132: 244-252. Person, P. and Philpott, D. E. (1967) On the occurrence and biological significance of cartilage tissue in invertebrates. Clin. Orthop. Related Res., 53: 185-212. Person, P. and Philpott, D. E. (1969) The nature and significane of invertebrate cartilages. Biol. Rev., 44: 1-16. Philpott, D. E. and Person, P. (1970) The biology of cartilage II. Invertebrate cartilages: Squid head cartilage. J. Morphol., 131: 417-430. Suzuki, S. (1968) Mucopolysaccharide-protein complexes: Elementary substances of connective tissues. Sci. Living, 19: 62-75. Meyer, K. (1947) The biological significance of hyaluronic acid and _ hyaluronidase. Physiol. Rev., 27: 355-359. Toole, B. P. and Gross, J. (1971) The extra- cellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation. Dev. Biol., 25: 57-77. ZOOLOGICAL SCIENCE 1: 471-476 (1984) © 1984 Zoological Society of Japan Variations in Squirrel Monkey in vitro Fertilization Responses with Seasonal and Environmental Conditions PHILIP J. CHAN! and W. RICHARD DUKELOW Endocrine Research Unit, Michigan State University, East Lansing, MI 48824, U.S. A. ABSTRACT — A squirrel monkey in vitro fertilization system was utilized to investigate reproductive variables. Monkeys were exposed to a controlled constant environment for the first, second and fourth quarters of the year and to a warm temperate outdoor environment for the third quarter of the year. (April-June) in a three year study (1980-1982). Peaks of high in vitro maturation and fertilization rate were observed in the second quarter Low embryo cleavage rates occurred in the second and third quarters. Degeneration of oocytes in culture was highest in the fourth but lowest in the second quarter. cultured oocytes. INTRODUCTION There has been very little research on the sea- sonal changes in gametes, a variable that may possibly affect the rates of in vitro fertilization and embryo transfer success. Squirrel monkeys, in their natural environment, have a mating season from June to September [1] but shift mating season to the winter months, January to March [2] when reared in North America. Jarosz et al. [3] reported that FSH treatment in the spring (April to June) resulted in increased eosinophilic and pycnotic vaginal cell counts (directly related to intensity of follicular or secretory phase) but this effect was not observed from July to September (anovulatory months). The number of corpora lutea in hormonally- induced animals were reported to be about 50% more from April to June compared to the July to September [3]. Similar findings were noted for the follicular response to an ovulation induction regimen [4]. The average number of animals ovulating from April to June (42.9%) can be Accepted February 10, 1984 Received October 15, 1983 Present address: University of Medicine & Den- tistry of New Jersey, New Jersey School of Osteopathic Medicine, Camden, New Jersey, U.S.A. The data show a seasonal effect, probably augmented by type of environment, on compared to July to September (13.9%) [5]. The breeding periods may also influence the responses of gametes in culture. Smith et al. [6] reported a higher rate of in vitro oocyte maturation during the breeding season (58%, October—Jan- uary) compared to the nonbreeding season (21 %, April-July) in Macaca mulatta. They hypothe- sized that low estrogen levels in the nonbreeding season decreased the maturity of oocytes. An earlier report [7] indicated a seasonal effect on in vitro maturation rates for squirrel monkey oocytes recovered laparoscopically in 1980. A higher maturation rate (up to 30% difference) was observed in the second quarter (April to June). The present report examines the oocyte maturation rate in the succeeding years. There have been no studies on the changes in in vitro fertilization and development of oocytes on a seasonal basis. The effect of seasonality on squirrel monkey in vitro fertilization is considered in the present study. MATERIALS AND METHODS Experimental animals and egg recovery The animals used in the experiments squirrel monkeys (Saimiri sciureus, 500 t of Bolivian origin (Charles River Res 472 P. J. CHAN AND W. R. DUKELOW mates, Port Washington, New York) housed indoors on a 12: 12 hr light: dark cycle at 21°+ 3°C. In the summer months (July through Sep- tember), the animals were housed in gang cages outdoors [8]. The diet consisted of a commercial high protein monkey feed (Ralston-Purina), slices of apples and fresh water. Gamete recovery Female squirrel monkeys received four daily im. injections of follicle stimulating hormone (1 mg, FSH-P, Burns-Biotec Labs, Inc., Omaha, NE) and a single i.m. injection of HCG (250 iu., APL® Ayerst Laboratories, Montreal) on the fourth day [9], nine hr after the last FSH treatment. During the anovulatory season (July to September), five days of FSH injections were given [10] followed by the HCG. At 15-16hr after the HCG treatment, the monkeys were anesthetized with sodium pentobar- bital (27 mg/kg body weight, i.p.) and examined with a laparoscope [11]. The ovarian follicles were punctured and the oocytes aspirated into a 1 ml tuberculin syringe fitted with a 25 gauge needle with about 0.05 ml of medium. The oocytes were then incubated in sterile 8-chamber tissue culture slides (Lab-tek Products, Napierville, IL) at 37°C in an atmosphere of 5% CO, in air. The volume of medium in the chambers was 0.25 ml. The oocytes were inseminated after 21 hr of incubation (i.e. 37 hr after HCG treatment). Semen was collected by electroejaculation. The ejaculate was diluted in medium (5 to 10 fold vol- ume) and incubated for 5-10 min at 37°C. The cultures were inseminated with 0.05 ml of sperm suspension (10° to 10° sperm/ml). Previous work from our laboratory has shown that squirrel monkey sperm capacitation requires 2—5 hr under these conditions [12]. Culture media The medium used was TC199 (with 2mM HEPES buffer, Earle’s salts and L-Glutamine, #380—2340, GIBCO Laboratories, Grand Island, NY) suppicmented with 20% heat inactivated fetal bovine serum (FCS, #210-6510, GIBCO Labo- ratories, Grand Island, NY) 100 yg per ml Gentamicin (Schering Corp., Kenilworth, NJ) and 1 unit per ml heparin. The medium was further supplemented with 1.05 mM pyruvate and sterilized by filtration through a 0.45 um Millex filter (Millipore Corp., Bedford, MA) and stored in 10 ml vacutainer tubes (4°C). Fresh medium was prepared every 3 weeks. Criteria of maturation and fertilization At intervals of 24 hr the cultures were examined and the stage of development noted. The presence of a polar body on an oocyte indicated a mature oocyte at metaphase II. The criteria for fertili- zation were: (1) two or more polar bodies in the perivitelline space (2) two or more polar bodies and two pronuclei or two or more equal sized blastomeres by 24 hr after insemination (3) verification of two or more sets of chromo- somes through Giemsa staining (4) observation of the sperm tail or midpiece within the cytoplasm. When an oocyte fulfilled one or more of the above criteria, it was designated as fertilized. An oocyte was considered degenerated when either the oocyte had become black or the oocyte had shrinken into an uneven shape. Statistical analysis of data The rates of maturation and fertilization were compared using 2x2 contingency chi-square analysis. For groups with less than 10 oocytes, a more stringent Fisher’s exact test was used. All analyses were computed using the Hewlett Packard 41C statistical packet and statistical programs on the Apple II plus computer. RESULTS In vitro maturation Follicular oocytes had a significantly higher maturation rate in the second quarter (April— June) (Table 1). The rate of maturation was significantly higher in the second quarter com- pared to the other quarters in 1980 (P<0.05). The increased maturation rate in the second Squirrel Monkey in vitro Fertilization 473 quarter was again observed in 1981, but in 1982, the maturation rate was the same throughout the year. The mean maturation rates for the three years were 52.4+5.1, 36.3+5.1 and 33.8+3.7 percent respectively. The variances of the mean maturation rates for the 3 years were not signifi- cantly different from each other. In vitro fertilization The mean fertilization rates for the three years, 1980 to 1982, were 54.5+6.1, 52.3+10.4 and 43.9+10.7 percent respectively (Table 2). In 1980, the rate of fertilization in the fourth quarter (P<0.20) was highest. However, in 1981, the highest fertilization rate was observed in the second quarter. In the following year, 1982, the fertili- zation rates were not significantly different among the four quarters. A summary of the data for the 3 years showed a higher fertilization rate in the second quarter when compared to the third quarter. In vitro cleavage The mean first cleavage rates for 1980 to 1982 were 19.8+6.9, 25+10.6 and 28.2+9.7 percent respectively (Table 3). There was considerable variability in the cleavage rates for each month Owing to low sample size. Fisher’s exact analysis revealed high cleavage rates in the first quarter of TABLE 1. A three year study of squirrel monkey in vitro maturation rates No. of matured oocytes/total oocytes (percent -+S.E.) Quarters* 1 2 4 Year (Jan—Mar) (Apr—June) (July—Sept) (Oct-Dec) 1980 40/86 53/76° 12/25 22/57 (46.5+ 13.2) (69.7+ 7.3) (48.0+ 1.1) (38.6+ 5.0) 1981 8/35 10/19 26/70 11/34 (22.9+ 1.4) (52.6+ 14.2) (37.14 4.3) (32.4+ 0.6) 1982 48/125 13/40 6/20 2/5 (38.4+ 7.4) (32.5+ 0.8) (30.0+ 12.1) (40.0+ 8.4) Total 96/246 76/135° 44/115 35/96 (39.0+ 6.9) (56.3+10.8) (38.3+ 3.3) (36.5+ 2.3) * Controlled environment for the first, second and fourth quarters; warm temperature outside environ- ment during the third quarter. © Significantly different from the other quarters of the same year (P<0.05). TABLE 2. A three year study of squirrel monkey in vitro fertilization rates No. of fertilized oocytes/No. of matured oocytes (percent-+S.E.) 1 2 4 Year (Jan—Mar) (Apr—June) (July—Sept) (Oct-Dec) 1980 23/40 29/53 6/12° 9/13" (57.5+19.5) (54.7+ 7.0) (50.0+ 18.0) (69.2+ 2.4) 1981 4/8 6/10° 11/26° 4/9 (50.0 + 28.9) (60.0 + 28.6) (42.3+ 5.6) (44.4) 1982 19/35 8/12 3/7 4/7 (54.3+ 1.9) (66.7 + 22.9) (42.9+25.0) (57.1 +33.4) Total 46/83 43/75” 20/45° 17/29 (55.4+ 2.2) (57.3+ 3.5) (44.4+ 2.5) (58.6+ 7.2) ment during the third quarter. Controlled environment for the first, second and fourth quarters; warm temperate Outside enviro ®.< Different superscripts indicate difference (P <0.20). 474 P. J. CHAN AND W. R. DUKELOW TABLE 3. A three year study of squirrel monkey first cleavage rates No. of 2-cell embryos/No. of fertilized oocytes (percent +S.E.) Quarters? 1 y 3 4 Year (Jan—Mar) (Apr—June) (July—Sept) (Oct-Dec) 1980 23° 5/29° 0/6° 2/9 (47.8 +29.2) (17.2+ 2.2) (0.0) (22.2+ 2.5) 1981 1/4 0/6° PAP e 3/4° (25.0+25.0) (0.0) (18.2+14.5) (75.0) 1982 8/19° 1/9° 1/3 0/1 (42.1+12.8) (11.1+25.0) (33.3) (0.0) Total 20/46? 6/44°-° 3/20° 5/14° (43.5+ 6.9) (13.6+ 5.0) (15.0+ 9.6) (35.7+22.3) @ Control environment in first, second and fourth quarters; warm temperate outside environment during the third quarter. *,¢ Different superscripts indicate difference (P<0.10). 4,e¢ Different superscripts indicate difference (P <0.05). TABLE 4. A three year study of squirrel monkey oocyte degeneration rates No. of degenerated oocytes/total oocytes (percent+S.E.) Quarters? 1 2 4 Year (Jan—Mar) (Apr—June) (July—Sept) (Oct-Dec) 1980 DT Te 13/76° 1/27 29/58" G5s122 78) (17.1+ 2.6) (40.7 + 14.4) (50.0+11.3) 1981 16/26° 29/33° 37/70° 19/28° (61.5+ 5.7) (87.9+ 8.0) (52.9+ 7.6) (67.9) 1982 50/94 14/30 13/20 9/20 (53.2+ 9.6) (46.7+ 6.3) (65.0+ 16.0) (45.0+ 6.9) Total 93/197 56/139°>4 61/117° 57/106° (47.2+ 7.8) (40.3 +20.6) (52.1+ 7.0) (53.8+ 7.0) a Controlled environment in first, second and fourth quarters; warm temperate oustside environment during the third quarter. ®.© Different superscripts indicate difference (P <0.05). ‘4° Different superscripts indicate difference (P<0.10). 1980, the fourth quarter of 1981 and again, the first quarter of 1982. In summing the data, the results showed that the second and third quarters of 1980 to 1982 had low cleavage rates. Degeneration rate The degeneration rate of the cultured oocyte (shrunken or blackened appearance) observed after 48 hr of culture in trials from 1980 to 1982, is shown in Table 4. These oocytes may or may not have been matured. The lowest rates were in the first year, 1980, with a mean degeneration rate of 31.3+5.4 percent. The mean degeneration rates for 1981 and 1982 were 67.8+6.4 and 51.9+ 5.7 respectively. Generally, the second quarter had the lowest degeneration rate. The lowest degeneration rate was recorded for the second quarter of 1980. However, in the following year, 1981, the second quarter had highest oocyte degeneration. There were no differences in the rates of degeneration in all four quarters of 1982. When the data for the 3 years were summed up, the second quarter had the lowest degeneration rate. Squirrel Monkey in vitro Fertilization 475 DISCUSSION A compilation of data on in vitro oocyte matura- tion yielded several observations. The maturation rate was higher in the second quarter (April—June) of 1980 and 1981. In 1982, the maturation rate was not different throughout the year. A sum- mary of the data for the 3 years indicates a signifi- cantly higher maturation rate in the second quarter months. Smith ef a/. [6] reported higher matura- tion rates for rhesus monkey oocytes during the breeding season. It was noted that circulating hormones, for example estrogen that influenced folliculogenesis, were higher during this period. This research would have implications in promot- ing breeding in monkey colonies. An analysis of the summarized in vitro fertili- zation rates for 1980 to 1982 demonstrated a significantly higher fertilization rate in the second quarter compared to the third quarter. This is consistent with the ovulation response to hormone induction in squirrel monkeys which also shows a peak ovulation rate in the second quarter followed by the lowest ovulation rate in the third quarter [4, 13]. The present studies show the existence of a seasonal or environmental difference in in vitro maturation and fertilization of squirrel monkey oocytes. Limited observations on embryos that underwent first cleavage showed low development rates in the third quarter of 1980, the second quarter of 1981 and the fourth quarter of 1982. Overall, the second and third quarters had the lowest cleavage rates. The low cleavage rates in the second quarter were unexpected since the data showed high in vitro maturation and fertilization rates for this quarter. The reason for this observation is unknown. However, it may help to explain the low conception rate of squirrel monkeys in cap- tivity [14]. The percentage of oocytes degenerating in culture was lowest in the second quarter of 1980, coinciding with high maturation rates observed. However, in 1981, the second quarter had highest oocyte degeneration even though more of the oocytes were matured. In the third year, 1982, the degeneration rates were relatively even with a slightly higher incidence of degeneration in the third quarter. Overall, the second quarter had the lowest incidence of oocyte degeneration in culture. The results presented in this report suggests that seasonal or environmental influences affect cultured oocytes and this should be given serious consideration in in vitro fertilization programs. REFERENCES 1 Baldwin, J. D. and Baldwin, J. I. (1971) Squirrel monkeys (Saimiri) in natural habitats in Panama, Columbia, Brazil and Peru. Primates, 12: 45—61. 2 Dumond, F. (1968) The squirrel monkey in a seminatural environment. In “The Squirrel Monkey”. Academic Ed. by L.A. Rosenblum and R. W. Cooper, Academic Press, New York, pp. 87-145. 3 Jarosz, S.J.. Kuehl, T.J. and Dukelow, W. R. (1977) Vaginal cytology, induced ovulation and gestation in the squirrel monkey (Saimiri sciureus). Biol. Reprod., 16: 97-103. 4 Dukelow, W. R., Theodoran, C. G., Howe- Baughman, J. and Magee, W. T. (1981) Ovulatory patterns in the squirrel monkey (Saimiri sciureus). Anim. Reprod. Sci., 4: 55-63. 5 Harrison,R.M. and Dukelow, W.R. (1973) Seasonal adaptation of laboratory-maintained squirrel monkeys (Saimiri sciureus). J. Med. Primatol., 2: 277-283. 6 Smith, D. M., Conaway, C. H. and Kerber, W. T. (1978) Influences of season and age on maturation in vitro of rhesus monkey oocytes. J. Reprod. Fertil., 54: 91-95. i eniam eo. “Lutz KR. J, “and. Dukelow, W. R. (1982) Nonhuman primate in vitro fertiliza- tion: Seasonality, cumulus cells, cyslic nucleotides, ribonucleic acid, and viability assays. Fertil. Steril., 38: 609-615. ‘ 8 Jarosz,S.J. and Dukelow, W.R. (1976) Tem- perate season outdoor housing of Saimiri sciureus in the northern United States. J. Med. Primatol., 5: 176-185. 9 Dukelow, W.R. (1970) Induction and timing of single and multiple ovulations in the squirrel monkey (Saimiri sciureus). J. Reprod. Fertil., 22: 303-309. 10 Kuehl, T.J. and Dukelow, W.R. (1975) Ovu- lation induction during the anovulatory season in Saimiri sciureus. J. Med. Primatol., 4: 23-31. 11 Dukelow,W.R. and Ariga,S. (1976) Lapa: oscopic techniques for biomedical resea Med. Primatol., 5: 82-99. 12 Kuehl, T. J. and Dukelow, W.R. (19% relations of squirrel monkey (Saim/ 476 sperm capacitation and ovum maturation in an in vitro fertilization system. J. Reprod. Fertil., 64: 135-137. Harrison, R. M. (1973) Ovulation in Saimiri sciureus: induction, detection and _ inhibition. Ph. D. Thesis, Michigan State University, East 14 P. J. CHAN AND W. R. DUKELOW Lansing, Michigan. Dukelow, W.R. (1983) The squirrel monkey (Saimiri sciureus) In “Reproduction in New World Primates”. Ed. by J. Hearn, MTP Press Ltd., Edinburgh, England, pp. 149-180. ZOOLOGICAL SCIENCE 1: 477-481 (1984) ©1984 Zoological Society of Japan Binding of Triiodothyronine by Nuclei of Bullfrog Tadpole Erythrocyte TSUNEO MoriyA!, CHARLES R. THOMAS and EARL FRIEDEN Department of Chemistry, Florida State University, Tallahassee, Florida 32306, U.S. A. ABSTRACT — Specific binding sites for triiodothyronine were demonstrated in the erythrocyte nuclei from the tadpole of Rana catesbeiana. The erythrocytes obtained from the tadpoles at stage XII had 220 binding sites/nucleus with an apparent Kd of 182 pM. The number of binding sites increased to 460 binding sites/nucleus at stage XVII, while the affinity remained unchanged. INTRODUCTION The existence of specific binding sites for thyroid hormones in the nuclei has been reported for target tissues including tail and liver of amphibia [1-3]. Recently, Boussios et al. [4] reported the presence of receptors for thyroid hormone in the nuclei of mammalian erythroid cells, precursor of erythrocytes, obtained from the spleens of adult hamsters. It is well known that in amphibia, erythrocytes retain their nuclei throughout their life cycle. In this study, we demonstrate the existence of specific binding sites for triiodo- thyronine (T;) in the nuclei of erythrocytes of bullfrog tadpoles for which we have developed a new system for the assay of T; receptor. This new assay method has been introduced briefly in our recent paper [5]. In this paper, details will be reported to stress the convenience of our technique and the advantages over methods that have been developed by other investigators for the T; receptor assay. MATERIALS AND METHODS Animals Accepted January 24, 1984 Received October 7, 1983 * Present address: Department of Biology, Sapporo Medical College, Sapporo 060, Japan. The tadpoles of bullfrog, Rana catesbeiana, were obtained from commercial sources. They were maintained at 17°C on a diet of collard greens. The developmental stages of tadpoles were deter- mined according to Taylor and Kollros [6]. Preparation of erythrocytes Tadpoles were anesthetized with 0.1% tricaine methane sulfonate (K & K Laboratories, Inc., Plainview, New York). The ventral skin near the heart was removed and the heart was exposed. The truncus arteriosus was cut near its bifurcation and the erythrocytes collected in heparinized capillary tubes attached to the cut end of the artery. The erythrocytes were pooled in Hank’s solution (GIBCO, 310-4060) which was modified for amphibian tadpoles by diluting to 70% of mammalian ionic strength and by adding 0.1% methylcellulose and 10 mM HEPES buffer, pH 7.6. The erythrocytes were washed twice in Hank’s solution and centrifuged at 700g for 5 min. The number of erythrocytes was counted in a hemocytometer. Assay of T, binding to nuclei In order to measure the total binding of T; to nuclei, the erythrocytes were incubated at 25°C with ***I-T,; (Triiodothyronine, L-3, 5, 3” 3,380 wCi/ug; New England Nuclear) in Hank’s solution using a 1.5 ml Eppendo: test tube with constant agitation (25 478 T. Moriya et al. 4hr). For determining nonspecific binding, an excess (10°’M) of unlabeled T; was added to the incubation mixture. After incubation, the erythrocytes were washed three times with cold Hank’s solution, and after each wash, the sus- pension was centrifuged at 9,000 rpm for 3 min to pellet the erythrocytes. In a preliminary experiment, the washed cells were lysed by adding cold ST-solution (250 mM sucrose, 20mM _ Tris-HCl buffer, pH 7.6 con- taining 0.5% Triton X-100 and 1.1mM MgCl). These conditions have been used by several investigators [1,2] to isolate the nuclei from liver or tail cells. However, we were unable to isolate intact nuclei from the erythrocytes. An aggregate formed after centrifugation, preventing the subsequent washes required to remove free 257_T,. We devised a new isolation technique in which 1 mM _ spermidine was added to the ST- solution containing 0.06% Triton X-100 and 2.5mM EDTA used for lysis of erythrocytes. Spermidine is known to inhibit DNase in nuclei [7] and may serve to prevent the degradation of nuclei. Triton X-100 (0.06%) proved ta be effective in reducing nonspecific binding of Ts. After lysis with the Triton solution, the suspension was centrifuged at 9,000rpm for 3 min, and the pellet was washed twice with cold ST-solution containing 1 mM _ spermidine and 2.5mM EDTA but without Triton X-100. The pellets obtained after the final washing were checked by light microscopy. For determination of *”°I-T; bound to the isolated nuclei, the pellet was dissolved in 1N NaOH and counted in an automatic gamma counter (Beckman, Gamma 4000). Specific bind- ing was calculated by subtracting nonspecific binding from total binding. RESULTS AND DISCUSSION The effect of spermidine on the isolation of nuclei from erythrocytes The erythrocytes were lysed by addition of vari- ous S71-solutions (e.g., with or without spermidine) containing Triton X-100. After lysis, the sus- pensions were centrifuged at 9,000 rpm for 3 min, and the pellets were washed twice with cold ST- solutions without Triton X-100. The final pellets were examined by light microscopy. The photo- graphs in Figure 1 demonstrate the integrity and homogeneity of nuclei prepared in the presence of spermidine. There appears to be no difference among B-1, B—2 and B-3 in Figure 1. However, in subsequent experiments, we concluded that the use of ST-solution containing 1 mM spermidine and 2.5mM EDTA (B-3) provides optimal conditions for the specific binding assay. The effect of erythrocyte concentration on T;- binding to nuclei We determined the optimal concentration of erythrocytes in the incubation medium in which the isolated erythrocytes were incubated with °1-T;. The result is illustrated in Figure 2 and demonstrates that a linear relationship between the specific binding of T; to nuclei and the cell concentration is observed only in the range of 7.0 x 10° to 20.0 x 10° cells/200 ul. Therefore, we used 12.5 10° cells/200 vl throughout our ex- periments. Figure 2 also shows that at higher concentration of cells (e.g., 29.0 x 10° cells/200 1), the total binding decreases whereas the nonspecific binding increases. The decrease in the total binding may be to the depletion of **°I-T; in the incubation medium, and the increase in the non- specific binding may be explained by contamina- tion of isolated nuclei with other cellular fractions including incompletely lysed erythrocytes; indeed, we observed an aggregated red pellet in the nuclei preparation, when a high concentration of cells was used for initial incubation. At low cell con- centrations, on the other hand, the total binding did not increase appreciably with the increase of the cell concentration. This may be due to the excess concentration of Triton X-100 (0.06%) compared with the cell concentration, which may induce a disintegration of nuclei during the washing process. The extrapolation of this curve to zero showed that some binding had ocurred. Time course of T3-binding to nuclei The specific binding of T; proceeds steadily for the first 1 hr of incubation and reaches a maximum level at about 4hr incubation. By contrast, T; Binding by Tadpole Erythrocyte Nuclei 479 Fic. 1. nuclei were stained with toluidine blue. Triton); A—2, ST-Triton with 1.1mM MgCl; Triton with 1 mM spermidine; Light micrographs of erythrocyte nuclei prepared using various solutions for lysis. B-2, ST-Triton with 1 mM spermidine and 1.1mM MgCl; The isolated A-1, ST-Solution containing 0.06% Triton X-100 (ST- A-3, ST-Triton with 2.5mM EDTA; B-1, ST- B-3, ST-Triton with 1 mM spermidine and 2.5mM EDTA. Bar: 10 um. nonspecific binding is low and the level does not change appreciably throughout incubation. Simi- lar binding experiments were performed with erythrocyte membranes and cytoplasm. However, no specific binding was observed for either the 10° CELLS membranes or cytoplasm (data not shown). Properties of T3-binding sites in the nucleus In order to test the nature of the binding sites for T; in the nucleus, the ratio of moles of bound hormone/free hormone was plotted against the molarity of free T; in the medium. The result given in Figure 3 demonstrates the bound/free decreases with the increase of free T; concentration indicating the presence of a limited number of specific binding sites with a high affinity for T;. Scatchard plots were made to determine the Fic. 2. The relationship between the cell number and the total binding of ‘'7*I-T, to nuclei. Two hundred pl of the erythrocyte suspension containing different numbers of cells (obtained from the tadpoles at stage X) were incubat: with '7°I-T, (2.5x10-'' M) for 4hr at For other experimental procedures see M Open circle, total binding; Solid line. binding; Cross, nonspecific binding. 480 T. Moriya et al. are = 20+ > e\e 6 & 15t = : 5 is Sy / S OS = 5 Ne —! — feral 5x10" 10° 5x10” 10° MOLARITY OF FREE T3 Fic. 3. Binding of ‘?°I-T; to nuclei at different concentrations of T;. The erythrocytes (12.5 x 10° cells/200 1) were prepared from tadpoles at stage XII and incubated with different con- centrations of ‘'7°I-T, for 4hr at 25°C. The ratio of moles of bound T; to the molarity of free hormone in the medium was plotted against the molarity of free hormone in the medium. Each point is the mean of duplicate deter- minations. affinity and the number of the specific binding sites in nuclei from tadpoles at two different stages, stage XII and XVII. The maximum number of binding sites and the dissociation constant (Kd) were then calculated from each Scatchard plot (Fig. 4). The results indicated that the erythrocytes have 220 binding sites per nucleus with a Kd of 182 pM at stage XII and 460 binding sites with a Kd of 207 pM at stage XVII, respec- tively. This implies that the number of binding sites increase as metamorphosis proceeds, while the affinity remains unchanged [5]. In tail fin cells, Yoshizato and Frieden [8] reported a dou- bling of T; receptor sites per nucleus during metamorphosis. Our data are consistent with their report. On the other hand, in liver cell nuclei, Galton [3] observed a constant level of receptor sites before metamorphic climax. The differences among these reports probably reflect the different assay systems, in vitro and in vivo, or different tissues. All the observations made in this study clearly reveal the existence of specific binding sites for T; in the nuclei of the erythrocyte of bullfrog 70 60 S \ & Stage XVI \ B x 10° moles Fic. 4. Scatchard plots of the specific binding of T; to nuclei at two different stages. Closed symbols, stage XII; Open symbols, stage XVII. Experimental condition as in Fig. 3. Squares, triangles and circles present the respective experimental series. Lines were obtained by least square calculations with a correlation coefficient of 0.96—0.98. tadpoles. The methods that have been developed for typical target tissues such as liver or tail include several complicated procedures to separate the cells and isolate the nuclei; e.g., digestion of the tissues by collagenase and homogenization. In contrast, it is not difficult to obtain sufficient amounts of pure erythrocytes. The subsequent isolation of nuclei is easily achieved after a simple hemolysis. In addition, it should be noted that unlike certain target tissues as the tail, erythrocytes with nuclei are present in the circulation through- out the life span of frog from early larval stage to the adult stage. This facilitates the investigation of changes that may occur in the binding sites for thyroid hormones during metamorphosis, although the physiological significance of spe- cific binding sites in erythrocyte nuclei demon- strated in this study remaines unexplained. T,; Binding by Tadpole Erythrocyte Nuclei 481 ACKNOWLEDGMENTS This work was supported by grant HD-01236 from the NICHHD. REFERENCES 1 Yoshizato, K., Kistler, A. and Frieden, E. (1975) Binding of thyroid hormones by nuclei of cells from bullfrog tadpole tailfins. Endocrinology, 97: 1030-1035. 2 Kistler, W., Yoshizato, K. and Frieden, E. (1975) Binding of thyroxine and triiodothyronine by nuclei of isolated tadpole liver cells. Endo- crinology, 97: 1036-1042. 3 Galton, V. A. (1980) Binding of thyroid hormones in vivo by hepatic nuclei of Rana catesbeiana tadpoles. Endocrinology, 106: 859-866. 4 Boussios, T., McIntyre, W. R., Gordon, A. S. and Bertles, J. F. (1982) Receptors specific for thyroid hormones in nuclei of mammalian erythroid cell proliferation. British J. Haematol., 51: 99-106. Moriya, T., Thomas, C. R. and Frieden, E. (1984) Increase in 3, 5, 3’-triiodothyronine binding sites in tadpole erythrocyte nuclei during spontaneous and T,-induced metamorphosis. Endocrinology, 114: 170-175. Taylor, A.C. and Kollros, J. J. (1946) Stages in the normal development of Rana pipiens larvae. Anat. Record, 94: 7-23. Harvey, W. and Atkinson, B.G. (1982) Chro- matin-associated deoxyribonuclease activity in liver nuclei isolated from Rana catesbeiana froglets and premetamorphic and T;-induced tadpoles. Can. J. Biochem., 60: 21-27. Yoshizato, K. and Frieden, E. (1975) Increase in binding capacity for triiodothyronine in tadpole tail nuclei during metamorphosis. Nature, 254: 705-706. ! iio apes lets. “Let, lin, , yete iw Ol 7 Yy ) allay bP x ty weed dl 4 ett ‘Ynext il ireks \ Ei if ‘i Tis eee ees Ta a ri. ; : ued’ Mm , , reps f ! 4 4,4 ‘ rte ive i 7 ity ; : wipes SS t 7 ¥ é as t "= ; i : ; hk a i A } pitti cae. £ ¢ > te a * i ¥ i & 1 . ’ ‘ 4 { et . ie Pie mall : pee * uPekhe sae be =) Pee ol i ~ wy i Asc 26, teky £ ik = cen Ys " . ‘ = Ae P - ‘ y a's 4 i ny a ‘ , J bs ek Peet eee LRT LY . bs a Ps — i , zy TE ; @ = a : 7 ; 4 ee ry a 3 “ae 1 nie Ss 5 ee - i 4 dle mee he } ; : a sz rar f j a j i = ae ae agers Ry : * Fic. 2. Freeze-fracture replicas of the proximal zone of the inner segmental layer of the retina. (a) A gap junction on a collateral. 126,000. (b) P face of a collateral (C) which bears a gap junction (arrow) is traceable to the plasmamembrane of a photoreceptor soma (S). 45,000. tors in the cephalopod retina [9, 10]. ACKNOWLEDGMENT I thank Mr. Nobuo Takasu for preparing freeze- fracture replicas and Dr. M. Yoshida for reading the 6 manuscript. This work was supported by a Grant-in- Aid from the Japanese Ministry of Science, Education and Culture (58540460). I 8 REFERENCES 9 1 Young, J.Z. (1962) Phil. Trans. Roy. Soc. B, 245, 1-18. 10 2 Patterson, J. A. and Silver, S.C. (1983) J. Comp. Physiol., 151, 381-387. Tonosaki, A. (1965) Z. Zellforsch., 67, 521-532. Gry. BG, (1970) ... J. Cell. Sei. 7, 203-215. Cohen, A. I. (1973) J. Comp. Neurol., 147, 379- 398. Yamada, E. and Usukura, J. (1982) In “The Structure of Eye’. Ed. by J.G. Hollyfield, Elsevier, North Holland, pp. 25-34. Yamamoto, M. and Takasu, N. (1984) Cell Tissue Res., in press. Staehelin, L. (1974) Int. Rev. Cytol., 39, 191-283. Tasaki, K., Norton, A. C. and Fukuda, Y. (1963) Nature, 198, 75-88. Lange, G. D. and Hartline, P.H. (1974) J. Comp. Physiol., 93, 19-36. ZOOLOGICAL SCIENCE 1: 505-507 (1984) [COMMUNICATION] © 1984 Zoological Society of Japan Changes in Level of DNA Polymerase a Activity during Oogenesis of the Toad, Bufo bufo japonicus TOKUKO HARAGUCHI!, CHIAKI KATAGIRI? and HirosHI NAGANO!:3 ‘Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, and *Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan ABSTRACT — Changes in the DNA polymerase a activity during oogenesis were studied with the toad. The DNA polymerase a activity increased logarith- mically as a function of the increase of oocyte diameter. However, massive increase was apparent after initiation of pigmentation during the last stage of oogenesis. INTRODUCTION It has been reported that unfertilized eggs con- tain a large amount of DNA polymerase required for DNA replication of rapid cleavage during early embryogenesis of the sea urchin [1, 2], Xenopus leavis [3] and Drosophila [4,5] and that DNA polymerase a activity increases several folds during oocyte maturation [3,6]. However, we have previously shown that full grown oocytes of the toad contain high level of DNA polymerase a activity in the germinal vesicle and no quantitative and qualitative change in the enzyme activity takes place during the maturational process [7]. There- fore, it is interesting to determine the stage when DNA polymerase a is synthesized in the cytoplasm and pooled in the germinal vesicle during oogenesis of the toad. In the present paper, we report that DNA polymerase a is exponentially accumulated in the germinal vesicle during oogenesis. Accepted January 12, 1984 Received November 12, 1983 3 Present address and for correspondence: Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Azusawa, Itabashi-ku, Tokyo 174, Japan. MATERIALS AND METHODS dATP, dCTP, dGTP and dTTP were purchased from Yamasa, Chiba. [methyl-*H]dTTP (specific activity; 43 Ci/mmol) was from Amersham International Ltd. Activated DNA was prepared according to the method of Fansler and Loeb [8] using calf thymus DNA (Miles Laboratories, Inc.). Adult females were collected monthly during May to August, and the ovary was removed and immersed in DeBoer’s solution. Thecal and follicular layers were removed from growing oocytes with forceps and rinsed in 0.25 M sucrose. Ten defolliculated oocytes at each stage were homogenized in 1 ml of a homogenizing solution containing 25% sucrose, 0.2 M potassium phos- phate buffer, pH 7.4, 1 mM EDTA and 2 mM 2- mercaptoethanol, and centrifuged at 10,000 g for 60min. Under these conditions, nearly all activities of DNA polymerase a and £ of frog oocytes are solubilized [7]. The supernatant was diluted with an equal volume of the homogenizing solution from which sucrose was omitted. Then, 0.3 ml of this crude extract was applied to 4.8 ml of 20-30% sucrose gradient column containing 0.2 M potassium phosphate buffer, pH 7.4,2 mM EDTA, 10mM _ 2-mercaptoethanol and 1 mg/ml bovine serum albumin and centrifuged for 17 hr at 50,000 rpm using a Beckman preparative ultracentrifuge and a SW 55 Ti rotor. DNA polymerase ac was determined in the reaction mixture containing 50 mM Tris-HCl, pH 7.5, 7 mV 50 uM each of dATP, dGTP and dC’ 506 dTTP, 0.2 #Ci of PH]dTTP, 40mM NaCl, 6 vg of activated DNA and 24 wg of bovine serum albumin in the presence or absence of 10mM N-ethylmaleimide. Incubation was carried out for 60 min at 37°C and the acid-insoluble radio- activity was counted as described previously [9]. RESULTS AND DISCUSSION The DNA polymerase activity in oocytes at various stages of oogenesis was determined using the supernatant of centrifugation at 10,000 xg. The result is shown in Figure 1. In the toad (Bufo bufo japonicus), yolk deposition begins in growing oocytes with a diameter of approximately 0.4 mm. Pigmentation begins in oocytes with a diameter of approximately 1.5mm. The diameter of full grown oocytes is 2mm. The DNA polymerase activity in the supernatant of oocyte homogenate No ras) . a — — © ©& (=n) i=) ~ = 2 pa) oO < i) QO. TA ey = 1 4 " ~ ; a ' i ; Z es i =) > ' Besides Analytical and Prepara- Seasiucn tive Ultracentrifuges, Hitachi SCR20BB Automatic with built-in Koki, a comprehensive centrifuge SCR20BA | 300 ~ 20,000 microprocessor control, Digital version Compact type for biochemistry Low cost type mo) g manufacturer, brings you a com- a 2 | B | S| Automatic control, plete line of High Speed Refrig- 20PR-52D| 300~20,000| = 5 X | Analog version erated Centrifuges, Table-Top scr” s\els | 7 Centrifuges and Miniature Cen- se eta 7,000 o 5 Large capacity, multi-place type ° = trifuges with wide-ranging te Ee speeds, capacities and _ tubes/ Satea SL bottles versatility for your SCT4BD 4,000 . s : SCT4BE clinical laboratory routine as well Sea 1a 1 Go as basic research. MC-200 MC-201 12,000 MC-202 MC-300 430 ~ 2800 HITACHI Refrigerated/ aur Table-Top/Miniature Centrifuges For clinical Automatic control laboratory test Automatic with microprocessor control Miniature : Exclusive Hematoerit Table-top type Dual purpose Cell washing © Nissei Sangyo Co.,Ltd. Hitachi Koki Co Instrument International Dept. Mori 17th Bldg., 26-5 Toranomon 1-chome, Minato-ku, Tokyo 105, Japan C.P.0O. Box: 1316, Tokyo 100-91, Japan Phone: 03-504-7282 Telex: J 22412 NISSEICOA Lz ZOOLOGICAL SCIENCE VOLUME 1 NUMBER 3 JUNE 1984 CONTENTS REVIEWS Yamada, T.: Developmental control of cell CHerentiation ic .c..csce ceskendacec coro eccaees 333 Chang, M.C.: Experimental studies of mam- malian fertiliZation: 23) chek. aca.e seo odeoeotn eA 349 ORIGINAL PAPERS Physiology Takasu, N. and M. Yoshida: Freeze-fracture and histofluorescence studies on photorecep- tive membranes of medusan ocelli Tomioka, K. and Y.Chiba: Effects of nymphal stage optic nerve severance or optic lobe removal on the circadian locomotor rhythm of the cricket, Gryllus bimaculatus ...375 Kobayashi, M.., M. Nakamura and _e£ =T. Hasimoto: Innervation of the heart of a prosobranch mollusc, Rapana thomasiana ...383 Kawakami, H. and M. Kobayashi: Pharma- cological approach to the analysis of regula- tion of molluscan heart activity ............... 389 Hashimoto, H.: Frustule movement of fresh- water polyp, Microhydra sowerbii (Lankester) Cell Biology Maruo, F. and M. Okada: Functions of a maternal gene are required for the synchro- nous nuclear division during early develop- ment of Drosophila melanogaster. Analyses Oi ca: ISM ant 5.55 350555c0co a eens ee 405 Mitsuhashi, J.: Isolation of a continuous cell line from larval fat bodies of an arctiid moth, Spilarctia seriatopunctata (Insecta, Lepidop- (eld, “ALCOIGAe) 3. cee... 2ect eka eee 415 Yamamoto, M.: Photoreceptor collaterals in the cuttlefish retina (COMMUNICATION) Biochemistry Sugita, H. and K. Sekiguchi: Lactate dehydrogenase of the horseshoe crabs and their hybrids Developmental Biology Sawai, T.: Surface movement in the first cleavage stage of newt eggs ........... Samal 427 Kinoshita, T. and K. Okazaki: Jn vitro study on morphogenesis of sea urchin larval spicule: Adhesiveness of cells ................. 433 Masuda, M. and H. Sato: Reversible resorp- tion of cilia-and the centriole cycle in dividing — cells of sea urchin blastulae ..................... 445 Itow, T. and K. Sekiguchi: Acid mucopoly- saccharide in embryos of the horseshoe crab, Techypleus tridentatus (Chelicerata, Arthro- POA) oo. ccvedescccssecs vocsdeeosen thee eee Eee 463 Haraguchi, T., Ch. Katagiri and H. Nagano: Changes in level of DNA polymerase a activi- ty during oogenesis of the toad, Bufo bufo japonicus (COMMUNICATION) .............. 505 Endocrinology Chan, P. J. and W.R. Dukelow: Variations in squirrel monkey in vitro fertilization responses with seasonal and environmental CONGITIONS ...55. cs dasccses cents oes cetera ee eee 471 Moriya, T., C.R. Thomas and E. Frieden: Binding of triiodothyronine by nuclei of bullfrog tadpole erythrocyte ..................... 477 Taxonomy Hasegawa, H.: Skrjabinelazia machidai sp. n. (Nematoda: Seuratidae) from Gekko japoni- cus on Okinawa Island, Japan .................. 483 Kawakatsu, M. and R.W. Mitchell: Oahu- hawaiiana kazukolinda gen. et sp. nov. (Tur- bellaria, Tricladida, Maricola), a new freshwater planarian from Honolulu, Oahu Island, Hawaii, UyS. A) icc... :..cn eee 487 Printed by Daigaku Letterpress Co., Ltd. Hiroshima, Japan fFOOLOGICAL SCIENCE tinuation of the “Zoological Magazine” and the “Annotationes Zoologicae Japonenses ZOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Nobuo Egami (Tokyo) Toshin-building, Hongo 2—27-2, Bunkyo-ku, Associate Editors: Tokyo 113, Japan. (03)-814-5675 Susumu Ishii (Tokyo) Officers: Seiichiro Kawashima (Hiroshima) President: Hideshi Kobayashi (Funabashi) Koscak Maruyama (Chiba) Secretary: Makoto Asashima (Yokohama) Assistant Editors: Treasurer: Kiyoaki Kuwasawa (Tokyo) Takeo Machida (Hiroshima) Librarian: Shun-Ichi Uéno (Tokyo) Kazuyoshi Tsutsui (Hiroshima) Editorial Board: Howard A. Bern (Berkeley, endocrinology) Aubrey Gorbman (Seattle, endocrinology) Robert B. 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New subscriptions and renewals begin with the first issue of the current volumes. All rights reserved. No part of this publication may be reprocuced or stored in a retrieval system in any form or by any means, without permission in writing from the copyright holder. © Copyright 1984, The Zoological Society of Japan - Publication of Zoological Science has been supported in part by a Grant-in-Aid for | E Scientific Publication from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 1: 509-522 (1984) © 1984 Zoological Society of Japan REVIEW Molecular Aspects of Intracellular Symbiosis in the Aphid Mycetocyte HAJIME ISHIKAWA Department of Biology. College of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan INTRODUCTION Aphids (Homoptera; Aphidoidea) form a group which includes many of the most successful insects in the present day. One important factor supporting this prosperity is intracellular symbionts present in the mycetocyte of these insects. All the aphid species except Phylloxeridae contain the intracellular symbionts which are harbored by the mycetocyte, a cell differentiated spcifically for this purpose [1]. The intracellular symbionts are transmitted from generation to generation of the aphid within the mycetocyte. The mycetocytes are huge, polyploid cells which are scattered in the fat body around the midgut of the aphid. Since a discovery of intracellular symbionts as Blochmann bodies in several insect tissues in the late 19th century, physiological roles pertinent to these structures have been searched for by many biologists [2]. One of the most exciting possibilities raised for a biological role of the aphid symbionts in the past was that they might fix atmospheric nitrogen to provide developing embryos with amino acids. However, this was disproved later by the experiments using stable isotope of nitrogen [3]. The aphid symbionts were once suspected to supply the host with sterols [4, 5]. The proposition was based on an obser- vation that the aphid could be raised upon a chemically-defined diet deficient in cholesterol, a precursor to ecdysterone, which is not synthe- sized by insects in common [6]. This observation Received March 31, 1984 is now questioned since the synthetic diet sucked by aphids is inevitably contaminated by fungi and bacteria [7]. At present it is believed that the aphid symbionts supply the host insect with several species of amino acids [8] and long-chained fatty acids [4] which the host insect is unable to produce. In the meantime, it is a molecular viewpoint that the intracellular symbiont serves a genetic element distinct from the genome of its host cell, which all these works invariably lacked. In this context, of importance is the fact that structural and functional analogies exist between the aphid symbiont and the DNA-containing organelles like mitochondria and chloroplasts in the eukaryotic cell [9]. Both appear to be sur- rounded with a host-derived membrane [10] and firmly integrated into the developmental cycle of the host. Neither the symbionts nor the organelles can successfully replicate themselves extracel- lularly. Both have their own systems of DNA, RNA and protein synthesis which differ from those of the host cell. They are not attacked by the host cell’s defence mechanism. Moreover, both preserve prokaryotic properties in common [11]. In view of these facts, intracellular sym- biosis in the aphid mycetocyte is one of the best systems in which to. study the _ interaction between two types of genomes in a single cell, the one of the eukaryotic host and the other of the prokaryotic symbionts [12]. Probably, in such an intracellular symbiosis not only the low molecular weight metabolites described above but also protein molecules are exchanged 510 H. IsHIKAWA between the symbiont and the host, just as be- tween the organelles and nuclear-cytoplasm in the eukaryotic cell. Even a gene transfer from the symbiont to the host cell and vice versa will be among the possible events involved in the intracellular symbiosis as revealed recently in the eukaryotic cells [13-15]. Therefore, this endosymbiotic system also will be useful in experimental search for the origin of cellular organelles [16]. DNA AND RNA SYNTHESIS Judging from the symbiont’s similarity to the subcellular organelles, it seemed feasible that a modification of the procedure for isolating chloroplasts might be applied to the isolation of the endosymbionts. Biologically active sym- bionts were successfully isolated at a large scale from the elder aphid, Acyrthosiphon magnoliae and the pea aphid, A. pisum, using a Percoll density gradient [17]. When the intracellular symbionts isolated from A. pisum were incubated in insect saline, they incorporated [°H]thymidine into the acid-insoluble fraction in proportion to times for, at least, the first 30 min (Fig. 1). dpmx104/ug DNA 0 60 120 180 minutes Fic. 1. Incorporation of [H]thymidine into the acid-insoluble fraction of symbionts isolated from A. pisum. The isolated symbionts equivalent to about 200 yg of DNA _ were suspended in 1 ml of Ephrussi-Beadle solution containing 0.1mCi of [methyl—*H]thymidine (50 Ci/mmole) and incubated with shaking at 30°C (From Ishikawa [11]). This was the first demon-_ stration that the aphid symbiont, whether in vivo or in vitro, can synthesize DNA [11]. The fact that the isolated symbiont incorporates thymidine actively suggests also that it depends upon a salvage pathway for the DNA synthesis using thymidine kinase. This is reminiscent of mitochondria and_ chloroplasts. In these organelles, as in prokaryotic cells, there are thymidine kinases that are not normally present in eukaryotic cell nuclei [18]. It is also remini- scent of those organelles that the symbiont does not replicate itself in vitro in spite of an active synthesis of DNA. However, the symbionts differ from the organelles in that the synthesis of DNA is insensitive to ethidium bromide [19]. The DNA synthesis in the symbiont in vitro is highly sensitive to nalidixic acid, a synthetic antibiotic which inhibits prokaryotic DNA _ polymerases [20]. It was also demonstrated that the isolated symbiont in vitro is capable of an active synthesis of RNA. While neither actinomycin D nor a-amanitin had an effect, rifampicin at 20 ug/ml exerted an 80% inhibition of symbiont RNA synthesis [11]. The rifamycin antibiotics in general are highly specific in that their action is dependent on binding to an apparently unique structural component of the prokaryotic RNA polymerase rather than to the DNA template [21]. High sensitivity of the symbiont to this antibiotic, therefore, provides one crucial piece of evidence for its prokaryotic nature. When the aphid mycetocytes were incubated in Grace’s medium, a culture medium for insect cells, they synthesized preferentially unique RNAs with molecular weight of 1.2x10° and 0.6x10° [17]. These RNAs were supposed to be ribosomal RNAs of the symbiont (Fig. 2). The RNA synthesis was completely inhibited by rifampicin at a concentration as low as 1 ug/ml [22]. Acti- nomycin D did not selectively inhibit the synthesis of these RNAs. Preferential synthesis of these RNA _ species were also observed when the aphid was injected with [’H]uridine [22]. These findings are taken to indicate that in the aphid mycetocyte the rRNA genes of the endosymbiont are selectively expressed. It was shown that the rRNAs of aphid symbiont is Intracellular Symbiosis 511 ) re) CPMx10°2 ( MIGRATION (cm ) Fic. 2. Polyacrylamide gel electrophoresis of high molecular weight RNA from the mycetocytes labeled in vitro. The mycetocytes, isolated from A. magnoliae, were incubated in Ephrussi- Beadle solution containing 0.1 mCi/ml of [>H]juridine at 30°C and the RNA extracted 3hr later. The labeled RNA was subjected to polyacrylamide gel (2.5%) electrophoresis for Shr. Arrows indicate the position of the 28S and 18S rRNA of host insect (From Ishikawa [17]). quite unstable in structure. Optical analyses suggested that this instability is due to a low content of guanosine-+cytosine in these RNAs. In particular, it is characteristic to the symbiont rRNAs that the G+C content in the bihelical region is even lower than that of the whole mole- cules [17]. The physical instability of the symbiont RNA forms a sharp contrast to the property of the aphid’s own rRNAs [23-25]. The latter are quite heat-stable, and the exceptional properties inherent in these molecules seem to stem from its physical stability [17]. Though biological significances of these properties of symbiont RNAs are still beyond speculation, it should be emphasized that mitochondrial rRNAs are also extremely unstable [26] and that their G-+C contents are invariably lower than those of rRNAs in the cytoplasm [27]. Nascent RNA species of the symbiont are more stable, which will suggest that in the symbiont the RNA under- goes a rapid turnover [17]. It is probable that some of the biological functions of the symbiont are exerted through this kind of metabolic process of RNA. PROTEIN SYNTHESIS The intracellular symbionts isolated from aphid species, when incubated in Grace’s medium, incorporated [**S]methionine very actively into the acid-insoluble fraction. The fact that the incorporation was sensitive to chloramphenicol but not to cycloheximide indicates not only that the incorporation is due to an actual synthesis of polypeptides but that the machinery of protein synthesis of the symbiont is of a prokaryotic nature [11]. As has been often pointed out, the aphid, viviparous females in particular, will need a large amount of amino acids to raise a number of embryos in the ovarioles. Under such stringent circumstances, the intracellular symbiont is given a share of amino acids probably because of the important roles it possesses. In this relation, it is of great interest what protein species the vi Sean al ee OO : : = ==— 69K —— - * a A ae : b Fic. 3. Polyacrylamide gel electrophoresis of proteins from the symbionts labeled in vitro. The isolated symbionts were incubated with one of five antibiotics below at 20 g/ml in Grace’s medium (methionine-less, serum-free) with shaking at 30°C for 30 min, and further incu- bated for 3hr after adding [*°S]methionine at 0.1 mCi/ml. a, Stained with Coomassie blue; b, fluorographed. iS, symbionin; A, actin. 1, Control (untreated with antibi- otics); 2, a-amanitin; 3, actinomycin D; 4, rifampicin; 5, chloramphenicol; 6, cycloheximide (From Ishikawa [12]). 512 H. ISHIKAWA symbiont has and _ synthesizes. The isolated symbionts were incubated with [°°S]methionine in Grace’s medium and the proteins were re- solved on polyacrylamide gel [12]. Upon staining the gel with Coomassie blue, it was indicated that the most abundant proteins in the symbiont are those with apparent molecular weight of 180 x 10’, 63 x 10° and 43x10° (Fig. 3a). While 180 and 43 kilodalton proteins are very probably heavy chain myosin and actin, respectively, 63 kilo- dalton species is unknown and tentatively named “‘symbionin.” None of these three proteins was labeled appreciably in vitro. In addition, most of the symbiont proteins labeled in vitro differed from those detected by staining (Fig. 3b). Syntheses of all the proteins seen above were insensitive to any of a-amanitin, actinomycin D and cycloheximide. In contrast, there were complete inhibitions of these syntheses by rifampicin and chloramphenicol. Based on these results, it was concluded that the proteins syn- thesized by the isolated symbionts are their own gene products. Conversely speaking, the three major proteins including symbionin above are not coded for by the symbiont genome. It is likely that most of the bands seen in Figure 3a La =<. ‘ey 2 ~ <= * te “@ * se o * o * 4 a : * al oa j Ladd #* am * a é e “ & beac ips . : - ff» y * * » se j «, 3 ee es Es ad. ‘ ae ie by Seg ig i a i ——_— Lae Pp Ws Wes ae Ce sigh % he es Lia A 6 Fic. 4. Two-dimensional separation of symbiont proteins synthesized in vitro. The isolated endosymbionts were incubated with [°*S]- methionine in Grace’s medium at 25°C for 1 hr and the proteins resolved two-dimensionally and fluorographed (From Ishikawa [28]). are due to proteins which are synthesized in the host insect and transferred to the intracellular symbiont. Two-dimensional gel separation sug- gested that the symbiont’s genomic products amount to several hundred species (Fig. 4) [28]. Protein synthesis by the aphid endosymbiont in vivo was studied by injecting [*°S]methionine into the host insect [12]. The symbiont was isolated from the injected insects, and the proteins separated on polyacrylamide gel. As shown in Figure 5, radioactive bands due to nascent pro- teins coincided with those of total proteins. When the insects received an injection of cycloheximide, nascent proteins found with the symbiont were almost solely symbionin described above. This suggests that all the other proteins including myosin and actin found with the sym- ke baal 234 ‘S) =e A— a b Fic. 5. Polyacrylamide gel electrophoresis of proteins from the symbionts labeled in vivo for 3hr. Young apterous, viviparous females were injected with one of the three antibiotics below at about 10ng/mg of body weight and [?°S]methionine at about 5 nCi/mg at an interval of Ihr. The insects were allowed to in- corporate the radioactive precursor for 3 hr. The intracellular symbionts were isolated from the injected insects and the proteins separated. a, Stained; b, fluorographed. S, symbionin; A, actin. 1, Control; 2, rifampicin; 3, chloramphenicol; 4, cyclo- heximide (From Ishikawa [12]). Intracellular Symbiosis 513 biont are synthesized by the eukaryotic host. That symbionin alone is synthesized by the intracellular symbiont was further evidenced by injecting chloramphenicol into the insect. This anitibiotic, effective selectively upon the prokaryo- tic ribosome, did not inhibit synthesis of any protein but symbionin. An injection of rifam- picin did not inhibit the synthesis of symbionin effectively [12]. This lends support to the possi- bility that symbionin is not coded for by the symbiont’s own genome (see later). Since symbionin was unambiguously evidenced to be synthesized by the intracellular symbiont, it became feasible to use the whole body of insect instead of the isolated symbiont in the studies of protein synthesis by the symbiont in vivo [12]. Judging from sensitivity to antibiotics, all the protein syntheses in the whole body but that of - + oe l<¢e 72 m - ~~. = es : -: ° Cc Fic. 6. Two-dimensional separation of the whole body proteins of pea aphids. of symbionin are undertaken by the eukaryotic machinery of the host. The symbionin synthesis was extremely sensitive to chloramphenicol but not to rifampicin. Two-dimensional gel sepa- rations of the whole body proteins indicated that symbionin is almost only one protein synthesized by the symbiont in vivo (Fig. 6) [28]. A bare possibility that the symbiont synthesizes other proteins not containing methionine residue was excluded by the experiment using [*H]leucine as a precursor to protein [28]. All these results clearly indicate that the symbionts confined in the mycetocyte are under a stringent control by the host insect, and concentrated on the synthesis of symbionin. When incubated under microaerophyllic con- ditions, the isolated symbiont synthesized several protein species which otherwise are not produced. Young insects were injected first with either distilled water or 200 g/ml cycloheximide and 1 hr later with [°*S]- methionine. proteins were separated two-dimensionally. b, control, fluorographed; position of symbionin (From Ishikawa [28)]). They were allowed to incorporate the radioactive precursor for 3 hr. a, Control (injected with distilled water), stained; c, injected with cycloheximide, fluorographed. Arrows indicate the The whole 514 H. IsHIKAWA Complete anerobiosis of the endosymbiont re- sulted in a cessation of all the protein syntheses [28]. It is yet to be known whether a presumed microaerophyllic environment in the mycetocyte is involved in mechanisms in the stringent control of the intracellular symbiont by the host. It is possible that the host cell controls the protein synthesis by the symbiont by means of some inhibitors. In fact, when the tissue extract of aphids included, DNA, RNA and protein syntheses by the symbiont in vitro were strikingly inhibited [29]. The inhibitors were heat-stable, Sy dialyzable and insoluble in hexane. Among several aphid species examined, the inhibitors were without species-specificity. | Moreover, tissues of termites and ladybirds were revealed to contain similar substances. By contrast, silkworm and honeybee larvae did not contain inhibitors of this kind. Since these substances are also inhibitory on the RNA synthesis in E. coli, it is still a matter of speculation whether these inhibitors are actually involved in the host’s mechanism controlling the intracellular symbiont in vivo [29]. Fic. 7. Protein synthesis in the whole body after an injection of cycloheximide. Young (15 days) (a), middle-aged (35 days) (b) and old (55 days) (d) insects were injected first with cycloheximide and 3 hr later with [?5S]methionine. Insects were allowed to incorporate the precursor for 3 hr. In (c), young insects were injected with the radioactive precursor 45 hr after the injection of cycloheximide. In (e), the same protein sample as in a was mixed with the proteins extracted from the symbionts which were incubated in vitro with [32S]methionine for 3 hr. Photographs represent part of twodimensional fluorograms of the proteins. Arrows indicate the position of symbionin while arrow-heads, that of symbionin-like protein (SLP) (From Ishikawa [30] and data submitted for publication). Intracellular Symbiosis 515 SENESCENT INSECTS Pea aphids older than about 40 days do not produce offspring, get darker in color and yet live, at least, up to 60 days after birth as far as they are kept solitary [30]. Of interest is whether the intracellular symbionts harbored in such an old insect are controlled by the host in the same way as in the younger aphids. An answer to this question will lead to an understanding of biological functions of the symbiont and _ its controlling mechanisms. It has been shown that in an old (45-50 days) aphid the symbionts synthesize protein species which are not produced in the young (13-20 days) ones [30]. Symbionin was not produced to any great extent in old hosts. A comparison of protein species synthesized by old insects with those synthesized by the symbiont in vitro indi- cated that in the old host the intracellular symbiont in vivo produces some of the same species as those synthesized in vitro (Fig. 7). Among these proteins the most conspicuous is the one similar to symbionin itself (symbionin-like protein, SLP). While symbionin is an acidic protein with mole- cular weight of 63,000, SLP is slightly more acidic and a little smaller in size than symbionin. SLP is one of the proteins which are synthesized most actively by the isolated symbiont in vitro [12, 28]. Synthesis of this protein by the symbiont in vivo became active in nearly inverse proportion to that of symbionin with age of the host insect [30]. It seems that controi over the intracellular symbiont by the host is weakened in old insects, Fic. 8. and the progeny produced between 24 and 48 hr post-injection were raised. c, RF insects, alatae; apterae; b, control, aptera; and Yamaji, submitted for publication). and that some of the symbiont’s own genes begin to be expressed (unpublished data). Of interest was that a similar situation can be reproduced by keeping young insects under the influence of cycloheximide for a prolonged period (Fig. 7). The intracellular symbiont in these injected insects began to produce, at 40 hr post-injection, some of the symbiont’s own gene products including SLP described above which otherwise are not synthesized in vivo. It is likely that a shutoff of protein synthesis in the eukaryotic machinery by cycloheximide brought about physiological state somewhat similar to that in senescent insects [30, unpub- lished data]. APOSYMBIOTIC INSECTS Symbionin, the only one protein synthesized in vivo by the intracellular symbionts, is not produced in old insects which have finished giving birth to offspring. This suggests that symbionin plays some roles in the embryonic development ongoing in the ovariole of the young insect. Prerequisite to test this possibility was to produce insects inherently deficient in symbionin. This was achieved by injecting an antibiotic into the insect (unpublished data). While the apid injected with cycloheximide or a-amanitin does not produce offspring, such antibiotics as_ chlor- amphenicol, rifampicin and nalidixic acid, if injected, allow the insect to keep producing progeny. Yet, since the latter three antibiotics are inhibitors of the prokaryotic system, those rtttTrret RF insects at the 20th day of birth. Viviparous females were injected with rifampicin a, RF insects, d, control, alata (From Ishikawa 516 H. IsHIKAWA ~ - + ~~ a : b Fic. 9. Two-dimensional separation of proteins of RF insects. RF insects at the 20th day of birth were injected with [°>°S]methionine and the proteins extracted 3 hr later. a, Stained; b, fluorographed. Arrows indicate the position of symbionin. For control, see Fig. 6a and b (From Ishikawa and Yamaji, submitted for publication). 0.6 A260 0.3 MIGRATION (cm ) Fic. 10. Polyacrylamide gel electrophoresis of high molecular weight RNA from the control and RF insects. RNA was extracted from the whole bodies and resolved on 3% polyacrylamide gel. a, Control; b, RF insects. Arrows indicate the peaks due to ribosomal RNA of the intracellular symbionts (From Ishikawa and Yantaji, submitted for publication). Intracellular Symbiosis 517 progenies produced by the insects injected with these drugs may carry some defects due to their selective actions upon the prokaryotic symbiont. It was demonstrated that rifampicin brought about the most significant effect on the progeny. Insects produced by the rifampicin-injected parents (RF insects) were significantly smaller in size than controls both at birth and 20 days later (Fig. 8). Moreover, the later born were these insects, the smaller they were in size. RF insects born 7 days after the rifampicin injection weighed at 20 days only one fifth as much as did the controls. It seemed that the alate progeny are more seriously affected than are the apterous one (Fig. 8). One important finding was that all of these RF insects do not at all produce progeny (unpublished data). Insects produced by the chloramphenicol- and nalidixic acid-injected par- ents, though somewhat smaller in size than (= Fic. 11. normal insect; b, 20 days RF insect. chondria; N, nucleus of a mycetocyte. Electron micrographs of the intracellular symbionts in normal and RF insects. S, symbiont (a) or symbiont-like structure (b); controls, were fully productive. Upon separating the total proteins of RF insects at 20 days, it was demonstrated that they neither contain nor synthesize symbionin (Fig. 9). Also, a resolution of high molecular wiehgt RNA from RF insects on polyacrylamide gel indicated that they lack the rRNAs of the intracellular symbiont (Fig. 10). As was expected from these results, RF insects are deficient in the symbiont, which was demonstrated by electron microscopy (unpublished data). No normal symbiont was observed with RF insects. Instead, there were many structures unique to these insects (Fig. 11). It is likely that these are degenerative forms of the symbiont. Presumably, injected rifampicin represses replication of the developing embryos by inhibiting the synthesis of RNA primer of the Okazaki fragment, which leads to an inhibition of DNA replication. a, Twenty days M, mito- Bars represent 1 #~m (From Ishikawa et al., submitted for publication; photographs through the courtesy of Dr. H. Hashimoto). 518 H. IsHIKAWA SYMBIONIN Though RF insects at 20 days are deficient in symbionin, yet those at the earlier stages contain, more or less, amounts of this protein (Fig. 12). Separation of the proteins of RF insects at various developmental stages showed that the symbionin content in these insects is decreased ex- ponentially over the course of post-embryonic development (Fig. 13). A semi-logarithmic plot- ting suggested that symbionin is decreased at a half-life of 3.5 days (unpublished data). The symbionin content in normal insects decreases also abruptly at birth, but later remains almost unch- anged. The symbionin content in newborn ny- mphs produced by the rifampicin-injected insects also depended on time of birth. The earlier born they were, the more symbionin they contained. The symbionin content seemed to decrease linearly Fic. 12. Diminution of symbionin during post- embryonic development of RF insects. Fifteen days insects were injected with rifampicin and their progeny produced between 24 and 48 hr later were raised. Proteins from these insects at various stages of development were sepa- rated and stained with Coomassie blue. 1, Full grown embryos whose parent received an injection of rifampicin 24hr before; 2, newborn RF insects; 3, 5 days RF insects; 4, 10 days RF insects; 5, 15 days RF insects; 6, control embryos. S, symbionin; A, actin (From Ishikawa et al., submitted for publication). with the period between the time of injection into the parent and that of birth of the insect (Fig. 14). RF insects born late must have been influenced by rifampicin since an early stage of their embryonic development while those born early only at a final stage. Naturally, this dif- ference will be reflected upon that in number of the symbiont present in these embryos because presumably injected rifampicin affects the insect primarily by inhibiting DNA replication of the symbiont in the embryo as mentioned already. Difference in the number of symbiont, in turn, will result in difference in the symbionin content of these embryos as described earlier. 1.5 = Cc fo) = £ > )) 1-0 re c 5 fo) £ o © 2 = 0.5 @ a 0 0 5 10 15 Days after birth Fic. 13. Plotting of symbionin contents versus developmental stage of insects. A densitometric. tracing of the protein bands on each lane in Fig. 12 was performed. Sizes of peaks of symbionin were estimated with a computer- aided analyzer and expressed as values relative to those of actin on the same lanes. Inset represents a semi-logarithmic plotting of the same data. Solid circles, RF insects; open circles, controls (From Ishikawa ef al., sub- mitted for publication). Intracellular Symbiosis 519 Here arises one important question: where is symbionin synthesized? One possibility is that 1.5 Cc T= 2 2a € my 1-0 Oo = s ° € oO & > = 0.5 Ss a ied 0 symbionin is synthesized only by the symbionts in the embryo growing in the ovarioles of the parent. An alternative is that this protein is synthesized by the symbionts in the maternal tissue and subsequently transferred to the embryo. In an effort to answer this question, syntheses and amounts of symbionin in the maternal and embryonic tissue were determined separately. As a result, most of symbionin, both nascent and preexisting molecules, were found with the embryonic tissue. Further, a chase with chlor- amphenicol did not suggest a change in locality of these nascent molecules. These results suggest that major site of the synthesis of symbionin is the embryonic tissue (unpublished data). Fic. 14. Amounts of symbionin in newborn nymphs. Fifteen days insects were injected with rifampicin and their progenies produced various times after the injection homogenized for protein analysis by one-dimensional gel electrophoresis. Symbionin content was estimated as described under Fig. 13. Solid circles, RF insects; open circles, controls (From Ishikawa et al., submitted for publication). Fic. 15. Electron micrographs of the intracellular symbionts in full-grown embryos. embryos from insects injected with rifampicin 48 hr before. a, Control; b, Bars represent 5 ~m (From Ishikawa et al., submitted for publication; photographs through the courtesy of Dr. H. Hashimoto). 520 H. ISHIKAWA It seems that symbionin molecules are ac- cumulated during embryonic development, and that over the course of post-embryonic develop- ment the insect consumes these preexisting mole- cules (Fig. 13). That the symbionin content in the normal insects is kept almost unchanged during post-embryonic development can be simply because a new production of this protein takes place in early embryos of the next generation developing in these nymphs. Symbionin can be a kind of storage protein to sustain post- embryonic development of the parent against embryonic development of the next generation ongoing inside. RF insects, particularly those born later, are undersized probably because symbionin accumulated in these insects during embryonic stages is less in amount than that in controls. It is also probable that symbionin is involved in a mechanism of initiating embryonic development since RF insects, whether born earlier or later, are completely sterile (unpublished data). It was shown that the embryos in the rifampicin- injected insect contain various structures which seem to represent intermediate forms from the normal symbiont to its degenerative forms (Fig. 15). The same photograph is also taken to indicate that DNA replication of the symbiont is more sensitive to rifampicin than that of mitochondria. The embryonic symbionts appear considerably different in shape from those in the adult. This be partly because in_ the former self-replication takes place much more frequently. Other thing relevant to this difference will be that at the embryonic stage the myce- tocytes enveloping the symbionts form a mycetome [31]. Also, it is probable that the surface of these embryonic symbionts is so rugged because of their high activity in exchanging substances with the host cell [unpublished data, 5]. This is compatible with a conclusion that the embryonic symbiont is active in synthesizing symbionin. will CONCLUSION AND PERSPECTIVES An only one symbiont that has been studied detailedly from molecular viewpoints is a Rhizobium bacteroid in the root nodule of the plant family Leguminosae [32]. The Rhizobium- legume symbiosis brings about many changes both in the host and in the intracellular symbiont. These include activation of nitrogenase gene residing on the Rhizobium genome [33], de- repression of leghemoglobin genes of the host cell [34] and development of a nodule as a result of a true cooperation between two genomes [32]. What should be emphasized in this context is that though the Rhizobium-legume interaction is so close, yet the symbiosis does not seem obligatory to both of them. In the presence of a plenty amount of soil nitrogen the legume does not require the symbiont while, needless to say, so many free-living Rhizobium and related bacteria are there in the soil [32]. This seems to be also true for intracellular symbioses in hemipterous insects other than aphids [35]. More than a hundred species of intracellular symbionts from leafhoppers and stinkbugs are now cultivated even at a large scale. All taken into consideration, a relationship between the host and symbiont in the aphid system is closer and more advanced evolutionarily than those in organisms mentioned above. As for the intracellular symbiosis in the aphid mycetocyte, of the most interest is to know the locality of gene coding for symbionin (Fig. 16). Circumstantial evidence suggests that it resides at the host genome. It can be that symbionin is coded for by the eukaryotic genome and is syn- thesized in the prokaryotic machinery of the intracellular symbiont [12, 36]. If this is the case, it is the first example of an information transfer across the cell at RNA level. Even if the gene is located at the symbiont genome, it is equally interesting. How is the symbionin gene alone expressed selectively where all the other symbiont genes are stringently repressed? Will a structural analysis of the symbionin gene answer this question? The first step of approach on this problem is being undertaken by examining whether the aposymbiotic insects contain mRNA of symbionin. In view of the number of protein species syn- thesized in vitro (Fig. 4), the aphid symbiont is supposed to have much more genes than needed for its intracellular life [28]. Probably, not a Intracellular Symbiosis 521 Nucleus Symbiont MYCETOCYTE Fic. 16. to the host-symbiont interactions. A schematic drawing of protein synthesis in the aphid mycetocyte in special reference S, symbionin; genome, synthesized in the eukaryotic machinery and transferred to the symbiont; same as A but not transferred to the symbiont; A, proteins, coded for by the host B, the C, proteins, coded for by the symbiont genome, synthesized in the prokayotic machinery of the symbiont and probably transferred to the host; D, the same as C but not transferred to the host. C and D are not synthesized to any great extent by the symbiont in young hosts (From Ishikawa [12]). few of them will not be expressed at all over the course of the symbiont’s life. In this very point, the intracellular symbiont is distinct from the DNA-containing cell organelles no matter how harmoniously the symbiotic system may be operated. At the present stage, it is somewhat moot to debate whether the intracellular symbiont of aphids should be following evolutionarily up the mitochondrion and chloroplast through loss of some genetic coding capacity to the host. However, the aphid symbiont is undeniably organelle-like, and perhaps its ambiguous position provides us with insight into the early stages of endosymbiotic evolution. REFERENCES 1 Biichner, P. (1965) Endosymbiosis of Animals Ww N . Griffiths, G. W. with Plant Microorganisms. New York, pp. 297-331. Lanham, U.N. (1968) Smith, J. D. (1948) Houk, E. J., (1976) Interscience, Biol. Rev., 43: 269-286. Nature, Lond., 162: 930—931. Griffiths, G.W. and_ Beck, S. D. Biochem. Physiol., 54B: 427-431. and Beck, S.D. (1975) Tissue Res., 159: 351-367. Robbins, W.E., Kaplanis, J. N., Svoboda, J. A. and Thompson, M.J.(1971) Ann. Rev. Entomol., 16: 53-72. Campbell, B. C. and Nes, W. D. (1983) Physiol., 29: 149-156. Srivastava, P.N. and Auclair, J.L. (1975) J. Insect Physiol., 21: 1865-1871. Schwemmler, W. (1980) 122, Hinde, R. (1971) 2050. Cell J. Insect BioSystems, 12: I11- J. Insect Physiol., 17: 2035- 522 23 24 Ishikawa, H. (1982) Insect Biochem., 12: 605- 612. Ishikawa, H. (1982) 622. Martin, J. P. and Fridovich, I. (1981) Chem., 256: 6080-6089. Gellissen, G., Bradfield, J. Y., White, B. N. and Wyatt, G.R. (1983) Nature, Lond., 301: 631-634. Timmis, J. N. and Scott, N.S. (1983) Nature, Lond., 305: 65-67. Margulis, L. (1981) Symbiosis in Cell Evolution. Freeman, San Francisco, pp. 1-62. Ishikawa, H. (1982) Comp. Biochem. Physiol., 72B: 239-247. Dasche, S. W. and Howell, S. H. (1976) J. Cell Biol., 69: 215-218. Keller, S. J. and Ho, C. (1981) 69: 157-190. Simon, T. J., Masker, W. E. and Hanawalt, P. C. (1974) Biochim. Biophys. Acta, 349: 271-274. Buss, W.C. and Kun, E. (1978) Biochem. Pharmacol., 27: 2139-2145. Ishikawa, H. (1978) Biochem. Biophys. Commun., 81: 993-999. Shine, J. and Dalgarno, L. (1973) J. Mol. Biol., 75: 57-72. Ishikawa, H. 435: 258-268. Insect Biochem., 12: 613- J. Biol. Int. Rev. Cytol., Res. (1976) Biochim. Biophys. Acta, 25 26 Zi 28 29 30 31 32 33 34 35 36 H. ISHIKAWA Ishikawa, H. in press. Grivell, L. A., Reijnders, L and Borst, P. (1971) Eur. J. Biochem., 19: 64-72. Ashwell, M. and Work, T. (1970) Ann. Rev. Biochem., 39: 251-290. Ishikawa, H. (1984) Insect Biochem., 14: 417-425. Ishikawa, H. (1984) Comp. Biochem. Physiol., 78B :51-57. Ishikawa, H.(1984) Insect Biochem., 14: 427-433. Lamb, K. P. and Hinde, R. (1967) J. Invertebr. Pathol., 9: 3-11. Verma, D. P.S. and Long, S. (1983) In “Intra- cellular Symbiosis.”’ Ed. by K. W. Jeon, Academic Press, New York, pp. 211-245. Bisseling, T., Van den Bos, R.C., Weststrate, M. W., Hakkaart, M. J. J. and van Kammen, A. (1979) Biochim. Biophys. Acta, 562: 515-526. Sullivan, D., Brisson, N., Goodchild, B., Verma, D.P.S. and Thomas, D. Y. (1981) Nature, Lond., 289: 516-518. Taylor, F.J.R. (1983) In “Intracellular Sym- biosis.” Ed. by K.W. Jeon, Academic Press, New York, pp. 1-28. Ishikawa, H. (1983) In “Endocytobiology II: Intracellular Space as a Oligogenetic Ecosystem.” Ed. by H.E.A. Schenk and W. Schwemmler, Walter de Gruyter, Berlin, pp. 759-769. (1984) Comp. Biochem. Physiol., ZOOLOGICAL SCIENCE 1: 523-532 (1984) © 1984 Zoological Society of Japan REVIEW Analysis of Respiratory Role of Haemoglobin in Daphnia magna MICHIYORI KOBAYASHI AND TAKEO HosHi1! Biological Institute, Faculty of Science Niigata University, Niigata 950-21, Japan INTRODUCTION The presence of haemoglobin in the haemolymph of Daphnia was shown by Lankester more than a hundred years ago [1]. This haemoglobin is characterized by a large variation in concen- tration; hence, the body colour of Daphnia changes from bright red to pale. Since Daphnia has no red blood cell, the haemoglobin is an extracellular substance dissolved in the hae- molymph. Svedberg and Erikson-Quensel [2] estimated on the basis of sedimentation rate that the molecular weight of Daphnia-haemoglobin is as high as those of many other invertebrate haemoglobins. The molecular weight of haemoglobin in three species of Cladocera has been hitherto known; 420,000 (daltons) [2] or 430,000-470,000 [3] for Daphnia pulex, 670,000 for Daphnia magna [4] and 670,000 for Moina macrocopa [5]. The subunit structure of Cladocera-haemoglobins has been studied, and the results obtained were not always the same [2-8]. Dangott and Terwilliger [3] proposed that the subunit of Daphnia-haemo- globin has two haems per polypeptide chain. In general, the structural properties of invertebrate haemoglobins show a spectacular diversity com- pared to the typical tetrameric structure of vertebrate haemoglobins. Those have been re- viewed by Terwilliger et al. [8]. Received October 1, 1983 ’ Present address: 8-6 Kamisugi 5-chome, Sendai- shi, Miyagi 980, Japan. Large variations in the haemoglobin concen- tration of Daphnia have attracted much attention to a key of the functional significance of this haemoglobin. In physiological studies the quan- titative estimation of the concentration of haemoglobin is of important significance. How- ever, the amount of haemoglobin in a single animal is too small to measure the haemoglobin concen- tration by ordinary spectrophotometry. Fox [9] was the first to attempt an estimation of hae- moglobin concentration in Daphnia in vivo. He deviced a haemoglobin index method and studied with his coworkers for ten years starting from 1945. The haemoglobin index method is basically to compare the pink colour of a living animal with the colour of diluted human blood in a wedge- shaped glass under microscope. By this method they found various factors accompanied with the changes in the concentration of haemoglobin, the functional significance and the breakdown of haemoglobin [10]. Using a large number of animals, Hildeman and Keighley [11] extracted haemoglobin and measured the concentration by spectrophotometry. Kring and O’Brien [12] re- ported the effect of both concentration of haemo- globin and oxygen concentration on the filtering rate of Daphnia. In our laboratory, the relationships between respiratory rate of Daphnia and_ surrounding oxygen concentration, physicochemical properties of purified haemoglobin and the functional signi- ficance have been studied for many years. In recent years, oxygen response of haemoglobin in living Daphnia has been investigated by micro- 524 M. KOBAYASHI AND T. HOSHI spectrophotometry [13-16]. Furthermore, the absolute values of the haemoglobin concentration in the haemolymph in a single animal have also been measured by microspectrophotometry [17-19]. In this paper, the results obtained by micro- spectrophotometry are mainly described, and the physiological function of this haemoglobin is discussed. Since male Daphnia is smaller than the female and the occurrence is less in the male than in the female, female Daphnia magna was used throughout the experiments. VARIATION IN HAEMOGLOBIN CONCENTRATION Haemoglobin and oxygen concentration of the habitats As pointed out by many workers [10, 18, 20], there is a close correlation between the dissolved oxygen in water and the haemoglobin concen- tration in haemolymph of Daphnia. This seems to be essential for understanding the nature of this haemoglobin. In connection with this, the upper and lower limits of oxygen concentrations were determined by the modified Winkler method at 16: 00 in the surface water, and at 7: 00 in the bottom water respectively. The range of fluctu- ation in the oxygen concentrations is also given. The relationship between the dissolved oxygen concentration of the water in which Daphnia lives and its haemoglobin concentration is shown in Figure 1. A large variation in the oxygen concentration was noted in the water in which pale animals lived. As the oxygen concentration of air- saturation corresponded to approximately 6 ml/I, the water in which pale animals lived proved to be supersaturated in the day time. The oxygen concentration of the water in which red animals lived, in contrast, was characterized by both the low and small diurnal fluctuations and the large vertical oxygen gradient. The haemoglobin con- centration was found to vary from 0.1 to 1.6g haemoglobin per 100ml of haemolymph. The highest haemoglobin concentration is about one- tenth that of human blood. It is clearly demon- strated in the Figure 1 that D. magna synthesizes 10.0 8-0 S 6-0 Cc o oO > x< fe) 5) 60 cD > re) 1) mn S220 0 Haemoglobin concentration, g/100ml Fic. | Haemoglobin concentrations of Daphnia magna (2.5mm in body length) in water with different concentrations of dissolved oxygen (From Kobayashi and Hoshi [18]). haemoglobin in response to oxygen deficiency, and that concentration of haemoglobin in the body and the concentration of oxygen in habitat are inversely related with each other. It is also found that even a small oxygen deficiency below air-saturation induces an increase in haemoglobin concentration. Accordingly, it may be said that the body colour of animals is a good indicator for the dissolved oxygen concentration in the water. Haemoglobin concentration and body size Although the body colour of large animal is mostly pale, the colour of small animal collected from the same habitat is pink. This suggests a large difference in the haemoglobin concentration between these two types of animals of different body colours. Figure 2 represents the relationship between the haemoglobin concentration and the body size of animals. The haemoglobin con- centration is generally low in young animals, in which the concentration increases as the body size increases, reaching a maximum level just before the sexual maturity followed by subsequent decrease after the maturity. This relationship Respiratory Role of Daphnia-Hb 525 — _ ° a Haemoglobin conc., g./100ml. oO an Length, mm Fic. 2. Relationship between body size and haemoglobin concentration in Daphnia magna from four populations. (a) Red population; (c) yellow population; (From Kobayashi [19]). (b) pink population; (d) pale population. is typically observed in a pink Daphnia population. One of the reasons for the decrease of hae- moglobin concentration in mature animals is already known, namely, the passage of haemo- globin from the haemolymph into the eggs [9, 21]. Fox et al. [9] reported that the mean body length after moulting was 1.1 times of the size before moulting. They pointed out that dilution of the haemolymph by moulting might cause a decrease in concentration of haemoglobin. In this con- nection, the increasing haemoglobin concentration with an increase in the body size as observed in immature animals suggests that small animals have high ability to synthesize haemoglobin. In a red population, not only a higher haemoglobin concentration but also small body size than those in other populations are noticed, as pointed out by Green [22]. Since the total amount of haemoglobin in each individual depends on both the concentration of haemoglobin and the volume of haemolymph, it is incorrect to discuss the change in haemoglobin content only in terms of the haemoglobin concen- tration in animals of different body sizes. At present, it is not always possible to measure the haemolymph volume in Daphnia especially of small body size. In the present study, therefore, haemoglobin was extracted from a large number of animals, and the concentration was measured spectrophotometrically to estimate the total a- mount of haemoglobin per individual. The relationship between body weight and haemoglobin content of D. magna is shown in Figures 3 and 4. The total haemoglobin content of red animals increased by 1.62 power of the body weight (log-log plot). The haemoglobin concentra- tion per unit dry weight in red animals varied from 29.2 to 80.3 mg Hb/g dry weight. Therefore, it means that in adult red animals the haemoglobin amounted to approximately 8% of the dry weight of animals. Kring and O’Brien [12] reported that Daphnia pulex exposed to a low oxygen concen- ee -2-0 y ye a red D.magna 0 3 e i ff PN > e ° = Cr x 07° Uv e ° c a 9 Be fe, E on-4:0 - pale D.magna ° ° re | ee ee ee -2-0 -1.0 log mg dry wt Fic. 3. Haemoglobin content per animal of Daphnia magna as a function of dry weight of animal. 526 M. KOBAYASHI AND T. HOsHI vie eae eae red D. magna ,MgHb/g Hb content pale D.magna Pe ° ue ogo oO——______o 0 0.05 010 0.15 Dry weight,mg 0.20 0.25 Fic. 4. Haemoglobin concentration of Daphnia magna as a function of dry weight of animal. tration for 15 days had 50 mg Hb/g dry weight. The haemoglobin concentration of pale animals is too low to be measured by the haemoglobin index method. It has been believed that Daphnia produces haemoglobin only under the water con- ditions of low oxygen concentrations. As to the haemoglobin, much attention has been paid to the red animals, but little to the pale animals. How- ever, it is seen in Figure 3 that even in pale animals the synthesis of haemoglobin does occur in accordance with the increase in the body size, and the haemoglobin concentration of pale animals varied from 1.4 to 13.9 mg Hb/g dry weight. On the comparison between animals of the same body size, it was found that the haemoglobin concen- tration of pale animals ranged from 1/5 to 1/28 of that of the red ones. A relatively high con- centration of haemoglobin of neonates in both pale and red animals seems to show the presence of a high concentration of haemoglobin in the egg. In pale animals, haemoglobin seems to be synthesized for the passage into the eggs. Red animals lay the eggs with slightly pink colour under microscope, which is in contrast to pale bluish eggs of pale animals. This may in- dicate a high haemoglobin content in the eggs. But, since red animals lay only a limited number of eggs (one or two eggs), the haemoglobin concen- tration in the haemolymph of mother does not undergo a large change before and after the egg laying. Fox [10] reported that in poorly aerated water Daphnia lays fewer eggs than in well aerated water, and the haemoglobin in the eggs accelerates the embryonic development in the conditions of oxygen paucity. OXYGEN CONSUMPTION IN WATER OF LOW OXYGEN CONCENTRATION Oxygen consumption rate and ambient oxygen concentration Many studies have analyzed the functions of this haemoglobin, most of which have been based on comparison between the normal and carbon- monoxide treated animals, or between the pale and red animals [10, 23-26]. In this paper particular attention is paid to the difference in oxygen consumption between pale and red animals in water of low oxygen concentrations. The type of respiration in Daphnia with decrease of oxygen concentration was classified into the following three kinds: normal respiration, critical respiration and lethal respiration. In the experiment for the time course of respiration, normal respiration is followed by critical and lethal respirations. At the lethal respiration, animals could never absorb pale D.magna fe oO £ & Cc iS a E =) w (‘Ss (e) O oO 1 2 3 4 5 D.O., mi/I Fic. 5. Effect of ambient oxygen concentration on oxygen. consumption rate of Daphnia magna at 20°C. Respiratory Role of Daphnia-Hb 527 any oxygen from the water of very low oxygen concentration and died. Figure 5 shows the effect of ambient oxygen concentration on the oxygen consumption rate in pale and red animals (2.5 mm in body length, 20°C). The horizontal bars in the figure indicate the initial and final oxygen concen- trations in the closed respiration chamber. The oxygen consumption rates were calculated from this difference in oxygen concentration. For the analysis of respiratory function of haemoglobin both the cirtical oxygen concentration with which respiration responsible for the critical respiration is possible and the lethal oxygen concentration for the lethal respiration were especially taken into account. In pale animals, the critical oxygen concentration was 0.9 ml/l and the lethal oxygen concentration was 0.5ml/l. In red animals, both the cirtical and lethal oxygen concentrations were lower than those in pale animals. It is sig- nificant that red animals could maintain a normal oxygen consumption rate even at a low oxygen concentration where pale animals were unable to survive. This gives the physiological importance of Daphnia-haemoglobin in picking up oxygen from water of low oxygen concentration. Haemoglobin concentration and lethal oxygen concentration The relationship between the lethal oxygen concentration observed at 25°C and the haemo- globin concentration is shown in Figure6. A remarkable decrease in the lethal oxygen concen- tration is found in proportion to the increase in the haemoglobin concentration. As to the range of oxygen concentration for the function of haemo- globin in red animals, it is demonstrated that the haemoglobin performs the function of intake of the major portion of oxygen from surrounding water under the low oxygen conditions which is below the lethal oxygen concentration for pale animals. These phenomena suggest that haemo- globin functions only in a low oxygen con- centration. This conclusion is supported by other experi- ments; Daphnia-haemoglobin has relatively high affinity for oxygen (Pso, 3.5 mmHg at 20°C and pH 7.2) [25]. Low levels of carbonmonoxide elevated the lethal oxygen concentration of red O° a — ation, mL/I io) > eee ae Oxygen concentr 0 0-5 1.0 1.5 2-0 Haemoglobin concentration, g./100mlI. Fic. 6. Relationship between lethal oxygen con- centration and haemoglobin concentration of Daphnia magna. (From Kobayashi [17]). animals, and at the CO concentration above 0.3 ml/l, where haemoglobin might probably have been saturated with carbonmonoxide, the lethal oxygen concentration maintained a constant level [28]. Deoxygenation of haemoglobin in vivo was observed only in a low oxygen concentration, which is given later in this paper. Resistance to low oxygen concentration and body size The relation between the body size and lethal oxygen concentration was investigated in animal samples collected from pale, yellow, pink and red populations (Fig. 7). The resistance of respective animal to low oxygen was almost constant before the sexual maturity, but it was greatly affected by body size after the maturity. It is especially found in a red population that the resistance to low oxygen was exceedingly high, and not affected by body size. Red animals are usually observed to appear in summer season and the small body size of red animals has been attributed to environmental factors such as temperature and poor nutrition. The decrease in resistance to oxygen deficiency as a result of an increase in body size must also be one of the important factors for the appearance of red population in summer. Since red animals can live in severe oxygen deficiency, they may have many chances to encounter, during longer or shorter periods, such a low oxygen concentration 528 M. KOBAYASHI AND T. HOsHI ray d/ Ut Oxygen conc., Length, mm Fic. 7. Relationship between body size and lethal oxygen concentration of Daphnia magna at 20°C. (a) Pale population; (b) yellow population; (c) pink population; (d) red _ population. (From Kobayashi [19]). as their critical or lethal oxygen concentrations. On the other hand, pale animals can live only in a high oxygen concentration, which does not exert a vital effect to the largest animals in spite of their very low resistance to oxygen deficiency. OBSERVATION ON IN VIVO OXYGENATION OF HAEMOGLOBIN Estimation of the percentage of oxyhaemoglobin in vivo The actual mode of oxygenation and deoxy- genation of haemoglobin in living pink Daphnia, the results of which give many definite evidences for the respiratory function of this haemoglobin, was investigated. The trans- parency of the carapace of Daphnia permitted the measurement of absorption spectra of haemoglobin in the haemolymph of single living pink animal with a microspectrophotometer. The animal was adhered on the carapace to the tip of a fine glass stick with an instantaneous adhesive agent, carefully inserted into an absorption cell and kept in a fixed position. The light absorbance was measured in the neighbourhood of the heart of Daphnia at 420 or 578nm, which are the ab- sorption maximum of oxyhaemoglobin in vivo. Figure 8 represents the time course of the change in optical density at 420 nm in accordance with the oxygen condition in surrounding water. In vivo response of oxygen to haemoglobin in single pink Daphnia was then obtained. It is seen that oxygenation and deoxygenation are reproducible, and the percentage of oxyhaemoglobin is affected by the oxygen concentration in the surrounding water; 100% oxygenation in air-saturated water, deoxygenation in N.-saturated water (oxygen-free water) and partial oxygenation (41% oxyhaemo- globin) were observed in a low oxygen concen- tration (11 % air-saturation). 100,- UI earn i O Wriee water = \ \ \ { 1083 Ate cab, Manor Fic. 8. Time course of change in optical density in living pink Daphnia magna at 420nm in various oxygen concentrations. A, Air-saturated water and oxygen-free water. B, 11% air-saturated water and oxygen-free water. Arrows indicate the time of exchange of water in the absorption cell. (From Hoshi e¢ al. [14]). In vivo oxygenation of haemoglobin and ambient oxygen concentration The in vivo percentage of oxyhaemoglobin was measured at various oxygen concentrations (Fig. 9). The curve of the oxyhaemoglobin percentage was plotted against oxygen concentrations. A steep curve was obtained. The oxyhaemoglobin in- creased with increase of oxygen concentration above 3% air saturation. The haemoglobin was 50% oxygenated at 15% air-saturation, and fully Respiratory Role of Daphnia-Hb 529 O Hbo2 € %) a) 106,26) 30 40 50 60. 70 80 ._90 100 Oz-concentration (%) Fic. 9. Relationship between ambient oxygen con- centration and percentage of oxyhaemoglobin of living pink Dapnia magna (From Hoshi et al. [28)). oxygenated at 30% air-saturation. The occurrence of deoxygenation only in a low oxygen concen- tration seems to support the respiratory function 100 90 80 70 60 50 40 O2-concentration( %) of this haemoglobin described above. Time for oxygen equilibration under different oxygen concentrations In Figure 8, it was noticed that oxygenation in air-saturated water took place very rapidly, while deoxygenation in oxygen-free water did slowly. To measure the time required for complete deoxy- genation, pink Daphnia whose haemoglobin had been equilibrated with various concentrations of oxygen (shown in the ordinate of Fig. 10) was then subjected to oxygen-free water. As shown in Figure 10, the time for complete deoxygenation was hardly affected by the initial oxygen concen- tration in the water, and deoxygenation was completed during only a few minutes. In con- nection with this short periods, one of the authors [29] measured the haemolymph volume of red Daphnia and found that the haemolymph volume of single red animal (2.5 mm in body length) was 0.7 wl. Based upon the following three factors, i.e., haemolymph volume, haemoglobin concen- tration and oxygen consumption rate of red animal Time( min) Fic. 10. Time required for oxygenation and deoxygenation of haemoglobin in single pink Daphnia magna. Closed circles: time required for equilibration with oxygen. Animal whose haemoglobin had been deoxygenated in oxygen-free water was then subjected to water with varying oxygen concentrations (shown in the ordinate of the figure). Triangles: time required for complete deoxygenation. Animal whose haemoglobin had been equilibrated with various concentration of oxygen (shown in the ordinate of the figure) was then subjected to oxygen-free water. Oxygen concentration was expressed as per cent of air saturation. Hoshi and Takahashi [16]). (From 530 M. KoBAYASHI AND T. HOosHI (2.5 mm in body length at 20°C), it was estimated that the haemoglobin could supply the animal with oxygen only during about 2min. The storage function of oxygen of this haemolymph is thus very low, and the estimated value seems to be in good agreement with the observed time in the figure. The absence of the effect of initial oxygen concentrations on the time for complete deoxy- genation may indicate that at all the initial oxygen concentrations studied, haemoglobin was almost completely in an oxygenated state. The striking aspect of the figure is that oxy- genation differs markedly from deoxygenation. This fact is significant for the analysis of re- spiratory function of haemoglobin in pink Daphnia lived in water of low oxygen concentrations, which will be mentioned later again in the experiment of respiratory inhibition. Animal whose haemo- globin had been deoxygenated in oxygen-free water was subjected to water with varying oxygen concentrations (shown in the ordinate of Fig. 10). It is noteworthy indeed that the time required for equilibration with oxygen is inversely related with the concentration of oxygen in water. Marked prolongation of the time for equilibration with oxygen in water of low oxygen may be most WA Wye de Seca ex iNaNa 4] Ties Pisses | aaa ae | mista iN a Oxygenation Sees aa ra ae OD an: probably ascribed to the following; the time used for replacement of oxygen-containing water in the absorption cell, temporary increase in oxygen demand after return to oxygen-containing water, and utilization of haemoglobin-bound oxygen for respiration during equilibration. Deoxygenation time of oxyhaemoglobin in pink Daphnia treated with NaN; Since oxygen released from haemoglobin in oxygen-deficient water must be utilized for cell- respiration, the time necessary for in vivo deoxy- genation may be protracted if animals were treated with a respiratory inhibitor such as KCN or NaN3. Figure 11 shows the time course of oxygenation and deoxygenation of haemoglobin in water with and without NaN; [15]. The results presented in the figure clearly show that the presence of NaN; had no effect on the time for oxygenation but had a remarkable effect on prolongation of the time for complete deoxygenation. KCN also induced the same results [13]. These findings give directly the experimental evidence for the utilization of oxygen released from haemoglobin in the haemo- lymph of pink Daphnia kept in oxygen deficient water for their cell-respiration [16, 28]. =o) He rH ae Vn pare fins =| min T= Se eae SARREANRTEEEEEEe O Fic. 11. Effect of NaN; on the oxygenation and deoxygenation of haemo- globin in single pink Daphnia magna. Arrows indicate the time of exchange of water in the absorption cell. Hoshi and Akuzawa [15]). (From Respiratory Role of Daphnia-Hb PERSPECTIVES In many ponds and diches in our districts, natural red populations of zooplanktons, mostly of Moina macrocopa, are frequently encountered. Their redness is very impressive and we have found that they always live in low oxygen con- ditions. In laboratory, we could understand the physiological significance of haemoglobin, that is to say, adaptation to low oxygen conditions, by measurements of both oxygen consumption rate of red animals and in vivo oxygen saturation of haemoglobin in Daphnia. We have realized, on the other hand, the importance of pale animals, in addition to red animals, as the pale animals may provide the valuable information on the production of haemoglobin in Daphnia. The results of studies on the uptake of radioactive iron into the electrophoresed components of haemo- globin, which was produced by pale animals cultured in low oxygen conditions, suggested the possible occurrence of continous turnover of this haemoglobin. Further studies are therefore expected for the mechanism of haemoglobin synthesis in Daphnia. The interesting topic connected with this haemoglobin synthesis is the persuit of synthetic site for haemoglobin. From the result of histochemical observations, it is very likely that gut caecum and fat body are the tissues of haemoglobin synthesis. More work is needed to establish this hypothesis. The physico-chemical properties of haemo- globins in Moina and Daphnia have been studied. The molecular weight estimated is 670,000 daltons. In connection with this, 38 subunits per molecule were estimated from the values of molecular weight and iron content (0.317%). An SDS-polyacry- lamide gel electrophoresis revealed the presence of smaller dissociation products with molecular weights of 11,700 and 8,900 daltons. Further analysis of the subunit structure is necessary. The amino acid composition of Moina- haemoglobin was also measured. Our next interest is to determine the sequence of amino acids, and the result of which will lead to the evolutional origin of this haemoglobin. 25 26 27 531 REFERENCES Lankester, E.R. (1871) Pfliig. Physiol., 4: 315-320. Svedberg, T. and Erikson-Quensel, I. (1934) J. Amer. Chem. Soc., 56: 1700-1706. Arch. gesam. Dangott,L.J. and Terwilliger, R.C. (1980) Comp. Biochem. Physiol. 67B: 301-306. Sugano, H. and Hoshi, T. (1971) Biochim. Biophys. Acta., 229: 349-358. Hoshi, T., Kobayashi, M. and Sugano, H. (1967) Sci. Rep. Niigata Univ. Ser. D., 4: 1-8. Hoshi, T. and Kobayashi, K. (1971) Sci. Rep. Niigata Univ. Ser. D., 8: 65-68. Hoshi, T., Kobayashi, M., Sugita, O., Hatayama, M., Sugano,I. and Sugano, H. (1976) Sci. Rep. Niigata Univ. Ser. D., 13: 7-13. Terwilliger, R. C. (1980) Amer. Zool., 20: 53-67. Fox, H. M., Hardcastle, S. M. and Dresel, E. I. B. (1949) Proc. Roy. Soc. (B), 136: 388-399. Fox, H.M., Gilchrist, B. M. and Phear, E. A. (1951) Proc. Roy. Soc. (B), 138: 514-528. Hildemann, W.H. and Keighley, G. (1955) Amer. Nat., 89: 167-174. Kring, R. L. and O’Brien, W. J. (1976) Ecology, 57: 808-814. Hoshi, T. and Yahagi, N. Niigata Univ. Ser. D., 12: 1-7. Hoshi, T., Yahagi, N. and Watanabe, T. (1977) Sci. Rep. Niigata Univ. Ser. D., 14: 7-13. Hoshi, T. and Akuzawa, K. (1979) Sci. Rep. Niigata Univ. Ser. D., 16: 13-18. Hoshi, T. and Takahashi, K. (1981) Sci. Rep. Niigata Univ., Ser. D., 18: 21-28. Kobayashi, M. (1981) Comp. Biochem. Physiol., 69A: 679-682. Kobayashi, M. and _ Hoshi, T. Biochem. Physiol., 72A: 247-249. Kobayashi, M. (1982) Comp. Biochem. Physiol., 72A: 599-602. Hoshi, T. and Kobayashi, K. (1972) Sci. Rep. Niigata Univ. Ser. D., 9: 55-62. Dresel, E. I. B. (1948) Nature, 162: 736-737. Green, J. (1955) J. Anim. Ecol., 24: 84-97. Hoshi, T. and Yajima, T. (1970) Sci. Niigata Univ. Ser. D., 7: 107-115. Kobayashi, M. (1974) Sci. Rep. Niigata Univ. Ser. D., 11: 1-10. Hoshi T., Kobayashi, M., Sugano, H. (1969) Sci. Ser. D., 6: 155-164. Usuki, I. and Yamaguchi, K. (1979) Sci. Rep. Niigata Univ. Ser. D., 16: 5-12. Kobayashi, M. and Hoshi, T. (1980) (1975) Sci. Rep. (1982) Comp. Rep. and Univ. Honma, M. Rep. Niigata Sci. Rep. 532 M. KOBAYASHI AND T. HosuHi Niigata Univ. Ser. D., 17: 1-6. 29 Kobayashi, M. (1983) Comp. Biochem Physiol., 28 Hoshi, T., Kobayashi, M. and Takahashi, K. 76A: 803-805. (1983) Sci. Rep. Niigata Univ. Ser. D., 20: 1-6. ZOOLOGICAL SCIENCE 1: 533-543 (1984) © 1984 Zoological Society of Japan Two Types of Antennal Hygro- and Thermoreceptive Sensilla of the Cricket, Gryllus bimaculatus (De Geer) TSUNAO ITOH, FUMIO YOKOHARI, and YOSHIYA TOMINAGA Biological Laboratory, Faculty of Science, Fukuoka University, Fukuoka 814-01, Japan ABSTRACT — Antennae of the cricket, Gryllus bimaculatus (De Geer) bear sensilla each of which is both hygro- and thermoreceptive. reveal two types of sensilla. apparatus (ca. 0.5 wm in tip diameter). apparatus (ca. | wm in apex diameter). type II in each antenna. Electrophysiological recordings and scanning electron microscopy Type I is coeloconic and has a blunt-tipped cone-shaped cuticular Type II is also coeloconic but has a cylindrical cuticular There are at least 20 sensilla of type I and 40 sensilla of The response of a dry receptor of type I is phasic-tonic and that of a moist receptor is phasic. The responses of both hygroreceptors of type II are phasic-tonic. The humidity-response curves of all four hygro-receptors are almost linear over 0-100% r.h. The curves from either class of hygrore- ceptor of Type II are not affected by flow-rates of the stimuli over the antenna. The cold receptors were identified in these sensilla but not analyzed quantitatively. INTRODUCTION Sensilla which are both hygro- and thermore- ceptive have been identified electrophysiologically in many insects (refer to[1]). Structural character- ization of the hygro- and thermoreceptive sensilla of several of the insects studied was carried out with scanning (SEM) and/or transmission electron microscopy (TEM). In _ some studies _ the coeloconic sensillum of Locusta and the basiconic sensillum of Periplaneta have been reported to possess stimulus-conducting pores similar to those of olfactory sensilla [2-4]. On the other hand, Yokohari et al. [5] showed with SEM that the hygro- and thermoreceptive sensillum of Peri- planeta is not perforated, and referred to it as a sensillum capitulum. Subsequently, the hygro- and thermoreceptive sensilla of some insects have been shown to be poreless (Carausius [6], Peri- planeta [7-10], Locusta [11], Leucophaea [12] and Apis [13, 14]). Thus, two essentially different results have been obtained on the structure of Accepted March 13, 1984 Received August 5, 1983 insect hygro- and thermoreceptive sensilla. Fur- ther, among the poreless hygro- and thermore- ceptive sensilla, some features of the external structures differ greatly. For example, i) some sensilla are coeloconic (Carausius [6], Locusta [11, Tominaga, in preparation] and Apis [13, 14]). The others are basiconic (Periplaneta [5, 7-10] and Leucophaea [12]). ii) The surface of the cuticular apparatus is smooth in Periplaneta [10], longitudi- nally sculptured in Locusta (Tominaga, in pre- paration), and irregular in Apis [14]. The characteristics of hygroreceptor responses have been investigated electrophysiologically in some insects. The responses of all hygroreceptors studied were phasic-tonic. The dry receptor of Periplaneta is bimodal, responding to both tem- perature and humidity [15]. The response of Locusta hygroreceptors was larger at higher temperature regardless of whether they plotted against relative or absolute humidity [16]. Re- sponses of Periplaneta hygroreceptors depend on relative humidity, and it has been proposed that a mechanical transformation occurs in the primary process of hygro-reception [8, 17]. In order to understand completely the proper- 534 T. ITOH et al. ties of hygroreceptors it is necessary to examine hygroreceptors of widely divergent species. In the present paper we will identify two types of antennal hygro- and thermoreceptive sensilla of Gryllus and characterize their responses. Fea- tures of structure and function common to the insect hygro- and thermoreceptive sensilla are discussed in comparison with those of several species of insects. MATERIALS AND METHODS Adult crickets, Gryllus bimaculatus (De Geer), were used throughout this study. They were reared in a laboratory colony at constant tem- perature (30°C), and fed solid rat food and water. Electrophysiological Recording Electro- physiological recording methods were similar to those described in the previous paper [17]. The animal was first anaesthetized by cooling and then fixed to an acrylic plate without disturbance of respiration. The antennae were firmly fixed to the plate with beeswax in such a way that the outer sides of the antennae were turned upward. The head of the animal was projected into a small pool which was filled with physiological saline [19]. An indifferent electrode was placed in this pool. The active electrode was an electrolytically sharpened tungsten wire which was inserted into the base of the cuticular apparatus just deep enough to make electrical contact. Electrical events were ampli- fied, displayed on a cathode ray oscillo-scope, and photographed. Humidity stimuli were applied by the same method as described previously [17]. The conditioning and stimulus air was prepared by mixing dry and fully moistened air. Humidity was monitored by an Ace PH-IP electric hygrom- eter. Morphological Observation The hygro- and thermoreceptive sensilla identified electro- physiologically were marked by advancing the active electrode into the sensilla, thus enlarging the hole or cleft which was the recording site on the antennal cuticle. The regions around the sensilla were sketched in order to facilitate reloca- tion in SEM. Then a small piece of flagellum which contained the identified sensillum was cut out with a razor and immediately immersed in 70% acetone. Following ultrasonic cleaning in 70°% acetone, it was dehydrated in an acetone series, dried, and coated with gold-palladium using an Eiko IB-3 ion coater. Observations were made with a Hitachi S-430 SEM. RESULTS Identification of the Hygro- and Thermoreceptive Sensilla Several types of sensilla have been identified by SEM on the antennal flagellum of the cricket, Gryllus bimaculatus (De Geer); a chaetic sensillum, a trichoid sensillum, a basiconic sensillum, and three types of coeloconic sensilla which have a specific cuticular apparatus different in each type (Fig. 1). Identification of hygroreceptive sensilla was as follows: Electrical responses to humidity changes were first recorded from each type of sensillum. Each sensillum was then marked and examined with SEM. The electrical activity from these two types of coeloconic sensilla is composed of at least three types of impulses (Fig. 2). The first of them responds to humidity increase with increasing impulse frequency. The second responds to humidity decrease with increasing impulse fre- quency. The third responds to temperature fall with increasing impulse frequency, while it is not shown in Figure 2. Thus, these sensilla contain moist, dry, and thermal receptors. Electron micrographs illustrating them are shown in Figure 3. The other types of coeloconic sensillum did not respond appropriately to hygric stimulation. External Structure of the Hygro- and Thermorecep- tive Sensilla Three types of coeloconic sensilla commonly consist of a broad pit (ca. 10-15 wm in diameter) having a central opening (ca. 4.0-6.0 ym in diameter) and a cuticular apparatus in the bottom of the pit. The outer surface of the circumfer- ence of the opening is smooth in comparison with other parts of the antennal surface and is a little elevated proximally so that the opening points slightly distally. One type of hygro- and thermoreceptive sensilla Hygro- and Thermoreceptive Sensilla of the Cricket 535 Fic. 1. seen. sensilla; tr, tricoids. x 900. has a blunt-tipped cone-shaped cuticular apparatus, which measures ca. 0.5 wm in tip diameter, ca. Typel O°%or.h. 100°%or.h. 100°%or.h. O°%or.h. ImV Type | D M 200msec om i biddenncl ‘ Te. Wu ae US See 100°%er.h. O%or.h. Scanning electron micrograph of a middle flagellar segment. An arrowhead indicates the cleft into which the recording electorode was inserted. Three coeloconic sensilla (co) are ch, chaetic 1.5-2.0 wm in base diameter and ca. 2.5 wm in length (Fig. 3a). Externally, the cuticular appa- ratus appears to arise from the bottom of the pit and to be surrounded basally by a cuticular collar. Its outer surface is smooth and appears to lack perforation. This type of sensillum is described henceforth as a hygro- and thermoreceptive sensil- lum of typeI. The other type of hygro- and thermoreceptive sensillum has a cylindrical cuticu- lar apparatus, which measures ca. 1 sm in diameter (Fig. 3b). arise from the bottom of the pit in a similar manner to that of type I. tured radially at the apex and longitudinally on The cuticular apparatus appears to Its proximal region is sculp- the sides and is depressed or covered by a button in the centre of the apex. In most specimens, there Fic. 2. Electrical activities recorded from hygro- and thermoreceptive sensilla of type I and type Il. M, impulse from a moist receptor; D, from a dry receptor; C, froma cold receptor. T. ITOH et al. Hygro- and Thermoreceptive Sensilla of the Cricket 537 is a wedge-shaped incisure (Fig. 3b). This type of sensillum is described henceforth as a hygro- and thermoreceptive sensillum of type Il. The third type of coeloconic sensillum has a tapered and grooved cuticular apparatus (Fig. 3c), and does not respond to hygric stimulation. Distribution of the Hygro- and Thermoreceptive Sensilla The antenna of the cricket consists of three parts, from proximal to distal, a scape, a pedicel, a flagellum. The flagellum comprises about 150- 200 segments and is ca. 3.5cm Jong and ca. 300 ym in diameter proximally. Each type of hygro- and thermoreceptive sensil- lum is distributed with a similar pattern in both sexes. Both types of sensilla are mostly located on the outer side of the flagellum. One to four type I sensilla are always found on a given segment; on the segment distal to it there is a putative proprio- mechanoreceptive structure (Fig. 4b) located ven- trally and one or two on every two to five segments on the other segments. The type II sensillum is distributed similarly on the segment distal to that on which the presumptive proprio-mechano- receptive structure is located, but is rarely found on the Ist and 2nd flagellar segments (Fig. 4a). As the segment, on which the mechanoreceptive struc- ture is located, occurs more frequently proximally than distally, both types of sensilla are usually larger in number in the proximal region. The numbers of type I and type II sensilla were esti- mated to be 20 and 40 on a single antennal fla- gellum, respectively. Electrophysiological Responses of the Hygrorecep- tors Three kinds of impulses have been recorded simultaneously from both hygro- and thermorecep- tive sensilla of type I and type II; one from a moist Fic. 4. sensillum typeI; @, sensillum type II; V, ventral side; <1, position of a putative proprio-mechanoreceptive structure. mechanoreceptive structure (arrow). 500. a: Schematic drawing of the distribution of hygro- and thermoreceptive sensilla. O, D, dorsal side; P, pedicel; b: A putative proprio- Fic. 3. The external structures of the cuticular apparatus of three types of coeloconic sensilla. a: A blunt-tipped cone-shaped cuticular apparatus of a hygro- and thermoreceptive sensillum type I. x 20,000. b: A cylindrical cuticular apparatus with a wedge-shaped incisure (arrow) of a hygro- and thermoreceptive sensillum type II. x 20,000. eto. | tapered and grooved cuticular apparatus of a probable olfactory sensillum. 5,000. 538 T. ITOH et al. receptor, one from a dry receptor and one from a cold receptor (Fig. 2). The spontaneous activity of the fourth kind of cell for which the adequate stiumulus is not known is recorded very rarely. 1. Time Courses of the Responses The Moist Receptor The moist receptors of type I and type II sensilla respond to relative humidity increase with increasing impulse fre- quency. Figure 5 shows the time courses of responses of the moist receptors. The instantane- ous impulse frequency was sequentially calculated every 200 msec from the impulse number. The moist receptor in type I responds to sudden in- crease in humidity in a phasic manner. When adapted to 0% r.h., the receptor discharges at 0-10 imp/sec. When stimulated with rectangular increase of humidity from 0% to 100% r.h., the impulse frequency increases suddenly to a maxi- mum of 70-80 imp/sec, then decreases immediately to the pre-stimulus level, despite the continuation of the stimulus. Thus, when the stimulation ceases, the impulse frequency does not change. On the other hand, the moist receptor in the type II : Moist receptor Imp/sec 100 Typel 0 1@ 20 0 4 50 imp/sec 100 Type I 80 Shes ine 0 1009207 30.540) 50.) 60m OnmGO sec O°%erh 100°%/erh. O°%or.h. Fic. 5. The time courses of responses of moist. receptors in a hygro- and _ thermoreceptive sensillum type I and type II. sensillum responds to sudden increase of humidity in a phasic-tonic manner. When adapted to 0% r.h., the moist receptor discharges at 10-20 imp/ sec. When stimulated with rectangular increase of humidity to 100% r.h. from 0% r.h., the impulse frequency increases suddenly to a maximum of 90 imp/sec. Within about 5 sec, it rapidly decreases to 30-40 imp/sec and then becomes nearly con- stant during continued stimulation. When the stimulation ceases, the impulse frequency decreases suddenly to a lower level than the pre-stimulus level and then gradually recovers to the pre- stimulus level. The Dry Receptor Figure 6 shows the time courses of the responses of the dry receptors. The dry receptors in both type I and type II sensilla respond to sudden decrease of humidity in a phasic-tonic manner. When adapted to 100% r.h., the dry receptors of type I and type II sensilla discharge at 5-15 imp/sec and 15-25 imp/sec, respectively. When the receptors are stimulated with sudden decrease of humidity from 100% mofeee Dry receptor 100 Typel 10 20 s@ 40 2) os 100°%or.h. | O%er.h. | 100°%er.h. imp/sec 160 Type I 140 120 100 80 0 10 20 30 20 50 GO 0 Go sec 100%rh| O%er.h. | 100°%or.h. Fic. 6. The time courses of responses of dry receptors in a type I and a type II sensillum. Hygro- and Thermoreceptive Sensilla of the Cricket r.h. to 0% r.h., the impulse frequencies increase to a maximum of 80 imp/sec for type I and more than 100 imp/sec for type II. In about 5sec, they rapidly decrease to 20-30 imp/sec in type I and 40 imp/sec in type II, and then remain nearly constant during stimulation. When the stimula- tion ceases, the impulse frequencies of the dry receptors of both types of sensilla suddenly de- crease to lower levels than those before stimulation, and then recover gradually. 2. The Relation Between Stimulus Intensity and Responses The Moist Receptor The moist receptors of type I and type II sensilla were stimulated by air of various relative humidities after complete adaptation to0% r.h. The impulse frequency for the type I sensilla was calculated from the number of impulses for the first 400 msec, between | sec and 2 sec, and between 2 sec and 4sec from the start of the stimulus. It was calculated with the same method for type II sensilla for the first 400 msec, between 2 sec and 4 sec, and between 10 sec and 12sec. Figure 7 shows the stimulus-response curves plotted against relative humidity, obtained by this method. Each point in this figure is the mean of three to six values measured from a single receptor. Vertical lines indicate standard 539 deviations. In type I, the frequency increases with increase in relative humidity for the first 400 msec and does not change for the other periods, while the moist receptor in the typeI responds very phasically. In type Il, the impulse frequency increases with increase of relative humidity in every period measured. The _ stimulus-response curves are almost straight and have no plateaux. The Dry Receptor The dry receptors in both types of sensilla were also stimulated by air of various relative humidities after complete adaptation to air of 100% r.h. The impulse frequency was calculated by the same method used for the moist receptor. Figure 8 shows the re- sponse curves of dry receptors plotted against relative humidity. The impulse frequency in- creases with decrease in relative humidity in every period measured in both types. Further, the curves appear almost straight and have no plateaux. 3. The Effect of Stimulus Flux on the Responses The stimulus-response relationships of the moist and dry receptors in type II were investigated at two different flow-rates. Figure 9 shows the stimulus-response curves at stimulus fluxes of | liter/min and 2 liters/min. The pairs of curves are almost coincident for each type of receptor. Therefore, the responses are independent of the Moist receptor Typel imp/sec 60 ae 0-400msec 40 30 20 1-2sec 10 0 2-45eC 0 20 40 60 80 100 *lorh. Fic. 7. imp/sec Type] 80 O-400msec 70 60 50 a0 2-4s5ec 30 20 10-12sec 10 0 Pe Ss aS a 0 20 40 60 80 100 */or KH. Humidity-response curves of moist receptors in a type I and a type II sensillum. 540 T. ITOH et al. Dry receptor Typel imp/sec Type I | 90 ee 80 0-400msec 70 70 at 0-400msec 60 50 507 2-4sec 40 40 30 30 20 20 10-12sec 10 10 aah gar ae Sa Se O's leenlee 0 20 40 60 80 100 0 20 40 60 80 100 *fer.h. *fer.h. Fic. 8. Humidity-response curves of dry receptors in a type I sensillum and type II. bata, Moist receptor Dry receptor imp/sec 40 80 70 30 60 50 20 40 30 10 20 10 0 (= Wea) eles oneal 0 [= ews ls os pe ea 0 20 40 60 80 100 0 20 40 60 80 100 *fer.h. . Fer.h. Fic. 9. Effect of stimulus flux on the responses of a moist and a dry receptor in type II sensilla. ©, 1 liter/min; @, 2 liters/min. flow-rate of the stimulus stream. electrode was advanced slightly so that the cuticular apparatus can be supposed to have bent slightly at the base, the impulse frequency of the moist receptor increased and that of the dry receptor The responses to mechanical deformation of the decreased. Opposite changes in responses were sensillum were studied in type II. Whentheactive induced by electrode retraction (Fig. 10). There- 4. The Responses to Mechanical Deformation of the Sensillum Hygro- and Thermoreceptive Sensilla of the Cricket 541 Fic. 10. Responses to mechanical deformation of type II sensilla. from a moist receptor; of recording electrode; | ImV 200msec M, impulse D, impulse from a dry receptor; push, slight advance pull, retraction of recording electrode. Deflection of the trace is artifact due to a movement of the recording electrode. fore, mechanical deformation may qualitatively simulate stimulation by humidity. DISCUSSION For the cuticular apparatus, some studies have shown that the hygro- and thermoreceptors can be identified electrophysiologically in structures mor- phologically similar to olfactory sensilla [7-14]. However, the most recent studies indicate that these receptors are found in poreless sensilla [5, 6, 8-14]. The present study also shows that both types of hygro- and thermoreceptive sensilla appear to be poreless and examination with transmission elec- tron microscopy also demonstrates that there is no open canal continuous with the lumen surrounding the sensory cilia of the receptors (Iwasaki, in preparation). Thus, a nonperforated cuticular apparatus can be regarded as a common, essential feature of the hygro- and thermoreceptive sensilla. On the other hand, some hygro- and thermore- ceptors are identified electrophysiologically as located in the basiconic type of sensilla and others in the coeloconic type (basiconic type: Periplaneta [4, 17] and Aedes [18] and coeloconic type: Locusta [11], Triatoma [20] and Apis [13]). The present study shows that the hygro- and thermoreceptors of Gryllus are in the coeloconic type of sensilla. Whether the hygro- and themoreceptive sensillum is basiconic or coeloconic seems to correlate with the antennal morphology: The basiconic type of hygro- and thermoreceptive sensillum is found on antennae on which bristles are distributed densely and the coeloconic hygro- and thermoreceptive sensillum are found on antennae on which bristles are distributed scarcely or are not present. These structural differentiations may be developed in order to protect the cuticular apparatus from harsh contact with solid components of the environment. This is significant because the present study shows that the hygroreceptors re- spond to mechanical deformation of the sensillum as has been previously demonstrated in Periplaneta [8]. Two types of the hygro- and thermoreceptive sensilla are found on the antenna of Gryilus; this is the first report that two types of hygro- and thermoreceptive sensilla exist together on the same antenna of an insect. The response of the moist receptor of type I is very phasic and that of type II is phasic-tonic. However, the responses of the dry receptors are phasic-tonic in both types of 542 T. ITOH ef al. sensillum. Thus, whether the responses are phasic or phasic-tonic does not correlate with the surface structures of the sensilla. This is also true for the sensilla of Periplaneta [17] and of Apis [13]; that is, the responses of the hygroreceptors are phasic-tonic in both species, though the surface of the cuticular apparatus is smooth in the cockroach and irregular in the honey bee. The response magnitudes of the receptors of type II are generally larger than those of type I, and the surface area of the cuticular apparatus is larger for type II than for type I. We infer that the sculpture of the cuticular apparatus makes the surface area larger, resulting in high sensitivities of the receptors. The stimulus-response curves of the hygrore- ceptors are almost linear in Gryllus, whereas those of Periplaneta and Apis are steep at low and high humidities but flattened somewhat at intermediate humidities in most fresh uninjured preparations [8, 13]. Those of the moist receptors of Locusta are almost linear in some preparations but some- what flattened at high humidities in the others [16]. These differences may be due in part to the experi- mental conditions and not to the receptor prop- erties. We investigated the effect of stimulus flux on the hygric responses and on responses to mechani- cal deformation of the sensillum for the receptors in type II, because the cuticular apparatus of type I is identical to that of Periplaneta [9, 10], while that of type II is a new type found in the present study. The responses of hygroreceptors in type II are independent of the stimulus flux as well as those of Periplaneta [17] and Apis [13]. The responses to mechanical deformation of the sensilla in type II are also similar to those of Periplaneta [8]. Thus, we infer that the hygroreceptors in type II sensilla of Gryllus have the same mechanism of stimulus reception as that proposed by Yokohari [8] in Periplaneta. The number of hygro- and thermoreceptive sensilla on an antenna is estimated in present study to be 60 and is similar to that of Locusta [11], Periplaneta [10] and Apis [13]. This suggests that this number of hygroreceptors may be required if insects are to perceive humidity in the environment, as indicated by Yokohari et al. [13]. ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. A.D. Blest (Dept. Neurobiology, Australian National University, Canberra, Australia) for cor- recting the English. This work was supported in part by a Grant-in-Aid for Special Project Research “‘Mechanism of animal behavior’ from the Ministry of Education of Japan. REFERENCES 1 Alter, H. and Prillinger, L. (1980) Ultrastructure of invertebrate chemo-, thermo-, and hygro- receptors and its functional significance. Int. Rev. Cytol., 67: 69-139. 2 Steinbrecht, R.A. (1969) Comparative mor- phology of olfactory receptors. In “Olfaction and Taste III’. Ed. by C. Pfaffmann, Rockefeller Univ. Press, New York, pp. 3-21. 3 Altner, H., Ernst, K. D., Kolnberger, I. and Loftus, R. (1973) Feinstruktur und adaquater Reiz bei Insektensensillen mit Wandporen. Verh. Dtsch. Zool. Ges., 66: 47-58. 4 Altner, H., Sass,H. and Alter, I. (1977) Re- lationship between structure and function of antennal chemo-, hygro-, and thermoreceptive sensilla in Periplaneta americana. Cell Tissue Res., 191: 389-405. 5 Yokohari, F., Tominaga, Y., Ando, M. and Tateda, H. (1975) An antennal hygroreceptive sensillum of the cockroach. J. Electronmicrosc., 24: 291-193. 6 Altner,H., Tichy,H. and Altner,I. (1978) Lamellated outer dendritic segments of a sensory cell within a poreless thermo- and hygroreceptive sensillum of the insect Carausius morosus. Cell Tissue Res., 191: 287-304. 7 Schaller, D. (1978) Antennal sensory system of Periplaneta americana L. Cell Tissue Res., 191: 121-139. 8 Yokohari, F. (1978) Hygroreceptor mechanism in the antenna of the cockroach Periplaneta. J. Comp. Physiol., 124: 53-60. 9 Yokohari, F. (1981) The sensillum capitulum, an antennal hygro- and thermoreceptive sensillum of the cockroach, Periplaneta americana L. Cell Tissue Res., 216: 525-543. 10 Tominaga, Y. and Yokohari, F. (1982) External structure of the sensillum capitulum, a hygro- and thermo- receptive sensillum of the cockroach, Periplaneta americana. Cell Tissue Res., 226: 309-318. 11 Altner, H., Routil, C.H. and Loftus, R. (1981) The structure of bimodal chemo-, thermo- and 12 14 15 Hygro- and Thermoreceptive Sensilla of the Cricket hygroreceptive sensilla on the antenna of Locusta migratoria. Cell Tissue Res., 215: 289-308. Schaller, L. (1982) Structural and _ functional classification of antennal sensilla of the cockroach, Leucophaea maderae. Cell Tissue Res., 225: 129-142. Yokohari, F., Tominaga, Y. and Tateda, H. (1982) Antennal hygroreceptors of the honey bee, Apis mellifera L. Cell Tissue Res., 226: 63-73. Yokohari, F. (1983) Thecoelocapitular sensillum, an antennal hygro- and thermoreceptive sensillum of the honey bee, Apis mellifera L. Cell Tissue Res., 233: 355-365. Loftus, R. (1976) Temperature-dependent dry receptor on antenna of Periplaneta. Tonic response. J. Comp. Physiol., 111: 153-170. Waldow, U. (1970) Elektrophysiologische Unter- 17 18 19 20 543 suchungen an Feuchte-, Trocken- und Kalterezep- toren auf der Antenne der Wanderheuschrecke Locusta. Z. Vergl. Physiol., 69: 249-283. Yokohari, F. and Tateda, H. (1976) Moist and dry hygroreceptors for relative humidity of the cockroach, Periplaneta americana L. J. Comp. Physiol., 106: 137-152. Kellogg, F. E. (1970) Water vapour and carbon dioxide receptors in Aedes aegypti. J. Insect Physiol., 16: 99-108. Fielden, A. (1960) Transmission through the last abdominal ganglion of the dragonfly nymph Anax imperator. J. Exp. Biol., 37: 832-844. Bernard, J. (1974) Etude électrophysiologique de recépteurs impliques dans |’orientation vers ’héte et dans l’acte hématophage chez un Hemiptére: Triatoma infestans. These Doct. Sci. Nat. Rennes, pp. 1-285. ai att to alleen roy? caietivte? 0h reo tong sot Qeupetecumty Wibke epee, iaerghd goa wk. sovtadioidy +g ebay bie inaeroaet’ 5 3 4 “= a @ i.e Hiri. 7 if ORY oh ee oe Var rw } 4] alec) wish 14 4 Jest en el t a 3 i t Ai ot) MAA EE ™ Sian - 1 i * Sony iF ey ; : co Rog. Boaein” OAS tee! ) a¢t 4 ; Boot hs ‘ ar 4 remy seal — Bhd ifs 4 bad} " : | ban di . . y i ' ; red ingen be F le o + ? y mt 5 f AS " \ * i S ] ay cg 4 Z i F§ - : Bh vanont® ene ii 2} Reet tec aeniaenele eri Thartse = aviv ht “SD ED rt weltlen \ “rt F © Peeed tie : mi ath Lag che! Brits velde rh Aaaerph: pees acinar 4 Mf ea PRC ety ke RO ! i mary Kya it: 5° Y ZOOLOGICAL SCIENCE 1: 545-552 (1984) © 1984 Zoological Society of Japan A Precision Photoelectric Method for Recording Chromatophore Responses in Vitro NoORIKO OSHIMA and Ryozo FUJII Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan ABSTRACT — A photoelectric method for recording quantitatively the motile response of chromato- phores is described. For the study of melanophores and other light-absorbing chromatophores, an ordinary transmission light microscope is most suitable, whereas a dark-field epi-illumination micro- scope should be used for investigating the motility of light-reflecting chromatophores; i.e., leucophores and motile iridophores. The output current of a silicon photo-diode, the chosen photo-sensor for the present purpose, is converted into voltage by means of I-V converter operated by a low bias current, high input-impedance operational amplifier. After proper amplification and cancellation of the DC component of the signal, chromatophore responses can be widely registered on a paper-chart recorder driven at an appropriate speed. INTRODUCTION Hill et al. [1] were the first to adopt the photoelectric method to assess skin color changes in animals. Using a photocell, they plotted galvano- meter deflections due to changes in light reflectance from the dorsal skin of the killifish, Fundulus heteroclitus, against time. By measuring light transmittance through an isolated scale of the common tautog, Tautoga onitis, Smith [2] was also able to plot the pulsation of melanophores. _ Since 1959, we have also been using photoelectric devices to record the responses of chromatophores in vitro, mainly of melanophores of teleost fishes, and have continually tried to improve them [3-8]. Concerned mainly with physiological analyses of chromatophores, though, our descriptions have only outlined methods, leaving unmentioned the details of practical arrangements and techniques. We have, however, been asked several times by readers of our papers and by researchers who actually engage in pigment cell studies to provide more precise information regarding our methods, particularly those employed in the electronic portions of the system. With the intention of Accepted March 27, 1984 Received March 19, 1984 satisfying these demands, we describe here a precision photoelectric method, which has been successfully used in our laboratory, and which can be carried out by hand without much difficulty and at low cost. Although our foregoing studies have been re- stricted to the pigment cells of vertebrates, the method described here is also applicable to those of invertebrates, if an appropriate preparation is adopted. MATERIALS Until now, isolated frog or toad skins have been commonly employed in measuring macroscopic darkening or whitening due to chromatophore activity, especially in the bioassay of some peptide hormones, such as MSH [9] and melatonin [10]. Skin pieces of tailed amphibians or reptiles may also be used for this purpose [11]. When these skin preparations are employed, though, measuring the intensity of light reflected from the skin may be more appropriate than quantifying the light transmittance through skin tissues. In either case, it is difficult to record the responses of individual chromatophores accurately when the tissue is thick. If the thin skins of small adult forms, youngs or 546 N. OSHIMA AND R. FUuSII larvae are used, the usual transmission microscope method can successfully be applied to study of the motile activities of melanophores and other light-absorbing chromatophores. Thus, we have repeatedly employed scaleless skin pieces of the silurid catfish, Parasilurus, [12, 13] or Kryptopterus [14, 15]. Since the pioneering work of Spaeth [16] on Fundulus melanophores, on the other hand, small isolated fish scales with a piece of integument at- tached have been very frequently used by a number of researchers. In this type of preparation, the epidermis, under which chromatophores usually lie, constitutes a strong diffusion barrier for solutes in experimental media and, therefore, stimulant chemicals or ions have to invade the epidermis centripetally from the torn margin of the skin tissue to affect the chromatophores successively. Then, the characteristics of the response, i.e., the reaction time, rate, maximal level attainable, etc., vary so greatly among chromatophores located at different parts of a scale that quantitative analyses are not worthwhile performing. In a few cyprinids such as dark chub (Zacco; [17]) and top-mouth gudgeon (Pseudorasbora; Oshima and Fujii, unpublished observation), melanophores suitable for observation lie in the dermis just under the scale proper, and thus are not covered by epidermal tissue. The chromatophore response therefore proceeds more quickly, and, moreover, at the same rate among the cells. Recently, split-fin preparations have become popular for physiological studies of teleost chro- matophores. These preparations can be made by splitting a piece of fin into two symmetrical halves [3, 4]. In such preparations, chemicals and drugs readily diffuse to reach the pigment cell layer, because there is no need for them to penetrate the epidermis. Therefore, the reaction time is very short, and the rate of response is very high. In addition, the responses develop almost simulta- neously among chromatophores. When split pieces from smaller fishes are adopted, chromato- phore responses proceed so quickly and are so reproducible that it is possible to detect the effect of a chemical or a drug at very low concentrations, and also to obtain very quantitative data. In this way, preparations from the tail fin of guppies [18-20] or from the dorsal fin of damselfish [21] have been successfully employed. Furthermore, the integrity of the peripheral nervous system is also well preserved in this type of preparation. Thus, to some extent, it has been possible by using them to study the peripheral nervous mechanisms controlling chromatophores [5, 18, 21, 22]. In addition, as we have already discussed elsewhere, this type of preparation is most suitable for in vitro studies of fish chromatophores [4]. METHODS Optical system For the photoelectrical assessment of changes in the state of one or more chromatophores, an ordinary transmission light microscope is most convenient (Fig. 1). A perfusion chamber, in which a thin skin preparation of an appropriate type (cf. Materials) is put, is set on the stage. Thereafter, pigment cells are focused for measure- ment of their activity. A diaphragm, usually with a circular aperture in the center is put on the plane of the intermediate image, i.e., the real image of the objective lens inside the eyepiece, above which the photoelectric transducer is placed. By changing the size and shape of the aperture, the dimensions of the field of observation may easily be restricted. A better effect can also be obtained when a finely frosted glass plate is placed between the aperture and the sensor, although current sensitivity must be in- creased at that time, owing to the loss of light intensity hitting the sensor (cf. Interface section). If the Kohler illumination system has been adopted, the effect of the field restriction may be somewhat improved by decreasing the diameter of the illuminator iris to fit the size of the field under measurement. In addition to the common upright microscope [3], an inverted one may also be employed, espe- cially when the chromatophore under examination is micromanipulated [5, 19]. If melanophore responses are to be recorded exclusive of the activities of xanthophores or erythrophores, a proper color filter which does not Recording of Chromatophore Responses 547 EP | OL es “2. | sl. SP aus mn eS ae, Se B&B Ko Fic. 1. Diagram showing the optical part of the system for quantitative recording of the response of light-absorbing chromatophores in vitro. AD: aperture diaphragm, CF: color filter, CL: condenser lens, EP: eye-piece, ES: electronic stimulator, FD: field diaphragm, FRD: field- restricting diaphragm, HCF: heat cut filter, IE: indifferent electrode, IP: inlet pipette for experimental media, MS: microscope stage, OL: objective lens, OP: outlet pipette for experimental media, PC: perfusion chamber, RM: reflecting mirror, SE: stimulating elect- rode, SP: split-fin preparation, SPD: silicon photo-diode, TLS: transmission light source. pass light with wavelengths to be absorbed by those pigment cells must be put across the light path. A sharp-cut red filter, Wratten 25, Corning 2424, Toshiba R-60, or equivalent filter, which blocks out light shorter than 600 nm in wavelength, fits this purpose. Assessment of the motile activities of xantho- phores or erythrophores is somewhat more difficult. At first, animal species, individuals, or skin por- tions without melanophores should be selected as easier materials with which to employ the present method. If a small area surrounded by melanophores but containing only brightly pig- mented cells can be selected and brought into the optical pathway of the measuring microscope system, their motile responses can be recorded. In any case, it may be better to restrict the wavelengths of illumination light to those of the color complementary to that to be absorbed by xanthophores or erythrophores to increase the contrast of these cells against the background. Blue monochromatic, or so-called band-pass filters, e.g., Wratten 45, Corning 4445, and Toshiba B-48, are useful in achieving this end. An interference filter may also be applicable. Because of its low light transmittance, however, the output current of the light-sensor should be strongly amplified at that time (cf. Interface section). It is rather natural to quantify the response of leucophores or motile iridophores by measuring light-reflectance, since intracellular inclusions responsible for their brightness are light reflective. An epi-illumination microscope is thus employed for this purpose [6, 23]. In such a case, the use of a microscope of dark-field type is also strongly recommended, since a usual bright-field type for metallographic studies has been found to be practically useless, because light reflection from the surface of cover slip strongly reduces the con- trast of the image of the cells. In our laboratory, a Nikon industrial microscope, XF-BD, with CF-BD plan objective lenses has been employed quite successfully. Having been installed with both transmission and incident-light illumination systems, the microscope has enabled us to study the responses of both light-absorbing and reflecting chromatophores when necessary. In any case, a voltage or current stabilizer should be employed as the power supply for the illuminator lamp in order to keep its luminous intensity constant. Photo-sensor Various semiconductor photoelectric elements are now commercially available, and some of them are being used for the present purpose. Among these, photoconductive cells, mostly cadmium- sulfide ones, have been commonly employed [5, 24-26]. In our laboratory, however, silicon photo-cells have recently been adopted, since they 548 N. OsSHIMA AND R. FUJII have better specific characteristics, including spectral sensitivity very similar to that of the human eye, and stability. These silicon photo- cells are commonly called “‘solar batteries’, or sometimes are referred to as “‘blue cells’. These elements are very easy to handle, since they are electrogenic themselves, being free from the ex- ternal current supply which is indispensable for photoconductive cells. Currently, we are using photo-cells produced by Hamamatsu Photonics (Hamamatsu), e.g., $1226-SBK, but similar photo- cells of high quality will suffice. When it is necessary to measure the light faintly reflected from light-reflecting chromatophores, especially from leucophores, a photomultiplier tube may be adopted as an almost perfect trans- ducer [6]. The weak points, though, are that it is rather troublesome to adapt to a microscope and to handle appropriately. Using a silicon photo- diode coupled with electronic circuit described below (cf. Interface section), we have succeeded in measuring such weak light as that reflected from part of a leucophore. Interface The electronic part of the recording system for chromatophore responses which is being used currently in our laboratory is roughly diagrammed in Figure 2. It is known that the linearity of the incident light intensity vs. the output current in a photo- electric transducer is best accomplished when its load impedance is null. Thus, the adaptation of an operational amplifier (OP-amp) for a current- to-voltage converter, or the so-called current amplifier, is quite reasonable, since one of the general and essential requirements of the OP-amp is that the potential difference between two input terminals must always be kept null, forming a virtual short in terms of the equivalent circuit. This requirement is almost perfectly met when we employ an OP-amp of a high quality hybrid type (e.g., ADS1I5K, 3431K, 3523K) in which the input bias current is extremely small and the input offset voltage is well balanced beforehand. Even if a cheaper monolithic OP-amp is used, quite good performance may be achieved, if the OP-amp is of a J-FET (e. g., wA740A, LF356A, TA7505M), or more properly of a MOS-FET input type (e.g., CA3140A, “PC152A), and, further, if the input offset potential is accurately balanced by means of a precision variable resistor, the so-called trimmer potentiometer, which is inserted between leads 1 and 5 of an OP-amp of very common metal-can type, as illustrated in Figure 3. The adjustment is especially important when it is necessary to measure very weak light such as that reflected from a leucophore. The lead wires that connect the light sensor with the OP-amp should be highly insulated and shielded. In this respect, it is wise to put the OP-amp very close to the sensor, and to connect Fic. 2. Gross diagram of the electronic portion of the photoelectric recording system. A,: OP-amp for current-to-voltage converter (current amplifier), inverting DC amplifier, CCS: constant current source, E,: recorder, constant current, A,: OP-amp for non- A3;: OP-amp for voltage follower for impedance conversion, voltage for DC component cancellation, produced by constant current (I) through its load resistance (R,), EPR: electronic paper-chart H: high impedance-side one of the differential input terminals of EPR, I: L: low impedance-side one of the differential input terminals of EPR, R;,: feedback resistance for A, to change the current amplification factor, back resistance for A, to change DC amplification, R;2: feed- R,: load resistance of CCS. Recording of Chromatophore Responses 50k2 10k9 Mie Fic. 3. 100k2 4 — 549 0.05pF Output ( BNC ) 0. 05pF 50ko 10k2 -15V Circuit of the current-to-voltage converter designed for measuring wide-range intensities of light so as to quantify the responses of either light-absorbing or reflecting chromatophores. OA: OP-amp of high photo-diode. them by short wires. The current sensitivity of the converter can be widely varied by selecting the feedback resistance of the OP-amp (R,;, in Fig. 2; cf. also Fig. 3). Special care should be taken in obtaining a high quality and well-insulated rotary switch for changing the resistors. Recorder To record chromatophore responses, an electronic paper-chart recorder is most convenient. Almost any kind of commercially available recorder will do, although those on which fine changes in the chart-driving speed around 5 through 40mm-°: min‘ may be made are the most suitable to register responses of the usual rate. If the recorder is equipped with a multistep changeover switch for selecting sensitivity, there may be practically no problem. However, usual recorders have only rough steps for sensitivity selection. As shown by A, in Figure 2, therefore, it is more convenient to have a simple DC amplifier, with which fine changes in amplification can be made, e.g., with step-wise factors of 1 through 10 by integers. Any proper DC amplifier on the market should suffice for such a purpose. If the recorder does not have a sensitivity selecting switch, but is sensitive enough, a simple attenuator consisting of only dividing resistors will function just as well. In order to record widely the trace of response on the chart, a certain DC component in the input input impedance type, SPD: silicon signal to the recorder must be properly canceled. Some commercial recorders are equipped with such a device, but most have only a narrow-range pen positioner. In our laboratory, we are making good use of a handmade apparatus in which multistep IR drops produced by a constant current through a series of precision resistors are converted into low-impedance output voltages by a voltage fol- lower using an OP-amp (Fig. 2). The output is inserted between the ground and the low im- pedance-side terminal of the differential input connections of the recorder. Similar electronic devices for canceling the DC component can be designed. RESULTS AND DISCUSSION The system described in this paper has enabled us to make very high quality recordings of the re- sponses of either light-absorbing chromatophores, i.e., melanophores, erythrophores and xantho- phores, or of reflecting ones, i.e., leucophores and motile iridophores. Some findings obtained by this method have already been published. These include results on the responses of melanophores of a Japanese silurid [27] and those of translucent glass catfish [14, 15]. By way of example, actual recordings of the responses of a melanophore and of a few motile iridophores just overlying the former on a split-fin 550 N. OSHIMA AND R. FuJII Fic. 4. Examples of actual records of the responses of chromatophores. the platy, Xiphophorus maculatus, to K*. method of electrical stimulation were identical to those described elsewhere [18]. A: response of a single melanophore on a split dorsal-fin preparation of the damselfish, Chrysiptera cyanea, to electrical nervous stimulation. B: response to electrical nervous stimulation of motile iridophores present just overlying the melanophore, on the response of which trace A in this figure was recorded. CC: response of a erythrophore on the scale of a tropical fish, The physiological saline solution and the Pulses of 20 V in strength and 1.0 msec in duration were applied. The frequency was 0.14 Hz. For K* stimulation, saline containing 50 mM K* was used, from which the equimolar Na* was compensatorily removed. preparation of the damselfish, Chrysiptera cyanea, are illustrated in Figure 4A and B, respectively. They were recorded in a system using a microscope in which either transmission (Fig. 4A) or incidence illumination (Fig. 4B) could be employed on command. The summation of small _ unit responses, each of them elicited by a single pulse, was clearly seen. Incidentally, we have been studying the motile mechanisms of these damselfish chromatophores, and part of the outcome of this study will appear soon [21]. Another example of recording is shown in Figure 4C, in which the pigment-aggregating response of an erythrophore of the platyfish to an elevated K* concentration is demonstrated. By this means, the motile activities of bright-colored cells can now be precisely recorded. Recently, a video-image analyser system has been employed for recording chromatophore response in vitro [23]. Installed optionally with a proper interface, the digital-to-analog (D-A) converter, to an electronic recorder, this apparatus should become popular in the near future. The weaknesses of such a system, however, are that it is rather expensive, and that, although short, a definite amount of time is required by the computer Recording of Chromatophore Responses 551 to calculate and to process the numerical data on area measurements. Thus, calculated values come out recurrently, leading to the registration of discontinuous and stepwise traces, when rapid responses of chromatophores must be recorded. Incidentally, the minimal period for proccessing an image is not less than one second in the systems presently available commercially. Thus, rapid reactions such as those melanophores of the guppy or damselfish, of motile iridophores of the latter, or of erythrophores of the squirrelfish Holocentrus (28) cannot be registered as they are. By using the photoelectric method described in the present study, the rapid changes in light transmittance and reflectance due to chromato- phore movements can be registered continuously with high fidelity. For precise investigations dealing with the motile response of chromato- phores, therefore, such a method should be adopted and effectively put to use. If it is, further development in the field of pigment cell research should be a certainty. ACKNOWLEDGMENTS This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan and also by a grant from the Ito Foundation for the Promotion of Ichthyological Research. REFERENCES 1 Hill, A. V., Parkinson, J.L. and Solandt, D. Y. (1935) Photoelectric records of the colour change in Fundulus heteroclitus. J. Exp. Biol., 12: 397-399. 2 Smith, D.C. (1936) A method for recording chromatophore pulsations in isolated fish scales by means of a photo-electric cell. J. Cell. Comp. Physiol., 8: 83-87. 3 Fupii, R. (1959) Mechanism of ionic action in the melanophore system of fish I. Melanophore- concentrating action of potassium and some other ions. Annotationes Zool. Japon., 32: 47-59. 4 Fujii, R. (1973) Responses of fish pigment cells to environmental changes. In ““Responses of Fish to Environmental Changes”. Ed. by W. Chavin, C. C. Thomas, Springfield, pp. 342-362. 5 Fujii, R. and Novales, R. R. (1969) The nervous mechanism controlling pigment aggregation in Fundulus melanophores. Comp. Biochem. Physiol., 29: 109-124. 6 10 11 13 Fujii, R. and Miyashita, Y. (1979) Photoelectric recording of motile responses of fish leucophores. Annotationes Zool. Japon., 52: 87-94. Fujii, R. and Oshima, N. (1984) Biological assay using chromatophore responses. In “Bioassay Methods for Organic Chemists”. Ed. by N. Ikekawa, S. Marumo and M. Hoshi, Kodansha Scientific, Tokyo, pp. 262-267. (In Japanese) Fujii, R. and Oshima, N. (1984) Movement of chromatophores. “Jikken Seibutsugaku K6za”, 10: “Undo Seibutsugaku” (Series in Experimental Biology, Vol. 10: Biology of Movement). Ed. by H.Sugi and Y.Hiramoto, Maruzen, Tokyo, pp. 255-270. (In Japanese) Shizume, K., Lerner, A. B. and Fitzpatrick, T. B. (1954) Jn vitro bioassay for the melanocyte stimulating hormone. Endocrinology, 54: 543-560. Mori, W. and Lerner, A. B. (1960) A _ micro- scopic bioassay for melatonin. Endocrinology, 67: 443-450. Hadley, M. E. and Goldman, J. M. (1969) Physi- ological color changes in reptiles. Amer. Zoologist, 9: 489-504. Fujii, R. and Miyashita, Y. (1976) Receptor me- chanisms in fish chromatophores — III. Neurally controlled melanosome aggregation in a siluroid (Parasilurus asotus) is strangely mediated by cholinoceptors. Comp. Biochem. Physiol., 55C: 43-49. Fujii, R. and Miyashita, Y. (1978) Receptor mechanisms in fish chromatophores — IV. Effects of melatonin and related substances on dermal and epidermal melanophores of the siluroid, Parasilurus asotus. Comp. Biochem. Physiol., 59C, 59-63. Fujii, R., Miyashita, Y. and Fujii, Y. (1982) Muscarinic cholinoceptors mediate neurally evoked pigment aggregation in glass catfish melanophores. J. Neural Transmission, 54: 29-39. Kasukawa, H. and Fujii, R. (1984) Potassium ions act to release transmitter from “cholinergic” postganglionic fiber to the glass catfish mela- nophore. Zool. Sci., 1: 553-559. Spaeth, R. A. (1913) The physiology of chroma- tophores of fishes. J. Exp. Zool., 15: 527-585. Iga, T. and Matsuno, A. (1980) Scale melano- phores of Zacco temmincki; A preparation suita- ble for physiological or pharmacological studies on fish melanophores. Zool. Mag., 89: 227-234. 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. Fujii, R. and Miyashita, Y. (1976) Beta adreno- ceptors, cyclic AMP and melanosome dispersion 552 22 23 24 in guppy melanophores. Pigment Cell, 3: 336—- 344, Miyashita, Y. and Fujii, R. (1975) Receptor mechanisms in fish chromatophores — II. Evidence for beta adrenoceptors mediating melanosome dispersion in guppy melanophores. Comp. Biochem. Physiol., 51C: 179-187. Oshima, N., Sato, M., Kumazawa, T., Okeda, N., Kasukawa, H. and Fujii, R. (1984) Motile iridophores play the leading role in damselfish coloration. Pigment Cell, 7: in press. Kumazawa, T., Oshima, N., Fujii, R. and Miyashita, Y. (1984) Release of ATP from adrenergic nerves controlling pigment aggregation in tilapia melanophores. Comp. Biochem. Physiol. 78C: in press. Fujii, R. and Miyashita, Y. (1978) Recording of chromatophore responses by means of an image analyzer system. J. Pre-med. Course Sapporo Med. Coll., 19: 129-140. Fujii, R. and Novales,R.R.(1968) Tetrodotoxin: 25 26 Zi) 28 N. OSHIMA AND R. Fusu Effects on fish and frog melanophores. Science, 160: 1123-1124. Iwata, K.S. and Fukuda, H. (1973) Central control of color changes in fish. In “‘Responses of Fish to Environmental Changes”. Ed. by W. Chavin, C.C. Thomas, Springfield, pp. 316— 341. Iga, T. (1975) Effects of sulfhydryl inhibitors on migration of pigment granules in the melanophore of Oryzias latipes. Memoirs Fac. Lit. & Sci. Shimane Univ., Nat. Sci., 8: 75-84. Fujii, R. and Miyashita, Y. (1982) Receptor mechanisms in fish chromatophores — V. MSH disperses melanosomes in both dermal and epidermal melanophores of a catfish (Parasilurus asotus). Comp. Biochem. Physiol., 71C: 1-6. Luby, K. J. and Porter, K. R. (1980) The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). I. Energy requirements. Cell, 21: 13-23. ZOOLOGICAL SCIENCE 1: 553-559 (1984) © 1984 Zoological Society of Japan Potassium Ions Act to Release Transmitter from “Cholinergic” Sympathetic Postganglionic Fiber to the Glass Catfish Melanophore HIROAKI KASUKAWA and Ryozo Fuji! Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan ABSTRACT — The mechanism by which K ions act to induce pigment aggregation within ‘‘cho- linergically”’ innervated glass catfish (Kryptopterus bicirrhi) melanophores was studied. cholinergic blockers interfered with the K* effect. Muscarinic An anticholinesterase, physostigmine, on the other hand, augmented the pigment-aggregating response caused by K*. Ca?* was required for the K* action, but not for melanosome aggregation per se, since acetylcholine or melatonin normally induced the action even in the absence of this cation. The conclusion was that K* does not act directly on the melanophore, but on the cholinergic nerve terminals to release the neurotransmitter, acetylcholine, which then causes pigment aggregation within the pigment cells. INTRODUCTION Since Spaeth [1] described the pigment-aggre- gating action of alkaline and alkaline-earth ions, including K*, on Fundulus melanophores, a number of researchers have pursued studies of the effects of electrolytes on vertebrate chromato- phores, especially on teleost melanophores [cf. 2-4]. In a later article, Spaeth [5] put forward his conviction that the melanophore might be a disguised type of smooth muscle cell, analo- gizing the characteristics of these cell types. Thereafter, the pigment-aggregating response was properly regarded to be akin to the K-contracture of the musculature. That is, K ions were thought to act directly on the melano- phore to aggregate its pigmentary organelles [6, 7]. Based on his observations on melanophores of the goby, Chasmichthys gulosus, on the other hand, Fujii [3] came to the conclusion that K* does not act directly on the effector cells, but does act on the peripheral nervous elements sur- rounding it to release adrenergic neurotransmitter, Accepted March 27, 1984 Received March 1, 1984 ‘ To whom reprints should be requested. which in turn gives rise to the cellular response. Having been confirmed by later studies [8-11], this conclusion has now become widely accepted. The pigment-aggregating fibers are known to be sympathetic postganglionic [2, 12-14]. Furthermore, recent pharmacological investiga- tions have indicated that the neurotransmitter is adrenergic [10, 15, 16], and that the adrenergic receptors concerned are of alpha nature [17]. Recently, however, it was disclosed that the pigment aggregation in melanophores of siluroid catfishes is peculiarly controlled by a cholinergic peripheral mechanism, although the fibers to the cells are sympathetic postganglionic as is the usual case [18, 19]. The postsynaptic receptors on the melanophore membrane are characterized as cholinoceptors of a muscarinic type. In the present study, therefore, we attempted to examine whether K* induces pigment aggregation in such a rare cholinergically controlled melanophore, and further to clarify the mode of action of the ion, if it did show the effect. A_ preliminary account of this work has already appeared [20]. MATERIAL AND METHODS The translucent glass catfish, Kryptopterus 554 H. KASUKAWA AND R. FudJII bicirrhi (Cuvier and Valenciennes), was used as the experimental material. The place of origin of this silurid is India. In the present study, young adult forms having body lengths between 35 and 45 mm were obtained from a commercial source. The method for preparing skin specimens was essentially the same as that described in a previous paper [19]. These skin pieces were prepared in a physiological solution of the fol- lowing composition (mM): NaCl 125.3, KCl 2.7, CaCl, 1.8, MgCl, 1.8, glucose 5.6, Tris-HCl buffer 5.0 (pH 7.2). Experiments were performed within 4 hours after the sacrifice of an animal. The photoelectric recording method for melano- phore responses was fundamentally the same as that described elsewhere [21]. All the measure- ments were done on a single melanophore by restricting the area of skin through which light was transmitted to 130 “wm in diameter. K*-rich salines, containing 10, 20, 50, 100 and 128 mM K* were used for chemical stimulation, although 50 mM K-saline was employed for the most part. In preparing them, an equimolar of KCl was substituted for NaCl in the primary saline. -In some _ experiments, Ca’*- and/or Mg’*-free salines to which 10mM EDTA (ethylenediamine tetraacetic acid, 3Na-salt; Wako Pure Chemical Ind., Osaka) was added were used. The drugs used were acetylcholine chloride (Daiichi Seiyaku, Tokyo), atropine sulfate (Tanabe Seiyaku, Tokyo), scopolamine hydrobromide (Yamanouchi Pharmaceutical, Tokyo), physo- stigmine sulfate (Tokyo Chemical Ind., Tokyo), 100 : 2 7 50 oO Ne ey ro.) < 0 K* 50 mM 50 mM rn (__/) Atr eed Mel Fia. 1. and melatonin (Nakarai Chemicals, Kyoto). These drugs were dissolved in normal saline or in one of the experimental solutions immediately before use. All the experiments were carried out at a room temperature between 22 and 25°C. RESULTS Effect of K* In the present study on the glass catfish, Kryptopterus bicirrhi, K* was found to be very effective in inducing pigment aggregation, as shown on the left in Figure 1. The pigment- aggregating action of K* depended on the concentration of the ion in the perfusing medium (Fig. 2). In normal physiological saline, in which [K*], was 2.7mM, melanosomes in the cells clearly remained in a dispersed state. When the concentration of K* was increased to 10 mM, a discernible pigment aggregation was observable. With a further increase in [K*], the magnitude of the response increased, and at 50mM, the maximal level was attained. Upon a further increase in [K*]., the magnitude of the response seemed to fall to some degree, although there was no Significant difference between the level induced by 50mM K* and that induced by 100 or 128 mM of the ion. Effect of Cholinolytic Agents on K* Action It has already been reported that in common 2min (L__; 50 mM (LJ | 10 pM ———— SSS) Typical recording showing the melanin-aggregating action of 50 mM K* and the effect of 1 uM atropine (Atr) on the K* action on a melanophore of the glass catfish, Kryptopterus bicirrhi. 10 uM melatonin (Mel) was used to induce the maximal pigment-aggregating response. K Releases ACh from Melanophore Nerve 555 100 | ~2 / ge / Cc ° 7% 50 / oO © = oO | oO < 0 2.7 10 20 50 100128 Concentration of K*, mM Fic. 2. Relationship between the concentration of K* and the magnitude of the aggregation response. Perfusion for 5 min with a saline containing various concentrations of K* rang- ing from 2.7 mM to 128mM was immediately followed by another 5 min application of 50 ~M acetylcholine to induce the maximal level of pigment aggregation. Abscissa, concentration of K* in mM (logarithmic scale). Ordinate, magnitude of the response as a percentage of the maximal level of the response. Each point is the mean of 7 measurements on different animals. Vertical lines indicate standard errors. species of teleosts the pigment-aggregating action of K* was inhibited by alpha adrenergic blockers [10]. In the present material, on the other hand, Fujii et al. [19] have lately shown that ad- renoceptors are not involved in the chemical transmission, and that cholinoceptors of muscarinic type are instead responsible for the pigment 100 Aggregation, %/o ul ro) Fic. 3. aggregation. Hence, the muscarinic blockers, atropine and scopolamine, were tested for their influence on the action of K*. As shown in Figure 1, atropine completely inhibited the K* action. Scopolamine had the same effect. These observations indicate that K* may not act on the effector cell directly, but that it does act on it through the liberation of a cholinergic trans- mitter, acetylcholine, eliciting the cellular response, which is mediated by the muscarinic receptors. Effect of Anticholinesterase An_anticholinesterase, physostigmine, which blocks the enzymatic destruction of acetylcholine, was used. A moderate melanin-aggregating response induced by K* was augmented under coexistence with this drug (Fig. 3). These results also suggest that liberation of acetylcholine at the nerve-melanophore junction is involved in the K* action. Such an augmentation of the response to electrical nervous stimulation or to exogenously applied acetylcholine has already been shown in the present material [19], and also in another silurid, Parasilurus asotus [18]. Ca** Dependence of K* Action In the present material, the pigment-aggre- gating reaction induced by K* was completely inhibited in Ca’*- and Mg’*-free, EDTA- containing saline (Fig. 4). Investigations were then made regarding whether Ca** or Mg’ could remove this inhibition. In the typical recordings shown in Figures 4 and 5, the skin Imin a | 10 uM Typical recording showing the augmentative effect of 10 »~M physostigmine (Phy) on the pigment-aggregating reaction to 20 mM K*. The maximal level of pigment aggregation was obtained by applying 10 4M acetylcholine after the illustrated part of the record. 556 H. KASUKAWA AND R. FUJII , 100 ss 4S. om 20 © 4min re) ro.) lO er ee 1.8 mM ; Mg?2* 1.8mM EDTA 1 mM Mel OT Fic. 4. Typical recording showing the effect of S50 mM K* in Ca?*- and Mg?*-free saline, to which 1.0mM EDTA was added. The action of melatonin (Mel) in the Ca?*- and Mg?t- free medium was finally examined. (e) (oe) Aggregation °/o Ul oO 2 min (Ea =) +O 50mM 50mM 50 mM 50mM | 50mM Ca’* \ 1.8mM 1.8mM Mg** 1.8mM 1.8mM EDTA 1 mM LM, mM ACh 50 uM Fic. 5. Typical recording showing the effect of Ca?*- and/or Mg?t-deficiency on the melanin- aggregating action of 50 mM K*. free medium was finally examined. preparations were first treated with Ca’*, Mg’*- free saline. When 1.8mM Meg’* was added to this saline, pigment aggregation did not take place (Fig. 5). When 1.8mM Ca’* was added, on the other hand, an apparent pigment- aggregating response was restored (Fig. 5). In contrast to K”, either melatonin or acetylcholine induced a remarkable pigment aggregation even in divalent cation-free saline (Figs. 4 and 5). DISCUSSION It is now widely accepted that an elevation of K* concentration in extracellular fluid, [K*]o, induces release of hormones from _ various endocrine cells. Among these, we may enumerate here a few instances of hypophyseal hormones: vasopressin [22], luteinizing hormone _ [23], thyrotropin and adrenocorticotropic hormone [24]. High [K*], also. stimulates hormone release from other endocrine organs, e.g., catechol- The action of acetylcholine (ACh) in the Ca?*- and Mg’t- amines from adrenal medullary cells [25] or insulin from pancreatic beta cells [26]. Exocrine glands are also stimulated by K’, as in, for instance, high [K*], augmented amylase secretion from rat parotid gland slices [27] and from isolated perfused pancreas of the cat [28]. According to the mode of action, furthermore, Argent et al. [28] classified K* effects into two categories: one in which K* acts directly on the secretory cells, and the other in which it acts indirectly through influence on the nervous components regulating these cells. Since K* affects both the effector cells and the controlling nervous elements, physiological analyses of the mode of action of K* in neuro- effector systems are rather difficult. Thus, to date, comparatively few reports have appeared in which K* has been shown to induce neuro- transmitter release from nerve terminals. It has, however, been observed to induce the release of the neurotransmitter, acetylcholine, at giant K Releases ACh from Melanophore Nerve 557 synapses of squid [29]. In the autonomic nervous system, neurotransmitter release has also been induced by K* from peripheral neural elements [30]. As mentioned early in this report, however, Fujii [3] and Iwata er a/. [8] presented clear results regarding K* action on the fish melanophore system more than a quarter of a century ago. Their conclusion was that K* acts on the chro- matic nerve terminals to release the neuro- transmitter, which then gives rise to the effector response. This hypothesis was later confirmed by detecting an inhibitory effect of alpha adrenolytic drugs on the K* action [10]. Very recently, Kumazawa and Fujii [31], applying radio-labeled norepinephrine to tilapia melano- phores, showed that the neurotransmitter is actually released from chromatic nerve terminals by the effect of K”. Strangely enough, it was recently found that sympathetic peripheral transmission to_ the melanophore in the glass catfish is cholinergic, and that the cholinoceptor involved is of a muscarinic type [19]. In the present study on the same material, we have recently shown that K* gave rise to a pigment aggregation in the melanophore, and that muscarinic cholinolytic agents blocked the response. It was further shown that an anticholinesterase augmented the K* effect. The conclusion that may be derived here is that K* acts on peripheral nervous elements to release the neurotransmitter, acetylcholine, which in turn causes pigment aggregation within the effector cells. In the end, the mode of action of K* was found to be quite common in both adrenergically and cholinergically innervated chromatophores. Many authors have reported that Ca’ is required in the process of stimulus-secretion coupling in neurosecretory cells [e.g., 24, 32, 33]. Such Ca’** dependence has also been shown in the neurotransmitter liberation at nerve-terminals in response to nervous stimuli [34, 35]. As to the nervous system controlling fish chromato- phores, Fujii and Fujii [36] first indicated that Ca** is required for catecholamine release from the sympathetic nerve terminals in the goby, Chasmichthys gulosus. In the present study on cholinergically controlled melanophores, it also became clear that the transmitter liberation by K* is dependent on [Ca**],. That is, the release of the neurotransmitter, either adrenergic or cholinergic, from the chromatic nerve terminals may be induced by the influx of Ca’* into them. Extracellular Ca** might flow into the cytoplasm through Ca** channels existing in the nerve terminal membrane. It is thought that Ca?* inflowed combines with a Ca** acceptor, pre- sumably calmodulin [37]. Later sequences leading to the exocytotic release of the neurotransmitter, however, still remain a matter of speculation. It has become widely accepted that the effect of Ca** on transmitter liberation is antagonized by excess Mg’* [32, 33]. Katz [38] explained the sequence of this inhibition as a competitive binding of Mg’* to the Ca’*-acting site on the membrane of nerve terminals. Working on the sympathetic nervous system controlling melano- phores of the goby, on the other hand, Fujii and Fujii [36] indicated that an excess concentration of Mg** (20 mM) can partly substitute for Ca’*. This result suggests that Mg?* may permeate the the nerve membrane through the Ca channels to some extent, substituting for Ca’* in the cytoplasm. In the present work, Mg’* did not substitute for Ca’*. Being the same as that in normal saline, i.e., 1.8 mM, the Mg** concentration might have been too low, and thus insufficient to take the place of Ca’**. Notwithstanding these observa- tions, we may conclude here that only Ca’** was effective in the depolarization-transmitter libera- tion coupling. Working on the goby, Chasmichthys gulosus, incidentally, Fujii and Taguchi [39] have already reported that some pigment-aggregating agents, e.g., norepinephrine, and _ dispersing ones, dibenamine and atropine, elicited the responses of melanophores quite normally in K’*-rich, Ca’*- and Mg?*-deficient saline. Similar results were Obtained on the cholinergically controlled glass catfish melanophores here, in which acetyl- choline or melatonin induced pigment aggregation without alkaline-earths in the medium. These observations suggest that the responsiveness or, more substantially, the receptors mediating motile responses of the chromatophores are rather independent of the ionic environment. By making 558 use of such characteristics, the adrenergic nerve- chromatophore system may be conveniently employed for physiological as well as phar- macological analyses of autonomic peripheral mechanisms. Thus, in some forms of study at least, merve-chromatophore preparations may replace the nerve-smooth muscle preparations from various animal sources very commonly used thus far. Working on the marine goby, Tridentiger trigonocephalus, Katayama [40] showed that melanophores on a split-fin preparation treated with norepinephrine beforehand responded to K* better than those on an untreated piece, sug- gesting neuronal uptake of norepinephrine. Intending to see how peripheral nerve-fibers make contact with a melanophore of the medaka, Oryzias latipes, Yamada et al. [41] lately observed on autoradiograms that norepinephrine is in- corporated into the neural elements surrounding the cell. Using ‘*C-labeled norepinephrine, the release in response to K* of adrenergic transmit- ter from sympathetic melanin-aggregating fibers has recently been substantiated in tilapia, in which melanophores are orthodoxly controlled by the adrenergic peripheral mechanism [31]. Also in the present material, where the pigment cells have been shown to be under the control of choliner- gics, ‘actual release of cholinergic transmitters has been lately ascertained by employing ‘*C- choline (Kumazawa and Fujii, to be published). Notwithstanding the earlier (cf. also In- troduction) and current results mentioned above regarding the peripheral mechanisms in the chromatic nervous system, the sequence of trans- mitter liberation by K* and also by nervous excitation remains largely unsolved. As is the case in other neuro-effector junctions, the initial part of the sequence may include first the de- polarization of presynaptic membrane, and then Ca’*-influx. However, later steps leading to transmitter liberation into the synaptic cleft remain totally unknown. Further studies are certainly needed to elucidate these problems. REFERENCES 1 Spaeth,R. A. (1913) The physiology of the 10 i 12 13 15 16 H. KASUKAWA AND R., Fusm chromatophores of fishes. J. Exp. 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(1982) Muscarinic cholinoceptors mediate neurally evoked pigment aggregation in glass catfish melanophores. J. Neural Transmission, 54: 29-39. Kasukawa, H., Kumazawa, T. and Fujii, R. (1983) Release by K* of the transmitter from cholinergic sympathetic postganglionic fiber to the glass catfish melanophore. Proc. 5th Ann. Meeting Jap. Soc. Gen. Comp. Physiol., 90. Oshima, N. and Fujii, R. (1984) A _ precision photoelectric method for recording chroma- tophore responses in vitro. Zool. Sci., 1: 545-552. Douglas, W.W. and Poisner, A.M. (1964) Stimuli-secretion coupling in a neurosecretory organ: The role of calcium in the release of vasopressin from the neurohypophysis. J. Physiol., 172: 1-18. Samli, M. H. and Geschwind, I. I. (1968) Some effects of energy-transfer inhibitors and of Ca**- free or K*-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology, 82: 225-231. Vale, W. and Guillemin, R. (1967) Potassium- induced stimulation of thyrotropin release in vitro. Requirement for presence of calcium and inhibition by thyroxine. Experientia, 23: 855-857. Douglas, W.W. and Rubin, R.P. (1961) The role of calcium in the secretory response of the adrenal medulla to acetylcholine. J. Physiol., 159: 40-57. Grodsky, G. M. and Bennet, L. L. (1966) Cation requirements for insulin secretion in the isolated perfused pancreas. Diabetes, 15: 910-913. Bdolah, A., Ben-zvi, R. and Schramm, M. (1964) The mechanism of enzyme secretion by the cell. II Secretion of amylase and other proteins by slices of rat parotid gland. Archs Biochem. Biophys., 104: 58-66. Argent, B.E., Case,R.M. and Scatcherd, T. (1971) Stimulation of amylase secretion from 29 30 31 32 33 34 35 36 37 38 39 41 559 the perfused cat pancreas by potassium and other metal ions. J. Physiol., 216: 611-624. Erulkar, S.D. and Weight, F. F. (1977) Extra- cellular potassium and transmitter release at the giant synapse of squid. J. 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(1965) Innervation of fish melanophores II. Transmission at melanin- aggregating nerve-terminals and divalent ions. Zool. Mag., 74: 351. DeLorenzo, R. J. (1982) Calmodulin in neuro- transmitter release and synaptic function. Fed. Proc., 41: 2265-2272. Katz, B. (1962) The transmission of impulses from nerve to muscle, and the subcellular unit of synaptic action. Proc. Roy. Soc., B 115: 455- 477. Fujii, R. and Taguchi, S. (1969) The responses of fish melanophores to some melanin-aggregating and dispersing agents in potassium-rich medium. Annotationes Zool. Japon., 42: 176-182. Katayama, H. (1978) Effects of catecholamines and inhibitors of catecholamine uptake on the melanophore-aggregating system of the marine goby, Tridentiger trigonocephalus. Zool. Mag., 87: 228-239. Yamada, K., Miyata, S. and Katayama, H. (1984) Autoradiographic demonstration of adrenergic innervation to scale melanophores of a teleost fiish, Oryzias latipes. J. Exp. Zool., 229: 73-80. EN ar Le ' : ’ oe “ Seine str. eo oe it t ‘plaisY!: Has De . wine? 2 be iy itt Ry Sk la | ‘ ; Ln Mi) CUP SLGe 2d é ha 7 ; » its f x sh 4 ve AG 1 at oy , a 7? i - 1 ' Bpéhaets m4 - “i ; : y \ Pee Paty BR ‘ ‘ _, wr i : Y y F AY gu &F y ar oS LIES te: ‘ie F J E sie ' ara tere LST SHR ogee as are P wr PES seat fe b awh 3 ; , oment - eiteiaee ¥ é 327 : eee er ee : wer ts ! 1% ¢ f Pica ju auc , . i ) 1 any * ' 1 i . - . c + 9 es es 7 4 5 ¥ i i, i ae Di a i: a = a ge is 7 t : aI Pe ee. «RS. Ge Te | i a yd ig if 1 MO { t : X\ | =) I } } is Y ' sl . j y f ‘ : } e + A ~ I 2. a 2 ‘ i mK: Y } : eT) EEL RR ED al & s he es i A i r = Ce eG Ah, ai CaN ‘ Z ; me Wii oy é : wees 1? whites | } CR Re PE chet mi i i ; Kone, 2 F ‘ bs oe in r or dye hes | i). 12.3" 4% awa i tf aN TD 4 ‘ fhet iia wi ne ws : | rhe : of i ; i as r tear ‘ is x - i) i ~ — ? 2 le . = = P ty ZOOLOGICAL SCIENCE 1: 561-570 (1984) © 1984 Zoological Society of Japan Choanocyte Differentiation and Morphogenesis of Choanocyte Chambers in the Fresh-Water Sponge, Ephydatia fluviatilis, after Reversal of Developmental Arrest Caused by Hydroxyurea KEIKO TANAKA!:2 and YOKO WATANABB!:3)4 ‘Department of Biology, Ochanomizu University, Otsuka, Tokyo 112, and *Tateyama Marine Laboratory, Ochanomizu University, Tateyama, Chiba 294-03, Japan ABSTRACT — The gemmules of Ephydatia fluviatilis were cultured in the hydroxyurea (HU)-con- taining medium, and its effects were examined. In 1.5 mM HU, among all types of cells constituting the sponge body only the differentiation of choanocytes was inhibited. The sponges developed without an aquiferous system because of the lack of choanocyte chambers. to the stage before choanocyte chamber formation in normal development. The situation corresponds After removal of HU, the sponges were released from the arrest and regained their ability of development: archaeocytes, which are presumptive choanocytes, began cell division and differentiated into choanocytes. The starting point of choanocyte differentiation was set at the time of HU removal. The process of the differentiation of choanocytes and the formation of choanocyte chambers was investigated in vivo in sections. By estimating the number of choanocytes in a choanocyte chamber, it was found that one archaeocyte differentiated into choanocytes after four cell divisions. were derived from one archaeocyte by dividing 4 or 5 times. About 25 choanocytes Large choanocyte chambers appeared to be formed by the fusion of mature choanocyte chambers. INTRODUCTION The choanocyte chamber, which consists of many small cells called choanocytes, is a special structure in the Porifera. The choanocyte is a cell type characterized by a flagellum and a microvillous collar that encircles the flagellum. In the normal development of sponges, the choanocytes appear in the last stage of cell differentiation. Brien [1] and Wintermann [2] showed in the fresh-water sponges developed from gemmules that a choanocyte chamber originates from a single archaeocyte by repeated division. It is now a popular view that one choanocyte chamber derives from a single archaeocyte. Borojevic and Lévi [3] Accepted February 10, 1984 Received September 7, 1983 * Present address; Shimoda Kita High School, 152 Rendaiji, Shimoda, Shizuoka 415, Japan. * To whom reprints should be requested. and Watanabe [4] reported that in the development of sexual reproduction in marine sponges, groups of small cells originating from a certain cell type transform into choanocytes and give rise to choanocyte chambers. However, they did not mention whether or not these cells derived from one archaeocyte. Although some studies have been made on the origin of choanocytes, no decisive answer is available regarding the time of deter- mination and the process of differentiation into choanocytes. This is due to the following difficulties. 1) There is no way to distinguish the archaeocytes which differentiate into choanocytes from those differentiating into other types of cells. 2) After repeated cell divisions, daughter cells are very small, and they aggregate in clusters, so that it is hard to follow the fate of individual cells. 3) The collar microvilli and flagellum of choanocytes are so fine that it is difficult to see them in vivo, but is able to observe only in sections. 4) Even in sections, since choanocyte chambers are formed 562 successively and asynchronously, various develop- mental stages of choanocyte chambers are met with simultaneously, but as has been said, ob- servations in wvo are extremely difficult and it is practically impossible to arrange observed stages in developmental order. It is reported that hydroxyurea (HU), which is an inhibitor of DNA _ synthesis, inhibits the differentiation of choanocytes in _ fresh-water sponges hatched from gemmules. Rasmont [5] and Rozenfeld and Rasmont [6] reported that in 1mM HU, more than 90% of the gemmules hatched, but they developed aberrantly. The resulting organism was a ‘hollow dome’ of pinacoderm, stretched on spicules, the bottom of the dome being strewn with embryonic ar- chaeocytes. The aquiferous system was com- pletely absent. However, this condition can be reversed toward normal development by removal of HU; an outburst of mitoses takes place and numerous choanocyte chambers are produced. Later Garrone and Rozenfeld [7] showed by electron microscopy that all cell types except choanocytes had developed in the hollow dome sponges treated with 100 ug/ml (1.31 mM) HU. We confirmed these facts by similar experiments. The same solution of HU did not inhibit the differentiation of the other sponge cell types, but inhibited only that of choanocytes. However, the internal structure of our sponges was not hollow, but was a network of endopinacocytes, with an appearance corresponding to the stage before the choanocyte differentiation in normal development. After rinsing away the HU, the arrested development started again; archaeocytes, presumptive choanocytes, differentiated into choanocytes through several mitoses, and finally organized themselves into choanocyte chambers. We consider that after removal of the HU, the sponge reinitiates its development and succeeds in choanocyte chamber formation synchronously throughout the whole body. With this in mind, we set the starting point of choanocyte development at the time of removal of HU. We attempted then to discover the origin of the choanocytes, the time of determination, the process of their dif- ferentiation, and the morphogenesis of the choano- cyte chamber as a whole, by fixing the materials K. TANAKA AND Y. WATANABE at regular intervals. MATERIALS AND METHODS Living materials The gemmule-bearing sponges Ephydatia fluviatilis were collected from the Yokotone-gawa River in Ibaragi Prefecture. The gemmules, attached to the parent sponge tissue, were stored in the dark at 4°C until the beginning of the ex- periments. Cultivation Gemmules were isolated from sponge tissue and cleaned with 1% hydrogen peroxide, and washed _ 10 times with M-medium [8]. Batches of 30 gemmules were incubated at 25°C in UV-sterilized polystylene petri dishes. HU was added to the M-medium at concentrations of 1mM, 1.5mM and 2mM. For reversal experiments, the gem- mules were cultured for 7 days in the 1.5mM HU medium, i.e. at the minimum concentration of HU that inhibits choanocyte differentiation. The treated sponges were washed with M-medium and incubated in the fresh M-medium. The cultivation medium was changed every 12 hr. Histological procedure Gemmules were incubated on 5% agar plates covering the bottom of petri dishes. The sponges were fixed in toto in 1% osmium tetroxide buffered to pH 7.2 with 0.025 M sodium cacodylate for 2 hr. The samples, washed in fresh buffer, were desilicated in 0.5% hydrofluoric acid for 10 min. They were dehydrated in graded ethanol solutions and embedded either in JB-4 resin for semithin sections and stained with toluidine blue for light microscopy or in Spurr’s resin for electron microscopy. The ultrathin sections were stained with uranyl acetate and lead citrate. All ob- servations were made with a Hitachi HS-7D electron microscope. Calculation of cell number in a choanocyte chamber We estimated the total number of cells making up a choanoblastic cluster or a choanocyte chamber from the cell numbers in the largest cross section Hydroxyurea and Choanocyte Differentiation Fic. 1. Diagrammatic schema of a _ choanocyte chamber. A. Primary choanocyte chamber before opening apopyle. B. Functional choanocyte chamber’ with apopyle. R: radius of a choanocyte chamber. r: radius of a choanocyte. ap: apopyle. 16 hr after release from HU. It is supposed that a choanocyte chamber is a sphere with radius R and that its surface is completely covered with choanocytes, with radius r (Fig. 1). The circum- ference of a choanocyte chamber in the largest cross section is 2zR. It can be expressed with the number of choanocytes (n) in the cross section. 2zR=2rn. The total cell number of choanocyte chamber, N, is expressed as 4*R’?=zr’N. Then we obtain N= el n’ from above formulae. ae On the other hand, the area of an apopyle amounts to one quarter of the surface area of the choanocyte chamber [9]. The above formula for N applies to the cell clusters before 35.5 hr, when apopyle was first observed after removal from HU. The cell number at and after 35.5hr, N’, 563 is obtained by multiplying three quarters, namely We ns, T RESULTS I. Effects of HU on the development of sponges from gemmules The gemmules were incubated in M-medium containing 1 mM, 1.5 mM, or 2mM HU. One hundred percent of the control gemmules hatched within 4 days of incubation. First, histoblasts, which migrated from the gemmule through its opened micropyle, immediately dif- ferentiated into exopinacocytes to form the pinacodermal envelope. Next, archaeocytes, which moved into the space surrounded by external epithelium, differentiated into sclerocytes, collencytes, endopinacocytes which constituted the wall of the lacunae, and later into choanocytes. The lacunae became canals by connecting with each other, and choanocyte chambers were formed along the side of the canals (Fig. 2A). The aquiferous system became functional 3 days after hatching when the osculum opened at the top of the oscular tube. HU had various effects on the normal develop- ment from gemmules. At the concentrations of 1 mM and 1.5mM HU, gemmules hatched 2 days later than the controls, but the hatching percentage was not affected: 98% in 1mM HU and 99% in 1.5mM HU. One mM HU-treated sponges developed normally and organized a complete aquiferous system including choanocyte chambers (Fig. 2B). In 1.5mM HU, the sponges looked like the ‘hollow dome’ of Rozenfeld and Rasmont [6]. In the histological sections, however, there were networks of lacunae composed of endo- pinacocytes inside the sponge. Archaeocytes were arranged along the walls of the lacunae. The choanocyte chambers were completely lacking (Fig. 2C). Eighty four percent of the gemmules in 2mM HU hatched with a lag of 4 days in comparison to the control gemmules. In histologi- cal observations all the cell types except choano- cytes developed in the sponges. The archaeocytes were filled with vitelline platelets and were less 564 K. TANAKA AND Y. WATANABE = after hatching. A. Control: choanocyte chambers are formed. Fic. 2. Histological sections of Ephydatia fluviatilis incubated in various concentrations of HU, on day 5 B. 1mM HU-treated sponge: a choanocyte chamber is seen in the mesohyle. C. 1.5mM HU-treated sponge: lacunae surrounded by archaeocytes which contain digested vitelline platelets. Choanocyte chambers are not formed. D. 2mM HU-treated sponge: archaeocytes rich in vitelline platelets surrounding lacuna. Scale bar shows 10 ym. Ch: choanocyte chamber. differentiated than those in 1.5 mM HU (Fig. 2D). In 2mM HU-treated sponges, the aquiferous system was completely lacking. This concen- tration of HU appeared to affect the cell. differ- entiation and the morphogenesis much more than 1.5 mM. 2. Differentiation of choanocytes and morpho- genesis of the choanocyte chamber after removal of HU HU-treated sponges resumed their differentiation and morphogenesis after removal of HU from the medium. In this experiment, we used the minimum concentration of HU that inhibited the choanocyte differentiation, 1.5mM. The gemmules cultivated in 1.5mM HU developed until the pre-stage of Ar: archaeocyte. lac: lacuna. choanocyte differentiation at the 7th day of incubation. After washing out the HU, these sponges were incubated in fresh M-medium, and then their development started again and choano- cyte chambers were formed. Just after removal of HU, cells in the sponge began to move more actively. In several hours, the archaeocytes began to divide. After the cell divisions, some archaeocytes, which became choanocytes, stayed at the periphery of the lacunae, and the others, which differentiated into other cell types, moved apart from each other. In early stages before 13.5 hr, cell clusters were not spherical but irregular, so that it was difficult to estimate the number of cells from the sections. Therefore, for the first 13.5 hr the cell number was counted in Hydroxyurea and Choanocyte Differentiation 565 oe OSes ak ut. Th ale ea Fic. 3. The process of choanocyte chamber formation after removal from 1.5 mM HU. Choanoblastic cluster at 9 hr. Primary choanocyte chamber at 16 hr. Choanocyte chamber at 33 hr. Functional choanocyte chamber at 38 hr. Arrow indicates an apopyle. Scale bar shows 104m. Cb: choanoblastic cluster. Ch: choanocyte chamber. Ar: archaeocyte. can: canal. OO > TABLE |. The cell number of a choanoblastic cell cluster or choanocyte chamber originating from an archaeocyte in relation to the time after removal of HU ‘ No. of choanocyte Average cell Estimated cell Paepen te chambers/No. of number in cross number constituting examined cell clusters section (n) a cluster (N or N’) fe 2 2 or 4* 9.0 2 4* 13.5 3-5 8* 16.0. 2/21 5.0+0.76 11 (N) 21.5 2/20 5.2+0.67 11 (N) 24.0 18/20 5.1+0.59 11 (N) 28.5 22/22 5.9+0.67 14 (N) 33.0 20/20 7.3+0.90 22 (N) 35.5 22/22 9.1+1.4 25 (N’) 38.5 22/22 11.7+1.7 42 (N’)** 38.5 22/22 15.8+1.2 76 (N’)** * The calculation formula cannot be applied to choanoblastic clusters before 13.5 hr. These cell numbers were actually observed in living sponges. ** Choanocyte chambers at 38.5 hr are thought to arise by fusion of 2 or 3 choanocyte chambers. 566 K. TANAKA AND Y. WATANABE the living sponges instead of in the sections. In living sponges, the first mitosis could be seen at 1.5 hr after removal of HU. Four hours after removal, most presumptive choanocytes had gone through the first mitosis. The second mitosis took place about 8 hr after the removal. Between 10 and 13 hr, the third mitosis occurred with a time lag among the cells in a cluster. In the his- tological sections, between 7.5 to 13.5 hr, clusters consisted of 2 to 5 cells which were in close contact (Fig. 3A). Jn vivo observations, at 13.5 hr, the cells in a cluster were more than 8. Therefore, an archaeocyte passed through at least 3 mitoses in the first 13.5 hr after removal of HU. After 16 hr cell size became smaller, and number of cells con- structing cell cluster could not be counted in the living sponges. On the other hand, as cell clusters became spherical at this time, cell number was presumed from the cross sections by the calculation mentioned in method. Therefore, after 16 hr, cell number was estimated by the calculation of the cell number in the largest cross sections of the cell clusters. The result is shown in Table 1. The first appearance of choanocyte chamber with a narrow central space was at 16hr after removal under the light microscope. At this stage, most of the choanocyte chambers consisted of 5 cells in the largest cross sections, 5.0+0.76 on the average. Total number was presumed to be 11. Each choanocyte was still larger than that of adult sponges (Fig. 3B). At 21.5hr and 24hr, there were 5 or 6 cells in the cross sections, 5.2+0.67 and 5.1+0.59 on the average, estimated total number was 11, which showed no remarkable increase. Among 20 counted clusters, two were choanocyte chambers at 21.5hr. At 24hr, 18 out of 20 cell clusters were choanocyte chambers. During this time cell numbers increased very little, but choanoblasts differentiated into choanocytes. After 28.5 hr, cell clusters had 5.9+0.67 cells on the average in cross section, estimatéd 14 cells in a whole cluster. At this time all the cell clusters had become choanocyte chambers. At 33hr, the choanocyte chambers possessed 7.3 +0.90 choano- cytes on the average in section, which means 22 choanocytes in a choanocyte chamber. The choanocyte chambers were formed at the periphery of lacunae, the latter of which became canals by joining each other (Fig. 3C). As the aquiferous system was formed, choanocyte chambers lay in the narrow mesohyle between the incurrent and excurrent canals. At 35.5 hr, the central space of the choanocyte chamber became larger because of the apopyle opening into excurrent canal. As the surface of apopyle occupied one third of that of choanocyte chamber, after this stage the calculation of cell number of the choanocyte chamber was ob- tained N’ by multiplying 3/4 as before mentioned. By this calculation, at 35.5 hr, each choanocyte chamber was estimated to be constituted by 25 choanocytes. At 38.5 hr, while most choanocyte chambers had 11.7+1.7 in section on the average, another group of choanocyte chambers composed of 15.8+1.2 choanocytes was found in the largest cross sections. It can be estimated that the former possess a total 42 choanocytes and that the latter contain 76 choanocytes. At 38.5 hr, 2 or 3 choanocyte chambers may have begun to fuse. We and others [2, 10] in fact observed the fusion of choanocyte chambers in living sponges. Electron microscopical studies were done on the choanocyte chamber formation. Figure 4A shows the choanoblastic clusters in close contact with each other, lacking the central space at 11.5 hr. The first appearance of choanocytes was 14.5 hr, although the number was small. At 17.5 hr, Fic. 4. Electron micrographs of choanocyte differentiation and choanocyte chamber formation after removal from 1.5 mM HU treatment. A. Choanoblastic cluster at 11.5 hr. B. Choanocyte (arrow) and choanoblasts in a cell cluster at 17.5 hr. C. Choanocyte in differentiation. can be seen. Choanocyte chamber at 33 hr. Functional choanocyte chamber at 48 hr. Scale bars indicate 24m. ap: apopyle. amo Well-developed Golgi apparatus and basal body of flagellum (arrow) Cell division in a well-developed choanocyte; dispersed chromosomes appear in the choanocyte. Hydroxyurea and Choanocyte Differentiation 567 ue, CG ae ~ 3 Ae: wus ya te - oe gar ~ - _ 568 K. TANAKA AND Y. WATANABE both choanoblasts and choanocytes were observed in a cell clusters (Fig. 4B). The process of mor- phogenesis from choanoblast to choanocyte could be observed by electron microscope; microvillous collars, well developed Golgi apparatus, and the basal body of a flagellum could be seen (Fig. 4C). At 33 hr, we observed the mitosis of a choanocyte with collar and flagellum (Fig. 4D). Cell division of choanocytes does not occur synchronously in a choanocyte chamber judging from variation of cell sizes (Fig. 4E). From these observations, an archaeocyte divides three times and forms a choanoblastic cluster by 13.5 hr after removal of HU. Afterward, it divides once more by 28.5 hr. The cell dividsion does not seem to be strictly synchronous, and the ceils which have passed through the 4th mitosis differentiate into choanocytes. Primary choanocytes undergo one more mitosis to form a functional choanocyte chamber with the apopyle connecting with the canal. DISCUSSION In the asexual reproduction of fresh-water sponges, the development from gemmules begins with a few mitoses of thesocytes in the shell. The resulting cells become either histoblasts or ar- chaeocytes [11, 12]. The former changes into exopinacocytes composing the external epithelium. Archaeocytes differentiate into choanocytes and other cell types. Buscema et al. [13] showed that an aggregate of pure archaeocytes, which had been dissociated from the adult sponge and cultured, reconstituted a functional sponge; all cell types including choanocytes arose from the archaeocytes. In the sponges treated with HU at the concen- tration of 1.5mM and 2mM, all the cell types except choanocytes were present. The appearance was superficially similar to the hollow dome de- scribed by Rozenfeld and Rasmont [6] owing to the undeveloped aquiferous system. However, from our histological preparations networks of lacunae made by endopinacocytes were stretched around inside the sponge. Canals were not formed because the lacunae were not joined. The ar- chaeocytes were arranged in the narrow space of the mesohyle among the lacunae. We consider that this appearance corresponds to that of sponge just before choanocyte differentiation in normal development. After removing HU from the medium, the archaeocytes accumulated in the mesohyle differ- entiate into choanocytes and form choanocyte chambers [6]. Their observations are in agreement with our results to this extent. We obtained the following results by examining this process. Some archaeocytes underwent repeated mitoses without growth and the resulting cell clusters formed choanocyte chambers. Consequently we think that the origin of choanocytes is the archaeocyte which had their division blocked by the HU. Presumptive choanocytes which were attached to the periphery of the lacunae differentiated into choanocytes simultaneously with the canal for- mation. Thus it is possible that the endopina- cocytes play some part in the differentiation of choanocytes. Rozenfeld [14] reported the incorporation of °H-thymidine into DNA. In the normal develop- ment of sponges from gemmules, three peaks of incorporation appear in the early stage. The first two peaks appear before the gemmule hatching. The third peak corresponds to the DNA synthesis proceeding cell division in archaeocyte and choanocyte differentiation [14]. It has been shown that 100 ug/ml (1.31 mM) of HU delays the two first peaks of incorporation and abolishes the third peak. The abolished peak must be responsible for DNA synthesis leading to the differentiation of choanocytes [6]. Rasmont and Rozenfeld made a microcinemato- graphical study on the formation of choanocyte chambers in E. fluviatilis whose gemmules were treated with 50 ug/ml HU [9]. They started their observation at 24hr after removal of HU. In our experiment, 1mM (76 yg/ml) HU-treated sponges developed normally with choanocyte chambers. We also confirmed that choanocyte chambers had already been formed in the 1.5 mM (114 ug/ml) HU-treated sponges 24hr after removal of HU. We think that 50 ug/ml HU may not be enough to inhibit the cell division of archaeocytes and the differentiation of choanocytes. Starting the experiment at 24 hr after removal of HU may have been too late. There may also be Hydroxyurea and Choanocyte Differentiation 569 difference in the sensitivity to HU among sponge strains. In living sponges, we observed cell division among archaeocytes after removal of HU, but some of the archaeocytes forming choanoblastic clusters stay at the periphery of the lacunae after the cell division. The other archaeocytes which differentiate into the other cell types move apart from each other with pseudopodia after the cell division. We could distinguish the presumptive choanocytes from the other to this extent. We presumed the number of mitosis and cell number during choanocyte differentiation from an archaeocyte, observing living sponges and calculation from histological sections (Table 1). Before 13.5 hr, we actually observed cell division in living materials. From 16 hr on, the counting was done on sections. Until 24hr the cells encircling a chamber increased very slowly, but number of choanocyte chambers increased rapidly (2/21 to 18/20). In sections, by the end of 16 hr all the archaeocytes had gone through 3 mitoses and form choanoblastic clusters. During the following several hours the cells passed through a 4th mitosis and differentiated into choanocytes. By 28.S5hr, all the cells in a cluster had passed through the 4th mitosis and differentiation of the primary choanocyte chambers was complete. Then choanoblastic clusters on the lacunae gradually transformed into choanocyte chamber in this period. It is likely that the choanocyte may undergo one more mitosis before the apopyle is formed and the choanocyte chamber becomes functional. Table 1 shows that the cell number doubles once more between 24 hr and 35.5 hr, and the cell size of choanoblasts or choanocytes decreases through cell division until 35.5 hr when apopyle is formed. The total cell number of choanocyte chambers does not double in a regular manner. However, as the fluctuation of the volume of choanocytes in a choanocyte chamber stays within the range of 1:2 a diversion from the regularity of two-fold number increase may be due to an asynchrony of mitosis. The cell size at and after 38.5 hr did not decrease, staying the same as at 35.5 hr. At 35.5 hr, the total cell number was about 25. At 38.5hr, there were two kinds of size in choanocyte chambers, one had 42 choanocytes and the other contained 76 choanocytes. It is considered that increase of the cell number of choanocyte chamber in 38.5 hr was not owing to the cell division but to fuse of established choanocyte chambers. We actually observed the fusion of mature choanocyte chambers in living sponges. Same observations have been reported by Wintermann [2] and Wierzejski [10]. Different estimates of the total cell number of a choanocyte chamber arising from a single ar- chaeocyte have been made [9]. According to these authors, the volume of a choanocyte in comparison with that of a mother archaeocyte of 15 wm diameter, is 27. In these rough estimates, however, the value calculated from the volume of the mother archaeocyte, approximately cor- responds to the value we calculated from the cell number in cross sections. We may conclude that choanocyte chambers are formed through the following differenti- ation and morphogenetic processes. An _ar- chaeocyte generates a choanoblastic cluster through three mitoses. The choanoblasts which go through a 4th mitosis differentiate into choanocytes. The choanocytes undergo one more mitosis with intact collar and flagellum before opening of the apopyle and functional choanocyte chamber formation. ACKNOWLEDGMENTS We express our great gratitude to Dr. Katsuma Dan for criticism of the manuscript. REFERENCES 1 Brien, P. (1932) Contribution a Il’étude de la régénération naturelle chez les Spongillidae, Spongilla lacustris L. et Ephydatia fluviatilis L. Arch. Zool. Exp. Gén., 74: 461-506. 2 Wintermann, G. (1951) Entwicklungsphysio- logische Untersuchungen an Siisswasserschwam- men. Zool. Jb. Abt. Anat. Ontog., 71: 427-486. 3 Borojevié,R. and Lévi,C. (1964) Etude au microscope électronique des cellules de |’éponge: Ophlitaspongia seriata (Gtant) au cours de la réorganisation apres dissociation. Z. Zellforsch. Mikros. Anat., 64: 708-725. 4 Watanabe, Y. (1978) Structure and formation 570 of the collar in choanocytes of Tetilla serica (Lebwohl). Develop. Growth and Differ., 20: 79-91. Rasmont, R. (1975) Fresh-water sponges as a material for the study of cell differentiation. Current Topics in Develop. Biol., 10: 141-159. Rozenfeld, F. and Rasmont, R. (1976) Hydro- xyurea: An inhibitor of the differentiation of choanocyte in freshwater sponges and a possible agent for the isolation of embryonic cells. Dif- ferentiation, 7: 53-60. Garrone, R. and Rozenfeld, F. (1981) Electron microscope study of cell differentiation and collagen synthesis in hydroxyurea-treated fresh- water sponges. J. Submicrosc. Cytol., 13: 127-134. Rasmont, R. (1961) Une technique de culture des éponges d’eau douce en milieu contrdlé. Ann. Soc. R. Zool. Belg., 91: 147-156. Rasmont, R. and Rozenfeld, F. (1981) Etude microcinématographique de la formation des chambres choanocytaires chez une éponge d’eau 10 11 14 K. TANAKA AND Y. WATANABE douce. Ann. Soc. R. Zool. Belg., 111: 33-44. Wierzejski, A. (1915) Beobachtungen tiber die Entwicklung der Gemmulae der Spongilliden und des Schwammes aus den Gemmulae. Bull. Intern. Ac. Polon., 2: 45-77. Berthold, G. (1969) Untersuchungen Uber die Histoblastendifferenzierung in der Gemmula von Ephydatia fluviatilis. Z. Wiss. Mikros., 69: 227-243. Hohr, D. (1977) Differenzierungsvorgange in der Keimenden Gemmula von Ephydatia fluviatilis. Wilhelm Roux’s Arch., 182: 329-346. Buscema, M., De Sutter, D. and Van de Vyver, G. (1980) Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes. Roux’s Arch. Dev. Biol., 188: 45-53. Rozenfeld, F. (1974) Biochemical control of fresh-water sponge development: Effect on DNA, RNA and protein synthesis of an inhibitor secreted by the sponge. J. Embryol. Exp. Morph., 32: 287-295. ZOOLOGICAL SCIENCE 1: 571-589 (1984) The Complete Larval Development of Caridina japonica De Man (Decapoda, Caridea, Atyidae) Reared in the Laboratory KEN-ICHI HAYASHI and TATSUO HAMANO! Shimonoseki University of Fisheries, Shimonoseki, Yamaguchi 759-65, and ‘Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812, Japan ABSTRACT — The Japanese atyid shrimp, Caridina japonica De Man, spawns small eggs, measuring 0.50 x 0.31 mm at spawning. The larvae show optimum growth under rearing conditions of 16.9% salinity, 25°C water temperature and by feeding an artificial diet, used in fish aquaculture, mixed with a small amount of rice bran. The hatched larvae develop to juveniles through nine zoeal stages, without a megalopa, over about one month. Zoeal stages and the first juvenile are described and illustrated in detail; these are compared, morphologically and ecologically in some important charac- ters, with those of several atyids reported previously. The present species shows the typical larval development of the group of atyids characterized by small eggs. Like C. weberi, the number of zoeal stages is nine, which is larger than that for other members of the same genus and smaller than © 1984 Zoological Society of Japan that for Atya innocous and Micratya poeyi. The stalked eye appears in the second zoeal stage as in almost all atyids bearing small or medium sized eggs. logically in the third stage and all pereopods in the seventh stage. The first pereopod is completed morpho- The pleopods are first recognized as buds in the sixth stage and the uropod is completed in the fourth stage. INTRODUCTION Based on egg size, the shrimps of the family Atyidae are divided into three groups [1]. Larval development has been described completely in several species of each group. The larvae of the “large egg”’ atyids are treated by Shen [2], Nair [3], Mizue and Iwamoto [4], Shokita [5, 6] and Benzie and Silva [7] and those of the “‘medium egg” ones by Yokoya [8], Babu [9], Glaister [10], Couret and Wong [11] and Benzie [12]. On the other hand, Gauthier [13], Chinnayya [14], Thomas ef al. [15], Pillai [16] and Zhang and Sun [17] described the larvae of the “‘small egg” group. Although Hunte [18, 19] did not mention the egg sizes of Micratya poeyi and Atya innocous, these two species probably belong to the small egg group, because the hatched larvae are small and there are many zoeal stages. In general, the large egg atyids usually have a landlocked life-cycle and spawn a comparatively small number of eggs which show direct develop- Accepted March 23, 1984 Received January 13, 1984 ment. By contrast, small egg species have numerous eggs and complex larval stages of long duration, which require saline media for develop- ment. The Japanese atyid, Caridina japonica De Man, belongs to the small egg group and occurs usually in the upper part of rivers in southern Japan and Taiwan [20-22]. Therefore, the likelihood of long development time in the larval stages has been predicted previously [23]. Complete larval develop- ment, to the first juvenile stage, was carried out by laboratory rearing, enabling the larval stages to be described fully. The general characteristics of the larvae of this species are discussed in relation to the atyid group. MATERIALS AND METHODS Specimens were captured during August, 1979 and May, 1981 from the Shiwagi River, Tokushima Prefecture, Japan. Measurements and counts were made of eggs from ovigerous females, pre- served in 10% neutral formalin, using a binocular microscope with an ocular grid. 572 Females were reared with males in aerated aquaria under the following conditions: water temperature, 20-++1°C; surface lumen, 1,400 lux; and photoperiod, 14L/10D. Daily feeding em- ployed an artificial diet for the ayu, Plecoglossus altivelis (No. 4C, Nippai Shrimp Feed Inc., Japan). After premating ecdysis and copulation, a female with newly attached eggs was placed alone in a 1,000 ml beaker under the aquarium conditions specified above. Within 24hr of hatching the larvae were individually separated into 100 ml beakers maintained without aeration at 25+1°C, 1,400 lux, 14L/10D. Every day the larvae were transferred by pipette to another set of beakers provided with fresh media and food. Experiments for food preference and for defining optimum salinity conditions of the rearing medium were carried out using the above rearing method. The larval developmental stages were studied from larvae reared under the most favourable feed and salinity conditions. Measurements of size and the duration of each zoeal phase were observed on larvae reared communally in large numbers in a large glass aquarium, 40x74x40cm, with a recirculating water supply system, in which the larvae fed on the diatoms, Cymbella and Navicula, adhering to the sand bottom and glass wall, as well as on the artificial diet. Specimens of zoeae and juveniles for examination were immobilized, in isotonic water, by chilling and dissected in 50% ethylene glycol. Drawings K-I. HAYASHI AND T. HAMANO measurements were made on five living individuals representing each larval stage using a binocular microscope with an ocular grid. The total length (TL) was measured from the rostral apex to the end of the telson, excluding setae, and the carapace length (CL) from the posterior margin of orbit, or the posterior margin of sessile eyes in case of the first zoea, to the posterior end of carapace. - RESULTS Females of C. japonica had an average of 1872.7 (range 747-4391, n=34) eggs, which were oval in shape and of dimensions 0.50 x 0.31 mm (n=55) soon after spawning and 0.54 0.35 mm (n=20) just before hatching. | A few eggs began to hatch sporadically from the 25th day (range 21st—28th, n=5) after spawning, but almost all eggs hatched out just before ecdysis of the mother shrimp, which took place on the night of the 39th day (26th-48th, n=5). Zoeae ingested all of five kinds of food proffered to them. However, green algae, mainly Chlorella, and a mixture of these algae and the rotifer, Brachionus plicatilis, could not maintain larvae in healthy condition. On the other hand, rice bran gave a longer survival time from hatching but the larvae never metamorphosed on it. Grated ~ powder of the artificial diet, mixed with a rice bran, appeared to be the best food for larvae producing 80% survival to metamorphosis (Table 1). were made with the aid of a camera lucida. All The effect of various salinity levels on the TABLE |. Larval rearing of Caridina japonica De Man on different foods at 25°C and 16.9% salinity Duration (days) Bead aM to () ae metamorphosis (% . atching to Hatching to death _ “metamorphosis Green algae 0 11.6 ( 8-16) ; oo Rotifer with situs PO green algae 0 8.3 ( 6-11) nea Rice bran 0 45.9 (15-60) ee. Artificial diet ' for ayu culture 50 19.2 ( 9-36) 36.2 (27-49) Artificial diet with rice bran 80 8.5 ( 8-9 ) 29.6 (26-34) Mean duration is shown with range in parentheses. Larval Development of Caridina japonica 373 TABLE 2. Larval rearing of Caridina japonica De Man on different salinities at 25°C Duration (days) Salinity Survival to (%o) metamorphosis (%) Hatching to Hatching to death metamorphosis 0 0 6.2 ( 4-9 ) a 8.5 0 10.0 ( 7-13) — 16.9 66.7 27.0 (25-30) 33.3 (23-38) 25.4 11.1 30.6 (21-31) 31.0 33.8 11 14.0 ( 7-30) 26.0 Mean duration is shown with range in parentheses. growth of zoeae is shown in Table 2. Normal development of zoeae was ensured by brackish or pure sea water but not by freshwater or low salinity media. After metamorphosis the juveniles grew normally in low salinities or freshwaters. In freshwater, at 20+1°C, the juveniles ecdysed every 6 days, and grew 0.2 mm in carapace length at each ecdysis. For juveniles with carapace length of more than 2.0 mm, ecdysis took place every 7 days and the average carapace length of 7 indi- viduals at the 8th ecdysis after metamorphosis was 2.6 mm. Description of larvae This species has nine zoeal stages, which are easily distinguished from each other by the number of segments of the antennular and antennal flagella and by morphological differences in the append- ages. No zoeae swim actively and the movement is carried out in reverse condition, using the exopods of the thoracic appendages. However, juveniles swim in the normal manner, using the pleopods for propulsion, and often walk on the bottom using the endopods of the pereopods. The first zoea (Fig. 1) Age 1 to 10 days. CL 0.35 (range 0.29-0.39) mm, TL 1.55 (1.48-1.76) mm. Rostrum short and unarmed; angle of carapace sharply pointed. Eyes large and sessile in carapace. Antennular peduncle unsegmented, with a long plumose seta at inner distal end; outer flagellum provided apically with a short plumose seta and 4 anterolateral long aesthetes. Peduncle of antenna with a small simple seta on outer distal corner; scaphocerite with 11 plumose setae along margin; flagellum unsegmented with a long plumose seta at apex. Mandible soft with a stalked long tooth on incisor process and many small teeth on molar process. Endopod of first maxilla palp-like with 4 setae; basal endite with one large and many small spines; coxal endite with 4, or 5 non-plumose setae. Endopod of second maxilla with 2 long setae at apex and with 4 to 6 setae on inner margin; endite 4-lobed with many simple setae on inner margin. Scaphognathite with 5 plumose marginal setae; posterior seta strong and directed posteriorly. Exopod of first maxilliped much longer than endopod and unsegmented with 4 long plumose setae at apex, often with 1 or 2 small simple setae near apex; endopod 5-segmented with 2 or 3 simple setae at apex, and a few short setae on some proximal segments; basicerite 4-lobed, each with two rather long setae. Exopod of second maxil- liped as in first maxilliped; endopod 4-segmented with 4 short setae at apex, and a few setae on each segment; inner margin of basicerite with some similar setae. Exopod of third maxilliped as in first two maxillipeds; endopod 5-segmented with 3 long plumose setae at apex and a few short setae on some proximal segments. Uniramous bud of first Other pereopods absent. All pleopods absent. Telson triangular with strong median notch, 6 pairs of plumose setae and a pair of short naked setae on posterior margin; uropods undifferen- tiated. pereopod present. 574 K-I. HAYASHI AND T. HAMANO Fic. 1. First zoea of Caridina japonica De Man. a, animal in lateral view, 6, animal in dorsal view, c, tail fan, d, antennule in ventral view, e, antenna in ventral view, f, mandible, g, first maxilla, h, second maxilla, i, first maxil- liped, j, second maxilliped, k, third maxilliped, J, first pereopod. Scales 0.2 mm. 575 Larval Development of Caridina japonica Fic. 2. Second zoea of Caridina japonica De Man. m, second pereopod. Scales 0.2 mm. a-/l as for Fig. 1, 576 K-I. HAYASHI AND T. HAMANO Body transparent with red chromatophores present on posterior margin of eyes and dorsolater- al parts of third, fifth and sixth abdominal somites. Much yolk present in central part of carapace. The second zoea (Fig. 2) Age 4 to 13 days. CL 0.41 (0.36—-0.46) mm, TL 1.76 (1.59-1.93) mm. Rostrum straight, reaching end of antennular peduncle. Suborbital angle and pterygostomial angle bluntly pointed. Eyes movable and stalked. Antennule and antenna as in first zoea. Mandible as in first zoea, but 3 large teeth on incisor process. First and second maxillae as in first zoea. All maxillipeds as in first zoea. First pereopod biramous. Uniramous bud of second pereopod present. Pleopods absent. Posterior margin of telson with 8 pairs of plu- mose setae. Additional chromatophores present on base of antennal peduncle. No food is taken by first and second stage zoeae. The third zoea (Fig. 3) Age 6 to 14 days. CL 0.47 (0.40-0.51) mm, Fic: 3. a-m as for Fig. 2, Third zoea of Caridina japonica De Man. n, third pereopod. Scales 0.2 mm. Larval Development of Caridina japonica 577 TL 1.78 (1.62-2.02) mm. Rostrum straight, reaching end of basal segment of antennular peduncle. Carapace as in second zoea. Antennular peduncle 3-segmented, distal seg- ment with 4 short and 2 long plumose setae on distoventral margin, median segment with a long plumose seta and often with 1 or 2 short plumose setae; basal segment as in median one, outer flagellum with 4 terminal aesthetes; inner flagellum with an aesthete at apex. Scaphocerite as in second zoea; the long, plumose setae of the anten- nal flagellum now absent. Mandible hardened and functional. First maxil- la as in second zoea. Endopod of second maxilla ve 4 A Dp c 7: ey , EL a | oa it ——_—— et a a, 22S, a’ with 3 long plumose setae at apex; scaphognathite broad with 7 plumose setae. All maxillipeds as in second zoea. Exopod of first pereopod longer than endopod, with 4 long plumose setae at apex, and often with 1 or 2 short plumose, or non-plumose, setae near apex; endopod 5-segmented with 3 long setae at apex. Second pereopod biramous. Uniramous bud of third pereopod present. Tail fan separated from sixth abdominal somite; pleopods absent. Shape and spination of telson as in second zoea, but small uniramous uropods present, provided with some plumose setae. Many chromatophores present, especially on base of telson. Fic. 4. Fourth zoea of Caridina japonica De Man. a-n as for Fig. 3, o, fourth pereopod. Scales 0.2 mm. 578 K-I. HAYASHI AND T. HAMANO The larvae begin to take food at this stage. The fourth zoea (Fig. 4) Age 10 to 18 days. CL 0.49 (0.43-0.55) mm, TL 2.10 (1.82-2.35) mm. Rostrum and carapace as in third zoea. Antennule as in third zoea, but with 4 long plumose setae on distal segemnt of peduncle; a short but stout plumose seta on ventral surface of basal segment. Scaphocerite broad with about 14 plumose setae and pointed at outer distal end; antennal flagellum broad and unsegmented. Mandible and first maxilla as in third zoea. Scaphognathite with about 10 plumose setae; posterior seta elongate with short plumes on inner margin only. All maxillipeds as in third zoea. First pereopod as in third zoea. Exopod of second pereopod longer than endopod with 4 long plumose setae at apex, and often with 1 or 2 small plumose, or non-plumose, setae near apex; endopod 5-segmented with 3 long setae at apex. Third pereopod biramous. Uniramous bud of fourth pereopod present. Pleopods absent. Telson rectangular; posterior margin medially notched with 5 pairs of inner plumose setae and 2 pairs of outer spines; lateral margin with another pair of spines posteriorly. Both exopods and endopods of uropods differentiated. Additional chromatophores scattered over ven- tral side of abdomen. The fifth zoea (Fig. 5) Age 12 to 22 days. CL 0.60 (0.52-0.66) mm, TL 2.21 (1.89-2.75) mm. Rostrum and carapace as in fourth zoea. Outer flagellum of antennule 2-segmented; inner flagellum as in fourth zoea. Distal segment of peduncle with 6 long plumose setae on distoven- tral margin. Scaphocerite with about 15 plumose setae; antennal flagellum 2-segmented. First and second maxillipeds as in fourth zoea. Exopod of third pereopod longer than endopod, with 4 long plumose setae at apex and often with 1 or 2 short plumose, or non-plumose, setae near apex; endopod 5-segmented with 4 long simple setae at apex. Fourth pereopod biramous. Uni- ramous bud of fifth pereopod present. Pleopod buds present on abdominal terga. Tail fan as in fourth zoea; outer distal end of exopod of uropod spinous. The sixth zoea (Fig. 6) Age 14 to 29 days. CL 0.66 (0.60-0.71) mm, TL 2.76 (2.42-3.10) mm. Rostrum and carapace as in fifth zoea. Antennule as in fifth zoea. Scaphocerite with about 17 plumose setae; antennal flagellum elongated and 3-segmented. Mandible with 3 large, stalked teeth on incisor process and many small teeth on molar process; one broad and 4 or 5 slender teeth present between incisor and molar processes. First maxilla as in fifth zoea. Scaphognathite with about 20 plumose setae along margin; posterior seta large and plumose. All maxillipeds as in fifth zoea. First two pereopods as in fifth zoea. Propodus of third pereopod long; exopod and endopod of fourth pereopod apparently distinguishable; exopod long, with 4 long plumose setae at apex and often with 1 or 2 short plumose, or non- plumose, setae near apex; endopod 5-segmented, with 3 simple setae at apex. Fifth pereopod biramous, endopod partly segmented. Uniramous buds of all pleopods present. Telson slender and rectangular; posterior margin medially notched with 5 pairs of plumose setae and 1 pair of spines; lateral margin with 2 other pairs of spines. The seventh zoea (Fig. 7) Age 15 to 31 days. CL 0.74 (0.72-0.83) mm, TL 2.95 (2.81-3.24) mm. Rostrum and carapace as in sixth zoea. Outer flagellum of antennule 3-segmented with 4 aesthetes at apex; inner flagellum 3-segmented with 2 apical aesthetes. Distal segment of pedun- cle with 7 long plumose setae; basal segment slightly expanded at outer proximal part. Scaphocerite with about 15 plumose setae; anten- nal flagellum 4-segmented. Mandible and first maxilla as in sixth zoea. Coxal endite of second maxilla single-lobed with many simple setae; scaphognathite with more than Larval Development of Caridina japonica 579 Fic. 5. a-o as for Fig. 4, p, fifth pereopod. Scales 0.2 mm. 25 plumose setae and its posterior part elongate, with a large plumose seta terminally. All maxillipeds as in sixth zoea. First four pereopods as in sixth zoea, but endopods of third and fourth pereopods longer than respective exopod. Exopod of fifth pereopod with 4 long plumose natatory setae and often with 1 or 2 small plumose, or non-plumose, setae near apex; endopod longer than exopod and 5-seg- ‘ Oo SINAN 2: SV 8 WN shy Fifth zoea of Caridina japonica De Man. mented with 3 long terminal setae; a few setae on some proximal segments. All pleopods biramous. Posterior margin of telson evenly convex without notch. The eighth zoea (Fig. 8) Age 16 to 33 days. CL 1.03 (0.91-1.09) mm, TL 3.90 (3.59-4.32) mm. 580 >>> noe SSS Fo aD — K-I. HAYASHI AND T. HAMANO Fic. 6. Sixth zoea of Caridina japonica De Man. a-p as for Fig. 5. Scales 0.2 mm. 581 Larval Development of Caridina japonica y < | Dari Y Seventh zoea of Caridina japonica De Man. Fic. 7. a-p as for Fig. 5, r, second pleopod. q, first pleopod, Scales for a and 6 0.5mm; those for c-r 0.2 mm. K-I. HAYASHI AND T. HAMANO SS > Fic. 8. Eighth zoea of Caridina japonica De Man. a, animal in lateral view, 6, animal in dorsal view, c, tail fan, d, antennule in ventral view, e, distal part of the same in dorsal view, /, antenna in ventral view, g, mandible, h, first maxilla, i, second maxilla, j, first miaxilliped, k, second maxilliped, J/, third maxilliped, mm, first pereopod, zn, second pereopod, o, third pereopod, p, fourth pereopod, q, fifth pereopod , r, first pleopod, s, second pleopod. Scales for a and b 1.0 mm; those for c—s 0.2 mm. Larval Development of Caridina japonica Rostrum and carapace as in seventh zoea. Outer flagellum of antennule 4-segmented with an aesthete on each inner margin; inner flagellum long and slender, composed of 3 segments, with a long terminal seta. Scaphocerite with about 20 plumose setae; antennal flagellum 12-segmented, reaching beyond scaphocerite. Mandible and first maxilla as in seventh zoea. Scaphognathite with more than 30 plumose setae, and its posterior elongated part extending beyond coxal endite, bearing 2 large plumose setae ter- minally. First and third maxillipeds as in seventh zoea. Endopod of second maxilliped with 5 or 6 short setae at apex; a few short setae on some proximal segments. Endopods of first two pereopods as long as respective exopod; propodi of those pereopods broadened near inner distal corner. Third and fourth pereopods as in seventh zoea. Endopod of fifth pereopod elongate, especially on propodus, with 2 long setae at apex and a few setae on distal articulation of each segment. Pleopods broad with some incipient setae near apex, except for endopod of first pleopod. Tail fan as in seventh zoea; two spines present on dorsolateral margin of telson. Many chromatophores present on base of pleopods and ventral side of body. The ninth zoea (Fig. 9) Age 22 to 35 days. CL 1.13 (1.05-1.28) mm, TL 5.01 (4.77—-5.35) mm. Rostrum and carapace as in eighth zoea. Pleuron of second abdominal somite broadened and overlapping those of first and third somites. Outer flagellum of antennule 5-segmented with an aesthete on each inner margin; inner flagellum 6-segmented. Distal segment of antennular pe- duncle with 10 long plumose setae on distoventral margin. Antennal flagellum 18-segmented and long carpocerite defined. Mandible as in eighth zoea. Coxal endite of first maxilla much expanded, with many setae. Scaphognathite with more than 25 plumose setae, elongated part with some short setae only. First and third maxillipeds as in eighth zoea. Endopod of second maxilliped with more than 6 583 short setae at apex; distal 3 or 4 segments curved inwards. Fixed fingers of first two pereopods elongated, and faced to each movable finger. Endopods of posterior three pereopods slender, propodi and meri especially long. All exopods of pleopods setose. Endopod of first pleopod small and oval without appendix interna; those of second to fifth pleopods setose, each with appendix interna. Telson tapering; posterior margin more convex with 5 pairs of inner setae and a pair of outer spines; two pairs of spines situated on dorsal sur- face near lateral margin. The first juvenile (Fig. 10) Age 24 to 38 days. CL 1.20 (1.09-1.29) mm, TL 4.83 (4.60-5.20) mm. Rostrum and carapace as in ninth zoea. Outer flagellum of antennule slender and 5- segmented; inner flagellum long and 9-segmented; stylocerite pointed distally. Scaphocerite with about 30 plumose setae; carpocerite of antennal peduncle elongate; flagellum with 31 segments, much longer than scaphocerite. Mandible very hard, with some lines of small teeth on molar process. Endopod of first maxilla reduced to palp-like process; basal endite with many small teeth on inner margin. Endopod of second maxilla now absent; basal endite single- lobed with many spines on median edge. setae of scaphognathite reduced. Exopod of first maxilliped reduced, often without long plumose setae at apex; endopod also reduced; basal endite broad with many setae arranged linearly on median margin; coxal endite broad, with several plumose setae. Endopod of second maxilliped broad and curved inwards, with many short setae along inner margin. Exopod of third maxilliped comparatively short; endopod stout and pediform with a strong spine at apex. Exopods of all pereopods highly reduced. Endopods of first two pereopods with complete chelae, with clusters of setae at apices of fingers. Endopods of posterior three pereopods stout, carrying 3 or 4 spines on posterior margin of dactyli and propodi, 1 or 2 on carpi, 4 or 5 on meri. All pleopods fully developed with many long Plumose K-I. HAYASHI AND T. HAMANO 584 Fic. 9. Ninth zoea of Caridina japonica De Man. Scales for a and b 1.0 mm; those for c—s 0.2 mm. a-s as for Fig. 8. 585 Larval Development of Caridina japonica Fic. 10. First juvenile of Caridina japonica De Man. Scales for a and 6 1.0 mm; those for c—s 0.2 mm. a-s as for Fig. 8. 586 plumose setae around margin, but endopod of first pleopod with vestigial setae at apex. Posterior margin of telson with 3 pairs of inner setae and 1 pair of outer spines; dorsal surface with two pairs of spines. Uropod longer than telson; exopod ending in an outer spine with apparent diaeresis. DISCUSSION Egg size in the freshwater decapods is well related to egg number and the duration of zoeal stages. The shrimps with large eggs are usually of low fecundity, and have abbreviated larval, or direct, development to juveniles, while the small egg species have high fecundities, and long larval lives. The medium egg species occupy an inter- K-I. HAYASHI AND T. HAMANO mediate position. Some atyids of the large egg group, such as Neocaridina denticulata sinensis [2], N. denticulata [4], N. brevirostris [5], N. ishigakiensis [6] and Caridina shinghalensis [7| have no zoeal stage and the hatched larva is referred to as a megalopa, which resembles the juvenile in general body form, but the setation and shape of the telson are different. On the other hand, Micratya poeyi [18], Atya innocous [19] and C. weberi [14] have many zoeal stages and develop directly to the juvenile without a megalopa. The present species, C. japonica, bears many eggs and belongs to the small egg group, most closely resembling the last mentioned two species in its larval development, i.e., in having many, as many as nine in its case, zoeal stages, and in the absence of a megalopa stage. TABLE 3. Developmental characteristics of Caridina japonica Species Atya innocous Micratya poeyi Caridina weberi Caridina japonica Caridina nilotica gracilipes Atyaephyra desmaresti Paratya compressa improvisa Caridina pseudogracilirostris Caridina wyckii Caridina nilotica aruensis Halocaridina rubra Caridina propinqua Caridina mccullochi Neocaridina ishigakiensis Neocaridina denticulata sinensis Neocaridina brevirostris Neocaridina denticulata Caridina shighalensis No. of Egg Egg Rearing zoeal size stages (mm) type’ medium? 12 ? (S) N) 10 ? (S) N) 9 0.47 x 0.37 N) F* 9 0.50 x 0.31 Ss S 8 0.45 x 0.30 N) L 8 0.60 x 0.40 S F 8 0.8 x0.5 M Be 6 0.32 x 0.28 Ny) N) 6 ? (M/L) [Br 4 0.64 x 0.40 M F 4 0.93 x 0.71 M S 3 0.49 x 0.36 M F 2 0.77 x 0.62 M F 0 1.07 x 0.67 L F 0 1.11 x 0.68 L F 0 1.24 x 0.79 L F 0 1.60 x 1.05 L F 0 1.80 x 1.24 L F + S: small egg type, M: medium egg type, 2 S: saline water, F: freshwater. 3M: megalopa, J: juvenile. L: large egg type. Egg type in parentheses is determined * These larvae do not develop well in freshwater [23]. ** Data based on field collection. Larval Development of Caridina japonica The newly hatched zoeae of the small and medium egg atyids, including C. japonica, usually share the following morphological characteristics: sessile eyes, an unsegmented antennular peduncle and antennal flagellum, developed exopods on all maxillipeds and a triangular telson with seven pairs of setae (Fig. 1 and Table 3). Similar morpho- logical changes tend to occur progressively through the same zoeal stages in these species. The eye is stalked in the second zoea in all species except for C. wyckii [24], the uropod is completed in the fourth zoea except for C. weberi [14] and the first pereopod is completed in the second, third or, rarely, fourth zoea. However, the completion of all pereopods and the appearance of the pleopod buds are more delayed in small egg atyids than in the medium egg ones. In C. japonica all pereopods are completed De Man in comparison with other atyid shrimps 587 in the seventh zoea as in M. poeyi and the pleopod buds appear in the sixth zoea as in Paratya com- pressa improvisa. Of course, absence of zoeal stages in the large egg atyids does not permit such comparison with the present species. As described by Benzie and Silva [7] the only difference is seen in the number of the telson setae. In the large egg atyids the hatched individual has a telson with eight or ten pairs of setae, instead of seven pairs as in C. japonica and other small and medium egg species. Various types of foods have been tried for rearing atyid larvae; ciliates and algal bloom for C. propinqua [9], detritus for C. nilotica aruensis [10], commercial fish food flakes for C. mcecullochi [12], algal layer and copepodites developed in aquarium for C. pseudogracilirostris [16] and particles of Stage? No. of appearing completed telson setae on References stalked pleopods first all uropods hatched eye buds leg legs individuals II IX ll VII IV Tapa Hunte [19] II VII IV Vil IV Uap) Hunte [18] II VII II Vill V Vee 7 Chinnayya [14] Il VI ll Vil IV Fla oa Present authors II Vv ll Vv IV T+1 Zhang and Sun [17] II Vv Ul VI IV 7+7 Gauthier [13] II VI II IV IV +7 Yokoya [8] Il IV lll V IV 74+7 Thomas et al. [15], Pillai [16] I IV Il V IV TT Daday [24] II II II Ill IV T+7 Glaister [10] II II Ill IV IV 74+7 Couret and Wong [11] II II Il Il J ET Babu [9] Il I II II J 1+7 Benzie [12] M M M M i 8+8 Shokita [6] M M M M J 8+8 Shen [2], Zhang and Sun [17], Shokita [23] M M M M J 8+8 Shokita [5, 23] M M M M J 8+8 Mizue and Iwamoto [4] M M M M J 10+10 Benzie and Silva [7] by larval characteristics published. 588 K-I. HAYASHI AND T. HAMANO wheat germ and grated Tetramin for M. poeyi [18] and A. innocous [19]. All of these shrimp larvae were developed successfully to juveniles; the first three species belong to the medium egg group and the last two species probably the small egg one, because their newly hatched larvae are small. Lakshmi [25] failed to rear larvae to juveniles using the Artemia nauplii. Judging from the size of the hatched larvae, his Caridina sp. seemed to be either a large egg or medium egg species. Adhesive diatoms, Cymbella and Navicula, or a grated artificial diet, mixed with a small amount of rice bran, were used to feed the zoeae of the present species. Poor swimming ability and bottom distribution in aquaria indicate that the zoeae of atyid shrimps are able to use non-sus- pended or attached organic matter as foods in nature as well as in an aquarium. Six species of large egg atyids, four species of the medium egg ones, including C. wyckii [24] and two species of small egg atyids [13, 14] are all landlocked and the larvae do not require high salinity for development. Halocaridina rubra [11], which has four zoeal stages in spite of having rather large eggs, and almost all small egg atyids, including A. innocous and Micratya poeyi, normally grow in saline media of 15-32%o. The zoeae of C. japonica also require 17-32%o to grow rapidly and normally (Table 2), and its juveniles have been observed migrating upstream near a river mouth at night (unpublished data). These facts show that C. japonica has a typical amphidromous life-cycle. M. poeyi [18], and A. innocous [19], whose zoeae metamorphose in saline waters only, are also thought to have a similar life style to that of C. japonica. The various species of the Japanese small egg atyids usually occur in the middle or lower parts of rivers or in both situations [21-23]. However, the present species is unique in its restriction to the upper parts of rivers in Japan, while retaining a high fecundity, many larval stages and a long larval life requiring high salinity waters for normal development. ACKNOWLEDGMENTS We wish to extend our sincere thanks to Dr. N. M. Morrissy of the Western Australian Marine Research Laboratories for reading and correcting our manuscript and Mr. Wang Yang, a student of Kyushu University for translating the Chinese literature. REFERENCES | Shokita, S. (1981) Life-history of the family Atyidae (Decapoda, Caridea). Aquabiol., 12: 15-23. 2 Shen, C. T. (1939) The larval development of some Peking Caridea-the Caridina (Atyidae), the Palaemonetes and the Palaemon (Pala- emonidae). 40th Anniv. Pap. Nat. Univ. Peking, 1: 169-201. 3. Nair, K. B. (1949) The embryology of Caridina laevis Heller. Proc. Ind. Acad. Sci., Ser. B, 29: 211-288. 4 Mizue, K. and Iwamoto, Y. (1961) On the deve- lopment and growth of Neocaridina denticulata De Haan. Bull. Fac. Fish., Nagasaki Univ., 10: 15-24. 5 Shokita, S. (1973) Abbreviated larval develop- ment of fresh-water atyid shrimp, Caridina brevirostris Stimpson from Iriomote Islands of the Ryukyus (Decapoda, Atyidae). Bull. Sci. Engin. Div., Univ. Ryukyus (Mathem. Nat. Sci.), 16: 222-231. 6 Shokita,S. (1976) Early life-history of the land-locked atyid shrimp, Caridina denticulata ishigakiensis Fujino and Shokita, from the Ryukyu Islands. Res. Crust., 7: 1-10. 7 Benzie,J.A.H. and Silva, P.K. (1983) The abbreviated larval development of Caridina sighalensis Ortmann, 1894 (Decapoda, Atyidae). J. Crust. Biol., 3: 117-126. 8 Yokoya, Y. (1931) On the metamorphosis of two Japanese freshwater shrimps /Paratya compressa and Leander paucidens, with reference to the development of their appendages. J. Coll. Agr. Tokyo Imp. Univ., 11: 75-150. 9 Babu, N. (1963) Observations of the biology of Caridina propinqgua De Man. Ind. J. Fish., 10: 107-117. 10 Glaister, J. P. (1976) Postembryonic growth and development of Caridina nilotica aruensis Roux (Decapoda: Atyidae) reared in the laboratory. Aust. J. Mar. Freshwat. Res., 27: 263-276. 11 Couret,C.L., Jr. and Wong, D.C. L. (1978) Larval development of Halocaridina rubra Holthuis (Decapoda, Atyidae). Crustaceana, 34: 301-309. 12 Benzie, J. A. H. (1982) The complete larval development of Caridina mccullochi Roux, 1926 (Decapoda, Atyidae) reared in the laboratory. J. Crust. Biol., 2: 493-513. 13 Gauthier, H. (1924) Recherches sur le dévelop- pement larvaire d’Atyaéphyra desmaresti (Millet, 15 Larval Development of Caridina japonica 1832) (Décapodes, Natantia, Caridea, Atyidés). Bull. Soc. Hist. Nat. Afrique Nord., 15: 337-342, 345-376. Chinnayya, B. (1974) The embryonic and larval development of Caridina weberi De Man in the laboratory (Decapoda, Atyidae). Broteria, 43: 119-134. Thomas, M. M., Pillai, V.K. and Pillai, N. N. (1973) Caridina pseudogracilirostris sp. nov. (Atyidae: Caridina) from the Cochin backwater. J. Mar. Biol. Ass. Ind., 15: 871-873. Pillai, N. N. (1975) Larval development of Caridina pseudogracilirostris reared in the labo- ratory. J. Mar. Biol. Ass. Ind., 17: 1-17. Zhang, J. and Sun, X. (1979) Studies on the larval development of six freshwater prawn species in the middle and lower Chang Jiang (Yangtze) Valley. Acta Zool. Sinica, 25: 143-153. Hunte, W. = (1979) The complete larval development of the freshwater shrimp Micratya poeyi (Guérin-Méneville) reared in the laboratory (Decapoda, Atyidae). Crustaceana, Suppl. 5: 153-166. 19 20 21 22 23 24 2 589 Hunte, W. (1979) The complete larval develop- ment of the freshwater shrimp Atya innocous (Herbst) reared in the laboratory (Decapoda, Atyidae). Crustaceana, Suppl. 5: 231-242. Kubo, I. (1938) On the Japanese atyid shrimps. J. Imp. Fish. Inst., 3: 67-100. Kamita, T. (1970) Studies on the freshwater shrimps, prawns and crawfishes of Japan. Sonoyama Shoten, Matsue, pp. 50—64. Yu, H. (1974) On the atyid shrimps (Crustacea, Decapoda, Atyidae) from Taiwan. Aquiculture, 2: 49-58. Shokita, S. (1979) The distribution and speciation of the inland water shrimps and prawns from the Ryukyu Islands-II. Bull. Coll. Sci., Univ. Ryukyus, 28: 193-278. Daday, E. (1907) Der postembryonale Entwi- cklungsgang von Caridina wyckii (Hicks). Zool. Jahrb., Abthandl. Anatomie, 24: 239-294. Lakshmi, S. (1975) On the early larval develop- ment of Caridina sp. (Crustacea, Decapoda, Atyidae). Ind. J. Fish. 22: 68-79. Coherieh paler PL ya tah - RN A gta eee | eal tee ri ih We Hen, ei Eoorre Ae yt PY Py FT we me ees AL” da ’) Pal ; a | ra it ( 4 : ‘ - . 7 . > . ane m | Whar sy ae i ae ¥ H ; = ; ote aL f hep § \ a th 3) > de yo m~ 5) * | - n. a) ; 4 i au ry yD —_ bee otra oe 7 ast) wt wahd, Sel vest - AOR — _ae wid Hed axd opeaes of) mm ' iia hes ; ne , kannst Wb is bien ioe \ D. § j, a uf Rote kaw 4y ‘ 4 2 eer Ss ee ee . £ 7B heugy! off el var et =o .u ‘ pn | + ur i his ./ rr z 3 ? ithe apes “rpc ae eaten peree is ahs M i f pul t dd ZOOLOGICAL SCIENCE 1: 591-600 (1984) Ultrastructural Study of Gonadal Development in Xenopus laevis HISAAKI IWASAWA and KoJIRO YAMAGUCHI Biological Institute, Faculty of Science, Niigata University, Niigata 950-21, Japan ABSTRACT — The development and sex differentiation of the gonads were observed light micro- scopically and electron microscopically in Xenopus laevis. The mode of sex differentiation belongs to the so-called differentiated type. The testicular or ovarian differentiation of the primordial gonads began between stages 50 and 53. The medulla of the sexually indifferent gonads was formed by the multiplication of the epithelial cells in the hilum region of the primordial gonads, i.e., both the medul- lary cells and the cortical ones are of coelomic epithelial origin. Cellular invasion from the mesonephric area or else to the primordial gonads was not found during earlier stages of gonadal development, or in the subsequent sex differentiation either. The contact between the irregularly shaped surfaces of the germ cells and their enveloping cells was very close in the primordial testes as well as in the sexually indifferent gonads, but in the primordial ovaries, the contact between these © 1984 Zoological Society of Japan cells was rather loose. INTRODUCTION For nearly a hundred years, embryological studies on amphibian gonads have been performed by many investigators. Among others, Witschi presented the cortico-medullary antagonism theory to explain the mechanism of gonadal sex differ- entiation [1]. He insisted that the somatic cells that form the cortex of the primordial gonads derive from the coelomic epithelium and the medullary cells are of mesonephric origin, and that the testicular differentiation of the sexually indifferent gonads is induced by the further invasion of the mesonephric blastemas from the mesonephric area. On the other hand Vannini and Sabbadin argued that the medulla has its Origin in the interrenal blastemas, not the mesonephric blastemas [2]. It may be said, however, that these two opinions belong to the same category in a sense, because both assert the extra-coelomic epithelial origin of the medullary cells. Recently, the coelomic epithelial origin of Accepted February 6, 1984 Received December 21, 1983 the medullary cells in Rana pipiens and Xenopus laevis was suggested by Merchant-Larios and his co-worker [3,4]. Concerning the larval gonads of Xenopus laevis, one of the materials used in their observations, a good many experi- mental studies have been carried out until now [5], and many electron microscopic studies on the primordial germ cells have also been reported [6-10]. Nevertheless, there are only a few brief descriptions of the normal development of the gonads [5, 11], and, on the basis of light micro- scopic observations, these papers say that the medulla of the primordial gonads derives from the mesonephric or interrenal blastemas. Ac- cordingly, we observed light microscopically and electron microscopically the gonadal development in Xenopus laevis, paying a special attention to the origin of the medullary cells, and obtained results which indicate that the traditional view of the origin of the medullary cells should be rejected. MATERIALS AND METHODS Many fertilized eggs were obtained from a few pairs of male and female Xenopus laevis 592 H. IWASAWA AND K. YAMAGUCHI TABLE 1. Relation between larval developmental stages and gonadal differentiation Gonadal Nieuwkoop and Faber’s developmental stages differen- tiation 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63° (64556s5mm6G I, 34 2 I, 29° 12 TS yas AR I; i tS 17 2) 1G = ¥ AR; 2 SoG 10 M25 8,013 T, 3. T"8 3 2 T; 2. 5 8 «49 ees O; 1 4 M1 SO): Md ta! O, D 8-13" 8° “6 7 Omer O; Permian, 5 1© frogs injected with a human chorionic gona- dotropin (Gonatropin, Teikoku Zoki Co.). The eggs and embryos were kept in dechlorinated tap water, and the water temperature was main- tained at 23°+1°C. After hatching out, the larvae were fed on a commercial diet for tropical fish. Under these rearing conditions, the larvae metamorphosed at the age of 68 days on an average. Animals at developmental stages 46 to 66 [12] were used in the present observation. For light microscopy, animals in each stage were lapatomized and immersed in Bouin’s fixative. Then the urogenital regions were taken out, sectioned serially at 10m according to the routine paraffin method and_ stained with Delafield’s hematoxylin and eosin. The number of animals used for light microscopic observation was 12-34 for each stage (see Table 1). For electron microscopy, the gonads and kidneys were fixed together in Karnovsky fixa- tive for 2hr at room temperature followed by postfixation in 1% OsO. buffered with 0.2 M phosphate buffer (pH 7.4) for 2hr at 4°C. After dehydration in a series of ethanol, the specimens were embedded in Epok 812. Semithin and ultrathin sections were cut with a Porter-Blum microtome MT2-B. The ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H—300 electron micro- scope. The semithin sections were stained with a 1% solution of toluidine blue in a 1% borax solution and examined with a light microscope. RESULTS 1. Light microscopic study The mode of gonadal sex differentiation in this species belongs to a typical differentiated type. The developmental process in the sexually indifferent gonads was divided into 3 stages (Stages I,-I;), and each process of testicular and ovarian differentiation observed until metamor- phosis was divided into 3 stages (Stages T,-T; and Stages O,—Os). Stage I, The coelomic epithelium on both sides of the dorsal root of the dorsal mesentery protruded in the ventral direction, and formed a pair of genital ridges. Then the ridges formed primordial gonads. Within the primordial gonads a small number of primordial germ cells, one or two in a cross section, were seen. Each of them was enveloped in single-layered epithelial cells. Stage [, The outlines of the primordial gonads appeared elongated oval in cross section. In the proximal region of the gonads, the so-called medullary cells appeared. While in the distal region, two or three germ cells were seen in a cross section. Stage [; The primordial gonads became more voluminous, and the so-called cortico-medullary structure was clearly seen. Several germ cells were observed in the distal cortical region. In Stages I, and I; the mitotic germ cells were numerous. Gonadal Development of Xenopus 593 Stage T, The gonadal volume continued to increase and the germ cells began to migrate from the cortical region into the medullary tissue. Stage T, The testicular volume _ increased remarkably. The germ cells were embedded in the medullary tissue. Formation of the sem- iniferous tubules was progressing. Stage T; The formation of the rudiments of the seminiferous tubules was definitely recog- nized, and spermatogonia, one to three in number, were seen in each section of the primordial tubules. The efferent testicular tubules were not yet developed at this stage. Stage O, The gonadal size increased slightly. The middle part of the medullary tissue became excavated and the ovarian cavity was formed. The mass of medullary cells changed into a simple epithelium and lined the ovarian cavity. In the cortical region, mitotic germ cells were noticeable. As already reported [13], the number of germ cells was greater in the ovarian primordia than in the testicular ones at this developmental stage. Stage O. The ovaries became remarkably voluminous and the ovarian cavities expanded considerably. In some parts of the cortex, the multiplying oogonia formed a mass which was enveloped by follicle cells. Stage O; The ovaries became more volumi- nous. The formation of the germ-cell nests was seen in most parts of the ovaries, and the number of oocytes in early prophase stages increased. Most oocytes situated at the inner part of the cortex entered their growth stages at the end of this stage of ovarian development. The relation between the larval developmental stages [12] and gonadal differentiation is shown in Table 1. 2. Electron microscopic study Formation of sexually indifferent gonads The primordial gonads were formed by outer folding of a simple epithelium covering the coelom (Fig. 1). Beneath this epithelium, a small number of scattered mesenchymal cells were seen. The basal membrane, under which a sheet of melanin granules was seen, lining the coelomic epithelium was pulled into the inner parts of the gonadal primordium (Fig. 2). The germ cells were enveloped by the epithelial cells (Fig. 3). The contact between the germ cells and the epithelial cells was very close at their irregularly shaped surfaces (Fig. 4). The germinal plasm was seen in the germ cells. The above description is of Stage I). From Stage I, to Stage I;, a notable change occurred at the proximal region (hilum) of the primordial gonads. At first, this region consisted of parallel sheets of monolayered epithelial cells, which were derived from the coelomic epithelium, in a cross section. Then the cells of both the epithelial sheets took on an undulating arrange- ment. Simultaneously, the sheet of melanin granules, which lines the basal membrane, penetrated towards the inside of the gonad (Fig. 5). These melanin granules were still seen within the gonads for a while thereafter, but they disappeared gradually during the following stages. The mitotic figures of the epithelial cells were noticeable. As the next step, the cells which are very similar to the epithelial cells in ultrastructure appeared between the two sheets of undulating epithelia (Fig. 6). The so- called medulla of the primordial gonads was formed by the proliferation of these cells. In the hilum region, it became difficult to follow the continuity of the basal membrane, owing to the invasion of the multiplied epithelial cells toward the inner part. The medullary cells increased remarkably in number at Stage I;, and the proximal half of the gonads was full of these epithelium-originated cells (Fig. 7). The medul- lary cells were connected to each other by the desmosomes. At this stage of gonadal de- velopment, a close similarity in ultrastructure was Observed between the cortical and medul- lary cells (Fig. 8). Thus, the medulla of the primordial gonads was formed by epithelial proliferation, and no cellular intrusion from the mesonephric region or else to the gonadal primordium was observed at all during the undifferentiated gonads. Invasion of the blood vessels was not detected in the sexually indif- ferent gonads. The vessels appeared at and after Stages T, and Q,. 594 H. IWASAWA AND K. YAMAGUCHI Gonadal Development of Xenopus 595 Fics. 5—7. Cross sections of sexually indifferent gonads (Stage I;). Epithelial cells of hilum region (arrow) multiply and penetrate inner part, and form medulla (M). i z Medullary formation pro- gresses in order of 5,6 and 7. C: cortical cells. Cells in the mesonephric region Three characteristic types of cells were found among the mesonephric tubules with respect to ultrastructure during Stages I,-I; (Fig. 9). (a) Amoeboid-like cell which contains irregularly shaped inclusions of medium electron-density. Cells of this type were mnumerously seen. (b) Heterochromatin-rich cell which consists of a large heterochromatin-rich nucleus and small amounts of poorly differentiated cytoplasm. (c) Chromaffin cell in which numerous very electron-dense small granules are seen within the cytoplasm. As for the ultrastructure, no similarity was seen at all between the medullary cells and mesonephros-distributed cells. Testicular differentiation Stage T, The germ cells were enveloped by Fics. 1-4. Gonadal primordia of Stage I,. Magnification of square shown in Fig. 1. epithelium is pulled into inner part of genital ridge. Another cross section of genital ridge. coelom. Arrow: hilum region. tightly with complicated surface. Unit of numerals written above bars is ym. Genital ridge suspending in coelom. No germ cells are seen in this cross section. Basal membrane (large arrow) lining coelomic medullary cells. The epithelial cells in the distal region of the primordial testes were rather round and arranged like the beads of a rosary (Fig. 10). Stage T, The rudiments of the seminiferous tubules were developing in all regions of the primordial testes, i.e., in a cross section, a small number of the spermatogonia enveloped by the medullary cells were seen to be surrounded together by other medullary cells, and the inter- stices among the tubular rudiments were filled with medullary cells also (Fig. 11). Unlike from the oogonia in the primordial ovaries at this age, the contact between the interdigitated surfaces of the spermatogonia and their enveloping cells was very tight as seen in the sexually indifferent gonads (Fig. 12). Stage T, Rudiments of the seminiferous tubules were formed. Epithelial cell strata were seen on the circumference of the testes (Fig. 13). Fig. 1. Fig. 2. Small arrow: desmosome. Fig. 3. Germ cell (G) enveloped by epithelial cells suspends in Fig. 4. Germ cell (G) and epithelial cells (E) contact 596 H. IWASAWA AND K. YAMAGUCHI Fic. 8. Cross section of sexually indifferent gonad (Stage I,). Medullary cells (M) and cortical cells (C) are very similar in ultrastructure. Fic. 9. Various types of cells found in mesonephric region of Stages I,-I C: chromaffin cell, H: heterochromatin-rich cell, N: nephrostome. . A: amoeboid-like cell, Gonadal Development of Xenopus 597 Fic. 10. Earlier stage of testicular differentiation (Stage T,). Germ cells are enveloped by medullary cells. Cellular intrusion from extra- gonadal region is not seen. Throughout the process of testicular differ- entiation, the intrusion of cells or tissues from the mesonephric region or else to the gonads was not seen excepting the blood vessels. Ovarian differentiation Stage O, Degenerative changes were occur- ring in some medullary cells, and the ovarian cavity appeared in the central part of the medulla. Shortly the ovarian cavity was lined with the monolayered cells derived from the medullary cells, but narrow spaces were seen between this cellular sheet and the cortex of the primordial ovary (Fig. 14). Stage O, Follicle cells, which arose from the somatic cells of the cortical region, enveloped oogonia. The ultrastructure of the follicle cells was very similar to that of the ovarian cavity- lining cells. The contact between the oogonia and the follicle cells was rather loose (Fig. 15). Stage O; Nests of oocytes, which were in the early prophase stages, were seen (Fig. 16). Intercellular bridges were observed between these oocytes. The characteristic ultrastructure of steroid- producing cells was not detected throughout the gonadal development described above. DISCUSSION The results of this study seem to indicate that the following two theories, which have been accepted by most investigators, are not correct: (a) the dual embryological origin of medullary cells and cortical ones in sexually indifferent gonads and (b) the invasion of blastemal cells from extra-gonadal regions during testicular differentiation. Recently, it was shown, in the tree frog Rhacophorus arboreus which belongs to a sexually semidifferentiated type [14], that the wall of the seminiferous tubules is not formed by blastemal cells which invade from extragonadal regions, but by reactivated epithelial cells which formerly lined the ovarian cavity [15]. It is conceivable that this phenomenon indirectly supports the present findings. It may be said that the elucidation of the mechanism by which the activation of the medullary cells occurs is one of the most fundamental problems in gonadal sex differentiation in anurans. The present results obtained by light microscopy are essentially the same as those described in previous papers [5,11] excepting those on the origin of the medullary cells. Witschi considered that all the steroid-producing tissues are derived from mesonephrogenic cells [16]. The previous descriptions that mesonephric [5] or interrenal 598 H. IWASAWA AND K. YAMAGUCHI Fic. 11. Cross section of testicular primordium (Stage T,). developing. Fic. 12. Stage T,- digitated surface. Fic. 13. Cross section of testicular primordium (Stage T;). Rudiments of seminiferous tubules are Germ cells (G) and medullary (epithelial) cells (E) contact very tightly with inter- Rudiments of seminiferous tubules are formed. Each germ cell is enveloped by medullary cells. blastemas [11] invade the primordial gonads might be influenced by the above-mentioned hypothesis. Lepori reported that the medullary cells of the primordial gonads originate from the hilum epithelium of the proximal side of the dorsal root of the mesentery (but from the epithelia of both sides of the hilum), and that the interrenal blastemas invade the primordial gonads in the process of testicular differentiation in anurans [17]. His opinion, however, is denied by the present electron microscopy findings. Thus, ultrastructural studies seem to be a powerful aid in the elucidation of events which occur during gonadal differentiation. In some mammalian species, it has been shown that blastemal cells, seen beneath the coelomic epithelium of the genital ridge, are of mesonephric origin [18, 19]. However, the process of gonadal development in anurans is much simpler than that in mammals. Furthermore, the participation of the mesonephrogenic cells in gonadal formation has been denied in teleosteans [20], and, needless to say, in lampreys [21]. Further extensive studies seem to be needed to formulate a coherent theory on the subject of gonadal differentiation in vertebrates. In the present observation, a difference according to sex in contact between the germ cells and their enveloping cells was recognized in the process of sex differentiation. In Xenopus laevis, gonial proliferation is remarkable in genetic females from just before the beginning of gonadal sex differentiation, so that, as far as larvae of the same age are concerned, gonia are noticeably more numerous in the ovaries than in the testes [13]. Furthermore, oocytes in the prophase of the first meiotic division appear at Stage O., as described in the present paper. It is conceivable that gonial proliferation and/or advance into meiosis are concerned with the grade of cellular contact between the gonia and their enveloping cells. REFERENCES 1 Witschi, E. (1957) The inductor theory of sex differentiation. J. Fac. Sci. Hokkaido Univ., Ser. VI, 13: 428-439. 2 Vannini, E. and Sabbadin, A. (1954) The relation of the interrenal blastema to the origin of the somatic tissue of the gonad in frog tadpoles. J. Embryol. exp. Morph., 2: 275-289. 3 Merchant-Larios, H. (1978) Ovarian differenti- Gonadal Development of Xenopus 599 Fic. 14. Part of cross section of ovarian primordium (Stage O,). Ovarian cavity (OV) is lined with medulla-derived cells. Fic. 15. Stage O,. Contact between germ cells (G) and epithelial cells (E) is rather loose. g: germinal plasm, Arrow: desmosome. Fic. 16. Cross section of ovarian primordium (Stage O,;). Nests of oocytes are seen. ation. In “The Vertebrate Ovary”. Ed. by 5 Witschi, E. (1971) Mechanisms of sexual dif- R. E. Jones, Plenum Press, New York, pp. 47-81. ferentiation. Experiments with Xenopus laevis. 4 Merchant-Larios,H. and Villalpando, I. (1981) In “Hormones in Development’. Ed. by Ultrastructural events during early gonadal M. Hamburgh and E. J. W. Barrington, Appleton development in Rana pipiens and Xenopus laevis. Century Crofts, New York, pp. 601-618. Anat. Rec., 199: 349-360. 6 Al-Mukhtar, K.A.K. and Webb, A.C. (1971) 600 10 14 An ultrastructural study of primordial germ cells, oogonia and early oocytes in Xenopus laevis. J. Embryol. exp. Morph., 26: 195-217. Kalt, M. R. (1973) Ultrastructural observation on the germ line of Xenopus laevis. Z. Zellforsh., 138: 41-62. Ikenishi, K. and Kotani, M. (1975) Ultra- structure of the ‘germinal plasm’ in Xenopus embryos after cleavage. Develop., Growth and Differ., 17: 101-110. Kamimura, M., Ikenishi, K., Kotani, M. and Matsuno, T. (1976) Observations on the mig- ration and proliferation of gonocytes in Xenopus laevis. J. Embryol. exp. Morph., 36: 197-207. Wylie, C. C. and Heasman, J. (1976) The for- mation of the gonadal ridge in Xenopus laevis. I. A light and transmission electron microscope study. J. Embryol. exp. Morph., 35: 125-138. Vannini, E. (1956) The development of the gonadal system and adrenal glands. In ““Normal Table of Xenopus laevis (Daudin)’. Ed. by P.D. Nieuwkoop and J. Faber, North-Holland Publ. Co., Amsterdam, pp. 137-139. Nieuwkoop, P. D. and Faber, J. (1956) Normal Table of Xenopus laevis (Daudin). North-Holland Publ. Co., Amsterdam. Yamaguchi, K. and _ Iwasawa, H. (1981) Histological studies on gonadal development and sexual difference in germ cell number in Xenopus laevis. Zool. Mag., 90: 456. Iwasawa, H. (1969) Variation of the mode 16 17 18 19 20 21 H. IWASAWA AND K.~ YAMAGUCHI of sex differentiation in the tree-frog, Rhacophorus arboreus. Sci. Rep. Niigata Univ. Ser. D, 6: 141-— 153. Takasu, T. and Iwasawa, H. (1983) Ultrastruc- tural studies on the testicular differentiation in the frog Rhacophorus arboreus. Zool. Mag., 92: 501. Witschi, E. (1962) Embryology of the ovary. In “The Ovary”. Ed. by H. G. Grady and D.E. Smith, Williams & Wilkins, Baltimore, pp. 1-10. Lepori, N. G. (1980) Sex Differentiation, Her- maphroditism and Intersexuality in Vertebrates Including Man. Piccin Editore, Padua. Wartenberg, H. (1981) Differentiation and devel- opment of the testes. In “The Testis’. Ed. by H. Burger and D. de Kretser, Raven Press, New York, pp. 39-80. Wartenberg, H. (1981) The influence of the mesonephric blastema on gonadal development and sexual differentiation. In “Development and Function of Reproductive Organs’. Ed. by A. G. Byskov and H. Peters, Excerpta Medica, Amsterdam, pp. 3-12. D’Ancona, U. (1956) Morphogénése et diffé- renciation sexuelle chez les poissons téléostéens. Bull. Soc. Zool. Fr., 81: 219-229. Hardisty, M. W. (1965) Sex differentiation and gonadogenesis in lampreys (Parts I and II). J. Zool., 146: 305-387. ZOOLOGICAL SCIENCE 1: 601-607 (1984) Differential Cell Proliferation and Morphogenesis in the Developing Adenohypophysis of the Fetal Rat YUICHI G. WATANABE Department of Anatomy, Sapporo Medical College, Sapporo 060, Japan ABSTRACT — A quantitative study was performed on the volume change and the rate of mitosis in the adenohypophysial primordium of rats. The volume of the primordium was measured on every other horizontal section using the paper weight method. On day 12.5 of gestation, the sectioned areas from the upper part of the adenohypophysial primordium were slightly larger than those from the lower part. The mitotic index was also higher in the upper part of the primordium. When Rathke’s pouch closes, its bottom undergoes a noticeable thickening from which the so-called lateral lobes grow outward on day 13.5. Between days 12.5 and 13.5, the sectioned areas of the lower part of the primordium increased 4- to 8-fold in contrast to a 2-fold increase in the upper half. Despite this pronounced volume increase, mitotic activity was very low in the lower part, and even more rare within the lateral lobes. Thus, local thickening, bulging and outgrowth observed at the bottom of the rat adenohypophysial primordium are not the direct results of cell proliferation at this site, but rather they seem to be formed through a translocation of cells or cell sheets such as described in other epithelial organ rudiments. On day 13.5, the posterior wall of the primordium showed a higher mitotic activity when compared to (©) 1984 Zoological Society of Japan the anterior wall. dium as well as the formation of Atwell’s recess. INTRODUCTION In general, the outgrowth of confined portions of an organ rudiment is very important not only for the determination of the future shape of the organ but also for the process of cytodifferentia- tion, since microenvironments surrounding such outgrowths often differ from those of other parts [20]. In the case of the adenohypophysis, the bilateral processes or the lateral lobes, are known to be formed at the bottom of Rathke’s pouch soon after its closure [2, 5, 9, 12, 16, 22]. The lateral lobes extend anteriorly to form tongue-like processes above which a rich amount of mesen- chymal tissue invades [17]. At present, little information is available as to the mechanism of such a morphological change. One _ possible explanation is that cell proliferation occurs ac- tively in the outgrowing tip and thus contributes to its extension. Available data, however, do not Accepted April 5, 1984 Received October 24, 1983 Such differential growth is likely to contribute to a bending of the entire primor- favor this assumption. Cell proliferation in the rat adenohypophysial rudiment in vitro has been known to be stimulated by the presence of the diencephalic floor [6, 18] which is located on the opposite side of the lateral lobes. In fact, Wilson [21] has shown that mitotic activity is higher in the portion in contact with the brain than in the lower part of the mouse adenohypophysial pri- mordium. As the lateral lobes grow outward, moreover, the distance between their tip and Rathke’s lumen becomes larger; this makes it difficult for the cells in the tip to reach the lumen where mitosis usually occurs [9, 12, 21]. There- fore, it seems likely that sites other than the out- growing tip undergo active cell division. To test this possibility, a quantitative study was undertaken to determine the relationship between the regional difference in the mitotic rate and the change in tissue volume in the rat adenohypophysial pri- mordium. In this study, rat fetuses at day 12.5 and 13.5 of gestation were used since the bottom of the primordium shows the most pronounced volume change during this period [12, 16, 17]. 602 Y.G. WATANABE MATERIALS AND METHODS Animals The present study was carried out using timed pregnant female rats of the Sprague-Dawley strain. Day 0.5 of gestation was considered to be the day when sperm were found in vaginal smears. Tissue preparation Three pregnant rats at day 12.5 and 13.5, re- spectively, were anesthetized and 35 fetuses were immediately fixed in Bouin’s solution for 3-6 hr. After fixation, the heads were cut and trimmed under a dissecting microscope to facilitate subse- quent sectioning along a fixed plane. They were then dehydrated, embedded in paraplast and cut serially at 8 um thickness along a plane as shown in Figure 1. They were referred to as “horizon- Ps eo ~< AR Fic. 1. Diagrams of midsagittal sections through the hypophysial primordium of fetal rats on days 12.5 (a) and 13.5 (b) of gestation. Lines (X, Y, Z) indicate the plane of sectioning. Anterior to the left. AR, Atwell’s recess; L, lateral lobe; N, neurohypophysial primor- dium. tal” sections in this study. Every other section was used to measure the adenohypophysial area and the number of mitotic cells. Three fetuses of both days 12.5 and 13.5 were processed for sagittal section. Among horizontally cut specimens, those which were at developmental stages too retarded or advanced were discarded. Measurements were finally performed on 12 hypophysial primordia from day 12.5 and 8 from day 13.5. For general observations of the developing hypophysis, several fetuses at day 13.0 were also fixed and their heads cut sagittally. Measurement of adenohypophysial area in sections The horizontal sections of the adenohypophysial primordium were thrown and drawn on white paper using a camera lucida at x400. After careful cutting, these drawings were weighed on a chemical balance. Obliquely sectioned areas (Fig. la, X and Y) of the day 12.5 primordium were corrected before weighing. At level X of Figure la, a line parallel to the inner contour was drawn and the outer area was removed (Fig. 2a). Below ta tp Fic. 2. Correcting method of obliquely cut portions of the adenohypophysial primordium of day 12.5. a) Diagram of a horizontal profile cut at level X of Fig. 1. Striated area was removed before measuring sectioned area. Anterior to the left. b) Part of a horizontal profile below level Y of Fig. 1 showing the corrected thickness of the anterior (t,) and posterior (t,) wall by com- paring the t, and t, values of the 20th section (Table 1) with those of the 26—40th sections. Stippled area was removed. c) Diagram of the rectangular area of Fig. 1 indicating how the thickness (t) of the adenohypophysial wall below level Y was measured in a sagittal section. level Y of Figure la, or beyond 208 vm from the top of the primordium, horizontal profiles were corrected using the following method: a repre- sentative median sagittal section of a day 12.5 primordium was photographed and enlarged to a final magnification of x 400. On this photograph, 20 horizontal lines corresponding to every other sectional plane were drawn at intervals of 16 ym. Then the relative thickness of the adenohypophy- sial wall (Fig. 2c, t) was measured (Table 1). By applying such values to the 20th and the 26—40th horizontal profiles of each specimen, correction of the thickness was performed as illustrated in Figure 2b. Drawings of the day 13.5 primordium were weighed without correction since their wall Mitosis and Morphogenesis of Hypophysis 603 TABLE |. The relative thickness of the anterior and posterior walls of sagittally cut rat adenohypophysial primordium on day 12.5 of gestation for correction of horizontal profiles Distance from Relative thickness oa ne top o n no. primordium (sm) bey wall acy wall in Lt eer 160 10g" 1.00 20 208 0.91 0.32 26 224 Olay 0.19 28 240 0.62 0.17 30 256 0.30 0.15 32 272 0.19 0.15 34 288 0.15 0.15 36 304 O15 0.15 38 320 0.15 0.15 40 * The order of the section numbers is from top to bottom of the primordium. ** The thickness of a perpendicularly cut wall in the middle part of the primordium, i.e., 160 ~m from its top, was expressed as 1.00. f (x 104 pm? ) of section Area be 80 160 240 320 Distance from the top of = primordium (pm) Fic. 3. Volume of the adenohypophysial primordia of fetal rats on day 12.5 (clear circle, n=12) and day 13.5 (solid circle, n=8) as expressed by the area of horizon- tal sections. Vertical bars indicate the standard error. LL shows where the lateral lobes are included. was cut almost perpendicularly (Fig. 1b). A 100 x 100 wm square was thrown and drawn on paper with the aid of an objective micrometer; this After staining with haematoxylin, the cells from served to calculate the actual area of each section. the late prophase to telophase were counted on The mitotic index 604 Y.G. WATANABE each section. In each section, the total number of mitotic figures was then expressed in terms of 100 profiles of cells. RESULTS Volume changes Figure 3 shows the area of every other section of adenohypophysial rudiment in 12.5- and 13.5- day-old rat fetuses. On day 12.5, the sectioned areas of the upper half of the primordium were slightly larger than those of the lower half. This is mainly due to differences in the thickness of the primordial wall between the two regions, i.e., the wall was composed of 2-3 layers of cells in the upper part, whereas it gradually decreased in thickness to a single layer of stomodeal epithelium (Fig. 4, a and b). On day 13.5, on the contrary, the lower half of the primordium became far thicker (Fig. 4, c and d). The sectioned area was largest at the bottom of the primordium where the lateral lobes were formed (Fig. 3). In this region, the area of the sections increased 4- to 8-fold between days 12.5 and 13.5. On the other hand, the sectioned areas of the upper part of the pri- mordium increased only 2-fold during the same period. The wall of the upper half of the pri- mordium remained almost unchanged in thickness between days 12.5 and 13.5 (Fig. 4, a and c). Mitosis The mitotic index of the adenohypophysial primordia on days 12.5 and 13.5 is given in Figure 5. At both stages of development, mitosis was frequent in the upper part of the primordium (Fig. 4, a and c). Mitotic figures were rarely observed within the lateral lobes on day 13.5 (Fig. Fic. 4. Horizontal sections of adenohypophysial primordia of fetal rats on day 12.5 (a, b) and day 13.5 (c, d) of gestation. in a and c, and 256 pm in b and d. The distances from the top of the primordium to these sections are 96 »m On day 12.5, the wall of the adenohypophysial primordium is thicker in a section from the upper part (a) when compared to that at a lower level (b). On day 13.5, a marked volume increase occurs in the lower region (d) of the primordium where the lateral lobes (arrows) grow outward. In contrast, only a slight volume increase is seen in the upper part of the primordium (c). x 113. Mitosis and Morphogenesis of Hypophysis index Mitotic no 80 160 240 320 Distance from the top of primordium (pm ) Fic. 5. The mitotic index of each plane of horizontal section through the adenohy- pophysial primordia of fetal rats on day 12.5 (clear circle, n=12) and day 13.5 (solid circle, n=8). Vertical bars indicate the standard error. 8 * p<0.05 r ** p<0.01 *** P<0.001 index Mitotic 80 160 240 320 Distance from the top of primordium (pm) Fic. 6. The mitotic index of the anterior (solid circle) and the posterior (clear circle) wall of the adenohypophysial primordia of fetal rats on day 13.5. Vertical bars indicate the standard error. n=8. 605 606 Y.G. WATANABE 4d). Figure 6 compares the mitotic index between the anterior and posterior wall of the adenohy- pophysial primordium on day 13.5. The mitotic index was higher in the posterior wall when com- pared to the anterior wall. Such a difference was not observed on day 12.5. DISCUSSION Various organ rudiments are known to change their shape during development. In some tis- sues, this may be ascribed to a change in the shape of the individual cell where microtubules or microfilaments accumulate in a localized region of cytoplasm [7]. In other tissues, on the other hand, cell proliferation itself may be responsible for morphogenetical alterations. In chick blasto- derms, Spratt [15] suggested that active cell pro- liferation of a localized region forced the entire cell sheet to extend radially from the growing center. Smuts et al. [14] postulated that thicken- ing and evagination of the thyroidal placode result from a pressure generated by differential cell proliferation of the neighboring pharyngeal epi- thelium. Observation of limb bud mesenchyme in vitro by time-lapse movies has demonstrated that no cellular activities other than cell division occur when limb buds grow outward [11]. In this study, the upper part of the adenohy- pophysial primordium exceeded the lower part in mitotic activity on both days 12.5 and 13.5. In vitro, | found that when the primordium was horizontally cut into two parts on day 12.5 and cultivated separately, the upper half grew far better than the lower one [18]. Jn vivo, on the contrary, the tissue volume increase was more pronounced in the lower half of the primordium. This discrepancy cannot be explained without postulating a translocation of cells or cell sheets as suggested by Spratt [15]. A rapid extension of the primordial wall may partly cause an upward rotation of the neurohypophysial rudiment [12]. It may be possible, however, that such epithelial growth is mostly related to a downward transloca- tion of primordial cells resulting in a thickening and budding at the bottom of the primordium (Fig. 7, A-C). After day 13.5, such a thickening and outgrowth of the lateral lobes are followed by —_ A B Cc Fic. 7. Diagrams showing possible translocation (arrows) of cells or cell sheets in the developing adenohypophyses of fetal rats on day 12.5 (A), day 13.0 (B) and day 13.5 (C). the development of the pars distalis and pars tuberalis [2, 5, 9, 12]. Mitotic figures, however, were only infrequently observed in this morpho- logically active site, i.e., the lateral lobe, in ac- cordance with the observation of Kerr [9]. Whether or not a localized cell proliferation occurs at the distal ends of outgrowing or branch- ing rudiments is different in various organs. For example, Bernfield and Banerjee [3] and Bernfield et al. [4] observed a greater concentration of dividing cells at the branching tips of developing salivary gland epithelium. However, mitotic ac- tivity was not enhanced at the growing tips of tracheal buds [19], or lung buds [1]. In growing chick limb buds, there was also no difference in mitotic activity between the distal and proximal regions [8, 13]. The infrequent occurrence of mitosis in lateral lobes growing outward may again support the importance of the afore- mentioned translocation of cells or cell sheets. If this assumption holds true, however, it cannot offer a sufficient explanation for the bending of the adenohypophysial primordium which begins from day 13.5 [12, 17]. In this study, mitotic activity was higher in the posterior wall than in the anterior on day 13.5 (Fig. 6). Such differential growth of primordial wall is likely to cause a bimetal-like force making the anterior wall concave (Fig. 7C). This seems to facilitate the formation and subsequent deepening of Atwell’s recess which is indispensable for the invasion of connective tissue including blood capillaries to the adenohy- pophysis [12, 17]. Of course, factors other than the differential growth of primordial wall should Mitosis and Morphogenesis of Hypophysis be considered. An ongoing study (Watanabe, unpublished) has demonstrated that mesenchyme associated with the posterior wall on days 12.5 and 13.5 is more adhesive than that surrounding the anterior wall. The present report extends my previous in vitro data [18] indicating a change in the localization of ACTH and LH cells from the upper to lower portion of the adenohypophysial primordium during their determination and hormone accumula- tion. In studying the developing adenohypophysis in Xenopus tadpoles, Nyholm and Doerr-Schott [10] have actually observed a shift in the immuno- reactive MSH cells when they are grouped to form the definitive pars intermedia. It remains unclear as to what extent such translocation of cells is necessary for the comprehensive development of the adenohypophyses in various species. REFERENCES 1 Alescio, T. and di Michele, M. (1968) Relation- ship of epithelial growth to mitotic rate in mouse embryonic lung development in vitro. J. Embryol. Exp. Morph., 19: 227-237. 2 Atwell, W. J. (1926) The development of the hypophysis cerebri in man, with special reference to the pars tuberalis. Am. J. Anat., 37: 159-193. 3 Bernfield, M. R. and Banerjee, S. D. (1972) Acid mucopolysaccharide (glycosaminoglycan) at the epithelial mesenchymal interface of mouse embryo salivary glands. J. Cell Biol., 52: 664-673. 4 Bernfield, M. R.,; Banerjee, S. D. and Cohn, R. H. (1972) Dependence of salivary epithelial mor- phology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. J. Cell Biol., 52: 674-689. 5 Brahms, S. (1932) The development of the hypo- physis of the cat (Felis domestica). Am. J. Anat., 50: 251-281. 6 Daikoku, S., Chikamori, M., Adachi, T. and Maki Y. (1982) Effect of the basal diencephalon on the development of Rathke’s pouch in rats: A study in combined organ culture. Dev. Biol., 90: 198-202. 7 Goldin, G. V. (1980) Towards a mechanism for morphogenesis in epithelio-mesenchymal organs. Q. Rev. Biol., 55: 251-265. 8 Jannes, M. Y. and Searls, R. L. (1970) Changes in rate of cellular proliferation during the differ- entiation of cartilage and muscle in the mesenchyme of the embryonic chick wing. Dev. Biol., 23: 10 11 12 13 15 16 17 18 19 20 21 22 607 136-165. Kerr, T. (1946) The development of the pituitary of the laboratory mouse. Q. J. Microsc. Sci., 87: 3-29. Nyholm, N. E. and Doerr-Schott, J. (1977) De- velopmental immunohistology of melanotrophs in Xenopus laevis tadpoles. Cell Tissue Res., 180: 231-239. Reiter, R.S. and Solursh, M. (1982) Mitogenic property of the apical ectodermal ridge. Dev. Biol., 93: 28-35. Schwind, J. L. (1928) The development of the hypophysis cerebri of the albino rat. Am. J. Anat., 41: 295-319. Searls, R.L. and Janners, M. Y. (1971) The initiation of limb bud outgrowth in the embryonic chick. Dev. Biol., 24: 198-213. Smuts, M.S., Hilfer, S. R. and Searls, R. L. (1978) Patterns of cellular proliferation during thyroid organogenesis. J. Embryol. Exp. Morph., 48: 269-286. Spratt, N. T. Jr. (1963) Role of the substratum, supracellular continuity and differential growth in morphogenetic cell movement. Dev. Biol., 7: 51-63. Svalander, C. (1974) Ultrastructure of the fetal rat adenohypophysis. Acta Endocrinol., 76: Suppl. 188: 1-113. Szab6, D. and Csanyi, K. (1982) The vascular architecture of the developing pituitary-median eminence complex in the rat. Cell Tissue Res., 224: 563-577. Watanabe, Y. G. (1982) An organ culture study on the site of determination of ACTH and LH cells in the rat adenohypophysis. Cell Tissue Res., 227: 267-275. Wessells, N. K. (1970) Mammalian lung develop- ment: Interactions in formation and morpho- genesis of tracheal buds. J. Exp. Zool., 175: 455-466. Wessells, N. K. (1977) Extracellular materals and tissue interactions. In ‘Tissue Interactions and Development”. Benjamin/Cummings, California, pp. 213-229. Wilson, D. B. (1980) Patterns of proliferation in the hypophysis of the mouse embryo. A quantitative autoradiographic study. Anat. Em- bryol., 159: 101-113. Yoshimura, F., Harumiya, K. and Kiyama, H. (1970) Light and electron microscopic studies of the cytogenesis of anterior pituitary cells in perinatal rats in reference to the development of target organs. Arch. Histol. Jap., 31: 333-369. “i /H ws { i ‘ ial teh rig gniierab ved Ath, Fig: | an, eeodsak ele. ON aT a | : en | mole ile 4 “i ee tie em | veuee ny eines "a = rr is bs tc ) | | é | | x e. Spy ty —~ : ae bo& t a. ae a. 3 hi 4 . ; 2 = fin qT fois Jere: of ipciiage rh nid é U ’ ; \ a = : | 1 % J ; . : ‘ 4 # | a — 7 Le | ZOOLOGICAL SCIENCE 1: 609-615 (1984) © 1984 Zoological Society of Japan Uptake of [*°S] Sulphate by Xenopus Cartilage: The Influence of Growth Hormone and Prolactin TAKBHISA IsHiI! and SAKAE KIKUYAMA2 Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 160, Japan ABSTRACT — Hypohysectomized juvenile Xenopus were injected with growth hormone (GH) or prolactin (PRL) of either ovine or bullfrog origin and the growth-promoting activity of these hor- mones was measured by monitoring the uptake of [?*S]sulphate by the xiphisternal cartilage in vitro. Analysis of the labelled cartilage revealed that the acid mucopolysaccharide fraction contained about 60-80 % of the label and that most of them were incorporated into chondroitin sulphates. All of the hormones tested enhanced the [**S]sulphate uptake dose-dependently. Among them bullfrog GH was most effective, then followed ovine GH and ovine PRL. Bullfrog PRL was far less effective than other three. The sensitive assay for frog GH developed in the present experiment may be applicable to the assay for somatomedin-like activity and contribute to the analysis of the mode of action of GH in amphibians. INTRODUCTION Since Etkin and Lehrer [1] reported that auto- transplantation of the pituitary gland of Rana pipiens tadpoles to an ectopic site resulted in an excess growth, hormonal regulation of growth in anuran larvae has received a considerable atten- tion. It is now widely accepted that both mam- malian and amphibian prolactin(PRL)s rather than growth hormone(GH)s have a potent growth- promoting effect especially on the larval tissue such as the tail [2-4]. In contrast, growth of postmetamorphic anurans seems to be regulated by GH rather than PRL judging from the effect of mammalian GH and PRL [5, 6] or electrophoretically separated amphib- ian GH and PRL [7] on the growth of juvenile anurans. In these experiments growth-promoting activity has been evaluated by the increase in body length, body weight or femur length after the hormonal treatment for a certain period. In mammals, extensive works on growth regu- lated by GH and its mediator, somatomedin have Accepted February 15, 1984 Received January 17, 1984 * Present acdress: Bioscience Laboratory, Mitsubishi Kasei Research Center, Yokohama 227, Japan. ? To whom all correspondence should be addressed been carried out [8]. In these experiments, [°*S]- sulphate uptake by cartilage in vitro is often adopted as a parameter of growth [9]. The purpose of the present experiment is to measure growth-promoting activity of GH and PRL of both mammalian and amphibian origin by monitoring the in vitro uptake of [°*S]sulphate by the xiphisternal cartilage from the hormone-treated hypophysectomized juvenile Xenopus. To assess the growth-promoting activity of these hormones and to establish the method for assaying growth- promoting activity in vitro seem to be necessary steps for the understanding of the mode of action of growth-promoting hormone(s) in anurans. MATERIALS AND METHODS Animals Juvenile African toads, Xenopus laevis weighing 3-4g were used. They were hypophysectomized and kept in tap water at 22°C. Food (Tubifex) was provided in surplus throughout the experiment. Hormones Ovine GH(NIH-—GH-S11) and PRL(NIAMDD-oPRL-15) were supplied from NIH. Amphibian PRL was purified from bull- frog anterior pituitary glands as previously de- scribed [10]. Amphibian GH was prepared from a fraction obtained during the purification of PRL. 610 Acid acetone extracts prepared from the acetone- dried pituitary powder were subjected to DEAE cellurose collumn equilibrated with 0.01 M Tris HCl buffer (pH 7.4). PRL was adsorbed on the column [10]. Non-adsorbed fraction exhibited an electrophoretic pattern on polyacrylamide gel which is similar to that of GH separated from bullfrog pituitary glands by Farmer et al. [11]. This fraction was applied to Sephadex G—100 column equilibrated with 0.1 M NH,HCO;. The protein eluted with Ve/Vo ratio of 2.0 was used as GH preparation in the present experiment. It has been acertained by the preliminary experiment that this preparation posessed as much an activity as ovine GH in increasing body weight of hypophysecto- mized juvenile Xenopus. Each hormone was dissolved in 0.6% saline and given to animals intraperitoneally at 10-11 a.m. every other day. The injection volume was 0.05 ml. Incorporation of [?*S|sulphate into cartilage Twenty hours after the last injection, animals were sacrificed. From each animal xiphisternal car- tilage was dissected out, cut into 0.8-1.0 mm sec- tions and kept in ice-cold sterile 67% Eagle’s minimum essential medium (Nissui Seiyaku Co.) supplemented with 18 mM NaHCO; and 5mM Hepes until use. Cartilage sections from 2 animals were pooled, transferred to a glass vial containing 2 ml medium added with 5 wCi [°*S]- sulphate (carrier-free, The Radiochemical Center, Amersham) and incubated at 23°C for varying time in a Dubnoff-metabolic shaking incubator gassed with 95% O.-5% CO,. After incubation, the cartilage sections were immersed in boiling water for 10min. Sections were then transferred into a small basket made of stainless steel and soaked in saturated sodium sulphate overnight. Then they were washed in running water for 2 hr and immersed in distilled water for 1 hr [12]. After drying in an oven at 55°C for 1 hr, the sec- tions were weighed and solubilized in 250 pl of 98% formic acid at 110°C in glass vials with plastic caps. After solubilization, 2.5 ml scintil- lation fluid (4 g PPO, 0.1 g POPOP, 1 liter toluene, 0.5 liter Triton X—100) was added to the vial. The radioactivity was measured in a liquid scintillation counter (Aloka, LSC-700). T. Isham AND S. KIKUYAMA Analyses of regression and variance were ap- plied to certain data and the slope (radioactivity/ log dose unit) of the dose response curve and the index of precision (standard deviation/slope) were determined. Identification of *°S-labelled acid mucopolysac- charides The acid mucopolysaccharide frac- tion was separated from the dried cartilages weighing 20mg which had been incubated with [°°S]sulphate according to the method of Iwata and Urist [13] presented below schematically. The acid mucopolysaccharide fraction (C) thus ob- tained was chromatographed according to the method of Marzullo ef al. [14]. C fraction was dissolved in 1 ml DW. An aliquot (10 wl) was spotted on a silica gel plate (Kiesel gel 60, Merck). Several standard acid mucopolysaccharides were also applied on the plate. The solvent consisted of n-propanol, concentrated ammonium hydroxide and water (40/60/5, v/v/v). The chromatography was performed at 4°C for 5hr. The area where the samples were run was cut in 1 cm width. From the strip the silica gel was scraped off by every 5mm from the spot to the buffer front and transferred to a counting vial. To each vial 0.5 ml DW was added and left overnight. The radio- activity was measured as described above after the addition of 3ml scintillation fluid. The standard acid mucopolysaccharides were detected by spraying orcinol (1 %)-H.2SO, (50%) solution on the plate. °S-labelled xiphisternal cartilages Pronase (4.5 PUK/mg cartilage) digestion Addition of NaOH (final 0.3 N) Neutralization with 6 N HCl Addition of TCA (final 5%) Centrifugation Precipitate (A) Supernatant Dialysis | Dialysate (B) Retaintate Addition of 3 volumes of ethanol saturated with CH,;COONa Centrifugation | Precipitate (C) Supernatant (D) Sulphate Uptake by Xenopus Cartilage 611 RESULTS Figure 1 shows the effect of hypophysectomy on the uptake of [*S]sulphate by xiphisternal carti- lages in vitro. During the first 3 days after hy- pophysectomy, incorporation of [**S]sulphate into the cartilage declined rapidly. After that the changes were very little. Accordingly, in the subsequent experiments the animals were used 3-4 days after hypophysectomy. x10° 3 3550, uptake (cpm/mg cartilage) nN Days after hypophysectomy Fic. 1. Effect of hypophysectomy on the uptake of [*S]sulphate by xiphisternal cartilages of Xenopus in vitro. Animals were sacrificed on various days after the operation. The cartilage sections were incubated in a medium containing [?*S]sulphate for 12 hr at 23°C. The uptake of the isotope by the cartilage sections was deter- mined as described in the text. Each point and vertical line represent average of 4 determi- nations and standard error of the mean. The cartilage sections from the ovine GH- treated and saline-injected Xenopus were incubated for varied time at 23°C in the presence of [°°S]- sulphate. During the first 12 hr, the incorporation of the isotope into the cartilage sections was much faster in the samples from the treated animals than in the samples from the controls. After that, the rate of incorporation in the hormone-treated samples did not differ from that in the saline- injected ones (Fig. 2). In the subsequent experi- ments incubation time was 12 hr unless otherwise stated. x10 o oD i) =o | ‘. eee oO oD E F E4 = o x = 5 2 I S | WY) w ~ 0 6 12 18 Incubation time (hr) Fic. 2. Time course of the uptake of [°°S]sulphate into xiphisternal cartilages hypophysectomized Xenopus received 3 injections of 3 wg ovine GH or saline every other day. Incorporation of [?°S]sulphate into the cartilages was deter- mined as described in the text. (1, GH-treated; O, saline-injected. Each point and_ vertical line represent the mean of 4 determinations and standard error of the mean, respectively. The changes in the uptake of [°*S]sulphate by the cartilage were examined during the successive treatment with ovine GH. As shown in Figure 3, the uptake of the isotope by the cartilage from the hormone-treated animals increased during the first 3 injections and then, the uptake reached maximum. In the samples from the control animals, the uptake was invariably lower than those from the hormone-treated specimens. In further experiments, animals received 3 injections of hormone or saline. The uptake of [°°S]sulphate by the cartilage in response to the dosage of ovine GH or PRL was shown in Figure 4. It was revealed that the uptake of the isotope by the cartilage from both ovine GH-treated and ovine PRL-treated animals increased dose-dependently. The effect of a given dose of PRL was a little less than that of the same weight of GH. The slopes of the dose-response curve for GH and PRL were 2714 and 2462, re- spectively, and the index of precision for GH was 0.35 and for PRL 0.21. The sensitivity defined as 2SD above the control value was estimated to be 0.20 wg for GH and 0.26 wg for PRL. LU) w SO, uptake (cpm/mg cartilage) 35 Fic. T. IsHi1 AND S. KIKUYAMA 0 1 2 3 4 5 6 7 Injections 3. The uptake of [*°S]sulphate by xiphisternal cartilages from the animals which received ovine GH injections for varied time. Hypo- physectomized Xenopus received injections of 3 wg ovine GH or saline every other day. The uptake of [*°S]sulphate by the cartilages was determined as described in the text. 0, hormone-treated; ©, saline-injected. Each point and vertical line represent the mean of 4 determinations and standard error of the mean, (cpm/mg cartilage) x ook @ oO WwW °S0, uptake respectively. o BSS Ls) 6) 0.3 3 30 Hormone (yg) Fic. 4. Effect of ovine GH or PRL administered to hypophysectomized Xenopus on uptake of [?*S]sulphate by xiphisternal cartilages in vitro. The animals received 3 injections of either GH or PRL. The uptake of [**S]sulphate by x 10° 8 3550, uptake (cpm/mg cartilage) pS O 0.3 3 30 Hormone (yg) Fic. 5. Effect of frog GH or PRL administered to hypophysectomized Xenopus on uptake of [?°S]sulphate by xiphisternal cartilages in vitro. The animals received 3 injections of either GH or PRL. The uptake of [?*S]suphate by the cartilages was determined as described in the text. Each point and vertical line represent the mean of 4-17 determinations and standard error of the mean, respectively. @, GH-treated; Wy, PRL-treated. All responses were significantly greater than in the saline-treated controls. a, p<0.02; b, p<0.002. As shown in Figure 5, the uptake of [°°S]- sulphate by the cartilage from the frog GH- treated and frog PRL-treated animals was also increased in response to the dosage of the hor- mones. However, the response to PRL was much lower than that to GH. The slope of the dose response curve and the index of precision for GH were 3491 and 0.19, respectively, whereas the slope and the index of precision for PRL were 692 and 0.61, respectively. The estimated sensitivity was 0.15 wg for GH and 0.30 wg for PRL. Acid mucopolysaccharide fraction was obtained from the *°S-labelled cartilages of ovine GH- treated animals and _ saline-injected animals. the cartilages was determined as described in the text. Each point and vertical line represent the mean of 4-17 determinations and standard error of the mean, respectively. @, GH- treated; m, PRL-treated. All responses were significantly greater than in the saline- treated controls. a, p<0.02; b, p<0.002. Sulphate Uptake by Xenopus Cartilage 613 TABLE |. Distribution of **S-radioactivity among the fractions obtained during the separation of acid mucopolysaccharide from 35§-labelled cartilages Fraction cpm/mg dry weight Control GH-treated A 8 14 B 280 500 .; 430 2101 D 49 98 Hypophysectomized Xenopus received 3 injec- tions of 3 wg ovine GH or saline. Xiphisternal cartilage sections were incubated for 10 hr in the medium containing [**S]sulphate. The acid mucopolysaccharide fraction(C) was obtained by the procedure described in the text. x10? Radioactivity 12) 0.2 0.4 0.6 0.8 1.0 Rf Fic. 6. Chromatographic pattern of **S-labelled acid mucopolysaccharide fraction. Xiphisternal cartilages in the hypophysectomized Xenopus injected with ovine GH or saline were incu- bated with [°*S]sulphate. From the cartilages, acid mucopolysaccharides were separated by the method of Iwata and Urist [13]. The chromatography was performed by the method described in the text. O, ovine GH-treated, @, saline-injected. ChS—A, chondroitin sul- phate A; ChS-—C, chondroitin sulphate C; HA, hyaluronate; Hep, heparin; HS, heparan sulphate. Arrow indicates Rf of each standard polysaccharide. Radioactivity was expressed in terms of cpm per mucopolysaccharides from mg cartilage. About 80 and 60% radioactivity were recovered in the mucopolysaccharide fraction from the car- tilages of the GH-treated and of the saline-injected animals, respectively (Table 1). Identification of the mucopolysaccharides by thin-layer chromato- graphy was carried out. In both cases, most of the radioactivity was present in the position of chondroitin sulphate A and C (Fig. 6). DISCUSSION In mammals, it is known that GH treatment enhances the incorporation of [**S]sulphate into the cartilage when incubated in vitro [15]. This was also observed in the juvenile Xenopus. Fur- thermore, analysis by thin-layer chromatography revealed that the increase in the uptake of [°*S]- sulphate by the xiphisternal cartilage of Xenopus is related to the elevation of synthesis of chon- droitin sulphates in the cartilage. It is not surprising that ovine prolactin stimu- lated the uptake of [°*S]sulphate by the cartilage, since unlike other species of anurans, Xenopus is known to respond to mammalian PRL to show a marked body-growth [3]. On the other hand, frog PRL exhibited a far less activity in stimulat- ing the [°°S]sulphate uptake by the cartilage as compared with frog GH. Frog PRL is known to stimulate the collagen synthesis in the tadpole tail fin [16]. It has been ascertained by the prelimi- nary experiment that the bullfrog PRL used in the present experiment retains the bioactivity to stimulate the collagen synthesis markedly. There- fore, it seems to be of an intrinsic nature that frog PRL has much less potency to stimulate the incor- poration of [°*S]sulphate into the cartilage as compared with frog GH. The fact that the bull- frog PRL is far less effective than other three hormones may indicate that in Xenopus, the receptor sites which mediate this effect can dis- criminate amphibian PRL from amphibian GH but hardly discriminate ovine PRL from ovine GH. In this respect, it should be mentioned that in the eel, ovine GH and PRL as well as a teleost GH are effective in enhancing the conversion of thyroxine to triiodothyronine, teleost PRL does scarcely affect the conversion [17]. When electrophoretically separated GH or PRL while a 614 from Rana catesbeiana and Bufo marinus was in- jected to Bufo boreas, the effect of PRLs on the body growth was not conspicuous, while GHs exhibited a marked growth-promoting effect [7]. We have also observed that the frog PRL used in the present experiment has little activity to enhance the body growth of the juvenile Xenopus, while the frog GH has a prominent growth-promoting activity (unpublished data). It has been demon- strated that the frog GH preparation is essentially free of PRL contamination, when tested with the frog PRL immunoassay system [18]. Although assays for other pituitary hormones in the GH preparation have not been performed, it is highly probable that the growth-promoting activity is derived from GH. Judging from the index of precision and sensi- tivity obtained from the dose-response curve for the frog GH, it is concluded that a precise and sensitive bioassay for amphibian GH was devel- oped. In amphibians, little work has been done to elucidate the mechanism of action of GH, although there is one report that Xenopus plasma enhances [**S]Jsulphate uptake by porcine carti- lages in vitro [19]. The assay for frog GH developed in the present experiment may be applicable to the assay for the somatomedin-like activity in amphibians and contribute to the elucidation of the mode of action of amphibian GH. ACKNOWLEDGMENTS We thank the NIAMDD for providing ovine GH and PRL. Thanks are also due to Dr. K. Yamamoto, Mr. T. Kato and Mr. T. Kobayashi for their help and interest. This work was supported by grants from the Seich6 Association and Waseda University and Grants-in-Aid from the Ministry of Education. REFERENCES 1 Etkin, W. and Lehrer, R. (1960) Excess growth in tadpoles after transplantation of the adeno- hypophysis. Endocrinology, 67: 457-466. 2 Bern, H. A. and Nicoll, C.S. (1968) The com- parative endocrinology of prolactin. Recent Prog. Horm. Res., 24: 681-720. 3. Dodd, M.H.I. and Dodd, J. M. (1976) Biology of metamorphosis. In “Physiology of Amphibia’’. 10 11 12 13 14 IS) 16 17 T. Isha AND S. KIKUYAMA Ed. by B. Lofts, Academic Press, New York, pp 467-599. White, B. A. and Nicoll, C. S. 1981) Hormonal control of amphibian metamorphosis. In ‘“‘Metamorphosis”. Ed. by L.I. Gilbert and E. Frieden, Plenum Press, New York, pp 363-396. Brown, P.S. and Frye, B. E. (1969) Effects of hypophysectomy, prolactin and growth hormone on growth of postmetamorphic frogs. Gen. Comp. Endocrinol., 13: 139-145. Zipser,R.D., Licht, P. and _ Bern, H. (1969) Comparative effects of mammalian prolactin and growth hormone on growth in the toads Bufo boreas and Bufo marinus. Gen. Comp. Endocrinol., 13: 382-391. Nicoll, C.S. and Licht, P. (1971) Evolutionary biology of prolactin and somatotropins. II. Electrophoretic comparison of tetrapod somato- tropins. Gen. Comp. Endocrinol., 17: 490-507. Van Wyk, J. J. and Underwood, L. E. (1978) The somatomedins and their actions. In “Bio- chemical Actions of Hormones Vol II’’. Ed. by G. Litwack, Academic Press, New York, pp 101-148. Daughaday, W. H., Phillips, L.S. and Herington, A. C. (1975) Measurement of somatomedin by cartilage in vitro. Methods in Enzymology, 37: 93-109. Yamamoto, K. and Kikuyama, S. (1981) Puri- fication and properties of bullfrog prolactin. Endocrinol. Japon., 28: 59-64. Farmer, S. W., Papkoff, H. and MHayashida, T. (1976) Purification and properties of reptilian and amphibian growth hormones. Endocrinology, 99: 692-700. Holder, A. and Wallis, M. (1977) Action of growth hormone, prolactin and thyroxine on serum somatomedin-like activity and growth in hypo- pituitary dwarf mice. J. Endocrinol., 74: 233-239. Iwata, H. and Urist, M. R. (1972) Protein poly- saccharide of bone morphogenetic matrix. Clin. Orthop., 87: 257-274. Marzullo, G. and Lash, J. W. (1967) Separation of glucosaminoglycans on thin layers of silica gel. Anal. Biochem., 18: 575-578. Hawkins, J. (1973) Sulphate incorporation by cartilage from dwarf mice: a possible new bioassay method for growth hormone. Proc. Endocr. Soc. Australia, 16: 8. Kikuyama, S., Yamamoto, K. and Mayumi, M. (1980) Growth-promoting and antimetamorphic hormone in pituitary glands of bullfrogs. Gen. Comp. Endocrinol., 41: 212-216. de Luze, A., Leloup, J. and Seugnet, I. (1983) Influence of fish prolactin and growth hormone on T, deiodination in the eel. Abstracts of Conference 18 Sulphate Uptake by Xenopus Cartilage 615 of European Comparative Endocrinologists, pp 12. Yamamoto, K. and Kikuyama, S. (1982) Radio- immunoassay of prolactin in plasma of bullfrog tadpoles. Endocrinol. Japon., 29: 159-167. 19 Shapiro, B. and Pimstone, B. L. (1977) A_ phy- logenetic study of sulphation factor activity in 26 species. J. Endocrinol., 74: 129-135. o C0 erisat-fas niet Qi | mn aay es etoin Thr Rergaal, ug ot » Lomi tools = l RD 2 é ‘ an P = x eS 7. Ny x + ~ a é 4 5 af _ seinhirrdcs aneguealll w ens a pe — Ring 7h, : rea 7 aft LAChy dl pevutalintag fc om ch, RRS at ; 2: it iy, en 1 - i it , “ j \ hillins, ite Flic” viet re Rag, rays HE Meotan j.)/ rod ye | it prepettes aie t S he a at 1 | i + ik : : 4th i SoM Fa, 5 a Ch oro and Rare ~ a the pet iett Ath s a ee as c. ZOOLOGICAL SCIENCE 1: 617-629 (1984) Purification of Bullfrog Gonadotropins: Presence of New Subspecies of Luteinizing Hormone with High Isoelectric Points Koj! TAKADA and SUSUMU ISHII Department of Biology, School of Education, Waseda University, Nishi-Waseda, Tokyo 160, Japan ABSTRACT — From bullfrog pituitaries, two subspecies and three subspecies of luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like gonadotropins, respectively, were purified. Gonadotropic activities of fractions obtained by chromatographies were surveyed with two radio- receptor assay (RRA) systems, using testicular receptors of Xenopus and Anolis. The relative activity of one of LH-like subspecies with p/ 10.2 (FL421B) was 5.18 x NIH—FSH-—P2 in Xenopus RRA and 1.02 x NIH—FSH-P2 in Anolis RRA, the activity ratio being 0.2. On the other hand, the activity of one of FSH-like subspecies with p/ 5.7 (FF1341B) was 0.925 x NIH—-FSH-—P2 in Xenopus RRA and 40.2 x NIH-FSH-P2 in Anolis RRA, the ratio being 43. The other basic and acidic subspecies also had similar activities to those of FL421B and FF1341B, respectively. .FL421B enhanced cyclic-AMP accumulation and androgen releases in bullfrog testis in vitro in dose-dependent manners. FFI1341B also enhanced cyclic-AMP accumulation and androgen releases but the cyclic-AMP accumulation and androgen releasing activities of this subspecies was far less than those of FL421B, being about 1/200. Molecular weights of LH- and FSH-like gonadotropins estimated by the gel filtration were 24000- 28000 and 30000-33000, respectively. Isoelectric points of LH-like and FSH-like gonadotropin subspecies, determined by the electrofocusing, were 10.2 and 9.8 for the former and 6.4, 5.7 and 4.4 © 1984 Zoological Society of Japan for the latter. The LH-like gonadotropin subspecies obtained in this study are different from those (p/ 8.8, 9.0, 9.1 and 9.3) reported by Takahashi and Hanaoka (1981) in their isoelectric points. INTRODUCTION Almost a decade has passed since Licht and Papkoff [1] first purified bullfrog gonadotropin and demonstrated the presence of FSH- and LH-like gonadotropins in the amphibia. Their results were confirmed later by several inves- tigators using different chromatographic and assay methods [2-5]. Introduction of radioreceptor assay (RRA) methods devised by our group [3, 6] enabled identification and quantification of a minute amount of frog gonadotropins. Using the electrofocusing and the RRA _ methods, Takahashi and Hanaoka [4, 5] showed that bullfrog gonadotropins, as well as gonadotropins of higher vertebrates [7], consist of multiple subspecies or components with different isoelectric Accepted February 23, 1984 Received February 6, 1984 points. In the present study, we isolated two new subspecies of bullfrog WLH-like gonadotropin that have higher isoelectric points than those of Takahashi and Hanaoka [4]. A chromatofo- cusing method was successfully applied to isolate the subspecies. Biological and receptor binding activities of the new bullfrog LH-like gonadotropin subspecies as well as those of bullfrog FSH-like gonadotropin subspecies are described. MATERIALS AND METHODS Material Ten thousand acetone-dried pituitary glands of adult bullfrogs, Rana catesbeiana, were used as the starting material of purification of LH- and FSH-like gonadotropins. 618 Radioreceptor assays LH-like potency was determined by a radioreceptor assay method [6] using testicular homogenate of Xenopus laevis as the receptor and rat FSH (NIAMDD-rat FSH-I-3) as the radioligand. This method (Xenopus RRA) has a high sensitivity to bullfrog LH-like gona- dotropin but a low sensitivity to bullfrog FSH-like Acetone dried powder (FA1B) K. TAKADA AND S. IsHIr gonadotropin [8]. FSH-like potency was assayed by a radioreceptor assay method, using homo- genate of Anolis carolinensis testes as the receptor and rat FSH as the radioligand, that was devised by Adachi ef al. [3]. The high specificity of the method to FSH-like gonadotropin of bullfrog has been confirmed [8]. Precision indices of Xenopus and Anolis RRA were —0.049-+0.005 and —0.042+0.007, respectively. Extraction with 6% ammonium acetate in 40% ethanol, pH 5.1 Precipitation by increasing ethanol concentration Glycoprotein (FAG11B) CM-cellulose; ammonium acetate gradient (30-500 mM) unadsorbed <170mM <260mM <400mM <500 mM Tonite —2B —3B —4B —5B DEAE-Sephacel; sodium chloride gradient (0-150 mM) unadsorbed 50-90 mM <145mM <150mM 220mM in P.B. FCD-11B —12B —13B —14B —15B | Chromatofocusing Chromatofocusing (pH 7-5) (pH 10-8) t | i pH 7.2-6.76 >6.32 5.98-5.6 5.48-5.22 FCDF-131B —132B —134B — 136B | | | Sephacryl S—200 S—200 S—200 Ve/Vo: 1.51 1.56 1.58 FCDFS-1321B —1341B —1361B (FF1321B) (FF1341B) (FF1361B) | aoe pH 10.3-10.2 >9.94 9.78-9.7 >9.55 9.64-9.6 FCF-41B —42B —45B —46B —46B’ | | | | Sephacryl S—200 S-200 S-200 S-200 Ve/Vo: 1.66 1.61 1.64 1.61 FCFS—421B —451B —461B —461B’ (FL421B) (FL451B) (FL461B) (FL461B’) Fic. 1. gonadotropin purification. Purification steps and names of fractions at each step of the bullfrog New Subspecies of Frog Gonadotropins 619 Purification Steps of purification procedures for LH- and FSH-like gonadotropins were summarized in Figure 1. Preparation of glycoprotein fraction: An ethanol precipitation method [9] was employed. Briefly, glycoprotein was extracted from acetone- dried powder with 6% ammonium acetate, pH 5.1, containing ethanol (40%). Then, glycoprotein was precipitated by adding absolute ethanol and raising the ethanol concentration up to 80%. Cation exchange chromatography: A_car- boxymethyl (CM) cellulose (Whatman) column of 15x280mm was used. It was equilibrated with 30mM ammonium acetate, pH 5.5, and eluted with a linear concentration gradient of ammonium acetate, from 30 to 500mM. The flow rate was 38.7 ml/hr and the fraction ‘size was 3 or 6 ml/tube. Anion exchange chromatography: A_ di- ethylaminoethyl (DEAE) Sephacel (Pharmacia) column of 16x200mm was used. The column was equilibrated with SmM_ glycine-NaOH buffer, pH 9.5, and eluted with a linear con- centration gradient of NaCl from 0 to 150mM. The flow rate was 22.8 ml/hr and fraction size was 2.3 or 4.6 ml/tube. Chromatofocusing: We followed the chroma- tofocusing method of Sakai and Wakabayashi [10], that was originally used for isolation of chicken gonadotropin subspecies. For purifi- cation of LH, the chromatofocusing gel, PBE118 (Pharmacia), was used. The column was equilibrated with 25mM__ triethylamine-HCl, pH 10.9, and then eluted with 45-fold diluted Pharmalyte-HCl buffer, pH 8.0. The pH range of Pharmalyte (Pharmacia) used was 8~-10.5. The column size was 10*412mm, flow rate 23.9 ml/hr and fraction size 3 ml/tube. The gel used for purification of FSH was PBE94 (Pharmacia). Starting and elution buffers were 25mM_ imidazole-HCl, pH 7.4, and eightfold diluted Polybuffer 74-HCl (Pharmacia), pH 5.0, respectively. The column size was 9235 mm, flow rate 20.4 ml/hr and fraction size 1.36 ml/tube. Gel filtration: A column, 15x900mm, of Sephacryl S—200 superfine (Pharmacia) was used. It was equilibrated and eluted with 0.1 M Tris- HCl, pH 8.0, containing NaCl, 0.5 M at the final concentration. The fraction size was 1.6 ml/tube, and flow rate was 3ml/hr. Estimation of the molecular weight was conducted by passing standard proteins through the same Sephacryl S—200 column [11]. Electrofocusing: We followed the method of Hattori and Wakabayashi [12]. A 25 ml capacity column containing 1.0% Ampholine (LKB) was used. The pH range was 9.0 to 11.0 for LH and 5.0 to 8.0 for FSH. Polyacrylamide gel electrophoresis: The me- thod of Davis [13] was used. The concentration of the gel was 7.5%. The electrophoresis was performed at pH 4.5 for LH-like gonadotropins and pH 8.9 for FSH-like gonadotropins. Gels were stained with Coomassie brilliant blue R. Bioassays Male bullfrogs were collected in the suburbs of Tokyo in the middle of September and used for assays within 3 to 5 days after capture. Incubation of testicular tissue in vitro: Testes were cut into small pieces with fine scissors, placed into a flask containing 1.0 ml of a medium described by Muller and Licht [14] with the following modifications: gelatin was removed and glucose was added at the final concentration of 0.1%. The tissue pieces weighing about 5 mg in total were incubated in a small flask under an atmosphere of 95% O, and 5% CO, with shaking. When an effect of hormone on the concentration of 3’-5’-cyclic adenosine mono- phosphate (cAMP) was examined, 3-isobutyl-1- methylxanthine was added into the medium at the final concentration of 2.6mM. The incu- bation was performed for 2 or 3 hr at 28°C in most cases. At the end of incubation, the medium and tissue pieces were separated. The medium was used for the radioimmunoassay (RIA) of testosterone and dihydrotestosterone released from the tissue. The tissue pieces were homogenized in 1.0 ml of 6% trichloroacetic acid and centrifuged at 1000xg. The super- natant was used for the RIA of cAMP and the sediment for the protein determination. 620 RIA of cAMP: The sample was shaken with ether in order to remove trichloroacetic acid. Then, the aqueous phase was used for the assay of cAMP using a RIA kit (Yamasa Shoyu). We followed the procedure described in the instruction of the company except that the volume of all the reagent was reduced to 1/2. RIA of androgen: Androgens in the medium were extracted with ether. Testosterone and Sa-dihydrotestosterone (DHT) were separated by a chromatography on a column (6.5 x60 mm) of LH-20 (Pharmacia) using hexane: benzene: methanol (90: 5: 5) as the eluant. Testosterone and DHT were determined by RIA methods using anti-testosterone- and anti-DHT-sera (Teikoku Hormone MFG.). Radioligands were [1, 2, 6, 7--H]testosterone and Sa-dihydro-[1, 2, 4,5, 6, 7--H]testosterone (Amersham). The in- itial incubation was at 4°C and for 18 hr. Then anti-rabbit-gamma globulin was added. Tubes were incubated again at 4°C and for TABLE 1. mediate and final preparations K. TAKADA AND S. IsHii 18 hr. After the centrifugation at 2500 x g radio- activity in the precipitate was measured. Protein determination Protein content of the tissue incubated was determined by the method of Lowry ef al. [15] with bovine serum albumin (fraction V, Wako) as the standard. RESULTS Purification of bullfrog LH-like gonadotropin The ethanol extraction and _ precipitation methods increased the specific gonadotropin activity of the sample more than tenfold as estimated by both Xenopus and Anolis RRAs. Recovery rates of the activities after the ethanol- extraction plus ethanol-precipitation were high, about 90 and 100% by Xenopus and Anolis RRAs, respectively (Table 1). Weight and relative potency estimates of bullfrog gonadotropins in starting, inter- Relative potencies? Relative potencies Anolis Protein’ Recovery Recovery f Recovery vs o Xenopus RRA 0 Anolis RRA 5 me) 2)" NIHEFSH=P2) 670)" ONT FSHEP2) © ee FA1B? (24700) 100 0.140 (0.120-0.165)* 100 0.651 ( 0.436— 0.972) 100 4.7 FAGII1B° 2100 8.5 1.44 (1.29 -1.60 ) 87 7.99 ( 4.57 -14.0 ) 100 3)45) FCIB 1020 4.1 0.706 (0.608-0.833) 21 16.4 (9.51 -26.7 ) 100 23 FC3B 250 1.0 3.97 (3.51 -4.49 ) 29 3.06 ( 2.32 — 4.03 ) 4.8 0.77 FC4B 230 0.93 5.62 (5.02 -6.28 ) 37 1.54 ( 1.25 — 1.91 ) ADD 0.27 FCD13B 191 0.77 0.475 (0.418-0.540) 2.6 ca. 30 ca. 40 ca. 60 FL421B 6.2 0.025 5.18 (4.15 -6.47 ) 0.93 1.02 ( 0.659- 1.59 ) 0.039 0.20 FL451B 6.2 0.025 3.96 (3.49 -4.50 ) 0.71 0.701 ( 0.370— 1.33 ) 0.027 0.18 FL461B 20.9 0.085 4.31 (3.62 —5.14 ) 2.6 0.785 ( 0.628— 0.981) 0.10 0.18 FL461B’ a7 0.0155 6:32 1651 .-7:25)) 0.68 1.17 ( 0.886— 1.56 ) 0.027 0.19 FF1321B 6 hs) 0.030 0.438 (0.329-0.584) 0.095 248 (19.8 -31.0 ) iL Sy) FF1341B 8.2 0.032 0.925 (0.752-1.14 ) 0.22 40.2 (366 -44.0 ) eI 43 FF1361B Dy e/4 0.021 0.639 (0.444-0.920) 0.096 28.2 (249 -31.9 ) 0.91 44 a: FAIB is acetone dried powder of the pituitary. b: FAGI1B is a glycoprotein preparation. c: Dry weight only for FAIB. The other fractions protein content in terms of the standard protein, bovine serum albumin. d: Relative potency estimation in the parallel line assay was based on the method of Bliss [30]. e: 95% confidence interval is given in parentheses. New Subspecies of Frog Gonadotropins 621 After the CM cellulose column chromatography, most of the Xenopus and Anolis RRA activities were detected in two portions of fractions (Fig. 2a and b). One was the void fraction, which contained substances not retained in the column and possessed higher Anolis RRA activity than Xenopus RRA activity. This portion was used as an acidic gonadotropin fraction (FCIB). 1.5 1 °o foe} < 0.5 0 : > 0.4 = ” ce = < c c 0.2 = ° Cc a Pad s 0 — Fer) +FC3B->+ The other was the fraction which was eluted over a wide concentration range of buffer (150 to 400mM) as a composite of several unclear peaks of the activities. This portion, a_ basic gonadotropin fraction, contained a high Xenopus and a slight Anolis activities. We divided the basic gonadotropin fraction further into two fractions of FC3B, eluted with buffer whose 500 3 = 250 7 0 s1]Ouy (>) (jw/6B HS4d) WHY 200 Fraction Number FC4B > Fic. 2. Chromatography of bullfrog adenohypophyseal glycoprotein (FAGI1B) on CM cellulose. with a continuous line. is shown with a dotted line. a: Optical absorption at 280 nm of eluate from the column is shown The NaCl concentration estimated with a conductivity meter b: RRA activities in fraction tubes. Anolis RRA activi- ty with triangles, Xenopus RRA activity with circles. Ranges of fraction tubes which were combined and renamed are indicated with arrows at the bottom of the figure. 622 K. TAKADA AND S. ISH pare) concentration was lower than 260mM, and FC4B, eluted with buffer whose concentration was higher than 260mM. Each of these two fractions also had a high Xenopus and a low Anolis RRA activities (Table 1). In order to avoid possible contamination of the other glyco- protein hormones, which is suggested by slight Anolis RRA activities (Fig. 2b), FC3B was omitted and only FC4B was used for further purification. After chromatofocusing of FC4B, three large protein peaks were obtained (Fig. 3). The Xenopus RRA activity was detectable in two of them. The first active peak (fraction FCF42B) was found around pH 9.95, and the second one around pH 9.65. As the second peak had a shoulder on its left side, the shoulder part (FCF45B) was collected separately from the main part (FCF46B). Furthermore, a part of the central portion of the main peak was also separately collected and named as FCF46B’. Each of these four fractions (FCF42B, FCF45B, FCF46B and FCF46B’) was further purified by A280 ( =) 25 FCF45B A FCF42B FCF46B +p FCF46B’ Fic. 3. 50 125 Fraction Number the gel filtration. Three of them (FCF42B, FCF45B and FCF46B’) showed a single peak and named as FL421B, FL451B and FL461B’ respectively (Fig. 4a, b and d). The other frac- tion, FCF46B, gave a main large and an additional small protein peaks (Fig. 4c). The small peak had no RRA activity. The main active peak was recovered and named as FL461B. These active fractions obtained after the gel filtration were used as final products of LH-like gonadotropin subspecies. Purification of bullfrog FSH-like gonadotropin The void fraction (FC1B) of the CM cellulose column, which had a high Anolis RRA activity and relatively a low Xenopus RRA activity, was analyzed by the DEAE Sephacel column chromatography (Fig.5a and b). After the chromatography, four major peaks and several minor peaks were obtained. One of the major peaks, which appeared first (FCD12B), had a high Xenopus RRA activity but a low Anolis = 0.5M NaCl (1w/Bw HS4d) yu sndouex@) 150 Chromatofocusing of a fraction (FC4B) after the CM cellulose chromatography. Optical ab- sorbance at 280 nm is indicated with a thick line, pH with a dotted line and the Xenopus RRA activity with a thin line. For details, see the legend for Fig. 1 and text. New Subspecies of Frog Gonadotropins 623 A280 (—) 9° ~ oO Ny uo os O | o5 e ithe > . > ur ow (qw/6uw HS4d) WHY SNdoUusy (+) 10 20 << FL461B’ 30 Fraction Number Fic. 4. Gel filtrations of LH-like gonadotropin fractions, FCF42B (a) FCF45B (b) FCF46B (c) and FCF46B’ (d), obtained after the chromato- focusing. Optical absorbance at 280nm is indicated with a thick line and the Xenopus RRA activity with a thin line. For details, see the legend for Fig. 1 and text. RRA activity. The second peak (FCD13B) that was eluted with a central part of the gradient had a high Anolis and a low Xenopus RRA ac- tivities. Accordingly the second peak (FCD13B) was considered to contain FSH-like gonadotropin. The remaining peaks had none of the activities. After chromatofocusing of FCD13B, four major protein peaks were obtained (Fig. 6). High Anolis activities were detected in three of them, these active peaks, which located at pH 6.6, 5.7 and 5.4, were named as FCDF132B, FCDF134B and FCDF136B, respectively. They were ana- lyzed further by the gel filtration. After gel filtration, large symmetrical peaks of protein and Anolis RRA activity were observed at the same location in each of them (Fig. 7a, b and c). An additional small clear or unclear protein peak with no RRA activity was observed in two of them. The main active peaks obtained from FCDF132B, FCDF134B and FCDF136B were named as FF1321B, FF1341B and FF1361B, respectively. They were used as final products of FSH-like gonadotropin subspecies. Physicochemical properties The analysis of the final products by the elec- trofocusing method revealed that isoelectric points of LH-like gonadotropin subspecies were 9.8 and 10.2 and those of SH-like gonadotropin subspecies 4.4, 5.7 and 6.4 (Table 2). The gel filtration analysis gave the molecular weight of 24,000 to 28,000 for LH-like gonadotropin subspecies and 30,000 to 33,000 for FSH-like gonadotropin subspecies (Table 2). The poly- acrylamide gel electrophoresis of LH- and FSH-like gonadotropin subspecies at alkaline pH showed that all protein of the FSH-like gona- dotropin subspecies moved to the anodal side as a single wide band (Fig. 8a, b and c). The LH-like gonadotropin subspecies moved to the other side and were lost from the gel. A faint band of a protein was detected at the location corresponding to FSH-like gonadotropin band but its optical density was as small as 0.3% of the FSH-like gonadotropin band (Fig. 8d and e). The polyacrylamide gel electrophoresis analysis of the fractions at acidic pH gave the following results: all of FSH- (Fig. 9e) and LH-like fractions (Fig. 9a, b, c and d) were divided into two bands and one of the bands had a common mobility but the other band had different mo- bilities between the FSH- and LH-like fractions. Biological potencies The receptor binding property differed clearly between LH-like and FSH-like gonadotropins. All the final LH-like gonadotropin products had high Xenopus RRA activities and low Anolis RRA activities. The activity ratio (Anolis RRA to 624 K. TAKADA AND S. IsHIt —~+—10mM P.B. with 0.22M NaCl (———) wu Sljouy (>) (m/6 HS43qd) vyuH 200 Fraction Number <——_—_> <—__> FCD12B FCD13B 2 o 1 rr) Sl ee fe) E SS rey) £ r 7) LL & < oc fea 5 ”) =) rol ° Cc o x O 0 Fic. 5. (FC1B). DEAE Sephacel chromatography of the acidic gonadotropin fraction a: Optical absorption at 280 nm is indicated with a continuous line, NaCl concentration estimated with the conductivity meter with a dotted line. b: Anolis RRA activity with triangles, Xenopus RRA activity with circles. For details, see the legend for Fig. 1 and text. Xenopus RRA) was about 0.2 in both of the two subspecies (FL421B and FL461B’). All the FSH-like gonadotropin subspecies had _ high Anolis RRA activities but slight Xenopus RRA activities. The activity ratio was 57, 43 and 44 in subspecies FF1321B, FF1341B and FF1361B, respectively. A LH-like gonadotropin subspecies (FL421B, pl 10.2) and also a FSH-like gonadotropin sub- species (FF1341B, p/ 5.7) could stimulate accumulation of cAMP in the testicular tissue in vitro (Fig. 10). The effect was dependent on the hormone dose. The minimum effective dose was approximately the same in two hormones but the slope of the dose response curve of the LH-like gonadotropin fraction (FL421B) was far steeper than that of the FSH-like gonadotropin fraction (FF1341B). A LH-like gonadotropin fraction (FL421B) enhanced the release of androgens significantly from the testicular tissue in a dose dependent manner (Fig. 11 and 12). The mini- mum effective dose was 19.5 ng/ml in both of testosterone and DHT release. In contrast, a FSH-like gonadotropin subspecies (FF1341B) could not increase the androgen release at dose levels below 1250 ng/ml. When we compared the activity at the minimum effective dose, the in yitro New Subspecies of Frog Gonadotropins 625 1.5 50 100 7 v = T i “ | rs) a oO z = z= © 150 5 °o 6 E : D 100 > .) wv ” =o =. 50 @ 5 3 fe) 150 Fraction Number =r FCDF132B FCDF134B FCDF136B Fic. 6. Chromatofocusing of a fraction (FCD13B) obtained after the DEAE Sephacel chroma- tography. Optical absorbance at 280 nm is indicated with a thick line, pH with a dotted line and the Anolis RRA activity with a thin line. text. TABLE 2. Molecular weights estimated by the gel filtration and isoelectric points deter- mined by the isoelectrofocusing of final products Molecular weight pl FL421B 24000 10.2 FL451B 27000 not tested FL461B (FL461B’) 28000 (25000) 9.8 FF1321B 33000 6.4 FF1341B 32000 = I FF1361B 30000 4.4 androgen releasing activity of FL421B was about 200 times of that of FF1341B. DISCUSSION As final products of purification of bullfrog pituitary protein, we obtained gonadotropin-like glycoproteins with five different isoelectric points, For details, see the legend for Fig. 1 and i.e., 4.4, 5.7, 6.4, 9.8 and 10.2. They are clearly divided into two of acidic and basic groups. Chromatographically as well as_ electrostati- cally, these acidic and basic groups are similar to FSH and LH, respectively, of reptiles [16, 17], birds [9, 18-23] and mammals [24, 25]. Fur- thermore, they are identical to FSH- and LH-like gonadotropins of the bullfrog isolated by previous investigators in their behaviors in ion exchange chromatographies [1, 3-5] and in molecular weights [4, 5, 26]. Isoelectric points of the acidic group were also identical to those of FSH-like go- nadotropins of Takahashi and Hanaoka [5]. On the other hand, isoelectric points of our basic group are clearly different from any of the isoelectric points of LH-like gonadotropins of Takahashi and Hanaoka [4], which were p/ 8.8, 9.0, 9.1 and 9.3. Accordingly, our basic gona- dotropins are considered to be distinct from those isolated by them at least electrostatically. Recently, Wakabayashi ef a/. [27] reported that 626 five peaks of the LH-like activity were detected after electrofocusing of a bullfrog pituitary extract. Isoelectric points of these peaks were 6.3, 7.6, 8.3, 9.1 and 9.8. The value of the last peak coincided well with that of one (FL461B) of our basic gonadotropins. Our FL421B had a com- pletely different pJ value but this substance, as well as FL461B, had biological activities which are the characteristic of LH. After the CM cellulose column chromatography, we used FC4B and not FC3B as the source of LH-like gonadotropin, whereas both contained LH-like RRA activities. FC4B is the retarded half of the active peak in the eluates from the CM cellulose column and hence protein in this peak may be more basic than protein in the earlier half of the peak (FC3B). Less basic 1 a 0.5 200 100 oe = FF1321B =~ ae = : > — } ° 2 N b nt P< ¢ 1 Boon = Pp») > 100 DO Tl w 10) ro) = <= FF1341B 3 To) SS 3 10 20 30 Fraction Number =. FF1361B Fic. 7. Gel filtrations of FSH-like gonadotropin fractions, FCDF132B (a) FCDF134B (b) and FCDF136B (c), obtained after the chromato- focusing. Optical absorbance at 280nm is indicated with a thick line and the Anolis RRA activity with a thin line. For details; see the legend for Fig. 1 and text. K. TAKADA AND S. ISHII Fic. 8. Polyacrylamide gel electrophoresis of final bullfrog gonadotropin preparations at pH 8.9. a, FF1321B; b, FF1341B; c, FF1361B; d, FL421B; e, FL461B. See Fig. 1 and the text for fraction names. Fic. 9. Polyacrylamide gel electrophoresis of vari- cAMP in tissue (nmol/mg protein) ous preparations of bullfrog gonadotropin at pH 4.5. a, FL421B; b, FL451B; c, FL461B; d, FL461B’; e, FF1361B; f, FAGI1B. See Fig. 1 and the text for fraction names. 3.0 2.0 1.0 Va ~o2o0 f_atenoent = —— (0) 19.5 78.0 313 1250 5000 20000 Dose of hormone (ng/ml) Fic. 10. Cyclic AMP accumulation in bullfrog testis treated with different doses of bullfrog LH-like (a continuous line) and FSH-like (a dotted line) gonadotropins (FL421B and FF134i1B) in vitro. Each point indicates the mean cAMP accumulated in tissues of duplicate tubes and each vertical line its standard error. New Subspecies of Frog Gonadotropins 627 = be ° = 10 \ f°] E Sz a = Uv o 2 | "55 . io 2 J : oof / Jf ° ee / o a 2 = , melee od bak Le eo 0 es 0 4.90 19.5 78.0 313 1250 5000 20000 Dose of hormone (ng/ml) F a . 11. dn vitro release of testosterone from the bullfrog testis treated with different doses of LH-like (a continuous line) and FSH-like (a dotted line) gonadotropins (FL421B and FF1341B) of the bullfrog. Each point indicates the mean testosterone released in mediums of duplicate tubes and each vertical line its standard error. 100 - | mn 7 DHT released (ng/mg protein) Oo —+— Le) 19.5 78.0 313 1250 5000 20000 Dose of hormone (ng/ml) Fic. 12. Jn vitro release of DHT from the bullfrog testis treated with different doses of LH-like (a continuous line) and FSH-like (a dotted line) gonadotropins (FL421B and FF1341B) of the bullfrog. Each point indicates the mean DHT released in mediums of duplicate tubes and each vertical line its standard error. LH-like gonadotropin subspecies which are identical to those reported by Takahashi and Hanaoka [4] may be contained in FC3B. Our bullfrog LH-like gonadotropin (FL421B) was more potent than our bullfrog FSH-like gonadotropin (FF1341B) to stimulate the ac- cumulation of cAMP and the release of androgens. These results are in good agreement with previous results by Muller [28] and Gavaud ef al. [29]. A low androgen releasing activity of FSH-like gonadotropin may be due to either the inherent property of frog FSH-like gonadotropin or a slight contamination of LH-like gonadotropin. In the present study, we employed the Xenopus RRA method to detect the MLH-like activity. In this RRA system, the radioligand was not LH but FSH of the rat origin. This seems not to be in accordance with our common knowledge of endocrinology. However, there is evidence to show that rat FSH is recognized as LH by the testis of the amphibia. We demonstrated by radioautography that the rat FSH _ specifically bound mostly to the interstitial tissue in the testis of Xenopus [6]. This result indicates that rat FSH is recognized as LH-like substance by the Xenopus interstitial cell and is bound specifically to binding sites for frog LH [8]. Hence the Xenopus RRA is useful for the detection of LH of the frog. The electrophoretic analysis of our final pro- ducts showed that the final FSH-like gonadotropin fractions are homogeneous. It also showed that the final LH-like gonadotropin fractions were slightly contaminated with an acidic component that had the same mobility with FSH. The quantity of the contaminated component was extremely low (about 0.3% or less). After electrophoresis in the acidic condition, all of the final LH- and FSH-like gonadotropin fractions were separated into two bands. This result may be considered to be due to the dissociation of the hormone molecules into subunits in the acidic condition. Judging from the biochemical, physicochemical and biological properties of our final FSH- and LH-like gonadotropin products, we may conclude that they are practically almost pure FSH- and LH-like gonadotropins. In addition, our LH-like gonadotropins are considered to be the new subspecies or components of bullfrog gonado- tropins. ACKNOWLEDGMENTS We are grateful to the National Institute of Arthritis, Metabolism, and Digestive Diseases (NIAMDD) Rat Pituitary Hormone Distribution Program and Dr. A. F. Parlow for the supply of rat gonadotropins. Porcine 628 hormones were also supplied by NIAMDD._ Thanks are also due to Dr. K. Tsutsui and Mr. M. Inoue for their help in bioassays. This study was supported by a grant from the Ministry of Education, Japan. REFERENCES Licht, P. and Papkoff, H. (1974) Separation of two distinct gonadotropins from the pituitary gland of the bullfrog Rana catesbeiana. Endocri- nology, 94: 1587-1594. Yoshida, T., Yamamoto, K., Ishii, S. and Iwa- sawa, H. 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Wakabayashi, K. (1980) Miultiplicity of lutein- izing hormone and its comparative aspects. In “Hormone, Adaptation and Evolution’. Ed. by S. Ishii, T. Hirano and M. Wada, Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin, pp. 271-279. Takada, K. and Ishii, S. (1984) Mammalian FSH acts as LH in Xenopus frog. In ‘‘Proceedings of the Ninth International Symposium on Comparative Endocrinology”. Ed. by B. Lofts, Hong Kong Univ. Press, Hong Kong, in press. Stockell Hartree, A. and Cunningham, F. J. (1969) Purification of chicken pituitary follicle-stimulating hormone and luteinizing hormone. J. Endocrinol., 43: 609-616. Sakai, H. and Wakabayashi, S. (1983) Is chro- matofocusing available for separation and puri- fication of chicken follicle-stimulating hormone? Transactions of Pre-dental Faculty, Nihon Uni- versity, 11: 22-27. 11 14 15 16 19 20 21 22 23 24 053) 26 K. TAKADA AND S. IsHII Whitaker, J. R. (1963) Determination of molecular weights of proteins by gel filtration on sephadex. 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Reprod., 14: 222-232. Scanes, C. G. and Follett, B. K. (1972) Fraction- ation and assay of chicken pituitary hormones. Brit. Poult. Sci., 13: 603-610. Furuya, T. and Ishii, S. (1974) Separation of chicken adenohypophysial gonadotropins. Endo- crinol. Japon., 21: 329-334. Farmer, S. W., Papkoff, H. and Licht, P. (1975) Purification of turkey gonadotropins. Biol. Reprod., 12: 415-422. Oosthuizen, M. M. J., Oelofsen, W., Schabort, J. C., Neitz, A.W. H. and Viljoen, C. C. (1980) Isolation and characterization of three lutrophin isohormones from the pituitary gland of the ostrich (Struthio camelus). Int. J. Peptide Protein Res., 15: 181-191. Sakai, H. and Ishii, S. (1980) Isolation and characterization of chicken follicle-stimulating hormone. Gen. Comp. Endocrinol., 42: 1-8. Papkoff, H., Licht, P., | Bona-Gallo, A., MacKenzie, D. S., Oelofsen, W. and Oosthuizen, M. M. J. (1982) Biochemical and immunological characterization of pituitary hormones from the ostrich (Struthio camelus). Gen. Comp. Endo- crinol., 48: 181-195. Stockell Hartree, A. (1966) Separation and partial purification of the protein hormones from human pituitary glands. Biochem. J., 100: 754-761. Yoshida, T. and Ishii, S. (1973) Gonadotropins I. purification. Zool. Mag., 82: 1-15. Papkoff, H., Farmer, S. W. and Licht, P. (1976) 27 28 New Subspecies of Frog Gonadotropins Isolation and characterization of luteinizing hor- mone from amphibian (Rana_ catesbeiana) pituitaries. Life Sci., 18: 245-250. Wakabayashi, K., Hattori, M., Tanaka, S. and Minegishi, T. (1984) Comparative studies on the nature of gonadotropins. In “Proceedings of the Ninth International Symposium on Comparative Endocrinology”. Ed. by B. Lofts, Hong Kong Univ. Press, Hong Kong, in press. Muller, C. H. (1977) Jn vitro stimulation of Sa- 29 30 629 dihydrotestosterone and _ testosterone secretion from bullfrog testis by nonmammalian and mam- malian gonadotropins. Gen. Comp. Endocrinol., 33: 109-121. Gavaud, J., Licht, P. and Papkoff, H. (1979) In vitro stimulation of cyclic-AMP production in Rana catesbeiana ovaries by homologous gonad- otropins. Gen. Comp. Endocrinol., 38: 83-92. Bliss, C. I. (1952) “The Statistics of Bioassay”’. Academic Press, New York. aread yet Poem ee wee i (te blr te rae ee ae vs ia all « : - a mY, ey it Pu ‘gh: . we: agi F uo Sie eR uae oo” yea ge a Lak > , xb Pale rZ j iM Bess o “4 7 ' ae iy 5 = a ama P % 6 ’ i Piece 7 i mi J yu f! 3 ee a = e NN = Ait \ as =) 3) Tat 6 Z ’ , i ZOOLOGICAL SCIENCE 1: 631-640 (1984) Temperature Preferences of Eleven Drosophila Species from Japan: The Relationship between Preferred Temperature and Some Ecological Characteristics in Their Natural Habitats AKIHIKO YAMAMOTO! and SHIGERU OHBA Department of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158, Japan ABSTRACT — Preferred temperatures of 11 Drosophila species of sympatric origin were determined using a temperature gradient from 5 to 35°C. The mean preferred temperature differed significantly among species, ranging from 14 to 28°C. About 50 to 70% of the variance was due to interspecific differences; the intraspecific variance was only approximately 10%. Different rearing temperatures (15 to 25°C) also affected the mean preferred temperature, but the effect was negligible (less than 3°C) compared to the interspecific difference at the same temperature, which often exceeded 10°C. A negative correlation between the mean preferred temperature and mean temperature of appear- ance of each species in natural habitats reveals that the species appearing in hot seasons prefer relatively cold temperatures. A negative correlation between the mean preferred temperature and the domes- ticity index indicates that domestic flies prefer lower temperatures than wild species. These correlations suggest that the temperature preference of Drosophila species is a secondary trait to deter- © 1984 Zoological Society of Japan mine their seasonal appearances and spatial distributions in natural habitats. INTRODUCTION Generally speaking, two different approaches can be considered in thermal adaptation of insects, physiological tolerance or resistance to extreme temperatures and behavioural responses to different temperatures (Yamamoto and Ohba [1]). On drosophilid flies, which have many advantages for this kind of study, there have been several studies of the former aspect [2-5], while a few reports exist on the latter aspect, i.e. behay- ioural responses to thermal environments [6, 7]. In a preceding paper, Yamamoto and Ohba [1] studied the heat resistance, cold resistance, and temperature preference of two geographically widespread Drosophila species, D. virilis and D. immigrans, concluding that temperature pre- ference was as important as physiological tolerance Accepted January 31, 1984 Received December 5, 1983 * Present address and for correspondence: National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709, U.S.A. in the strategy of adaptation to thermal environ- ments. However, little is known about the general importance of temperature preference in other Drosophila flies, and nothing is known about the relationship between the thermal preference of each species and its seasonal ap- pearance and/or spatial distribution in natural habitats. The aim of this study is to determine the degree of inter- and intraspecies variation in thermal preference in several Drosophila species and to analyze the relationship between this characteristic and the seasonal occurrence or micro-distribution of the flies in their natural habitats. MATERIALS AND METHODS Flies were collected in a suburban area of Oiso (Kanagawa prefecture), which is located about 55km southwest of Tokyo. The area includes both a domestic housing area and a small valley with a creek covered with a protected natural forest. Collections were made from December, 1978, to June, 1980, using fermenting 632 A. YAMAMOTO AND S. OHBA TaBLe |. List of 11 Drosophila species used in the present study Taxon species Subgenus Sophophora melanogaster species-group suzukii species-subgroup suzukii takahashii species-subgroup lutescens melanogaster species-subgroup melanogaster simulans montium species-subgroup rufa Subgenus Drosophila robusta species-group lacertosa repleta species-group hydei bizonata species-group bizonata immigrans species-group immigrans curviceps Subgenus Dorsilopha busckii All were collected from Oiso, Japan, during the period from December, 1978 to June, 1980. banana traps. Collection records will be given elsewhere (Yamamoto, in preparation). From the thirty five drosophilid species collected in the above area, the eleven common species belonging to three subgenera of the genus Drosophila were used in the present study (Table 1). D. (Sophophora) melanogaster, D. (S.) simulans, D. (Drosophila) hydei, D. (D.) immigrans, and D. (Dorsilopha) busckii are cosmopolitan domestic species [8, 9]. The other six species are endemic to Japan or the Oriental region [10-12]. All flies were from isofemale strains, each derived from a wild-caught mated female. Before the present study began, flies were maintained in the laboratory for several generations as small mass cultured stocks. For both stock and experimental cultures, the standard yeast-sugar medium con- taining 8% dried brewer’s yeast, 5°% sucrose, 0.8% agar powder, and antimicrobiotics was used. Preferred temperature was examined using the same temperature gradient incubator (Toyo Kagaku Sangyo Co., Model TN-3) and experi- mental procedures as described in a previous study [1]. The range of experimental temperatures was from 5 to 35°C. Twenty flies of either sex were introduced into the high temperature end of a glass tube 78 cm in length, which forced them to move quickly to their preferred temperature range. When introduced from the other end, most of the flies did not move because of the cold. After 30 min under 10001x illumination, the number of flies in each of 16 sections of the tube was recorded and a mean preference score and variance were calculated. The preference score as a function of temperature is shown in Figure 1. For more details see Yamamoto and Ohba [1]. Temperature (°C) Score Fic. 1. Temperature gradient of the apparatus. Bars show the ranges over which temperature fluctuated with a cycle of 2 to 3 min. RESULTS 1. Sexual difference and age-associated changes in temperature preference Sexual differences in temperature preference were anlayzed for six species of flies raised and kept at 20°C. The effect of adult age from eclosion to 2 or 3 weeks old was also examined. As a typical example, the case of melanogaster is shown in Figure 2. The mean preference score was almost Temperature Preference of Drosophila 633 constant throughout the period examined for both females and males. In most cases the mean score of both sexes was between 8 and 9, which cor- responded to about 22 and 19°C, respectively (Fig. 1). Except for the cases in which only 1-2 measurements were made, analyses of variance 10 Mean score Le *) were made on the mean and variance of the scores for each test. The results (Table 2) in- dicate neither significant sexual difference nor age-associated effects on mean scores. However, the score variance changed markedly with age. The variance was large in the first few days 6 12 SY i 12) U VY) 8 — 1) i) c = 4 ‘ie \s) a 0 Days after eclosion Fic. 2. Changes of mean and variance of score with age in D. melanogaster. TABLE 2. Analyses of variance of ageing effect on temperature preference in D. melanogaster Source of re Mean score Variance of score variation F MS F Age 6 0.708 1.67 25.11 1G. 12"?* Sex l 0.065 0.03 255 1.65 Experiment/Sex 4 2.259 D.doe 1.42 0.57 Age x Sex 6 0.806 1.19 6.00 2.42 Error 24 0.423 2.48 MS: Mean square. **: Significant at the 1 % level. ***- Significant at the 0.1% level. 634 A. YAMAMOTO AND S. OHBA TABLE 3. Temperature preference scores of 11 species raised at three temperatures eae) Rearing Isofemale strain Temper- Species temper- Nera ature ature 1 Dy, 3 4 5) (°C) suzukii 2I1€ 4.82 1.05 3.48 0.22 4.45 0.71 6.28 0.37 5.23 0.35 4.85 26.0 20°C 6.07 0.92 5.62 1.32 2.80 0.60 6.02 0.34 6.22 1.46 5.35 24.9 ISAC 8.00 0.36 5.80 0.23 4.00 0.69 — 4.88 0.27 5.67 24.5 mean 25.1 lutescens ISXC 4.43 0.40 3.95 0.48 4.52 0.37 6.90 1.30 3.87 0.85 4.73 26.1 20°C 4.87 0.20 4.65 0.58 5.53 0.92 8.33 0.22 4.62 0.42 5.60 24.6 ISLS 5.80 0.60 7.05 0.73 7.45 0.35 6.82 0.72 6.85 1.39 6.79 22.9 mean . 24.5 melanogaster 25.€ 7.92 0.87 6.82 0.82 7.38 0.97 7.45 0.26 7.98 0.31 eon 21.8 20°C 9.05 0.55 7.80 0.68 8.08 1.11 7.30 0.10 9.10 0.63 8.27 20.8 153€ 7.55 0.74 8.53 0.29 7.50 0.45 7.12 0.52 9.22 0.09 7.98 21.3 mean 21.3 simulans ISAC 8.83 0.12 9.75 0.28 4.48 1.21 8.68 0.71 9.43 0.26 8.30 20.8 20°C 6.62 0.44 9.83 0.59 9.45 0.51 9.98 0.65 9.42 0.35 9.06 19.6 15°C 7.77 0.70 9.08 0.29 9.30 0.31 7.30 0.43 7.40 1.05 8.17 21.0 mean 20.5 rufa KE 3.82 0.39 3.72 0.31 2.63 0.39 3.37 0.88 4.72 0.55 3.65 28.0 20°C 5.23 0.41 7.27 0.75 5.30 0.28 7.37 1.17 5.33 0.38 6.10 23.9 ISS 6.37 0.19 7.92 0.19 7.82 0.33 7.15 0.68 6.80 0.15 7.21 Mid, mean 24.7 lacertosa DIAC 7.58 0.78 — — 6.57 0.37 — 7.07 22.4 20°C 5.82 0.65 7.95 0.39 6.58 0.42 5.87 0.61 — 6.56 235 i5.€ — — 4.10 0.16 6.70 1.70 — 5.40 25.0 mean 23.6 hydei 25°C —_: 11.50 0.74 11.30 0.41 12.80 0.96 12.50 0.50 10.52 0.17 11.72 14.0 20°C 8.97 0.49 11.25 0.43 11.40 1.28 10.57 0.50 11.50 0.28 10.74 16.1 ISAC 8.33 2.15 10.63 0.46 9.75 0.68 9.57 0.92 10.95 0.43 9.85 18.0 mean 16.0 bizonata 25.€ 5.58 0.19 5.05 0.56 4.45 0.38 4.67 0.62 6.73 0.25 5.30 Dal 20°C 4.77 1.05 4.65 0.55 5.35 0.88 6.02 0.58 6.05 0.64 5.37 25.0 ISS 6.13 0.08 6.03 1.03 6.50 0.40 — 6.90 0.51 6.39 23.5 mean 24.5 immigrans Dae 8.78 0.72 8.07 0.43 7.67 0.33 7.60 0.55 7.92 0.40 8.01 Dien 20°C 8.62 1.37 7.88 0.67 11.70 0.85 9.15 0.28 9.82 0.92 9.43 18.8 152€ 8.55 0.20 9.65 1.08 9.78 0.68 8.57 0.44 9.43 0.23 9.20 19.3 mean 19.8 curviceps 20°€ 8.10 0.65 8.95 0.35 10.07 0.89 9.30 1.45 — 9.10 19.5 busckii D5 ae 5.15 1.44 4.48 1.01 4.08 0.24 7.58 1.09 7.18 1.37 5.69 24.5 20°C 6.88 1.08 6.98 0.77 7.45 1.62 9:77 0.87 7.25 1.75 7.67 21.6 St 6.93 0.15 6.98 1.00 5.83 0.75 7.25 0.34 6.43 0.35 6.68 23.0 mean 23.0 Each score is the mean with standard error calcultated from three replicate tests. shows the preferred temperature which corresponds to the average score. The last column Temperature Preference of Drosophila 635 after emergence, decreased gradually until 4 or 5 days after emergence, then maintained a constant level. The peak of egg-laying activity, at which the following experiments were carried out, corresponded to the beginning of this stable phase of variance. In the case of melanogaster, 4-day-old flies raised at 20°C were chosen for further analyses of temperature preference. The mean scores did not seem to differ significantly between sexes, although males tended to show larger variation among replications than females. For the remaining 5 species tested (suzukii, lutescens, simulans, bizonata, and immigrans), the results were almost the same as for melanogaster. 2. Interspecific and intraspecific differences in temperature preference Temperature preference scores of female flies raised at 15, 20 and 25°C are listed in Table 3. Each estimate is the mean and the standard error of three replicates. It is clear from the table that considerable variation exists among 11 species examined here. Analyses of variance of the mean scores given in Table 3 revealed highly significant differences both among species and among iso- female strains within the same species at all culture temperatures (Table 4). The analyses include 11 species for 20°C and 9 species (excluding (lacertosa and curviceps) for 25 and 15°C. For every culture temperature, interspecific differences were significant at the 0.1 % level, always exceeding half of the total variance (50 to 72%). Intra- specific differences among isofemale strains from the same population were not as large as inter- specific ones, although they were significant at the 0.1% level for 25 and 20°C and the 5% level for 15°C. They shared 10 to 12% in the total variance. Further analyses of variance for each species (data not shown) also showed significant intraspecific differences for mean preferred tem- perature at the level of 0.1% in busckii, 1% in simulans, lutescens, suzukii and lacertosa, and 5% in rufa and bizonata. In the remaining four species hydei, immigrans and curviceps) no significant difference among strains was detected. Interspecific differences in preferred temperature are also seen in Figure 4 as well. Generally speaking, species preferring higher temperatures were suzukii, lutescens, rufa, lacertosa and bizonata, in which mean preferred temperature for each culture temperature was always above 22.5°C. Averaging the data for three temperatures, the overall mean preferred temperature was 25.1°C (suzukii), 24.7°C (rufa), 24.5°C (lutescens and bizonata) and 23.6°C (lacertosa). On the other hand, Aydei preferred lower temperatures, always below 18°C, giving an overall mean preferred temperature of 16.0°C. The other four species fell between these two groups, with overall mean (melanogaster, TABLE 4. Analyses of variance of preference scores Rearing Source of df. temperature variation sda Species 8 Strain/Species 36 Error 89 20°C Species 10 Strain/Species 42 Error 106 SC Species 8 Strain/Species 34 Error 82 Ms F Se 88.91 ONO hadi TAS 3.94 2:16°** 10.6 1.43 17.9 52.16 12.46*** a8 4.19 FS dada 12:3 1.97 32.6 Aas Fa 19.06*** 49.9 PA be, 1.69* 10.2 1.33 39.9 At 20°C, the 11 species in Table 2 were analyzed; at 25°C and 15°C, 9 species excluding D. lacertosa and D. curviceps were used. * Significant at the 5% level. *** Significant at the 0.1 % level. 636 A. YAMAMOTO AND S. OHBA preferred temperatures between 23.0°C (busckii) and 19.8°C (immigrans). Two sibling species, melanogaster and simulans, had similar preferred temperatures (21.3 and 20.5°C, respectively). 3. Effects of culture temperature on temperature preference The effects of temperature during development and maturation upon the preferred temperature of adult flies are seen in Tables3 and 4. Averaging ten species (curviceps excluded), the mean preferred temperatures were 23.0, 21.7 and 22.1°C for the respective rearing temperatures, 25, 20 and 15°C. Although the differences among rearing temperatures were statistically significant in 7 species out of 10 (0.1% level in lutescens, rufa and busckii; 1% in lacertosa, hydei and immigrans; 5% in bizonata), they were not as conspicuous as species differences. In most cases, the species characteristics were maintained over the three rearing temperatures. 4. Strictness of temperature preference According to Yamamoto and Ohba [lI], the strictness of temperature preference can be expressed as the variance which was calculated simulans ae eee ae Tyee e eee L— MMNNUHUOAUNCAONUAHUOUAARGALOUAUOGAAUGLOUAAUGALUAAUT E==))20%¢ mani ie immigrans aa a) INTVAVNNVNNUTUNUUOUAUAAAUNUTUAUAUVALUUHCHAUAI Pe EEE ere ee eee melanogaster ..(fenmasaanean pane CNINTHANHAAVALNUAAULALARAAAAALA ALANA ae ar a el) rufa Pe ae IVNHIUUUUSAAHNGANAUNAOAAUAERELOUAUUAUUAUGARASUAAUAAUAOOAUUAUAAULL : eae ee Cee ome bizonata aon Se eee any QUDLSQOOLHUOOANAUOGAUOOUGAONUGEREGAAAGGOOTOGOUATOOOTUGOATUGAOTTTGAAT a ae ae lutescens aa Ae Ne ee os SE iii MM suzukii UVIUUIVNNUUOUUNUUAUUAUAOUALUAOLAOLNELAUUELUEUAEU ARVANA busckii 7 IUNHNAUOAVOTATAVOALTSNAATCAGUAAUASOAAAAUAAUAUUAUAAUAAUAUG hyde: HVIVNUVALVNOAUNUUVUOVUUVACGUEVAAAARATEHAEAAAAALAEA AAO Ries. Tish ka lacertosa MEO. Oa ee UOAVAUAOAVAUATHAVAUATVAUALARAAAAALATARAUAAUAAAAALANAUAAULUAAA UH ALAA curviceps na a 0 0.5 1.0 1.5 Relative variance Fic. 3. Relative variance values of 11 species raised at 25, 20 and 15°C. Temperature Preference of Drosophila 637 from 20 flies’ scores in each tube. However, the variance was not completely independent of the mean score, because, in the plugged tubes used in this study, the variance tended to be smaller at both ends than in the middle of the tube. To correct this, a mean score-variance curve was obtained from the data of all trials, and the ratio of the variance for each trial to the mean variance from the curve (relative variance) was used as a measure of the strictness of temperature preference. Figure 3 shows graphically the relative variances of 11 species grown at three different temperatures, 25,20 and 15°C. The rearing temperature did not change the relative variance in most species, except for busckii in which the relative variance differed as a function of temperature. Judging from the relative variances at three temperatures, simulans, immigrans, and melanogaster were classified as the species with a narrow temperature preference range, in which the average relative variance was less than 0.8. On the other hand, curviceps, lacertosa, hydei, busckii and suzukii seemed to have a wide temperature preference range, with an average relative variance greater than 1.2. 5. Relationship between temperature preference and seasonal appearance and micro-distribution of flies in natural habitats In the same area from which the flies in this study were obtained, drosophilid flies were col- lected periodically from December, 1978, to June, 1980. From the collection records of this period, the following ecological indices were calculated for each species: 1) The domesticity index of species j is defined as follows: DW) D,+W;, where D, and W, are numbers of trapped flies belonging to species 7 in domestic area and wild area, respectively. Domesticity ranges from +1 for a completely domestic species to —1 for a purely wild species. 2) The niche breadth in space index, which represents the width of micro-distribution. 3) The niche breadth in time index, which indicates the duration of appearance. These two niche breadth indices were estimated from an Domesticity j= equation for weighted relative niche breadth which was presented by Colwell and Futuyma [13]. They vary from 0 to 1 according to the niche breadth of each species. A indicates a broader niche. 4) The mean temperature of appearance index, which is the overall average temperature of the days on which each fly was collected. 5) The mean relative humidity of appearance index. Indices 4 and 5 were estimated from the weather records at Yokohama Local Meteoro- logical Observatory, which is the nearest weather station to Oiso, using the following equation: i ae Ni; X; Temperature 7 (Humidity / peewee larger value Ni; where N;; is number of species j which were col- lected an ith collection and X; is the mean temperature (the mean relative humidity) of the day of ith collection. The details of these five indices will be explained elsewhere (Yamamoto, in preparation). The above five indices are listed in Table 5, the last column of which shows the mean weight of mature females raised and kept at 20°C in the laboratory. Among the eleven species in Table 5, the five cosmopolitan ones (melanogaster, simulans, hydei, immigrans and busckii) had the highest domesticity index values, 0.67 to 1.00. Three endemic species (suzukii, lacertosa and curviceps) had the lowest values, less than —0.53. They were typical examples of wild species in the Oiso area. These were a compatible observation with Kawanishi and Watanabe [14]. In terms of niche breadth, /utescens had the largest index values both in space and in time. It can be called a broad-niche species. The nar- rowest niche index in space belonged to Jacertosa and that in time to bizonata. One of the cosmo- politan species, hydei was a narrow-niche species in both space and time. The weight of laboratory-cultured females demonstrated considerable variation among Drosophila species. With the marked exception of bizonata, species belonging to the subgenus Drosophila were far larger (3.2 to 5.4mg) than those belonging to the subgenera Sophophora (1.3 to 2.1 mg) and Dorsilopha (1.4 mg). 638 TABLE 5. A. YAMAMOTO AND S. OHBA Domesticity, niche breadth and climatic indices of 11 species estimated from the appearance of flies in Oiso and body weight of matured flies raised at 20°C in the laboratory i a ee Index of appearance in nature — i Female Species Domes- Niche breadth Mean Mean body ticity temper- _ relative weight Space Time ature humidity (mg) CC) (%) suzukii —0.53 0.73 0.63 14.0 56.7 2.05 .15 lutescens 0.03 0.95 0.76 20.0 68.2 1.76 .11 melanogaster 1.00 0.42 0.36 24.3 74.4 1.32 .09 simulans 0.76 0.58 0.37 21.4 72.9 1.40 .06 rufa 0.03 0.85 0.42 23.9 74.8 1.88 .09 lacertosa —1.00 0.15 0.64 18.2 73.5 5.41 .08 hydei 0.99 0.36 0.26 25.4 1385 Bali eal bizonata 0.00 0.72 0.14 22 72.8 1.33 .08 immigrans 0.75 0.68 0.35 21.6 70.0 4.31 .41 curviceps —0.60 0.75 0.44 eS 54.3 4.77 .29 busckii — 0.67 0.62 0.47 20.6 rls) 1.44 .12 TABLE 6. Correlation coefficients between preferred temperature or relative variance and indices of appearance or body weight Rearing temper- Domes- ature ticity Preferred temperature 2G —0.53 20°C —0.60* LS4€ —0.84** Relative variance AIAG —0.07 20°C —0.52 lee 0.74* * Significant at the 5% level. ** Significant at the 1% level. Table 6 shows the correlations between preferred temperature and relative variance of flies reared at three different temperatures and the five ecological indices and mean body weight given in Table 5. The domesticity index had a significant negative correlation with the preferred temperature of flies reared at 15 and 20°C, and the correlation coefficient at 25°C (—0.53) was nearly at the 5% level of significance. These indicate that domestic species preferred lower temperature than wild ones. Both niche breadth indices in space and in time showed positive correlations with preferred tem- Index of appearance in nature Female Niche breadth Mean Mean body temper- _ relative weight Space Time ature humidity 0.64* 0.42 —0.50 —0.31 —0.35 0.42 0.48 —0.36 —0.08 —0.31 0.10 0.47 —0.77** —0.36 —0.06 —0.21 0.07 —0.14 —0.10 —0.20 —0.50 0.15 —0.36 —0.37 0.72* —0.08 0.51 —0.47 —0.49 0.43 perature, indicating that wide-niche species tended to prefer higher temperature ranges, although only one case was significant statistically. In contrast, negative correlations were observed between pre- ferred temperature and mean temperature of appearance (r= —0.36 to —0.77). This is graphi- cally shown in Figure 4, in which the mean preferred temperature of flies reared at 20°C is plotted against the mean temperature of ap- pearance for 11 species. The correlation coeffi- cient was —0.69 and significant at the 5% level when curviceps was removed, although when Temperature Preference of Drosophila 639 Preferred temperature 25 (°C) 10 15 20 Temperature of appearance Fic. 4. Preferred temperature and mean _ tem- perature of appearance in nature of 11 species. Preferred temperatures are the values for flies grown at 20°C. sz; suzukii, lt; lutescens, m; melanogaster, sm; simulans, 1; rufa, \c; lacer- tosa, h; hydei, bz; bizonata, 1; immigrans, c; curviceps, b; busckii. curviceps was included the 11 species showed a non-significant coefficient value of —0.36. Some selection resulted from the difficulties to rear the exceptional species, curviceps, may have affected the preference of this species. The correlation between relative variance and the above indices gave contradictory results. Correlation coefficient values for relative variance differed greatly among the rearing temperatures. For example, the coefficient between relative variance and the domesticity index was —0.74 at 25°C and 0.07 at 15°C. Therefore rearing temperature seemed to affect the relative variance of temperature preference, but not the mean value. DISCUSSION Flies in the temperature gradient distributed themselves in a dynamical equilibrium for several hours, the length of which was different among species. After that, flies concentrated gradually near the low temperature end to avoid dessication and starvation. Prince and Parsons [6] observed similar movements of three Drosophila species (melanogaster, simulans and in a temperature gradient from 16.5 to 36.5°C. How- ever, their interests were mainly on time needed for entering the second phase, in relation to the effect of humidity. In this paper we studied the temperature which flies selected in the first phase immigrans) and it was defined as ‘“‘preferred temperature’. The effect of varying temperature during pre- adult stages on the preferred temperature of adults was not conspicuous. Concerning the average of the ten species examined in this study, the difference in preferred temperature was less than 1°C between flies raised at 15 and 25°C. This result agrees with that reported by Fogleman [7]. He studied the preferred temperature of D. melanogaster based on the temperature of ovipo- sition sites and found that changing the rearing temperature from 25 to 18°C changed the preferred temperature by only 1.8°C. However, the results given in this paper showed that the rearing tem- peratures from 15°C to 25°C changed the preferred temperatures of some species such as rufa and lacertosa by more than 4°C, although the preferred temperatures of melanogaster were stable. The correlation matrix between preference score and ecological indices and body weight demonstrated that statistically highly significant correlations at the 1 % level occurred in only two cases involving flies raised at 15°C. These facts indicate that the rearing temperature may affect the temperature preference of some Drosophila species. It should be noted that although all species used in this study were collected from the same locality and presumed to be under the same climatic conditions in their natural habitat, the importance of tem- perature preference seemed to be different among species. Little has been reported about the relationship between the preferred temperature and ecology of adult flies in natural habitats. In this study some ecological indices based on the collection data in the area from which the materials of this study were obtained were considered in relation to the preferred temperature. Even in the same locality different Drosophila species appear in different seasons and their micro-distributions also differ from each other. Their pattern of appearance in nature can be analyzed in the form of the following indices: Domesticity index; niche breadth in time and in space; mean tem- perature and mean relative humidity of appearance. The results of this study indicate that the preferred temperature of each species is inversely related to the mean temperature of appearance. In 640 other words, Drosophila species which appear in hot seasons prefer relatively low temperatures. The only exceptional case was found in curviceps, which may be due to the technical difficulties of culturing this species under laboratory conditions A negative correlation be- index and preferred in the present study. tween the domesticity temperature is also evident. Domestic species prefer lower temperatures than wild ones. Since habitats of domestic species contain poor vegeta- tion, it is hotter and less humid than those of wild species, especially in hot season. Therefore, the low temperature preference of domestic species may lead them to more favorable environments for their survival and reproduction in the domestic area when they must reproduce there. The negative relationships between the preferred temperature and the mean temperature of ap- pearance is an unexpected result. The low temperature preference, however, leads flies which appear in hot season to comfortable place, like as in the domestic area. Each species has its own genetically-determined hatibitat and season of appearance corresponding to some ecological need(s) such as resource utilization [15]. The temperature preference of adult flies seems to develop as a secondary characteristic and has significance in selecting more suitable places in the inherently determined habitat. ACKNOWLEDGMENTS The authors thank Dr. Y. Fuyama for his helpful comments on the manuscript and to Drs. A. Kaneko and S. Nishiharu for their help with the identification of Drosophila species. They also wish to thank Drs. K. Elliott and B. L. S. Pierce for their kind reading of the manuscripts and Miss K. Suzuki for her typing. REFERENCES 1 Yamamoto, A. and Ohba, S. (1982) Strategic differences in thermal adaptation between two Drosophila species, D. virilis and D. immigrans. Oecologia (Berlin), 52: 333-339. 2 Timofeeff-Ressovsky, N. M. (1940) Mutations and geographical variation. In “The New Sys- 10 11 WZ 13 14 115) A. YAMAMOTO AND S. OHBA tematics. Ed. by J. Huxley, Oxford Univ. Press, London, pp. 73-136. Lowe, C.H., Heed, W.B. and Halpern, E. A. (1967) Supercooling of the Saguaro species Drosophila nigrospiracula in the Sonoran desert. Ecology, 48: 984-985. Levins, R. (1969) Thermal acclimation and heat resistance in Drosophila species. Amer. Naturalist, 103: 483-499. Parsons, P. A. (1979) Resistance of the sibling species Drosophila melanogaster and Drosophila simulans to high temperatures in relation to humidity: Evolutionary implication. Evolution, 33: 131-136. Prince, G. J. and Parsons, P. A. (1977) Adaptive behaviour of Drosophila adults in relation to temperature and humidity. Aust. J. Zool., 25: 285-290. Fogelman, J.C. (1979) Oviposition site pre- ference for substrate temperature in Drosophila melanogaster. Behav. Genet., 9: 407-412. Carson, H. L. (1965) Chromosomal morphism in Geographically widespread species of Drosophila. In “Genetics of Colonizing Species”. Ed. by H. G. Baker and G.L. Stebins, Academic Press, New York, pp. 503-531. Parsons, P. A. and Stanley, S. M. (1981) Do- mesticated and widespread species. In “The Genetics and Biology of Drosophila, Vol. 3a’. Ed. by M. Ashburner, H. L. Carson and J. N. Thomp- son Jr., Academic Press, New York, pp. 349-393. Throckmorton, L. H. (1975) The phylogeny, ecology and geography of Drosophila. In ““Hand- book of Genetics, Vol. 3”. Ed. by R.C. King, Plenum Press, New York, pp. 421-469. Bock, I. R. and Wheeler, M. R. (1972) The Dro- sophila melanogaster species group. Univ. Texas Publ., 7213: 1-102. Okada, T. (1956) Systematic study of Drosophi- lidae and allied families of Japan. Gihodo, Tokyo. Colwell, R. K. and Futuyma, D. J. (1971) On the measurement of niche breadth and overlap. Ecology, 52: 567-576. Kawanishi, M. and Watanabe, T. K. (1977) E- cological factors controlling the coexistence of the sibling species Drosophila simulans and D. mela- nogaster. Jap. J. Ecol., 27: 279-283. Nishiharu, S. (1980) A study on ecology and evolution of drosophilid flies with special regard to imaginal and larval feeding habits and seasonal population fluctuations. Doctoral Thesis, Tokyo Metropolitan Univ., Tokyo. ZOOLOGICAL SCIENCE 1: 641-652 (1984) Heat and Cold Resistances of Sixteen Drosophila Species from Japan in Relation to Their Field Ecology AKIHIKO YAMAMOTO! and SHIGERU OHBA Department of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158, Japan ABSTRACT — The abilities to resist high temperature, dessication, and low temperature with and without acclimatization were compared in 5 isofemale strains of each of 16 Drosophila species collected from the same locality in central Japan. In every test, about half or more of the total variance was derived from interspecies differences. Interstrain differences within species comprised less than 15%. Based on the species differences, the following conclusions are obtained. The abilities to resist high temperature and dessication were correlated significantly to each other. Species belonging to sub- genus Sophophora were usually more dry resistant than those of subgenus Drosophila. Acclimatiza- tion at 10°C increased the ability to withstand low temperature strikingly, the rate of increase ranging from three times in D. hydei to 30 in D. lacertosa. Domestic species such as D. melanogaster, D. simulans, D. hydei, D. immigrans, and D. busckii were resistant to both high temperature and dry conditions, but they were sensitive to low temperature even after cold acclimatization. The situation was the reverse for the typical wild species such as D. pulchrella, D. lacertosa, and D. annulipes, which had the ability to withstand low temperature, together with the acclimatizing abilities, while they were sensitive to high temperature and dessication. In the genus Drosophila different strategies of thermal adaptation were found between domestic and wild species despite their habitation in the same area; © 1984 Zoological Society of Japan the former were resistant to hot-dry conditions and the latter to cold. INTRODUCTION Thermal adaptation of insects consists of many factors, such as resistance to extreme temperatures, both high and low, and preference for optimal temperatures. From the viewpoint of evolu- tionary ecological genetics, it is of interest to compare several principal components of thermal adaptation among sympatric species belonging to a related group of insects. The genus Dro- sophila contains many species, more than 100 in Japan alone. Past records of seasonal collection reveal that 20 or more species were found during the period of one year in a given narrow area in central Japan [1-3; Yamamoto, in preparation]. These species may have different niches according Accepted March 5, 1984 Received January 5, 1984 1 Present address and for correspondence: National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709, U.S. A. to their different seasons of emergence and their different food utilization [3, 4], although they occur in the same locality. In comparing the strategies of thermal adapta- tion between two Japanese Drosophila species, Yamamoto and Ohba [5] found that D. virilis was much more resistant to extreme temperatures than D. immigrans, while immigrans selected the nar- rower temperature range than virilis. Yamamoto and Ohba [6] also studied temperature preference of 11 Drosophila species using isofemale strains derived from a restricted area in Oiso. They analyzed the relationship between preferred tem- perature and pattern of emergence in nature and indicated that the temperature preference was closely related to the ecological natures of Dro- sophila. In this paper, 16 sympatric species including the 11 species stated above were collected from the same locality in Oiso and were analyzed for their abilities to resist high and low temperatures. The effects of humidity on heat resistance were analyzed 642 A. YAMAMOTO AND S. OHBA simultaneously. Ability to acclimatize to cold stresses was also studied, since there have been some disagreements about the importance of acclimatization in resistance to extreme tempera- tures [5, 7, 8]. Although these analyses were performed on species which belonged to one genus and lived under the same climatic conditions in nature, differentiation in characters concerning thermal adaptation were expected among the ecologically diversified species. MATERIALS AND METHODS Flies The 16 species used in the present study are listed in Table 1. They belong to 7 species-groups of three subgenera of the genus Drosophila. All materials were collected in a narrow suburban area in Oiso, Kanagawa prefecture, during the period from 1978 to 1980 (Yamamoto, in prepara- tion). For each species 5 isofemale strains were randomly chosen for this study. Flies used for the experiments were raised at 20°C on the standard yeast-sugar medium containing Brewer’s dry yeast (8%), cane sugar (5%), agar (0.8%) and antimi- crobiotics. For some species (Jacertosa, bizonata, sternopleuralis, curviceps and annulipes) the tradi- tional cornmeal-molasses medium was used because it was proven better. In most experiments only females were investigated because of the similarity between sexes both in heat and cold resistance. Emerged females which had grown under optimal larval densities were kept in new culture vials until their egg-laying activity reached maximum. Since the period for maturation differed widely among TABLE 1. Mean survival time (hr) in wet or dry condition at 20, 25, and 30°C with two resistance indices Heat Dry Species Wet (RH'>95%) Dry (RH! <30%) resist- _resist- 20°C 30°C 20°C PKC 30°C ance ance index index Subgenus Sophophora bifasciata 135.8+19.0 39.44 4.8 37.1+44.1*** 25.4+1.4 6.1+0.6* 0.29 0.27 suzukii 103.74 5.8 40.8+ 4.5* 27.9+1.0* 21.5+0.6 9.4+0.1 0.39 0.27 pulchrella 109.0+12.7 39.8+ 7.0 32.4+2.3*** 21.9+0.9** Uae: 0.37 0.30 lutescens 106.6+ 9.3 61,0+ 9.4 37.441.7** 21.5+1.1* 4.8+0.4* 0.57 0.35 melanogaster 97.4414.5 50.4+ 5.6 63.944.9*** 44.14+2.2*** 17.4+1.2 0.52 0.66 simulans whisae BO) sae ob Sot se yet We Gsbilot Oi Osedx 0.42 0.41 ficusphila 113.8+ 7.0 485+ 4.3 36.8+1.8 23.1+1.4 7.9+0.7* 0.43 0.32 rufa 232.4+21.6 861+ 8.8 42.8+1.7* 21.5+0.2 6.0-+0.2 0.37 0.18 mean 135 50.3 39.3 MS) 8.5 0.42 0.35 Subgenus Drosophila lacertosa 186.6+27.2 75.00+ 6.8 24.1+1.0 16.0+0.6 4.6+0.2 0.40 0.13 hydei 78.2+ 8.7 542+ 6.7 33.941.6** 23.4+0.7* 9.6+0.7 0.69 0.43 bizonata 255.8+37.8 111.2+14.8 39.54+3.5** 24.7+0.9 7.0+0.2 0.43 0.15 sternopleuralis 236.4+14.6 83.0+ 3.4 32.1+1.0 20.6+0.4 5.10.2 0.35 0.14 immigrans 149.8+11.7 81.5+ 3.4 38.441.9** 261+1.2* 7.4+0.3 0.54 0.26 curviceps 152.24+15.3 43.2+ 4.0 34.3+1.2 21.2+0.5 5.9+0.1 0.28 0.23 annulipes 230.0+26.4 101.6+19.5 33.9+2.1 21.4+0.6 8.1+41.0* 0.44 0.15 mean 184.1 78.5 Ball 21.9 6.8 0.45 0.21 Subgenus Dorsilopha busckii 170.4+13.7 104.24+10.0 46.2+3.1* 26.7+1.3 9.4+0.2 0.61 0.27 The standard error by strain difference accompanies each mean survival time. Dry and heat resistance indices are the ratio of mean survival time values the former dry-20°C/wet-20°C and the latter wet-30°C/ wet-20°C. +; Relative humidity. *, **, ***; Difference among isofemale strains was statistically significant at the 5%, 1% or 0.1% level, respectively. Heat and Cold Resistances of Drosophila 643 species, the age of females tested ranged from three days (melanogaster and simulans) to two weeks (lacertosa and curviceps) at 20°C. Heat and dry resistance Parsons [9] insisted that humidity controlled the survival of Drosophila under heat stresses. In the present study, therefore, heat resistance was tested at two different levels of relative humidity (RH): wet (RH>95%) and dry (RH<30%). For dry and wet conditions, three (30, 25 and 20°C) and two (30 and 20°C) temperatures were used, respectively. Since the age of flies may affect the ability to withstand high temperatures [10], changes in heat resistance during ageing were preliminarily ex- amined in an isofemale strain of each species. In general, mean survival time reached a maximum several days after eclosion, from three to 8 days depending on the species. Flies slightly older than this age were used in the following experi- ments. Twenty flies were put in a glass tube (2.5 cm in diameter <6 cm high) without food, both ends of which were capped with gauze and sponge plugs. The tubes were kept in a box controlled for given combination of temperature and hu- midity. The number of survivors was checked every three hours (dry-30°C) or twice (dry-25°C and dry-20°C) or once (wet-30°C and wet-20°C) a day. Based on these data, the mean survival time was calculated for 20 flies. There were three replicates in the dry-30°C test, 6 in the dry-25°C and dry-20°C tests, and one in both tests under the wet condition. To estimate the degree of resistance to high temperature and desiccation sep- arately, the following two indices were calculated: Heat resistance index —wet-30°C mean survival time/wet-20°C mean survival time. Dry resistance index—dry-20°C mean survival time/wet-20°C mean survival time. Cold resistance In acclimatization tests, flies raised at 20°C were acclimatized to 102°C with new media from two weeks to two months, then their 50% lethal time values (LT;,.) at —4 and —8°C were determined where females of 5 isofemale strains of each species were tested. In no-acclimatization experiment, LT;. at —4°C was examined in flies of both sexes from one isofemale strain of each species, which had been raised and matured at 20°C. To determine LT, values, 10 flies kept at 10 or 20°C were put into a vial (2.5 cm in diameter x 10 cm high) with a sponge plug, and then exposed to —4+0.1°C or —8+0.1°C for various periods. LT;. was estimated from percent survival follow- ing 5 different periods of cold treatment. Five isofemale strains of each species were investigated simultaneously. There were three to 7 replicates in the no-acclimatization test and three replicates in the acclimatization test both at —4 and —8°C. RESULTS Resistance to Heat and Desiccation 1. Survival time under the wet condition Mean survival time values of 16 species in two cases under the wet (RH>95%) condition, wet- 20°C and wet-30°C, are given in the second and third columns of Table 1. Under the wet-20°C condition, the mean survival time ranged from 78 hr in Aydei to 256 hr in bizonata, giving rise to an overall average of about 153 hr. Under this condition the main cause of death of flies was assumed to be starvation. In the wet-30°C condition the mean survival time was markedly reduced, from 36 hr in simulans to 111 hr in dizonata, the overall average of which was only 66 hr, a little more than 40% of the wet- 20°C average. In both cases bizonata was the longest-lived species and simulans was among the two shortest-lived species. Analysis of variance revealed that 58.8 % and 67.7 % of the total variance were due to interspecific differences in the wet-20°C and wet-30°C tests, respectively. The mean survival time of each species was generally parallel under the two wet conditions. The correlation coefficient between them, 0.82, was significant at the 0.1% level. In spite of this general parallelism at two different temperatures under the wet condition, the heat resistance index (wet-30°C mean survival time/wet-20°C mean survival time) varied considerably among species (7th column, Table 1). The index was highest in hydei (0.69) 644 A. YAMAMOTO AND S. OHBA and lowest in curviceps (0.28), resulting in an overall mean value of 0.44. It can also be seen in Table 1 that 8 species belonging to the subgenus Sophophora have generally shorter survival time values under the wet condition than 7 species of the subgenus Drosophila. The average mean survival time of the former was 123.3 hr and 50.3 hr at 20°C and 30°C, respectively, while that of the latter was 184.1 hr (wet-20°C) and 78.5 hr (wet-30°C). That is, Drosophila species could survive about 1.5 times longer than Sophophora species at both tem- peratures under the wet condition. However, the heat resistance index which demonstrates the effect of high temperature under the wet condition did not differ between the two subgenera, with an average of 0.42 in Sophophora and 0.45 in Dro- sophila. A Dorsilopha species, busckii, seemed to be close to the pattern of Drosophila species. But the heat resistant index of this species (0.61) differed from those of the above two subgenera, indicating a high ability to resist the high tempera- ture condition. 2. Survival time under the dry condition Columns 4, 5 and 6 in Table 1 show mean survival time values of 16 species under three different dry (RH<30%) conditions; dry-20°C, dry-25°C and dry-30°C. In dry-20°C, the mean survival time of each species ranged from 24 hr (lacertosa) to 64 hr (melanogaster), and the average of the 16 species was 37.3 hr. The mean survival time at 25°C was reduced by about one third, from 16 hr in Jacertosa to 44 hr in melanogaster, with an average of 24.0hr. At 30°C, the reduc- tion of survival time was even more remarkable, giving rise to an overall average of 7.8 hr. Throughout these three temperatures, melanogaster was the longest-lived one. Analysis of variance at each temperature revealed that about half of the total variance was derived from interspecific differences (40.5% at 20°C, 64.1% at 25°C and 48.4% at 30°C). On the other hand, intraspecific differences among isofemale strains contributed only 6.3% to the total variance at 20°C, 1.4% at 25°C and 14.1% at 30°C, although interstrain and interspecific differences were significant at the 0.1% level, in all three temperature conditions. Based upon the results of analyses of variance for each species, however, it seemed that the intra- specific differences became insignificant with increasing temperature. Significant intraspecific differences at the 5% level were detected in 11 out of 16 species at 20°C, 7 at 25°C, and only 4 at 30°C. The correlation coefficients between two out of three of the temperature conditions were always highly significant at the 0.1% level (r=0.92 be- tween 20 and 25°C; 0.89 between 25 and 30°C; and 0.74 between 20 and 30°C). These correla- tions demonstrated that under the dry condition the effect of temperature upon survival time was almost parallel in the 16 species examined. Intersubgeneric differences in mean survival time under the dry condition were not as con- spicuous as those under the wet condition. Species belonging to the subgenus Sophophora tended to survive a little longer than those of the subgenus Drosophila. Average mean survival time values under the dry condition were 39.3 (20°C), 25.5 (25°C) and 8.5hr (30°C) for 8 species of Sophophora, and 33.7 (20°C), 21.9 (25°C) and 6.8 hr (30°C) for 7 Drosophila species. The difference, however, was mostly due to one _ species, melanogaster, which was extraordinarily resistant to desiccation at any temperature tested. When this species was excluded, the intersubgeneric difference between Sophophora and Drosophila almost disappeared. A species of the subgenus Dorsilopha, busckii, had relatively longer survival time values under the dry condition. 3. Relationship between resistance to heat and desiccation It is evident from Table 1 that the effect of humidity upon mean survival time differed con- siderably among species. This was confirmed by correlation coefficients between the mean survival time values under the dry and wet conditions: r=—0.10 at 20°C and —0.18 at 30°C, both statis- tically insignificant. The dry resistance index (dry-20°C mean survival time/wet-20°C mean survival time) is listed in the last column of Table 1. The value of this index ranged from 0.13 to 0.66, with an average of 0.29. Judging from this index, melanogaster (0.66), hydei (0.43) and simulans (0.41) could be considered dry-resistant species Heat and Cold Resistances of Drosophila and Jacertosa (0.13), sternopleuralis (0.14) and annulipes (0.15) dry-sensitive ones. Intersub- generic differences in the dry resistant index were also evident. The average of 8 MSophophora species was 0.35, while that of 7 Drosophila species was 0.21. The dry resistance index of busckii (0.27) was intermediate between the above two subgenera. In terms of the heat resistance index described earlier, Aydei (0.69), busckii (0.61) and /utescens (0.57) were the most heat-resistant species. The relationship between the heat resistance and dry resistance indices is shown graphically in Figure 1. 30 O Sophophora 0.6 A Drosophila } Dorsilopha r = 0.50 (p<0.05) Dry resistance index 1°) 0.2 0.4 0.6 Heat resistance index Fic. 1. Heat resistance index (the ratio of mean survival time values, wet-30°C/wet-20°C) and dry resistance index (the ratio of mean survival time values, dry-20°C/wet-20°C). Species be- longing to different subgenera are distinguishable by © (Sophophora), 4 (Drosophila), and © (Dorsilopha). a; annulipes, bf; bifasciata, bs; busckii, bz; bizonata, C; curviceps, f; ficusphila, h; hydei, i; immigrans, Ic; lacertosa, \t; lutescens, m; melanogaster, p; pulchrella, r; rufa, sm; simulans, st; sternopleuralis, sz; suzukii. Highly dry resistant species tended to show high heat resistance index values: the correlation coefficient between the two indices (0.50) was significant at the 5% level. The figure also demonstrates the intersubgeneric differences in the dry resistance index stated above. 645 Resistance to Low Temperature 1. Cold resistance without acclimatization In this experiment flies emerged and matured at 20°C were immediately transferred to —4°C to test their cold resistances. LT 5. (50% lethal time) of each sex is summarized in Figure 2, in (hr) 30 20 1 2 5 10 20 30 40 FEMALE (hr) Fic. 2. Sexual differences of 16 species between LT;. values at —4°C without acclimatization. Bars indicate the extent of standard errors by three replications. Abbreviations of species are the same as in Fig. 1. which standard error was calculated from three replicates of an isofemale strain of each species. Survival time values of males were linearly cor- related with those of females, with a correlation coefficient of 0.90 (significant at the 0.1% level). The average survival time of males expressed as the LT;. was 77% of that of females. These facts indicated that females were generally more resistant to low temperature (—4°C) than males, although the difference was small. In further experiments in which effects of acclimatization on cold resist- ance were tested, only females were used. 2. Cold resistance at —4 and —8°C with acclima- tization Newly emerged female flies raised at 20°C were transferred to new culture vials and kept at 10°C for periods varying from two weeks to two months 646 TABLE 2. A. YAMAMOTO AND S. OHBA LT’, (lethal time for 50% death) at —4°C without acclimatization and —4 and —8°C after acclimatization at 10°C LT (hr) Cold Species —4°C without —4°C with —8°C with mre acclimatization acclimatization acclimatization ineex bifasciata 30.9 162.6+24.8** 272.3 +60.0*** 5.3 suzukii 8.3 42.5+ 3.2 4.6+ 0.2 Soll pulchrella 4.6 85.0+10.5 6.4+ 0.9 18.5 lutescens 8.1 44.6+ 7.5 6.4+ 0.5 S52) melanogaster 5.8 23.3== 0:6 2.4+ 0.2 4.0 simulans 5.4 21.4+ 0.5 1.8+ 0.2 4.0 ficusphila 3.0 Was ae iol 452 )L3) es rufa 11.1 57.4+ 4.1 7.3+ 1.9* Sy mean 97 57.4 38.1 6.9 lacertosa 25 74.5+16.3 12.0+ 1.8 29.8 hydei 10.6 Siose O7/ 6.1+ 0.4 2.9 bizonata 26.5 216.9+28.5 153.3+36.2* 8.2 sternopleuralis Sy) ola 17 30.3+ 4.0 16.5 immigrans 1.4 13.0+ 0.7 1.5+ 0.2 9.3 curviceps 12.6 147.5+25.8* 5.3+ 1.1 11.7 annulipes 6.3 154.3+ 9.8 5.9+ 1.9* 24.5 mean 9.4 104.9 30.6 14.7 busckii 4.4 Byes Lats} 4.4+ 0.3 7.4 The standard error by strain difference accompanies each LT59._ The cold acclimatization index, listed in the last column, is the ratio of LT;, values, --4°C with acclimatization/—4°C without acclimatization. * **) #**: Difference among isofemale strains was statistically significant at the 5%, 1% or 0.1% level, respectively. according to the rate of maturation of each species. After this acclimatizing treatment, the LT;, value was determined at both —4 and —8°C. Means of the LT; values of 5 isofemale strains and the standard error derived from strain dif- ferences are given in Table 2, together with the female’s LT;) value at —4°C without acclimatiza- tion described in the preceding section. With acclimatization at 10°C, the ability of female flies to withstand the low temperature increased strikingly. Averaging 16 species, the mean LT:, at —4°C changed from 9.2 hr (without acclimatiza- tion) to 76.6 hr (with acclimatization). Even when acclimatized flies were tested under a more severe condition, —8°C, the average LT;, was 32.7hr, much longer than the value at —4°C without acclimatization, 9.2 hr. Relationships among the LT;. values of each species under the three test conditions (—4°C without acclimatization, —4 and —8°C with acclimatization) are graphically shown in Figure 3. It should be noted that the two species which were the most resistant to —4°C in the non-acclimatized experiment, bifasciata and bizonata, showed the highest values of LT; at both —4 and —8°C under acclimatized conditions. Likewise, immigrans, which was the most sensitive to cold under non-acclimatized condition, gave rise to the lowest LT;.s in both experiments after acclimatization. Although the correlation coeffi- cients between two out of three test conditions were always significant (r=0.65 between —4°C without acclimatization and —4°C acclimatization; 0.73 between —4°C and —8°C with acclimatization; and 0.91 between —4°C without acclimatization and —8°C with acclimatization), Figure 3 showed that these correlations were mostly due to bizonata and bifasciata, which were the two most resistant Species commonly in the three conditions. In both the experiments at —4 and —8°C with acclimatization, analyses of variance demonstrated Heat and Cold Resistances of Drosophila -4°C without acclimatization c bto 2 g (hr) = 200 £ rs) bzo U o £ 100 3 oO st 7 ‘ l ° a ; £.os tor 0 oP cgoa 0 smmhsz 100 200 : ? : : (hr) -4°C with acclimatization Fic. 3. The relationships among LT;, values from three cold resistance experiments. a) —4°C with acclimatization and —4°C without ac- climatization. b) —4°C with acclimatization and —8°C with acclimatization. Abbreviations ’ are the same as in Fig. 1. that the interspecific differences as well as the interstrain differences within species were signifi- cant at the 0.1% level. At —4°C, interspecific variation comprised 13.8% of total variance, while interstrain variation was 53.3%. At —8°C, however, 85.1% of the total variance was derived from interspecific variation and only 4.8% from interstrain differences. In the experiment at —4°C without acclimatization interspecific variance was 15.4%, almost the same as the corresponding value for the —4°C experiment on acclimatized flies. 3. Interspecific differences in the ability of cold acclimatization To analyze the ability to acclimatize to low temperature, a cold acclimatization index (LTso at —4°C after acclimatization at 10°C/LTso at —4°C without acclimatization) was calculated for 647 each species (last column in Table 2). Although the average of this index was 10.3 for the 16 species examined, differences among species were remark- able. In /acertosa and annulipes the highest values were recorded (29.8 and 24.5, respectively), indicating their greater abilities to acclimatize to low temperature. Two species, pulchrella (18.5) and sternopleuralis (16.5) also gave high acclima- tization indices. On the other hand, the species with poor acclimatizing ability were Aydei (2.9), melanogaster (4.0) and simulans (4.0). Relationship between Thermal Resistances and Some Ecological Characteristics in Natural Habitat Yamamoto (in preparation) analyzed a Dro- sophila community in Oiso, and the materials of this study were derived from a part of his collec- tion. During regular monthly collection from December, 1978, to June, 1980, he recorded 26 Drosophila species. Based on the data concerning seasonal appearance and microdistribution in nature, he estimated several indices which might have close relationships to ecological character- istics of Drosophila flies in natural habitats. For the 16 species used in the present study, some of these indices (domesticity, niche breadth in space, niche breadth in time, mean temperature of ap- pearance, and mean relative humidity of appear- ance) are shown in Table 3. Female body weight and mean preferred temperature reported by Yamamoto and Ohba [6] are also listed. Relationships between heat and dry resistance parameters (mean survival time values in five different combinations of temperature and humidi- ty, heat and dry resistance indices) and the ecological indices shown in Table 3 are summarized in Table 4 in the form of correlation coefficients. Corresponding values between cold resistance and the ecological indices are given in Table 5. From these tables the following conclusions can be drawn: 1) Mean survival time under dry-20°C showed a negative correlation with body weight (r= —0.48, p<0.05), while that under wet-20°C, which mainly reflected tolerance to starvation, did not correlate significantly with body weight (r=0.06). The correlation coefficients between body weight and mean survival time values under dry-25°C and 648 A. YAMAMOTO AND S. OHBA TABLE 3. Domesticity, niche breadth, and climatic indices estimated from appearance of 16 Drosophila species in Oiso, Japan | eae of appearance in nature Fem: ale pee Species am Niche breadth Mean Mean weight temperature Domesticity S = temper- _ relative (ae) C) pace ime ature humidity bifasciata 0.34 0.32 0.16 10.6 51.1 1.49+ .13 os suzukii —0.53 0.73 0.63 14.0 56.7 UAVS) SE a)! 3) 24.9 pulchrella —1.00 0.44 0.35 10.8 SZ 2.49 + .08 30.5 lutescens 0.03 0.95 0.76 20.0 68.2 WSs oI 1 24.6 melanogaster 1.00 0.42 0.36 24.3 74.4 Lo 32ae (OY) 20.8 simulans 0.76 0.58 0.37 21.4 V2) 1.40+.06 19.6 ficusphila 0.52 0.68 0.18 15.6 60.7 1.66+.14 — rufa 0.03 0.85 0.42 23.9 74.8 1.88+.09 23.9 lacertosa —1.00 0.15 0.64 18.2 WB 5.41+.21 MS) hydei 0.99 0.36 0.26 25.4 (BES Zo] Tae oll y/ 16.1 bizonata 0.00 0.72 0.14 V7 72.8 1.33+.08 25.0 sternopleuralis —0.55 0.80 0.52 18.5 73.0 1.86+.09 — immigrans 0.75 0.68 0.35 21.6 70.0 4.31+.41 18.8 curviceps —0.60 0.75 0.44 11.5 54.3 4.77+.29 19.5 annulipes —1.00 0.41 0.34 8.6 49.8 2.76+.08 23.4 busckii 0.67 0.62 0.47 20.6 T5 1.44+ 12 21.6 Body weight and preferred temperature of females raised at 20°C in the laboratory are listed in the last two columns. TABLE 4. Correlation coefficients between heat-dry resistance characters and indices of appearance, body weight, or preferred temperature Index of appearance in nature Female Preferred Domes- Niche breadth Mean Mean body temper- ticity tempera-__ relative weight ature Space Time ture humidity Mean survival time Wet-20°C —0.44 0.12 —0.10 —0.16 0.12 0.06 0.25 Wet-30°C —0.11 0.15 0.00 0.14 0.35 —0.01 0.06 Dry-20°C 0.63** 0.08 —0.19 0.45 0.33 —0.48 —0.21 Dry-25°C 0.63** —0.11 —0.26 0.37 0.23 —0.42 —0.26 Dry-30°C 0.53* —0.27 —0.24 0.34 0.17 —0.34 —0.28 Ratio of mean survival times Dry resistance index 0.68** —0.11 —0.04 0.48 0.24 —0.37 —0.30 Heat resistance index 0.59* 0.04 0.16 0.66** 0.53* —0.13 —(0.39 * Significant at the 5% level. ** Significant at the 1% level. dry-30°C, and the dry resistance index were also negative, although they were statistically insignifi- These results indicate that smaller species cant. were more resistant to desiccation than larger ones and that there were no significant differences in tolerance to starvation between large and small Heat and Cold Resistances of Drosophila 649 TABLE 5. Correlation coefficients between cold resistance characters and indices of appearance, body weight, or preferred temperature Index of appearance in nature SE OTL 8 TE ee Le ee, ee Prefer- Domes- Niche breadth = Mean = Mean body guts, ticity .————————————— tempera- relative weight Space Time ture humidity ae LT 55 at —4°C without acclimatization 0.02 —0.07 —0.41 ~0.21 —0.20 —0.39 0.09 —4°C with acclimatization —0.66** —0.13 —0.35 —0.65** —0.45 0.05 0.47 —8°C with acclimatization —0.23 —0.21 —0.42 —0.34 —0.15 —0.21 0.40 Cold acclimatization index —0.78*** —0.39 0.14 —0.46 —0.24 0.56* 0.37 + *#. *#*- Significant at the 5%, 1% or 0.1% level, respectively. species. Since average body weight was sSig- nificantly different among subgenera (1.76 mg in Sophophora, 3.37 mg in Drosophila, and 1.44 mg in Dorsilopha), these phenomena agreed with the intersubgeneric differences in dry resistance index described in the former section. 2) In Table 4, there were significant positive correlations between mean survival time under the dry condition and the domesticity index (r= 0.63 at 20°C, 0.63 at 25°C, and 0.53 at 30°C). Under the wet condition, however, mean survival time was not significantly correlated with domes- ticity (r= —0.44 at 20°C and —0.11 at 30°C). Furthermore, the domesticity index was positively correlated with both the dry resistance index (r=0.68) and the heat resistance index (r=0.59). These results suggested that flies of the genus Drosophila had adapted to domesticated environ- ments by acquiring resistances to high temperature and desiccation. 3) Positive correlations between the _ heat resistance index and two indices describing seasonal appearance in nature (mean temperature and mean relative humidity of appearance) meant that species which appeared in summer, the hot and wet season in Japan, had relatively high resistance to heat. The dry resistance index had a weaker correlation with the seasonal appearance of flies in nature. 4) The preferred temperatures measured by Yamamoto and Ohba [6] showed little correlation with mean survival times and resistance indices in heat and desiccation experiments, although they suggested that heat and desiccation themselves make flies prefer lower temperatures. 5) From Table 5, it is evident that statistically significant correlations were found in only the cases involving cold resistance at —4°C with acclimatization at 10°C. A negative correlation between LT;, at —4°C with acclimatization and domesticity was highly significant (r= —0.66, p<0.01). The situation was the same with mean temperature of appearance (r=—0O.65). These correlations indicated that wild species which appeared in cold seasons had a high ability to withstand low temperature, when the temperature dropped gradually. The cold acclimatization index also had similar negative correlations with domesticity and mean temperature of appearance. When flies were subjected to a sudden decrease in temperature (LT; without acclimatization) or to temperatures lower than those which they might experience in the natural habitat (LT;. at —8°C with acclimatization), significant correlation coeffi- cients between LT;. values and ecological indices disappeared. 6) The significant positive correlation between cold acclimatization index and female body weight (r=0.56) indicates that larger species had higher ability of cold acclimatization. 7) The wild species which appeared in winter and had strong cold resistance at —4°C with acclimatization preferred relatively high tempera- tures, since the correlation coefficient between the LT;. values and preferred temperature was 0.47, p<0.05. Correlation coefficients listed in Tables 4 and 5 demonstrate that the domesticity index has the 650 A. YAMAMOTO AND S. OHBA 30 Alc Domestisity index Aa 10- 0.8 @ 20 oS 0 F@ “asl Ap 0 --08 A -0.8--1.0 A Sz r= -0.67 (p< 0.01) Cold acclimatization index 0 0.2 0.4 0.6 Dry resistance index Fic. 4. The relationship between the dry resistance and cold resistance indices with references to their domesticity indices. The correlation coefficient between two indices (r=—0.67) and the regression line, y=0.827x+0.038, was also shown, where y is cold resistance index and x is dry resistance index. Abbre- viations are the same as in Fig. 1. closest relationship with thermal resistances of the 5 ecological indices, body weight, and prefer- red temperature. This index had high correlation coefficients especially with dry resistance index (r=0.68, p=0.01) and cold acclimatization index (r= —0.78, p<0.001). Figure 4 shows the rela- tionships among these three indices. Wild species like lacertosa, annulipes, and pulchrella had large cold acclimatization index and small dry resistance index. For typical domestic species as melano- gaster and hydei, the situation was the reverse. The figure also shows the highly significant negative correlation (r=—0.67, p<0.01). between dry resistance index and cold acclimatization index. DISCUSSION In the analysis of heat resistance of Drosophila, the problem of relative humidity must be con- sidered at the same time. At various temperatures from 20 to 34°C, Parsons [11] observed drastic decreases in survival time of two Drosophila species, melanogaster and simulans, when they were exposed to dry conditions. He emphasized the importance of ability to resist dry-heat con- ditions in the survivorship of Drosophila flies. In this study, the resistances to heat and desiccation were estimated separately as the heat resistance index (wet-30°C mean survival time/wet-20°C mean survival time) and the dry resistance index (dry-20°C mean survival time/wet-20°C mean survival time) and subgenus Sophophora was more dry-resistant than the other two subgenera species. However, the heat resistance index was positively correlated with the dry resistance index, and, generally speaking, domestic species tended to have higher ability to resist both heat and desicca- tion than wild species. These correlations indi- cated the important roles of high ability to resist dry-heat conditions in the life of domestic species. — Ability to acclimatize to heat was discussed by Levins [8] in relation to the niche breadth of Drosophila species. He found the higher accli- matization ability in broader niche domestic species like melanogaster. In the cosmopolitan species, immigrans, Yamamoto and Ohba [5] reported that the LT;. value at 38°C increased about 30% after acclimatization at 25°C for two days. As for the acclimatizing ability to low temperature, the present study evidenced drastic increases in the LT; value at —4°C after acclimatization at 10°C for more than two weeks. A 30-fold increase was reached in an extreme case in Jacertosa, one of the wild species. No significant correlation was found between the cold-acclimatizing ability and the niche breadth of each species, although the cold-acclimatizing ability showed a highly signifi- cant negative correlation with the domesticity index. These results disagree with those of Levins [8], in which domestic species showed high accli- matizing ability to heat. This difference may reflect the different nature of acclimatization in heat and cold resistances and their different roles in the field life of Drosophila species. Little is known about the wintering places of Japanese Drosophila species. From the published local climatic data (Rikanenpyo, 1982, Maruzen, Tokyo), however, it seemed that Drosophila species would not be exposed to temperatures lower than —8°C in the Oiso region, from which all materials used in the present study were collected. At this temperature, —8°C, most species could Heat and Cold Resistances of Drosophila 651 survive for only one to 6hr. At a slightly higher temperature, —4°C, even the most cold-Ssensitive species, immigrans, showed an LT; value of 13.0 hr after acclimatization. This suggests that most Drosophila species examined in the present study can hibernate sufficiently at this temperature. It can be concluded from these results that, among the three kinds of cold resistance tests in this study, survival time at —4°C after acclimatization is the best index from the viewpoint of field ecology of Drosophila. According to Parsons and Stanley [12], the survival time at —1°C correlated positively with that at 25°C under dry conditions (RH=0%). Their results seem to disagree with those of the present study. In the present results, the correla- tion coefficients were always negative between LT;so value at —4°C with acclimatization and mean survival time at different temperatures in the dry condition (r= —0.31 at 20°C, —1.36 at 25°C, and —0.47 at 30°C). One of the reasons for this discrepancy may be due to the different low tem- peratures at which the cold resistances were tested. Since little is known about the temperature of hibernating places, no one can say which is the more appropriate condition, —1°C or —4°C, for testing the cold resistances of Drosophila flies. Another possible reason may be the difference between the species used by Parsons and Stanley and those used in the present study. Parsons and Stanley used species from various parts of the world, including tropical species such as bipectinata and subarctic ones like funebris, which are dis- tributed allopatrically. On the other hand, sym- patric species in the temperate zone were selected in the present study. However, the results for five species examined in both studies (melanogaster, simulans, hydei, immigrans, and busckii) are not parallel. The differences may be explained by experimental conditions and origin of materials. From all the results obtained in the present study together with the previous work [6], it can be concluded that domestic species and wild ones have different strategies of thermal adaptation. Domestic species have high ability to withstand dry-heat conditions and prefer low temperatures. Wild ones are resistant to cold and prefer high temperatures. The domestic and wild environ- ments clearly differ for Drosophila flies. The origin of domesticity still remains to be solved. This study suggests, however, that domestication of Drosophila is based on the tolerance to heat and desiccation. The cold sensitive nature of domestic flies indicates their physiological change from cold resistant to heat-dry resistant and the change of their overwintering habits. ACKNOWLEDGMENTS The authors thank Drs. Y. N. Tobari and Y. Fuyama for discussions of data and critical comments on this manuscript. They also wish to thank Drs. K. Elliott and B.L.S. Pierce for their kind reading of the manuscripts and Miss K. Suzuki for her typing. REFERENCES 1 Nozawa, K. (1955) A _ statistical study on the natural population of genus Drosophila. Japan. J. Ecol., 6: 1-6. 2 Wakahama, K. (1964) Some ecological studies in Drosophila. Bull. Shimane Univ. Natur. Sci. (Matsue), 13: 50-122. 3 Nishiharu, S. (1980) A _ study on ecology and evolution of drosophilid flies with special regard to imaginal and larval feeding habits and seasonal population fluctuations. Doctoral thesis, Tokyo Metropolitan Univ., Tokyo. 4 Kimura, M. T. (1980) Evolution of food prefer- ences in fungus-feeding Drosophila: An ecological study. Evolution, 34: 1009-1018. 5 Yamamoto, A. and Ohba, S. (1982) Strategic differences in thermal adaptation between two Drosophila species, D. virilis and D. immigrans. Oecologia (Berlin), 52: 333-339. 6 Yamamoto, A. and Ohba, S. (1984) Temperature preferences of eleven Drosophila species from Japan: The relationship between preferred tem- perature and some ecological characteristics in their natural habitats. Zool. Sci., 1: 631—640. 7 Maynard Smith, J. (1956) Acclimatization to high temperatures in inbred and outbred Dro- sophila subobscura. J. Genet., 54: 497-—S0S. 8 Levins, R. (1969) Thermal acclimation and heat resistance in Drosophila species. Amer. Naturalist, 103: 483-499. 9 Parsons, P. A. (1974) Genetics and resistance to environmental stresses in Drosophila populations. Ann. Rev. Genet., 7: 239-265. 10 Hollingsworth, M. J. and Bowler, K. (1966) The decline in ability to withstand high temperature with increase in age in Drosophila subobscura. 2 A. YAMAMOTO AND S. OHBA Exp. Geront., 1: 251-257. Parsons, P. A. (1979) Resistance of the sibling species Drosophila melanogaster and Drosophila simulans to high temperatures in relation to humidity: Evolutionary implications. Evolution, 33: 131-136. 12 Parsons, P. A. and Stanley, S. M. (1981) Domes- ticated and widespread species. In ‘“The Genetics and Biology of Drosophila’, Vol. 3a. Ed. by M. Ashburner, H. L. Carson and J. N. Thomson, Jr., Academic Press, New York, pp. 349-393. ZOOLOGICAL SCIENCE 1: 653-663 (1984) Experiments on Interspecific Hybridization between Oryzias latipes and Oryzias celebensis TAKASHI IWAMATSU, HIROSHI UWA*, AKEMI INDBN and KENJI HIRATA Department of Biology, Aichi University of Education, Kariya 448, and *Department of Biology, Shinshu University, Matsumoto 390, Japan ABSTRACT — Artificial interspecific fertilization of Oryzias latipes eggs by Oryzias celebensis sperm and the reciprocal cross were successful. The hybrids, O. latipes celebensis, which had a single set of each parental genome, were reared until they reached the adult stage with a survival rate similar to that of the controls. In 63 hybrids, some morphological characteristics were grossly intermediate between the parental types while others were O. celebensis in type. Only the rate of increase in ray node numbers and notches in the anal and dorsal fins of the male were like O. latipes. Two of the 33 hybrid females were induced to spawn every day by O. celebensis males. Ten of the hybrid males examined were sterile (testes lacked spermatozoa) and the remaining 20 were neuters (gonads not recognizable). Some sexual characteristics became conspicuous in the hybrids upon administration of sex hormones, but formation of horny processes in the anal fin ray was not induced. All digenomous triploid hybrids resulting from backcrossing of O. latipes celebensis (eggs) to O. celebensis © 1984 Zoological Society of Japan (spermatozoa) died at the embryonic stage of initial blood circulation. INTRODUCTION Berg [1] classified the medaka to the suborder Cyprinodontoidei, family Cyprinodontidae. Sub- sequently, Rosen [2] established a new family Oryziatidae for this fish. The genus names, Aplocheilus [3], Poecillia [4], and Haplochilus [5] have been used for this fish by early investigators. Since Jordan and Snyder [6], Oryzias has generally been used as the genus name. So far eleven species, O. celebensis, O. curvinotus, O. javanicus, O. latipes, O. luzonensis, O. marmoratus, O. matanensis, O. melastigma, O. minutilus, O. timorensis [7] and O. carnaticus [8] have been described. We collected live O. celebensis [9] which inhabited in fresh-water in the South Celebes, and examined the morphology [10] and karyotype [11]. However, we have little knowledge as to the origin and interspecific relationships among these species or the evolution and species differ- entiation in this genus. Usually, hybridization Accepted April 12, 1984 Received December 23, 1983 has been used for genetic analysis of the phylogenetic relationships among species. We succeeded in obtaining adult hybrids, O. latipes celebensis, between O. latipes eggs and O. celebensis sperm. The present paper will report observations on their morphology, karyotype and behavior. MATERIALS AND METHODS Live Oryzias latipes (orange-red variety) were purchased from a fish farm (Yatomi, Aichi Pref.) and O. celebensis were collected from Ujung Pandon, the South Celebes (1975). The following procedures were used to achieve hybridization between O. latipes and O. celebensis so that fertilization and the course of development could be observed. Females that had spawned every day were laparotomized after their brains were pithed and their ovaries were removed into a saline solution [12]. Unfertilized eggs were released from the lumen of the ovaries and kept in saline until used. A sperm suspension was prepared by squeezing spermatozoa out of the testes of mature males into the same saline. The 654 eggs were inseminated by immersing them in the sperm suspension. After hatching, the fry were raised in glass dishes (30cm in diameter, water depth ca. 15cm) under continuous light from a tungsten lamp (150 lux). When they had grown to more than 10mm in standard length, they were transferred to glass aquaria (ca. 60x35 x 30cm, 28-30°C, 14 hr light period) equipped with a water filter. Feeding and mating behaviors were ob- served in these aquaria. In order that their mating behaviors were might be observed, two hybrid females and one hybrid or O. celebensis male were kept together in a glass aquarium under reproductive conditions (26-28°C, 14hr light period). In order to examine the effect of sex hormones on sexual characteristics of 104 young hybrids (14-18 mm in standard length), the hormones were mixed with the powdered diet according to Yamamoto’s method [13] and orally administered as follows. Young fish in the experimental group were continuously reared with a hormone-diet of either 20 ug/g ethynyl estradiol (42 fish) or 50 yg/g methyl testosterone (44 fish). Fourteen fish in the control group were reared with a normal diet (mixture of shrimp and roasted wheat powder). The karyotype was examined using the culture technique described by Uwa and Ojima [14]. The CSB method [15] was applied for C-band staining of some preparations. Fish anesthetized with 10% saturated phenyl urethane and ethanol (3: 1) were measured under a binocular dissecting microscope (x20); fin length and body depth were expressed as a per- centage of the standard length. Large pit organs in fish fixed with Bouin’s fixative were observed T. IWAMATSU et al. after they were stained with 0.004% fast green- eosin (room temp.) for 1 hr. To allow observation of the skeleton fish were treated with 1-2 N NaOH (room temp.) for 6-12 hr and stained with 0.1% Arizalin S overnight. Sex was determined by dissecting the gonads, and oocytes and spermatozoa from which were examined with high magnification ( x 600). Data were statistically analysed by the Student’s t-test. RESULTS AND DISCUSSION Development and viability Unfertilized eggs of O. latipes or O. celebensis were artificially inseminated by spermatozoa of the reciprocal species. Eggs of O. latipes were slightly smaller than those of O. celebensis (Table 1). The results of their development are presented in Table 2. There was no remarkable difference between the developmental velocity of fertilized O. celebensis and O. latipes eggs. Eggs of O. latipes inseminated with O. celebensis spermatozoa developed normally up to the morula stage in the same time course as for O. latipes eggs inseminated with homologous spermatozoa. The percentage of normally developing embryos was somewhat less than in the control group, however. A very similar tendency was observed in the O. celebensis eggs inseminated with O. latipes spermatozoa. Some of the hybrids, O. latipes celebensis, developed to fry, but the reciprocal hybrids, O. celebensis latipes, did not. This failure to obtain the reciprocal hybrid may have resulted because O. celebensis eggs were TABLE 1. Size of O. celebensis, O. latipes and O. latipes celebensis eggs before and after fertilization Diameter (um) of Species ee Head unfertilized eggs fertilized eggs** (chorion) (chorion) (vitellus) O. celebensis 9) (Be 1202+7 1326+5 1123+14 O. latipes 4 (40) 1189+6 1235+4 1058+25 O. latipes celebensis 2 (17) — 1170+17 1005+12 * Numbers in parentheses indicate numbers of eggs examined. ** Shortly after activation. Interspecific Hybridization in Oryzias 655 TABLE 2. Development of interspecific hybrids between O. /atipes and O. celebensis % Of eggs showing NG.G) sein Women) activation morula SHAS y Gaya Me eeaaaes total normal abnormal O. celebensis X O. celebensis 75 (5) 97.2 97.2 69.5 52.8 16.7 O. latipes x O. latipes 168 (5) 91.2 91.2 2 i ee 87.7 in O. celebensis x O. latipes 53 (5) 79.2 66.7 56.0 46.0 10.0 O. latipes x O. celebensis 970 (21) 94 94.6 1Gn" 62.6 13.0 * Fry were obtained. Number of ray nodes 15 20 25 30 35 Standard length (mm) Fic. 1. Number of ray nodes of the anal fin relative to standard length in O. celebensis (C), O. latipes (O) and O. latipes celebensis (LC). Symbols @, © and x represent female, neuter and male, respectively. 656 T. IWAMATSU et al. Number of ray nodes 15 20 25 Standard length Fic. 2. Number of ray nodes of the dorsal fin relative to standard length in O. celebensis (C), O. latipes (O) and O. latipes celebensis (LC). 30 35 40 45 (mm) Symbols @, © and xX represent female, neuter and male, respectively. overripe or poor in developmental capacity in nature, as the developmental rate of the control eggs was also poor (Table 2). The number of ray nodes of the anal and dorsal fins increased in proportion with the standard length, particularly in females (Figs. 1 and 2). The rate of increase in ray node numbers in the dorsal fin relative to standard length in hybrids, O. latipes celebensis, differed significantly from that in O. celebensis but not from that in O. latipes. However, in the anal fin ray it was intermediate between the parents (P> 0.05). Characters, proportional measurements and counts of the adult body Eight proportional characters, the length of snout (LS), head (LH), body depth (BD), anal fin base (AFB) and of caudal (LC), pectoral (LP), dorsal (LD) and ventral (LV) fins were measured. In addition, longitudinal scales and rays of each Interspecific Hybridization in Oryzias fin were counted. These results are summarized in Table 3. The ranges of lengths of snout, head, caudal and ventral fins in proportion to standard length in hybrids were significantly smaller than those in their parents (P<0.05). The numbers of longitudinal scales and rays for all fins except the caudal were significantly greater than those in the mother QO. /atipes, and the number of caudal fin rays (Cr) was also greater than in their father O. celebensis. Large pit organs distributed on the head along the orbit and preperculo-mandibular lines and on the top of the head were of the same open type as in the parents [16]. Their body color was like the wild type O. celebensis. In color pattern, they possessed a fine dark lateral line branching to two conspicuous dark stripes around the 9th (from the dorsal side) ray in the truncated caudal fin, and a black stripe running along the base of the anal fin (Fig. 3) as seen in O. celebensis (Fig. 4) [10]. A fin line with a light greenish (metalic) color was observed along the base of the dorsal fin, as seen TABLE 3. and their hybrids 657 in O. celebensis. The body had a straight black stripe on its side extending from over the ventral fins to the base of the caudal fin, and continuing to the dark stripes in the caudal. Behavior O. latipes usually ate food floating on the water surface, whereas O. celebensis ate food suspended in a deeper region of the water. On the rare occasions when the latter caught food at the surface, they dived immediately by beating the water with their tails and bending their bodies. Feeding behavior of hybrids was intermediate in type. They usually ate food in deeper water as well as at the water surface, but behaved rather similarly to O. celebensis. In the mating behavior of both O. celebensis and O. latipes, the male exhibited a so called “courting round dance” [cf.17] just before copulation, at which time both sexes began to sink down to the bottom, maintaining a crossed posture. During copulation, both the dorsal and Proportional measurements and counts of fixed specimens of O. Jatipes, O. celebensis O. latipes No. of specimens 52 TL (mm) 35.5+0.4 SL (mm) 29.0+0.3 (24.8-34.0) Snout (LS)* 0.13 Head (LH)* 0.27 Body depth (BD)* 0.21 Anal fin base (AFB)* 0.29 Caudal (LC)* 0.27 Pectoral (LP)* 0.21 Dorsal (LD)* 0.19 Ventral (LV)* 0.13 30.3+0.1 (28-32) 9.5+0.1 ( 8-11) 6.1+0.0 ( 6 7) 20.9+0.1 (17-23) 18.7+0.2 (17-22) 6.1+0.0 ( 5— 7) L. tr. scale Pectoral fin rays** Dorsal fin rays** Caudal fin rays** Anal fin rays** Ventral fin rays** O. celebensis O. latipes celebensis 53 52 26.6+0.4 41.2+0.7 22.0+0.4 34.1+0.6 (15.9-28.4) (26.6-44.9) 0.14 0.12 0.29 0.25 0.27 0.25 0.30 0.30 0.27 0,23 0.24 0.22 0.25 0.20 0.14 0.12 31.6+0.1 (29-33) 31.7+0.1 (30-33) 9.8+0.2 ( 7-11) 8.7+0.2 ( 7-11) 21.4+0.2 (19-24) 21.3+0.2 (19-23) 6.1+0.0 ( 5— 7) 10.1+0.1 ( 9-11) 7.9+0.1 ( 7- 9) 22.7+0.3 (19-24) 21.3+0.1 (19-24) 6.1+0.1 (4 — 8) Numbers in parentheses indicate ranges of each counting number. * Length to standard length (SL). ** Ray number (including spines in caudal fin). 658 T. IWAMATSU et al. Fia. 3. the dorsal and anal fins of the male. Photographs of hybrid O. latipes celebensis. 2.3 Arrows indicate notches in Fic. 4. Drawing of O. celebensis showing urinogenital eminences (UGP) and sexual differ- ences in fins. anal fin. A whole drawing: male. anal fins of the male bent toward the female. Hybrid females would spawn upon mating stim- ulation by O. celebensis males but not by their brothers or males of O. latipes. Male hybrids displayed the dancing behavior for tempting females to mate and could induce spawning (only A: Female ventral fins and UGP, B: Female dorsal fin, C: Female unfertilized eggs) of O. latipes but could not induce spawning of sisters or females of O. celebensis. Therefore, it is doubtful that mating behavior of the male hybrids is the same as that of O. celebensis. The hybrid males, which possessed no horny processed on any anal fin rays, seemed to be able Interspecific Hybridization in Oryzias to stimulate spawning of O. /atipes females. Spawning in O. /atipes is normally stimulated by the male with the anal and dorsal fins during mating behavior [17, 18]. Sexual dimorphism In both O. celebensis and O. latipes, the anal fin rays of the male rarely branched dichotomously, while all the fin rays of the female were bifurcated dichotomously at their distal ends, as reported for O. latipes by early investigations [19-23]. In O. latipes, the most prominent of the male sexual characteristics is the presence of small horny processes on the internodes of the posterior rays of the anal fin [24, 25]. No such processes were recognized in any anal fin rays of the male O. celebensis. In the male, the longer dorsal fin reached the base of the caudal fin in O. Jatipes, and went beyond it in O. celebensis. The dorsal fin was shaped like a broad sward in O. celebensis. In both species, the ventral fins of the female were large and went beyond a pair of urinogenital eminences (UGP, Fig. 4), which were described by Oka [24] for O. latipes. In O. latipes males, the posterior margin of both the anal and the dorsal fins had a shallow notch, as shown in the figures of Oka [24], while O. celebensis (Fig. 4) did not have such a feature. In hybrids, sexual dimorphism was recognized in the shapes of body, fins (Table 4, Fig. 3) and 659 urinogenital eminences as observed in O. celebensis and O. latipes. The average number of nodes in a ray in both the anal and dorsal fins was greater in the male than in the female (Table 4). The posterior margin of each anal and dorsal fin of the male had a shallow notch by the hindmost ray, which was separated from the rest (Fig. 3). In the reproductive season (conditions), melano- phores in the ventral, anal and caudal fins were prominent in the males of hybrids as well as of O. celebensis and O. latipes. This feature in O. latipes was described earlier by Niwa [26, 27]. Females were characterized by both long ventral fins and well-developed urinogenital eminences. Anatomical observations of adult hybrids revealed that those showing typical male or female characteristics possessed testes or Ovaries, respec- tively. However, no spermatozoon was detected in any testes. Many hybrids were neuters in which no gonads were found. These exhibited indistinct male characteristics as seen in Figures | and 2. The sex ratio among the fully grown hybrids examined is shown in Table 4. Nearly equal numbers of females and males were counted in O. celebensis and O. latipes, but among 52 O. latipes celebensis, there were 33 females, 10 males and 20 neuters. Judging from the sex ratio and male characteristics, the neuters appear to have been completely sterile males with extremely poor or degenerated testes. Imperfection of the male TABLE 4. Sexual differences in O. celebensis, O. latipes and O. latipes celebensis O. celebensis female Number of each sex (%) Standard length (mm) (Length*) Body depth 0.27 0.27 Longest dorsal ray 0.19 0.32 Longest ventral ray 0.15 0.13 Anal fin base 0.28 0.31 (Number) Anal fin rays branched Rays with papilla processes 0 0 Ray nodes in an anal fin ray Ray nodes in a dorsal fin ray male 28 (50.9) 27 (49.1) 28 (53.8) 24 (46.2) 22 (42.3) 21.9+0.5 22.1+0.5 30.0+0.5 28.140.4 33.84+0.8 35.64+0.3 33.5+0.8 6.841.0 0.7+0.2 14.6404 0.940.1 12.9+1.1 O. latipes celebensis female male neutral O. latipes female male 10 (19.2) 20 (38.5) 0.20 0.23 0.25 0.24 0.25 0.17 0.22 0.18 0.23 0.21 0.14 0.12 0.13 0.12 0.13 0.27 0.30 0.29 0.29 0.30 4743.2 654+1.4 0 73£0.5 0 0 0 9.2+0.3 22.440.9 14.640.3 22.340.6 15.94+0.4 26.341.9 20.740.8 9.8+0.3 24.740.9 17.540.4 24.940.5 17.2+0.5 28.0+1.0 23.440.8 * Expressed as a percentage of the standard length. 660 T. IWAMATSU et al. LV (14) (25)L Ds (01)LD~™ LA(35) Fic. 5. Effect of sex hormones on sexual dimor- phism of hybrids O. latipes celebensis. C: Control, E: Treated with ethynyl estradiol, M: Treated with methyl testosterone. Aj, Dj and Vj indicate the ray node numbers in the anal, dorsal and ventral fins, respectively; LA, LD, LV and LD’ indicate the lengths of the anal, dorsal, ventral fins and the base of dorsal fin; BD indicates body depth. characteristics in the hybrid was possibly due to a deficiency in the sex hormones required to fully manifest these characteristics. Formation of the horny processes can be induced in O. latipes females by oral administration of a diet containing androgen [28]. In order to ascertain this possibility, ethynyl estradiol and methynyl testosterone were admini- stered orally to young hybrids for 45 days. Their sexual characteristics were carefully examined at the end of the administration period, and the data are presented in Figure 5. In the group adminis tered ethynyi estradiol, the number of ray nodes (Vj) and length (LV) of the ventral fin and the base of the dorsal fin (LD’) appeared to increase com- pared to those of the control group. On the other hand, the ray node numbers (Aj, Dj) in the anal and dorsal fins and the length of the dorsal fin (LD) conspicuously increased as a result of methyl testosterone administration. However, appea- rance of the horny processes in the anal fin rays and increase in body depth were not induced by administration of androgen to hybrids. This suggests that the administration of androgen was insuficient to induce manifestation of the male characteristics and that the characteristics are recessive. Karyotypes Karyograms of O. latipes celebensis are displayed in Figure 6. Their karyotypes contained 42 chromosomes, one set (n=24 chromosomes, 14) from O. latipes and one set (n=18 chromosomes, 11) from O. celebensis, consisting of 3 pairs of metacentric, 5 pairs of submetacentric, a sub- telocentric and 25 acrocentric chromosomes (FN=58). Therefore, this hybrid karyotype was apparently a digenomous diploid type. Reproductive ability Two-hundred-forty-two eggs were obtained from two female hybrids mated to O. celebensis males. Eggs of hybrids had a chorion 1170+16.5 ym in diameter and a vitellus 1005+12 wm in diameter. These eggs were significantly smaller than those of the parents (P<0.01). Some of them were capable of initiating development but could not develop to fry (Table 5). In some embryos blood circulation commenced, but soon stopped because of blood coagulation in the veins on the yolk sphere. These embryos were digenomous tri- ploids with one O. latipes genome and two O. celebensis genomes (Fig. 7). This indicates that the unfertilized eggs of O. latipes celebensis matured without meiosis before they were fertilized. It has been demonstrated that oocytes in O. latipes [29, 30] can reach full size before completion of meiosis and mature independently of the presence of the germinal vesicle. Judging from these facts, no pairing of chromosomes of inter- ruption for meiosis seemed to take place during gamete formation in the present hybrid. | Skeletons Seven skeletons of the hybrids were compared with those of the parents (Table 6). Numbers of vertebrae in hybrids varied from 29 to 32. Num- bers of precaudal vertebrae ranged from 12 to 14. These numbers were slightly larger than those of the parents. A pair of first pleural ribs stemed from the second vertebra, as reported previously [10]. Therefore, the number of vertebrae with a pair of long slender ribs was one less than the Interspecific Hybridization in Oryzias o> ~ ast i wo wD ela S OF wD Se OO 415 »** we eB ap 6 De - F- ap | a> » Oc 2 20 4 i Byes - mpeg ~o a> ‘o> & 2¥ ' “Se ane aS =» > op — = @&o oo & 661 eed ©£O< 5 um Fic. 6. The karyotype (upper of each 2 rows) and C-banding pattern (lower of each 2 rows) of QO. latipes celebensis. Biarmed chromosomes of paternal O. celebensis origin (the lower 4 rows) are discriminated from those of maternal O. J/atipes origin (the higher 2 rows). Arrows indicate characteristic C-bands. TABLE 5. Development of eggs of O. latipes celebensis females mated by O. celebensis (26—27°C) Ta No. of eggs No. (%) of or showing | oe oe ee Aug. 31, 1981 10 (1) 8 (80.0) 3 (30.0) 0 2 (20.0) 0 Sept. 1, 1981 28 (2) 18 (64.3) 9 (32.1) 0 Me get 8 0 4 17 (1) 13 (76.5) 8 (47.1) 4 (41.2) 6 (35.3) 0 13 (1) 8 (61.5) 7 (53.8) 4 (30.8) 6 (46.2) 0 36 (2) 22 (61.1) 22 (61.1) C28) rt 2.8)" 0 8 zr) 19 (90.5) 19 (90.5) 3 (14.3) 14 (66.7) 1 (4.8)** 14 (1) 8 (57.1) 7 (50.0) 0 3 (21.4) 0 15 8 (1) 5 (62.5) — — as i. 21 29 (2) 25 (86.2) 25 (86.2) 0 18 (62.1) 0 22 13 (1) 12 (92.3) 12 (92.3) 9 (69.2) — — 23 25 (2) 25(100) 21 (84.0) 0 10 (40.0) 0 25 11 (1) 1 ( 9.1) 1 ( 9.1) 0 1 ( 9.1) 0 17 (1) 15 (88.2) 11 (64.7) 0 11 (64.7) 0 Aug. 31-Sept. 25, 1981 242 179 145 21 74 I (2) (71.5+6.3) (58.447.4) (31.746.6) (34.1+6.5) (0.4) * With blood circulation. ** Normal in size and shape without blood circulation. 662 T. IWAMATSU et al. Dnannanandnh Ah AL OO a 9M9nManhANHRQAhA ANB OnannhANROB&AAA 86 i SO xz. $66 AQ 5um Fic. 7. A karyogram of back-crossed embryos resulting from insemination of O. latipes celebensis eggs by O. celebensis sperm. They had (basically) one genome of O. latipes (upper 2 rows) and two genomes of O. celebensis (lower 3 rows). TABLE 6. Skeletons of O. latipes, O. celebensis and O. latipes celebensis O. celebensis O. latipes O. latipes celebensis No. of specimens 165 19 8 Vertebrae 29.2+0.2 (28-30) 29.8+0.1 (29-30) 30.5+0.3 (29-32) Vertebrae with ribs 10.9+0.2 (10-12) 11.0+0 (11) 11.8+0.3 (11-13) Pterygium* 3.9+0.1 ( 3- 4) 3.6+0.1 ( 2-4 ) 3.0+0.4 ( 1- 4) Fan-shaped neural spine 8.1+0.2 ( 7-9 ) 8.7+0.2 ( 7-10) 8.1+0.5 ( 7— 9) Branchial sieve cartilage 13.1+0.6 (10-16) 17.9+0.1 (17-19) 13.0+0.7 (11-18) Branchial filament 43.0+1.7 (36-56) 40.9-+0.5 (37-44) 43.1+1.5 (39-51) Branchiostegal ray 5.2+0.1 (S— 6) 5.3+0.1 ( 5-6 ) 5.5+0.2 ( 5-6 ) * Including bones fused with scapula. number of precaudal vertebrae. Broad (fan- relation with feeding materials and behavior, for shaped) neural spines extended from each of the first to seventh or ninth centra. The mean number in hybrids was the same as in O. celebensis. Pterygia (the pectoral girdle consisting of a series of dermal bones) of hybrids often fused with the scapula so that the mode (3) was less than that (4) of their parents. There was no significance be- tween hybrids and O. celebensis in the number of branchial sieve cartilage structures (gill rakers) that aid in food gathering. Their number was significantly smaller than in O. latipes. In general, gill rakers may be few in number and small in fish that consume large prey but may bear rough prominences or denticles that aid in holding and swallowing. Thus, the similarity suggests a cor- feeding behavior of hybrids closely resembled that of O. celebensis, which consume large prey. Among O. celebensis, O. latipes and their hybrids, the mean numbers of branchials and branchiostegal rays that supported the branchiostegal membrane were in the same ranges. ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid for General Research (project No. 56340037) from the Ministry of Education, Science and Culture, Japan. REFERENCES 1 Berg, L.S. (1940) Classification of fishes, both 11 16 17 Interspecific Hybridization in Oryzias recent and fossil. Travaux. Inst. Zool. Acad. Sci., Russia, Tome V, Livre 2. Rosen, D. E. (1964) The relationships and tax- onomic position of the balfbeaks, Killifishes, Silversides, and their relatives. Amer. Mus. Natl. Hist., 127: 219-267. McClelland, J. (1839) Indian Cyprinidae. Asiatic Researches, 19 (2): 301, 427. Temminck, C.J. and Schlegel, H. (1842-1950) Pieces. Siebolt’s Fauna Japonica. 323 pp. Giinther, A. (1866) Catalogue of the fishes in the British Museum, 6: 311. Jordan, D.S. and Snyder, J. O. (1906) A revier of the poecilidae or killifishes of Japan. Proc. U.S. Natl. Mus., 31: 287-290. Yamamoto, T. (1975) Systematics and Zooge- ography. MEDAKA (killifish) Biology and Strains. Keigaku Publishing Co., Tokyo, pp. 17-29. Jerdon, A. (1849) Madras Journal of Literature and Science, 15: 331. Weber, M. and _ Beaufort, L. F. D. (1922) The fishes of the Indo-Australian Archipelago, 4, E. J. Brill, Leiden, pp. 1-410. Iwamatsu, T. and Hirata, K. (1980) Compar- ative study of morphology of three species of the medaka Oryzias. Bull. Aichi Univ. Educat., 29 (Nat. Sci.): 103-120. (in Japanese) Uwa, H., Iwamatsu, T. and Ojima, Y. (1981) Karyotype and banding analyses of Oryzias celebensis (Oryziatidae, Pisces) in cultured cells. Proc. Jap. Acad., 57, Ser. B: 95-99. Iwamatsu, T. (1974) Studies on oocyte matura- tion of the medaka, Oryzias latipes. Il. Effects of several steroids and calcium ions and the role of follicle cells on in vitro maturation. Annot. Zool. Japon., 47: 30-42. Yamamoto, T. (1953) Artificially induced sex reversals of male genotype (XY) in the medaka (Oryzias latipes) with special reference to YY- male. Genetics, 40: 406-419. Uwa, H. and Ojima, Y. (1981) Detailed and banding Karyotype analyses of the medaka, Oryzias latipes in cultured cells. Proc. Jap. Acad., 57, Ser. B: 39-43. Sumner, A. T. (1972) Simple technique for de- monstrating centromeric heterochromatin. Exptl. Cell. Res., 75: 304-306. Iwamatsu, T., Ohta, T. and Saxena, O. P. (1983) Morphological observations of large pit organs in four species of freshwater teleost, Oryzias. Medaka, 2: 7-14. Ono, Y. and Uematsu, T. (1957) Mating 20 23 24 25 26 27 28 29 30 663 ethogram in Oryzias latipes. J. Fac. Sci., Hok- kaido Univ., Ser. VI, Zool., 13: 197-202. Egami, N. and Nambu, M. (1961) Factors initi- ating mating behavior and oviposition in the fish, Oryzias latipes. J. Fac. Sci., Tokyo Univ., Sec. IV, 9: 263-278. Ishihara, M. (1916) Inheritance of body color in Oryzias latipes. Zool. Mag., 28: 177-194. (in (Japanese) Aida, T. (1922) Sex-limited inheritance in Aplocheilus latipes. Japan. J. Genet., 1: (in Japanese) Ono, Y. (1927) The behavior of the cells in tissue cultures of Oryzias latipes with special reference to the ectodermic epithelium. Annot. Zool. Japon., 11: 145-149. Goodrich, H. B. (1927) A study of the develop- ment of Mendelian characters in Oryzias latipes. J. Exp. Zool., 49: 261-280. Kamito, A. (1928) Early development of the Japanese killifish (Oryzias latipes), with notes on its habits. J. Coll. Agric. Tokyo Univ., 10: 21-38. Oka, T. B. (1931) On the processes on the fin rays on the male of Oryzias latipes and other sex characters of this fish. J. Fac. Sci., Tokyo Univ., Sec. IV, 2: 209-218. Yamamoto, M. and Egami, N. (1974) Fine stru- cture of the surface of the anal fin and the pro- cesses on its fin rays of male Oryzias latipes. Copeia, No. 1, 261-265. Niwa, H.S. (1955) Effects of castration and administration of methyl-testosterone on nuptial coloration of the medaka, Oryzias latipes. Jap. Jour. Ichthyol., 4: 193-200. (in Japanese) Niwa, H.S. (1965) Effects of castration and administration of methyl testosterone on the nuptial coloration of the medaka (Oryzias J/atipes). Embryologia, 8: 289-298. Uwa, H. (1975) Formation of anal-fin processes at the anterior margin of joint plates induced by treatment with androgen in adult females of the medaka, Oryzias latipes. Zool. Mag., 84: 161-165. (in Japanese) Iwamatsu, T. (1966) Role of germinal vesicle materials on the acquisition of developmental capacity of the fish oocyte. Embryologia, 9: 205-221. Iwamatsu, T. and Ohta, T. (1980) The changes in sperm nuclei after penetrating fish oocytes matured without germinal vesicle material in their cytoplasm. Gamete Res., 3: 121-132. 159-177. ZOOLOGICAL SCIENCE 1: 665-671 (1984) © 1984 Zoological Society of Japan Hynobius takedai (Amphibia, Urodela), a New Species of Salamander from Japan MASAFUMI MATSuI! and KOJI MIYAZAKI? ‘Biological Laboratory, Yoshida College, Kyoto University, Sakyo, Kyoto 606, and "Ishikawa Prefectural Board of Education, Kanazawa 920, Japan ABSTRACT — A new species of salamander, Hynobius takedai, is described from the lowland of Ishikawa Prefecture, on the Japan Sea side of the Chubu District, Central Japan. It belongs to the lichenatus group of Hynobius, and is characterized by the intermediate adult morphology between H. lichenatus and H. abei, large clutch size, absence of striations on the egg sac envelope, and unique electrophoretic pattern of serum proteins. lichenatus of northeastern Japan. Salamanders of the genus Hynobius are roughly divided into two types, lowland still-water one and mountain stream one, chiefly by the breeding habit and the shape of adult tail [1]. The members of the former type are further split into the nebulosus and lichenatus groups [2]. With the exception of MHynobius nigrescens, which has peculiar egg sac and adult morphology, all the known forms of these two groups from Honshu, the mainland of Japan, particularly preserved specimens, are very difficult to identify because of their remarkable morphological uniformity. Although members of the snebulosus and lichenatus groups having transparent “banana”’ shaped egg sacs are widely distributed in Honshu, they have been reported to be absent in some areas. The Hokuriku District had been among such areas until Mr. Toshio Takeda found a salamander with the characteristics mentioned above from near Koshijino Elementary School, Chijimachi, Hakui-shi, Ishikawa Prefecture on April 10, 1971. Subsequent surveys made by one of us (KM) proved that the salamander widely occurred in the lowland of Ishikawa Prefecture and had morphological and reproductive characteristics similar to those of H. abei. He then identified Accepted January 28, 1984 Received October 17, 1983 The new species is considered most closely related to H. it with the latter species [3, 4]. AMynobius abei, a member of the /ichenatus group, has been recorded only from a confined area of the Kinki District and is well known for its unique morphology and early breeding season [5]. Miyazaki’s [3, 4] identification was chiefly based on the peculiar secondary sexual characters developed in males of the salamander from Ishikawa Prefecture. However, ample specimens of H. abei and H. lichenatus for comparison were not available for him at that time. In recent years, we have been intensively studying the salamander in question, and have reached the taxonomic conclusion that the salamander is closer to H. lichenatus of northeastern Japan than to abei, but is still distinguished at the species level from the former by several characteristics which are considered taxonomically important. Hynobius takedai M. Matsui et Miyazaki sp. nov. [Japanese name: Hokuriku-sansyouo] Figure | Hynobius abei: Miyazaki, 1977 [3], Miyazaki, 1978 [4], p. 21, figs. 1-2. p. 46. — Holotype — National Science Museum, Tokyo 666 M. MATSUI AND K. MIYAZAKI FIGgl: (NSMT)-H-03990, an adult male, collected in a small pool, Chiji-machi, Hakui-shi, Ishikawa Prefecture, on February 5, 1983, by T. Takeda. Paratypes — NSMT—-H-03991, one female, from the same locality as the holotype, March 4, 1977, by K. Miyazaki; Osaka Museum of Natural History (OMNH) Am 7693, one male, from the same locality as the holotype, April 16, 1978, by K. Miyazaki; NSMT—H-03992, OMNH Am 7694, 2 males, from Shiotsu, Nakajima-machi, February 25, 1979, by K. Miyazaki; OMNH Am 7695, one male from Shiotsu, Nakajima-machi, February 24, 1980, by K. Miyazaki; NSMT-—H-03993, one female, from Sohama-machi, Nanao-shi, April 8, 1983, by J. Yamamoto; OMNH Am 7696, one female, from Hosoguchi-machi, Nanao-shi, August 5, 1978, by M. Sakai; NSMT—H-03994—03997, OMNH Am 7697-7701, 9 males, NSMT-—H-03998— 03999, OMNH Am 7702, 3 females, Akakura- yama, Tatsuruhama-machi, February 6, 1983 by T. Takeda, Y. Akita, K. Miyazaki and M. Matsui. Diagnosis — A member of the Hynobius lichenatus group [2]: breeding in still-water; adapted to cool climate; differing from others in the following combination of morphological characters: from H. nigrescens and H. sadoensis in smaller body size, less depressed head, shorter tail and limbs, and transparent egg sacs; from A. lichenatus in Dorsal and ventral views of holotype of Hynobius takedai (NSMT—H-03990). having shorter head and limbs, adpressed limbs usually separated, in having shorter and higher tail, more uniformly dark color of back, usually without yellowish speckles, larger clutch size and in lacking evident longitudinal striations on the envelope of egg sacs; from H. abei in having shorter and narrower head, longer limbs, less keeled lower tail with more pointed tip, in the presence of dark spots on the back of some females, and in lacking striations on the envelope of egg sacs. Description and variation — Morphometric data on 24 males and 7 females are summarized in Table 1 with those on the allied species, H. lichenatus and H. abei (all measurements taken on preserved specimens). Head moderately depressed, distinctly longer (% length/SVL 22.0-25.9) than broad (% width/SVL 17.8-21.3). Males with relatively great head both in length (HL) and width (HW) than in females, when each dimension converted to percentage ratio to snout-vent length (SVL) (Mann-Whitney’s U-test: Z,.4=3.52, p<0.001, and Zz, .,=2.67, p<0.01, respectively; Table 2). Contrariwise, females with relatively long trunk (Z7,24=3.50, p<0.001). Number of costal grooves 12 to 13 (Table 3), modal number being 12 in both sexes. Limbs short and stout, and when adpressed, they overlap at most one costal were A New Salamander from Japan 667 TABLE 1. Comparisons of measurements (means +2SE, followed by ranges in parenthesis, in mm) in the three forms of the Hynobius lichenatus group Form Sex N SVL HL TRE TAL HW MTAH H. takedai M 24 57.2+2.0 13.8+0.4 43.4+1.6 44.0+2.4 11.4+0.4 7.6+0.5 (45.6-66.8) (10.8-15.9) (34.8-51.2) (30.2-51.3) ( 9.9-12.8) (5.3-11.6) F 7 57.8+2.0 13.2+0.3 44.5+1.5 38.1+2.9 10.9+-0.5 6.0+0.6 (53.5-60.0) (12.6-13.8) (40.9-46.8) (32.7-44.8) ( 9.8-11.8) (5.2-6.8) H. lichenatus M 37 58.5+1.9 14.7+0.5 43.8+1.5 49.7+2.3 11.3+0.5 7.1+0.5 (42.6-68.5) (10.9-17.3) (31.7-52.3) (32.3-58.8) ( 8.8-13.8) (4.2-9.2) F 2 53.8 13.6 40.2 38.5 10.5 6.2 (53.4-54.1) (13.2-13.9) ( 40.2 ) (36.1-40.8) (10.1-10.9) (5.7-6.6) H. abei M 24 57.8+2.1 15.0+0.4 42.8+1.8 42.94+2.2 12.3+0.4 10.0+0.8 (48.5-68.0) _ (13.2-16.9) (37.2-51.2) (34.8-52.8) (10.8-14.2) (7.3-14.3) Bo 34) -55.0+1.2 13.9+0.3 41.2+1.0 35.2+1.2 11.0+0.2 7.0+0.3 (45.3-64.4) (12.2-15.5) (32.6-48.9) (28.8-41.8) ( 9.9-12.1) (56.-8.8) SVL=snout-vent length; HL=head length; TRL=trunk length; TAL=tail length; HW-=head width; MTAH=maximum tail height. TABLE 2. Comparison of percentage ratio of each character dimension to SVL (medians, followed by ranges in parenthesis) in the three forms of the Hynobius lichenatus group Form Sex N HL TRL TAL HW MTAH H. takedai M 24 24.2 Woes 78.3 Mobi) 13.0 (22.9-25.9) (74.1-77.1) (56.8-88.9) = (17.8-21.3) =(11.4-17.4) F ji 23.0 77.0 64.7 18.7 10.3 (22.0-23.7) (76.3-78.0) (61.1-78.0) (18.3-19.7) ( 8.9-12.1) H. lichenatus M 37 25-3 74.7 85.1 ike 12.1 (23.3-26.8) (73.2-76.7) (69.4-102.0) (15.9-22.0) ( 8.7-15.2) F 2 da 74.8 71.6 19.5 i Ws) (24.7-25.7) (73.4-75.3) (66.7-76.4) (18.9-20.1) (10.7-12.2) H. abei M 24 26.1 74.0 dict 21d 17.4 (23.4-28.2) (71.8-76.6) (59.2-97.9) (17.9-23.1) (12.2-21.7) F 34 25.4 74.6 64.5 20.1 12.8 (23.5-28.0) (72.0-76.5) (50.9-74.3) (18.0-22.3) (10.5-15.1) Abbreviations as in Table 1. fold and are usually separated by up to 2.5 folds. Separation is greater in females (median=2 folds) than in males (median=0.5 folds; Table 3, 7 =9.34, dF=1, 0.0010.5). Width of vomerine teeth series (VTW) larger in males than in females (t=2.97, dF=19, 0.0050.8). Shape of vomerine teeth series not sexually different as shown by 668 M. MATSUI AND K. MIYAZAKI TABLE 3. Variation in the number of costal grooves and of costal folds between adpressed limbs in the three forms of the Hynobius lichenatus group Number of Form Sex costal grooves 11 12 13 -3 H. takedai M 20 4 F 4 3 H. lichenatus M 31 7 F yD) H. abei M Deen: i F 30) 4° «33 Overlap of adpressed limbs shown by number of costal folds 2,5 =2) 941.55 =) 205° 0 OS ioe To8hG 3° 55) Se An tty mn A 1 1 Av 6: Oe mt {Gat t85 OO Figures indicate the number of specimens. TABLE 4. Comparisons of number of vomerine teeth (mean -+2SE) and of size (mean-+-2SE, in mm) and shape (median) of vomerine teeth series in the three forms of the Hynobius lichenatus group Vomerine Form Sex N teeth N number H. takedai M 23 37.1+2.8 14 (28-54) F 7 35.6+4.7 7 (27-44) H. lichenatus M 37 34.2+12.4 16 (23-49) F 2 38.5 (36-41) H. abei M 24 36.3+1.9 20 (28-46) Jer By4t 36.0+1.1 19 (31-43) Vomerine teeth Vomerine teeth series width series length VTW/VTL (VTW) (VTL) 3.13+0.10 1.69+0.16 1.76 (2.85-3.58) (1.07-2.17) (1.46-2.66) 2.88+0.11 1.69+0.20 1.68 (2.65-3.11) (1.23-1.98) (1.38-2.38) . 3.43 +0.12 1.78 +0.12 1.97 (2.98-3.77) (1.44-2.28) (1.44-2.41) 2.76+0.16 1.97+0.10 esh/ (2.09-3.72) (1.62-2.43) (1.21-1.62) 2.57 +0.07 1.78 +0.07 1.46 (2.28-3.03) (1.35-2.10) (1.25-1.69) Figures in parenthesis indicate variation range. comparison of the ratio of VTW to VTL (U, 4= 39, p>0.05). Color in life—Dorsum uniformly dark brown or yellowish brown, females with well-scattered obscure spots; underside paler, usually with bluish white mottling in females. Females and non-breeding males often with small bluish white spots on sides and limbs. Color in preservative —Dorsum uniformly dull brown with minute pale speckles; females often lighter with small darker spots scattered; under- surface grayish, with white speckles in some specimens, especially in females. Measurements of the holotype (in mm) — Head width 12.8, snout to gular fold (head length) 15.6, head depth at posterior angle of jaw 7.2, eyelid length 3.6, anterior rim of orbit to snout 4.3, horizontal orbit diameter 2.7, interorbital distance 4.0, snout to insertion of forelimb 22.6, distance separating internal nares 3.6, distance separating external nares 3.7, projection of snout past mandible 1.1, snout to anterior angle of vent (SVL) 66.8, axilla to groin 33.8, anterior angle of vent to tip of tail (tail. length, TAL) 49.3, tail width at base 10.2, tail height at base 10.1, tail height at middle 11.1, maximum tail height (MTAH) 11.6, axilla to tip of out- stretched forelimb 16.8, groin to tip of out- stretched hindlimb 20.0, width of vomerine teeth series 3.6, length of vomerine teeth series 2.5. The holotype has 78 upper jaw teeth, 37 vomerine teeth, 12 costal grooves between axilla and groin, 1.5 costal folds between adpressed limbs, and A New Salamander from Japan 669 5 digits on both hindlimbs. Etymology — The specific name “takedai’’ is given after Mr. Toshio Takeda, former head- master of Inoyama Elementary School, Hakui-shi, who first discovered the new form and has been continuously making efforts for its conservation. KY ype tocauiry 3830 Fic. 2. Map of Ishikawa Prefecture showing locality records for Hynobius takedai. Range—Known so far from lowland of Ishikawa Prefecture, on the Japan Sea side of the Chubu District, Central Japan (Fig. 2). Yanagida-mura: G6roku; Nakajima-machi: Ka- wazaki, Shiotsu; Tatsuruhama-machi: Akakura- yama; Nanao-shi: Sohama-machi, Hosoguchi- machi; Kokubu-machi; Togi-machi: Fukurami- nato; Shika-machi: Yaguradani; MHakui-shi: Shibagaki-machi, Taki-machi, Kashimaji-machi, Chiji-machi, Yanaida-machi; Tsubata-machi: Tsubata. Morphometric comparisons — For comparison, 37 males and 2 females of Hynobius lichenatus from various localities of its range (Aomori, Akita, Niigata, Fukushima, Tochigi, Gunma), and 24 males and 34 females of H. abei from Kyoto were examined. H. takedai was not different from H. lichenatus and H. abei in SVL in both sexes (Table 1), but males of H. takedai had smaller HL than males of the latter two species (t=2.29, dF=59, 0.020.5). The 5th toe is well developed in the majority of observed specimens in the three species. The number of vomerine teeth was not different ‘between WH. takedai and other two species, either, but the size of vomerine teeth series showed slight variation (Table 4): males of H. takedai had the teeth length similar to that in males of H. lichenatus, but had smaller width than in the latter (t=3.55, dF=28, 0.0010.2). Thus, H. takedai was judged to have shallower vomerine teeth series than H. abei in both sexes as shown by significantly larger ratio of VITW to VTL (Uys 2=12, p<0.05, for males, and U,, 1.=21.5, p<0.05, for females). No difference was found in this ratio between H. takedai and H. lichenatus in males (Ui4, 16 =78.5, p>0.05). Serum proteins and isozymes — To assess genetic diversity among three related species, we have examined, electrophoretically, the phenotypes of serum proteins, serum lactate dehydrogenase > Fic. 3. Representative electrophoretic patterns of the serum proteins of Hynobius takedai (A-C), H. lichenatus (D-F) and H. abei (G-I). The arrows indicate moderately migrating fractions (see text). + (LDH) and serum malate dehydrogenase (MDH) of 14 H. takedai from Akakurayama, Tatsuruhama-machi, 10 H. lichenatus from Niigata, and 33 H. abei from Kyoto. Electrophoresis was conducted in vertical polyacrylamide gels (7.5%) using Tris-glycin buffer (pH 8.3), for 2.5 hr at 1.5mA DC/cm. Representative phenotypes re- solved in this manner are shown in Figures 3-4. Inspections of the electrophoretic patterns of the three species revealed several significant findings: Moderately migrating serum protein fractions, which appeared as thin dark bands (arrows in Fig. 3), moved in H. takedai about the same rate as in some individuals of lichenatus, and were significantly in advance of the presumably ho- mologous bands of H. abei. H. takedai was not polymorphic at the serum LDH-A locus and the band migrated identically with that of one electromorph of H. lichenatus, which showed polymorphism in this locus. Serum LDH-A of H. abei migrated significantly slower than in H. takedai (Fig. 4). Serum MDH bands showed less rapid electrophoretic migration in H. takedai than in H. lichenatus, which in turn exhibited significantly smaller mobility than in AH. abei (Fig. 4). Clutch size and egg sac— Another difference between H. takedai and the other two species is in the egg number and the appearance of egg sacs: Hynobius takedai overlapped H. lichenatus and H. abei in the variation range of clutch size (Table 5), but the mean (of 4 localities =89.7+8.4 - : AGB CD €£ F Fic. 4. Representative electrophoretic patterns of the serum LDH (left) and the serum MDH (right) of Hynobius takedai (A, B), H. lichenatus (C, D) and H. abei (E, F). A New Salamander from Japan TABLE 5. Comparison of clutch size in the three forms of the Hynobius lichenatus group Form Locality N Range Mean+2SE H. takedai Ishikawa Chiji-machi 115 26-148 88.1+ 3.7 Yanaida-machi 47 40-108 67.7+ 4.8 Akakurayama 16 35-139 95.0+12.0 Shiotsu 11 90-124 107.8+ 8.5 H. lichenatus Yamagata Nezugaseki 10 36— 5] 43.0+ 3.4! Fukushima Hinoemata 6 25-— 37 31.0+ 3.6 Tochigi Okushiobara 3 34— 53 46.0+ 12.0 Gunma Minakami 29 25-— 58 41.7+ 2.8 Tanigawadake SB) 24— 50 38.2+ 2.0? Niigata Tagami 60 18— 62 34.0° Kamo 92 33-— 74 46.7+ 1.6* H. abei Kyoto Ohmiya-cho 10 43-— 72 61.8+ 5.4! Ohmiya-cho 4 52-109 87.5+32.3 Mineyama-cho 5 45-— 89 77.6+16.5 Takeno-gun | 26— 94 56.7+19.7 671 ‘Sato [2], *lizuka [6], *T6jio [7], values twice the original data reported for one egg sac (=1/2 clutch size), *Akiyama [8]. (2SE)) is evidently larger than those of the latter two species (mean of 7 localities=40.1+2.3, and of 4 localities=71.3+6.9, respectively). In addition, there are at most weak longitudinal wrinkles on the surface of egg sac in H. takedai, whereas the other two species have strong lon- gitudinal striations which can easily be observed even with the naked eye. ACKNOWLEDGMENTS We are particularly grateful to Toshio Takeda for leading us to conduct this study and providing valuable specimens. We also thank the following people for their help in the collection of specimens: Y. Akita, K. Ban, H. Fujii, I. Hamano, T. Hikida, T. Hongo, A. Itoi, H. Iwasawa, M. Kakegawa, M. Kanamori, K. Kinebuchi, Y. Kokuryo, S. Mori, O. Murakami, M. Sakai, S. Segawa, T. Sugiki, S. Tanabe, M. Tanaka, and J. Yamamoto. K. Nishio kindly provided assis- tance in electrophoretic analysis, and Y. Shibata provided literature. REFERENCES 1 Sato, I. (1937) A_ synopsis of the family Hynobiidae of Japan. Bull. Biogeogr. Soc. Japan, 7: 31-45. Sato, I. (1943) A monograph of the Tailed Batra- chians of Japan. Nippon Shuppan-sha, Osaka, pp. 24, 119, 500. (In Japanese) Miyazaki, K. (1977) Notes on Hynobius abei from Hakui, Noto Peninsula. Jap. J. Herpetol., 7: 46. (Abstract, in Japanese) Miyazaki, K. (1978) Hynobius abei from Ishikawa Prefecture. The Nature and Animals, 8: 21-24. (In Japanese) Nakamura, K. and Uéno, S.-I. (1963) Japanese Reptiles and Amphibians in Colour. Hoiku-sha, Osaka, pp. 10-11. (In Japanese) lizuka, M. (1964) On a salamander, MHynobius lichenatus, in Gunma Prefecture. Collecting and Breeding (Tokyo), 26: 250-253. (In Japanese) Tdjio, Y. (1976) Number of eggs deposited in one egg sac in the salamander Hynobius lichenatus. Jap. J. Herpetol., 6: 103-104. (In Japanese with English abstract) Akiyama, K. (1982) Number of eggs in a pair of egg sacs and the difference within a pair in the salamander Hynobius lichenatus. Nippon Herpetol. J., 23: 19-21. (In Japanese) ZOOLOGICAL SCIENCE 1: 673-675 (1984) [COMMUNICATION] © 1984 Zoological Society of Japan Differentiation of Hatching Gland Cells in the Explants from Anuran Embryos ZUTARO HASEGAWA and Erjy1 Ounzu! Department of Biology, Faculty of Science, Yamagata University, Yamagata 990, Japan ABSTRACT — Differentiation of hatching gland cells (HGCs) was studied on the explants from the super- ficial epidermis in various embryonic portions at various developmental stages of anuran embryos. Obtained results were as follows; (1) HGCs were differentiated only from the superficial epidermal portions localized on the neural crest in stage 14 embryos. (2) HGCs were autonomously differentiated in the explants isolated as early as stage 13a. The origin of HGCs was discussed on the basis of these experimental results. INTRODUCTION Hatching gland cells (HGCs) have been con- sidered to provide a good model for investigating early embryonic cell differentiation; as for their unique morphological and biochemical character- istics, pioneer works by Yanai [1-3] in this field have revealed that HGCs were differentiated from the neural crest under the superficial epidermal tissues at neural stage. More recently, Yoshizaki [4,5] studied the differentiation in epidermal explants from various embryonic portions at various developmental stages and revealed that HGCs are derived from cells localized on the neural crest in the neural stage. In view of this growing interest in the problem, our attempts were made to trace the development of HGCs in anuran embryos with special regards to both embryonic stage and specific portion of embryo with applica- tion of culture technique provided by Barth and Accepted February 27, 1984 Received November 18, 1983 * To whom requests of reprints should be addressed. Barth [6]. MATERIALS AND METHODS Developmental stages of the embryos in the present material, Rana ornativentris, were deter- mined according to the normal table of Rana japonica [7]. The operation and culture of em- bryos of embryonic explants were performed in the sterilized Barth’s X-solution [6]. The embryos at stages 11-16 were freed from their jelly coats by treatment with 0.1% papain (Difco) activated by 2% cystein-HCl (pH 7.0 with Tris), and rinsed thoroughly with Steinberg’s solution. The remain- ing vitelline membrane was removed manually with watchmaker’s forceps. Denuded embryos were transferred to Petri dish covered with 2% agar base, and then, the super- ficial epidermis (ca. 0.6 x 0.6 mm in stages 11-12, ca. 0.30.3 mm in stages 13-16) was extirpated with a glass needle and a hair loop from the em- bryonic portions as described in Figure 1 according to the experimental plan as shown by Yoshizaki [4]. Then, the explants were transferred to Ca-free DeBoer’s solution (110 mM NaCl, 2.2mM KCl, pH 7.2 with NaHCO,) in a Petri dish, and the inner surface of explant was scraped carefully with a hair loop to remove contaminating inner epidermal cells. Culturing of the explants was made in Barth’s medium at 20°C. The culture medium containing about 20 explants were changed daily. A number of unoperated embryos of the same stage were allowed to develop along with the ex- plants and served as controls. 674 Z. HASEGAWA AND E. OHZU Fic. 1. (dotted areas) which supplied epidermal explants for cultivation. Schematic representation of the portions C: stage 13a, F: stage 16. A: stage 11, B: stage 12, D: stage 14, E: stage 15, Scale line=1 mm. After the culture periods, explants were sub- jected to histological observations according to the methods of Yoshizaki and Katagiri [8]. RESULTS AND DISCUSSION HGC differentiation in epidermal pieces isolated from several portions of the stage 14 embryos were summarized in Table 1. As shown clearly in Table 1, HGCs were differentiated from the epidermal cells on the neural crest (portions 1 and 2). The explants from the other portions except for the portion 5 were composed of com- mon epidermal cells (CECs) and cilia cells (CCs). TABLE 1. Differentiation of hatching gland cell (HGC) in epidermal explants from various portions of stage 14 Rana ornativentris embryo after 75 hr culture Epidermal No. of explants No. of HGC portion examined withHGC %&% per explant 1 14 9 64 34+ 13* 2 20 3 15 6+3 3 21 0 0 0 4 19 0 0 0 5 14 0 0 0 6 13 0 0 0 Epidermal portions used were depicted in Fig. 1. * Mean-+standard deviation. TABLE 2. Differentiation of hatching gland cell (HGC) from explants of Rana ornativentris embryos at different developmental stages Duration of No. of explants Stage culture (hr) examined withHGC % i 94 10 0 0 12 91 14 | 7 13a 87 13 8 62 14 15 14 9 64 15 69 17 1 65 16 65 25 17 68 Extirpated portions of superficial epidermis are shown in Fig. 1A—F. The explants from portion 5 were exclusively occupied by the neural tissue. HGC differentiation in explants isolated from stage 11-16 embryos were summarized in Table 2. HGCs are autonomously differentiated in the explants isolated as early as stage 13a. The dif- ferentiation both of CECs andCCs was observed in all explants examined. Cement gland cells (CGCs) were occasionally found in some explants isolated at stages 11 and 12. In general, the results of the present experiments concerning the study of explants in superficial epidermis showed the same results with those of Yoshizaki [4] in following points; (1) HGCs are autonomously differentiated in the explants iso- lated from as early as stage 13a embryos. (2) HGCs are differentiated from the epidermal cells localized in the neural crest. In the present experiments, however, the frequency of induced HGCs are lower than those in the experimental results of Yoshizaki [4]. Low frequency of HGC in portions | and 2 may be caused by the different histological criteria of HGC differentiation in explanted tissues between Yoshizaki and ours. Mention should be made on the different potency for HGC differentiation in different species. Concerning the origin of HGCs, Yanai ef al. [9-11] have proposed that the precursors of HGCs lie beneath the superficial layer of epidermal cells and differentiate into HGCs after moving into the superficial layer. These findings differ from the results of ours showing HGCs originate from the superficial epidermis on the neural crest. Thus, Hatching Gland Cell Differentiation it seems worth a further experiments whether possibility cannot be still excluded that the pre- cursor of HGCs is the constituents of the neural crest cells, because underlying neural crest cells are also involved in epidermal cells of the intact embryo [9-11]. ACKNOWLEDGMENTS We wish to express our sincere thanks to Professor Mamoru Kusa of Yamagata University for his direction of this experiment. Further thanks are offered to Dr. Norio Yoshizaki of Gifu University for his interest, and also to Mr. Norio Abe for his kind help in various ways. REFERENCES 1 Yanai, T. (1951) Annot. Zool. Japan., 24: 103- 107. he 675 Yanai, T. (1952) Zool. Mag., 61: 155-158. Yanai, T. (1966) In “Vertebrate Embryology”. Ed. by M. Kume, Baifukan Shuppan Co. Ltd., Tokyo, pp. 49-S8. Y oshizaki, N. (1976) fer., 18: 133-143. Yoshizaki, N. (1979) fer., 21: 11-18. Battie G. and Barth, L.J. (1959) iJ. bryol. Exp. Morph., 72: 210-222. Tahara, Y. (1959) Ann. Rev. Exp. Morph., 61: 64-68. Yoshizaki, N. and Katagiri, Ch. (1975) Zool., 192: 202-212. Yanai, T., Ouji,M. and Kobayashi, K. (1953) Annot. Zool. Japon., 26: 193-201. Yanai, T., Ouji, M. and Iga, T. (1955) Annot. Zool. Japon., 28: 227-232. Yanai, T., Ouji, M. and Zool. Japon., 21: 11-18. Develop. Growth and dif- Develop. Growth and Dif- Em- Js Xp; Iga, T. (1956) Annot. - — 4 © - f : 4 ‘ uu = x ‘ i * * f m 7 é sr ih a y ae y Wo i ! i iI { a wo o } ‘f ; ‘ . e f 1 wv = os t i ‘ y Z : i” . = t, 1 | f | iS — ; j 5 ; y ; | i s -! ey ” Se walk itn dh with srovonie ui ‘ | x See Ae dag et | nies 8 ¥ ad rh + n, j 3 ( 7" tt i ‘ i “ iw 1 { ZOOLOGICAL SCIENCE 1: 677-680 (1984) [COMMUNICATION] © 1984 Zoological Society of Japan Pseudabbreviata yambarensis sp. n. (Nematoda: Physalopteridae) from the Tree Lizard, Japarula polygonata polygonata (Agamidae), on Okinawa Island, Japan’ Hipgo HASBGAWA and MASAMITSU OTSURU Department of Parasitology, School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-01, Japan ABSTRACT — Pseudabbreviata yambarensis sp. n. (Nematoda: Physalopteridae) is described from male specimens found in the duodenum of the tree lizard, Japarula polygonata polygonata, on Okinawa Island, Japan. This species is readily distinguishable from other members of the genus Pseudabbreviata and from some closely-related representatives in the genus Abbreviata by the disposition of caudal papillae, the arrangement of teeth and denticles on the pseudolabia and body dimensions. This is the first species of the family Physalopteridae recorded from Japanese reptiles. Until now, there has been no report of nematodes of the family Physalopteridae from Japanese reptiles. We have detected a new species of the genus Pseudabbreviata in the family from the duodenum of two of four tree lizards collected on Okinawa Island, Japan. The following description is based on seven male adults fixed in 70% ethanol at 70°C and cleared in a glycerin-alcohol solution or in gum-chloral solution. DESCRIPTION Pseudabbreviata yambarensis sp. 0. (Fig. 1) Physalopteridae, Pseudabbreviata. Body small, 7.35-9.23 mm long and 0.23-0.28mm wide at midbody, semitransparent when alive. Posterior Accepted January 28, 1984 Received September 29, 1983 1 Helminth Fauna of the Ryukyu Archipelago, Japan V. part of body bent ventrad in fixed condition. Head 53-58 wm in diameter, with cuticular collar and cervical inflation. Mouth elongated dorso- ventrally, encircled by two lateral pseudolabia, each of which has an externo-lateral tooth, an interno-lateral tooth and submedian teeth. Externo-lateral tooth large, sclerotized. Interno- lateral tooth small, weakly sclerotized. Sub- median teeth two on each side. Denticles present, 6 or 7 between externo-lateral and submedian teeth and 4 or 5 between submedian teeth and angle of mouth. Each pseudolabium with two submedian papillae. Area around amphid defined from pseudolabium. Buccal cavity developed, 25- 35 um long, consisting of protorhabdion and telorhabdion not surrounded by _ esophagus. Esophagus divided into anterior muscular portion, 188-245 wm long by 45-50 um wide, and posterior glandular portion, 1.63-1.86 mm long by 90-128 vm wide. Deirids and excretory pore at about same level with junction of two portions of eso- phagus. From cephalic apex to nerve ring, excretory pore and deirids 155-200 wm, 0.28- 0.33 mm and 0.26—0.30 mm, respectively. Caudal alae asymmetrical. Spicules markedly unequal: left one 0.24-0.28 mm long and right one 93- 125 4m long, and both with pointed tip. Gubernaculum absent. Pericloacal region with many verrucae of various sizes. Ten pairs of papillae (pairs I-IV pedunculate, and V—VII and IX—XI sessile) and one unpaired sessile papilla (VIII) present ventrally on caudal area. Papillae II in middle between I and III; III slightly anterior H. HASEGAWA AND M. OTSURU 78 6 f qo X | Bee i NIINNAATENUC TRADES LaF TLL = | TS iil Nl i a = n cs Pseudabbreviata yambarensis sp. n. 679 to cloaca; X and XI attached to each other, just behind pericloacal verrucose area; IV at same level with X and XI but more lateral; VI midway between IV and caudal tip: V in front of VI; VII in middle between V and caudal tip; IX and VIII at same level, slightly posterior to level of II (IX not adherent to pericloacal verrucose region, but VIII adherent to it in four specimens). Phasmids situated between papillae VI and VII. Tail conical, 0.25—0.28 mm long. Type host: Japarula polygonata polygonata (Hallowell) Site: Duodenum. Locality: Ada, Kunigami Village, Okinawa Island, Japan. Date of collection: July 31, 1981. Specimens deposited: Holotype (male) in the National Science Museum, Tokyo, Coll. No. NSMT-As 1771; and 6 paratypes (males) in the Department of Parasitology, School of Medicine, University of the Ryukyus, Nishi- hara, Okinawa, Japan. COMMENTS The genus Pseudabbreviata was erected by Lichtenfels and Quigley [1] based on a new species, P. nudamphida, from a “lizard” in Ghana, Africa. This genus is closely related to the genus Abbreviata Travassos, 1920, but is distinguishable from it in having a developed buccal cavity consisting of a protorhabdion and telorhabdion which are not surrounded by the esophagus. Sharpilo [2] transferred Abbreviata markovi Annaev, 1972 and A. pallaryi (Seurat, 1917) Morgan, 1945 to Pseudabbreviata and described a Pseudabbreviata sp. nematode. n. differs from P. nudamphida in having a smaller body, shorter spicules and caudal papilla X attached to XI instead of separated as in the latter species [1]. Pseudabbreviata yambarensis sp. Since P. markovi has caudal papillae Fic. 1. A: Anterior part, ventral view. view. D: Distal ends of spicules. tooth, ep; excretory pore, ph; phasmid, rs; right spicule, B: Cephalic extremity, subapical view. am; amphid, il; interno-lateral tooth, sp; submedian papillae, 1 and II on the same level and V midway between IV and the caudal tip [2] and P. pallaryi has submedian teeth of almost the same size as its denticles [3], P. yambarensis is distinguishable from them. It also differs from Pseudabbreviata sp. of Sharpilo, 1976, which has caudal papilla X apart from XI, a larger body, a longer left spicule and shorter right spicule [2]. Lichtenfels and Quigley [1] thought that several representatives of Abbreviata might belong to Pseudabbreviata. Among reptilian Abbreviata species, A. golvani Le-Van-Hoa, 1961 [4], A. euzebii Le-Van-Hoa, 1961 [4], A. nyassae Fitzsim- mons, 1964 [5], A. cyanogasteri H6rchner and Weissenburg, 1965 [6] and A. legendrei Caballero, 1968 [7] resemble P. yambarensis in having Type IV arrangement of teeth and denticles on the pseudolabium [3], the left spicule shorter than 1 mm in length and more than five denticles between the lateral and submedian teeth. However, they differ from P. yambarensis in that A. cyanogasteri and A. legendrei have a wider verrucose region extending to the caudal alae, A. golvani has a unique submedian tooth, A. euzebii has more denticles (more than ten) between the lateral and submedian teeth, and A. nyassae has more ante- riorly located deirids on the level with the nerve ring. ACKNOWLEDGMENTS We are greatly indebted to Prof. I. Miyagi, Uni- versity of the Ryukyus, for his kindness in giving us the opportunity to collect materials, and to Dr. M. Machida, National Science Museum, Tokyo, and to Dr. A. Ichihara, Meguro Parasitological Museum, Tokyo, for their kindness in allowing us access to many papers on the subject. REFERENCES 1 Lichtenfels,J.R. and Parasitol., 54: 1092-1094. 2 Sharpilo, V. P. (1976) [Parasitic Worms of Rep- Quigley, D. (1968) te Pseudabbreviata yambarensis sp. n., male. C: Posterior part, ventral c; cloaca, ca; caudal ala, el; externo-lateral Is; left spicule, m; mouth, nr; nerve ring, I-XI1; papillae numbers. 680 H. HASEGAWA AND M. OTSURU tilian Fauna of USSR.] Izdatel’stvo “‘Naukova 24. Dumka”, Kiev, pp. 1-288. (In Russian) 6 Horchner, F. and Weissenburg, H. (1965) Z. Par- Chabaud, A.G. (1956) Ann. Parasitol., 31: 29-52. asitenk., 25: 492-500. Le-Van-Hoa (1962) Explor. Parc. Nat. Upemba 7 Caballero, R.G. (1968) Ann. Parasitol., 43: 149- Miss. de Witte, No. 65, 3-58. 200. Fitzsimmons, W. M. (1964) J. Helmintol., 38: 21- Vil ANNOUNCEMENT CURT P. RICHTER PRIZE IN PSYCHONEUROENDOCRINOLOGY Through the generosity of the Institute of Clinical Pharmacology, Dublin, Ireland, an Annual Prize has been established for meritorious research in the area of Psychoneuroendo- crinology. The sum of US $ 1,000 will be awarded annually at the International Congress of Psychoneuroendocrinology for the best essay or manuscript (original research or review including original research) submitted by a scientist or physician under 40 years of age by January Ist, 1985. In support of the winner’s travel to the Congress, a travel grant of max. US $ 1,000 is also provided. The winning paper will automatically be considered for publication in the journal Psychoneuroendocrinology and should be prepared according to the journal’s instructions to the authors. The aim of the prize is to encourage younger scientists to contribute to this interdisciplinary field. Manuscripts should be submitted in quadruplicate to: Gerhard Langer, M.D. Associate Professor of Psychiatry Department of Psychiatry University of Vienna Lazarettgasse 14 A-1097 Vienna, AUSTRIA Deadline for submission: November 30th, 1984. All submissions will be screened by a broad committee of established psychoneuroendo- crinologists. The prize wiil be awarded at the 1985 meeting of the Society in Kyoto, Japan (April 15th to 18th, 1985). TOMY MICRO HIGH SPEED REFRIGERATED CENTRIFUGE MIR-15A HIGH-SPEED, FUE B@yAy ere wis CENTRIFUGE FOF, herstota fe] Bor 7/3 SAMPLES, WITH ‘\qim@athe ter Wileys QUICK, ILT at START | REFRIGERATOR h C QUICK STOP {| Integrated with a Re- ‘S Quick Start / Quick frigerator : Stop ; EASY ee \ mS © «¢, ® High Quality a. _ © Easy Operation SPA: () SAVE SPACE ] oe DRAIN TRAP S Maximizing Floor i, @ Convenient Drain Space Utilization . Trap me. | SPECIFICATION Co - Max. Speed 15,000rpm ; Lo y _ Max. Centrifugal Force Al Safe Design _— ~~ mee 15,860 4 a manufacturer, brings you a com- Seusga Tao 27 D001 & | E | | Automatic control, plete line of High Speed Refrig- 20PR-52D| 300~ 20,000] =| & | = | Analog version erated Centrifuges, Table-Top iad ne 8|e|z . ae, a ee 4 ® arge capacity, multi-place type Centrifuges and Miniature Cen- Sarena Mice trifuges with wide-ranging ee ae ne 8 | Compact type for biochemistry ‘48 - i ell Low cost type speeds, capacities and tubes/ soles sus Eee For clinical Automatic control bottles versatility for your sted 4,000 a igarate feat | a 6 . 4B > __| Automatic with microprocessor clinical laboratory routine as well scT158 [2,000~ 15000] | Miniature | control ; as basic research. MC-200 3 | Serna te MC-201 12,000 2 | Hematoerit [So as a bama. MC-202 ed a 2 | ia pipers 694 1. Non-spiking communication among spiking MeCUrONS .............ccceeeeeeeeeneeeneenees 695 2. Functionai difference between spiking and non-spiking mode of neuron Rs Las hc cthaesivaiths Leb Nie wv anti h hehe nattea deh tsb bes cdihh « chin wace DAawTduben eu dxdee 696 i i iia ll eile ae slhireloien deed unlalinionne eipdbinenanasibacanabes 696 ee aetna ee acs lio gk dualten a eaeln uns VR PATA rAteaous GURL URECIDO AAA CERA TS AREEEEN CAMA TRAN esSau ancy ote 698 appreciated in the sensory systems of both I. INTRODUCTION vertebrate and invertebrate animals. Photore- Neurons which function without generating ceptors of vertebrate retina, for example, respond (action potentials) have been well with hyperpolarization [1] while those of spikes invertebrates (sometimes called retinula cells) Received May 14, 1984 usually respond with depolarization when they 682 M. HisapA, M. TAKAHATA AND T. NAGAYAMA are illuminated [2]. These neurons never generate spikes irrespective of the presence or absence of the stimulus. Some of the interneurons in the vertebrate retina and in the invertebrate optic ganglions also function without spikes [2, 3]. Furthermore, some types of neurons in other sensory systems have been reported to function without spikes [4]. As has been dis- cussed by several authors [5, 6], the particular significance of this non-spiking mode of neuron operation seems to lie on the point that spikeless communication can be more precise than that with spikes. In the last decade, however, there have been growing numbers of reports on the local non- spiking interneurons which function in motor systems. The studies have so far been con- centrated on arthropod species [7-10]. As we will see below, these interneurons receive a variety of peripheral and central input and can effectively control the motoneuron activity without generating spikes. Large proportion of the total neurons in a ganglion is found to be these non-spiking interneurons. These facts suggest that the non-spiking interneurons might play important roles in the motor control, but their functional significance is not altogether clear at present. The aim of this paper is to review the accumu- lating data on both the physiology and the morphology of pre-motor non-spiking interneurons in arthropod species and to critically examine the possible roles of these interneurons proposed by many investigators. This task will inevitably include some references to the novel mode of operation in the ordinary spiking neurons, leading to the re-evaluation of the classical view on neuron operation. In a comparative approach contrasting the non-spiking interneurons with dendrodendritic networks of the vertebrate brain, we will make clear the generality and specificity of these interneurons in the animal kingdom for further understanding their functional sig- nificance in the nervous function. Reviews by Pearson [11], Shepherd [4] and Wilson and Phillips [12] will complement the present attempt. II. GENERAL FEATURES OF NON-SPIKING INTERNEURONS A. Backgrounds Neurons without spikes in the motor control system were first reported by Mendelson [13] in the ventilatory system of hermit crab and lobster. One particular neuron in each half of the subesophageal ganglion shows an oscillation in its membrane potential (from —15mV to —25mV, around —20mV) in accordance with the ventilatory reciprocal activation rhythm in the levator and depressor motoneurons of the second maxilla which shows rhythmic movements for gill ventilation. Intracellular current injection of either polarity into this neuron caused a change in the spike activity of these motoneurons also in a reciprocal way depending on the polarity of injected current. No spike was observed in this neuron during the free-running oscillation nor during the current injection. Mendelson thus first demonstrated that the activity of maxilla motoneurons could be controlled not by spikes but by the graded change in the membrane poten- tial of a pre-motor neuron which has no ability to generate spikes. The structure of this interneuron, however, remained unknown. In 1975, Pearson and Fourtner [7] have demonstrated using the intracellular staining technique with cobalt-filled microelectrodes that the leg motoneurons of cockroach are effectively controlled by local non-spiking interneurons. These interneurons, as Mendelson’s “oscillator” non-spiking interneurons, show rhythmic change in the membrane potential corresponding to the rhythmic leg movements. Artificial membrane potential change of these interneurons by current injection (8-10 nA) caused either an increase or a decrease in the spike activity of leg motoneurons depending upon the current polarity. Even the injection of 50nA depolarizing current was ineffective for eliciting a spike in these interneurons. The most important in their study was the finding that, morphologically, these interneurons lack an axon structure, being entirely confined to within the metathoracic ganglion. A number of pre-motor non-spiking interneurons Local Non-spiking Interneurons 683 have been reported in many other arthropod species following this report ((8] in locust; [9] in crayfish Pacifastacus; [10] in crayfish Procambarus; [14] in lobster; [15] in crab). Many of them show similar locally confined (i.e., intra- ganglionic) structure [8-10]. It seems that the finding of non-spiking interneurons in_ these animals has been greatly accelerated by the intensive analyses of arthropod nervous function with increasing rigorousness, assisted with intra- cellular recording and staining techniques. B. Non-spiking and Spiking Local Interneurons The number of local interneurons in a ganglion has been estimated by subtracting the number of cell bodies of projection neurons revealed by cobalt or nickel backfilling from the total number of cell bodies. Pearson [16] estimated that about 1500 of the total 2000 neurons in the cock- roach mesothoracic ganglion belong to local interneurons. Siegler and Burrows [17] suggested on the basis of works by Sbrenna [18] and Lewis et al. [19] that about 65% of total neurons are local interneurons in locust ganglions. In the terminal abdominal ganglion of crayfish, Reichert et al. [20] determined the total number of cell bodies to be 630-651. The number of projection neurons was found to be 323, thus yielding 307-328 as the total number of local interneurons in the ganglion. Kondoh and Hisada (in prep- aration) estimated the number of local inter- neurons to be about 270-310 in the same ganglion of the same species of crayfish. In sum, about a half of the total neurons in a ganglion seem to belong to local interneurons in both insect and crayfish. It should be pointed out immediately, however, that not all the local interneurons are non-spiking. Spiking local interneurons have so far been reported in various arthropod species ({21, 22] in cricket; [23, 24] in locust; [20, 25-27] in crayfish). Many of them were found in sensory pathways, but some spiking local interneurons in crayfish are also reported to have motor output as revealed by intracellular current injection ({20, 26); Nagayama and Hisada, in preparation). At present, the functional role of spiking local interneurons is not yet established. Although Siegler and Burrows [24] suggest that the spiking local interneurons are the major elements in sensory integration, the findings in crayfish (20, 26] indicate that their role as the pre-motor elements should also be investigated. The exact number of non-spiking interneurons is thus still an open question. It should be also pointed out here that not all the non-spiking local interneurons are pre- motor in their function. Reichert et al. (20, 25] have reported that a pair of LDS interneurons in the crayfish terminal abdominal ganglion mediate the lateral inhibition of mechanosensory interneurons. These LDS interneurons seem to have no significant connection with any moto- neurons. Similar mechanosensory non-spiking interneurons have also been reported in insect [28]. In this paper, we shall confine the subject to pre-motor non-spiking local interneurons and review first their physiological and morphological features in the following sections. C. Physiology and Mode of Operation Non-spiking interneurons can exert motor effects according to their membrane potential change. Most direct demonstration of this is to penetrate one of them and study the moto- neuron response to current injection into the interneuron. Because it is technically impossible to aim and penetrate the target neuron selectively and intentionally, there must be several criteria with which to judge whether the microelectrode is within the target neuron or not. Usually, the electrode is considered to be within a non- spiking interneuron when (1) no injury spike discharge can be observed on penetration, (2) no spike discharge can be observed either at the resting potential level or during the current injection of either polarity and (3) no rebound discharge can be observed on the termination of hyperpolarizing current injection [10, 29]. Using the microelectrode filled with staining solution such as 0.25 M cobaltous chloride or 5% Lucifer yellow CH, the penetrated cell was stained after the experiment to be confirmed morphologically as the local interneuron. They show in common significantly lower resting potential than that of spiking neurons (see section II-C-3). 684 M. Hisapa, M. TAKAHATA AND T. NAGAYAMA 1. Effect on Motoneuron Activity When the activity of motoneurons is monitored by the spike discharge frequency, there is a linear relationship between the intensity of current injected into the non-spiking interneuron and the spike frequency of the motoneurons (Fig. 1A; [29-31]). When the motoneuron is also pene- trated by another microelectrode simultaneously with the interneuron, a linear relationship has been obtained between the membrane potential change in the motoneuron and the current injected into the interneuron [8, 31, 32]. In some cases, this linear relationship was limited to be observed within a certain range of current intensity. Outside this range, there was no further change. in the motoneuron activity [28-32]. Since the current-voltage relationship of non- spiking interneuron membrane shows the outward rectification [32], it is difficult to know the exact relationship between the membrane potential change of the interneuron and that of the moto- neuron. But it would be safe to assume at least in a certain range (Fig. 1B) that the effect which the interneuron exerts on motoneurons is almost proportional to its membrane potential change. In some interneurons, intracellular injection of current of either polarity was effective in A Motoneuron discharge (Imps/sec) ® 12 changing the motoneuron’s activity. For example, when injection of depolarizing current into an interneuron caused an increase in the motoneuron activity, injection of hyperpolarizing current into the same interneuron caused a decrease in the activity of the same motoneuron(s). Other interneurons had an effect on motoneurons only when the depolarizing current was _ injected [10, 29, 30, 32]. 2. Transmission from Non-spiking Interneurons to Motoneurons Here the questions could be summarized into the followings: (1) Is the connection between the non-spiking interneuron and the motoneuron monosynaptic or polysynaptic? and (2) What is the nature of the transmission between them? These questions were examined most intensively in locust by Burrows and Siegler [8, 32, 33]. One non-spiking interneuron and a fast moto- neuron innervating the tergotrochanteral muscle were penetrated simultaneously. When a brief pulse of 1 msec duration was injected into the non-spiking interneuron, a voltage change in the hyperpolarizing direction was observed in the motoneuron within | msec. Exact synaptic . delay cannot be obtained. The transmission is graded so that the accurate time remains B (mV) e Current 8 -3 9 e c71-30 e 2 a ® C ) Y -50 Cc ° 2 O+_ -25 15 [OMS 15 25 . ela Hyperpolarization Depolarization e s Current (nA) “790 Fia. 1. A. Effect of current injection into a non-spiking interneuron on the spike activity of a closer (reductor exopodite) motoneuron of the uropod. Current was injected into the inter- neuron through the recording electrode. The spike activity of the motoneuron was recorded extracellularly from the axon bundle (Modified from Takahata et al. [10}). potential change of a non-spiking interneuron in response to current injection. B. Membrane Current was injected through the recording electrode with a bridge balance circuit (Nagayama, unpublished). Local Non-spiking Interneurons 685 unknown when the potential change of the inter- neuron becomes effective for releasing the trans- mitter sufficient to affect the motoneuron. But since the delay of less than 1 msec was comparable to that in the monosynaptic link between spiking neurons in locust, Burrows and Siegler [8] con- cluded that the connection between non-spiking interneurons and motoneurons is monosynaptic. The mode of transmission was concluded to be chemical from the following reasons [8, 32]. (1) Injection of current of either polarity into the motoneuron had no effect on the mem- brane potential of the interneuron. (2) A brief current pulse (1 msec) injected into the interneuron caused a long-lasting potential change (45 msec) in the motoneuron. (3) Depolarization of some interneurons caused a depolarization of moto- neurons while that of other interneurons caused a hyperpolarization. (4) The motoneuron hyper- polarization evoked by the interneuron was reduced and even reversed by injection of hyperpolarizing current into the motoneuron. (5) The amplitude of a compound EPSP evoked in the motoneuron by unknown spiking meurons was decreased when its membrane potential was hyperpolarized by the interneuron. (6) Fluctuations in the mem- brane potential of the motoneuron were frequently observed during the depolarization caused by current injection into the interneuron. These fluctuations could be indicative of the synaptic noise due to transmitter release from the pre- synaptic interneuron. 3. Control of Motoneuron Activity By interpreting the result of intracellular current injection experiment (section II-—C-1) according to the conclusions shown above (section II-C-2), the mode of motoneuron control by non-spiking interneurons can be understood in the following way. Membrane potential of motoneurons is affected by non-spiking in- terneurons through monosynaptic, chemically mediated pathways. The observation that the effect exerted on motoneuron activity depends upon the intensity of current injected into the interneuron suggests that the amount of trans- mitter released from the interneuron is dependent on the amount of its depolarization as in the presynaptic terminal at the neuromuscular junc- tion [34]. Since either the depolarizing or hyper- polarizing response can be evoked within 1 msec in motoneurons by the brief depolarization of non-spiking interneurons [8, 32], it is likely that these interneurons can be classified into at least two groups each of which exerts opposite effect on the same motoneuron by different kind of transmitters. The observation that some non-spiking inter- neurons could also change the motoneuron activity when they were hyperpolarized [8, 10, 31] can be accounted for by postulating that they are con- tinuously releasing transmitters at their “resting” potential: Depolarization of the interneuron further increases the amount of transmitter release whereas hyperpolarization decreases it. This postulate is supported by the fact that non-spiking interneurons show lower value of resting potential than that of ordinary spiking neurons [7, 10, 13, 29, 32]. These tonic interneurons must be extremely fatigue resistant. The plateau in the presynaptic current-postsynaptic activity relation- ship, which was occasionally observed when excessive hyperpolarizing current was injected into the interneuron, can be explained by assuming that the membrane potential was brought below the threshold for transmitter release. The plateau which was observed with excessive depolarizing current injection seems to be due partly to the outward rectification of the membrane of the interneurons [32]. Other interneurons had no effect on moto- neurons when hyperpolarizing current was injected. Non-spiking interneurons thus can be function- ally classified into those which are continuously releasing transmitters at their resting level and those which have resting potential well below the threshold for transmitter release. A question may be raised as to how much the interneuron should be depolarized to be able to release sufficient transmitter for changing the motoneuron activity. Depolarizing current less than 1 nA can be sufficient for affecting the post- synaptic motoneuron activity [10, 29]. Burrows and Siegler [32] have shown by penetrating the same interneuron with a pair of microelectrodes that 2mV of depolarization was sufficient for 686 M. HisApA, M. TAKAHATA AND T. NAGAYAMA changing the motoneuron activity. Hyperpolar- ization of the interneuron had no output effect on the motoneuron activity. Since the electrotonic distance between the site of output synapses to motoneurons and the site of electrode pene- tration is unknown, however, the exact value of threshold for transmitter release remains still to be an open question. The threshold would be further lower in the tonically active non- spiking interneurons. An important point, however, is that a very small fluctuation in the membrane potential of non-spiking interneurons could be effective for changing the activity of postsynaptic neurons. One functional advantage of non-spiking interneurons in comparison with spiking neurons is thus their ability to control the postsynaptic neuron activity according to a very small change in the membrane potential (see section III). D. Structure and Functional Implications Knowledge on the structure of non-spiking interneurons is indispensable for interpreting the obtained result of electrophysiological analysis as well as for understanding their function in the intact animal. The structure of local inter- neurons has been reported as early as in 1890 by Retzius for crayfish [35] and in 1924 by Zawarzin for dragonfly larva [36]. It was not until the intracellular staining technique was fully developed, however, that the morphology of such local neurons could be related to their function. Although the intraganglionically confined structure is very characteristic to the pre-motor non-spiking inter- neurons, not all the intraganglionic interneurons function without generating spikes (see section II-B). Morphological difference between spiking and non-spiking interneurons, however, is still to be established ((23, 24]; Nagayama ef al., in preparation). In the following sections, we will review the morphological characteristics of non- spiking interneurons relevant to their function, many of which, however, also apply to spiking local interneurons. 1. Gross Morphology In the terminal abdominal ganglion of crayfish, most of the pre-motor non-spiking interneurons are unilaterally confined to each half of the ganglion [10, 37]. Small number of bilateral type non-spiking interneurons are also present. Of these, however, an identifiable pair of LDS interneurons [20] were shown to have no definite motor output [20, 25]. The unilateral type interneurons extend their branches only within the ipsilateral half of the ganglion to their cell bodies. According to the position of cell bodies in the ganglion, these interneurons can be classified into two major groups [37]. Cell bodies of PL type interneurons are located in the ventral ridge of the postero- jateral portion of the ganglion. The cell body is connected to the thickened part of the neurite with a fine process. They have no discernible axon structure, extending their branches toward the anterior portion of the ganglion. The AL type interneurons have their cell bodies in the ventral ridge of the antero-lateral portion of the ganglion. They have no axonic structure, extending branches both antero-medially and postero-laterally. Com- mon features to both types of interneurons are: (1) Size of cell bodies is significantly smaller than that of fast motoneurons and ascending inter- neurons. (2) Only a single process emerges from the cell body. (3) Their branches extend both ventrally and dorsally. (4) Their branching density is higher than that of other neurons. Heitler and Pearson [9] also reported unilateral type non-spiking interneurons in the anterior abdominal ganglions of crayfish. In the locust metathoracic ganglion, Siegler and Burrows [17] reported several non-spiking interneurons with the cell bodies on the opposite side to their branches as well as many those with ipsilateral cell bodies. In any species, cell bodies are usually located outside the neuropilar region of the ganglion. The branches of non-spiking interneurons closely overlap those of motoneurons which they affect when current is injected. As will be discussed in the following section, however, it is dangerous to infer the functional connection of non-spiking interneurons solely on the basis of their structure. 2. Correlation between Structure and Function Although various types of non-spiking inter- neurons have been reported regarding their Local Non-spiking Interneurons 687 postsynaptic effect and mode of transmitter release (see section II—C-—3), no structural correlate to these functional types has so far been found [17, 37]. Conversely, structural variation of non-spiking interneurons (see section II—D-1) bears no functional implication. Those inter- neurons with almost the same structure excited, when depolarizing current was_ intracellularly injected, particular set of motoneurons in one preparation and suppressed them in another preparation [31, 37]. Identified motoneurons were shown to be under control of many non- spiking interneurons which showed a variety of structures different from each other [17, 37]. There are two difficulties, however, in inter- preting the results obtained in these studies. One is that the interneurons of similar structure are closely packed together to form a cluster in the ganglion (Fig. 2). The cell bodies join closely together and their major branches are A also closely apposed with each other (Takahata, unpublished observation). It is therefore very likely that the authors of above reports penetrated different neurons with a similar structure. The consequence of this argument would be that the neurons with a similar structure have output connections which are different from each other. Another point, however, lies on the very fact that these interneurons function without generating spikes. Since there is no spike to propagate over the neuron branches, output synapses to be activated are dependent on the electrotonic spread of the current generated either by synaptic input or by intracellular stimulation through a microelectrode and this will be confined in a limited area. Immediate consequence of this argument would be that we cannot know which output synapses are activated so far as the site of current source remains unknown. The different motor effect which the interneurons of similar Fic. 2. Non-spiking interneurons closely packed into clusters in the terminal abdominal ganglion of the crayfish Procambarus. extracellularly with a glass microelectrode filled with 0.25 M NiCl,. A. A cluster of PL type non-spiking interneurons were stained After the electrode penetrated a non-spiking interneuron, it was pulled out from the cell and left at its proximity. The positive current (10 nA, 500 msec, 1 Hz) was applied for 30 min. The preparation was then treated with rubeanic acid but not intensified. Six cell bodies can be discriminated. B. Three AL type interneurons were stained simultaneously with the intracellular electrode filled with 5% Lucifer yellow CH. Hyperpolarizing current (15 nA, 500 msec, 1 Hz) was injected for 10 min. stained with 0.25 M CoCl, by intracellularly applying depolarizing current. indicate 100 wm (Takah ita, unpublished). These cells seem to have been dye-coupled. C. Two PL type interneurons were Bars in A-C 688 M. HisapA, M. TAKAHATA AND T. NAGAYAMA structure exhibited in response to intracellular current injection might be hence due to the different site of electrode penetration into the same interneuron. One way to overcome these difficulties would be to concentrate on some particular identifiable interneurons. The validity of identified-neuron approach in the analysis of central neuron circuit of arthropods has been well established [38]. As can be seen from Figure 2, however, identification of non-spiking interneurons would be much more difficult than in motoneurons and interganglionic interneurons which are smaller in their numbers, having larger cell bodies and definite dendritic arborizations. Of a large number of non-spiking interneurons, only several interneurons have been reported to be identifiable in the locust mesothoracic ganglion [31, 39]. Pearson and Fourtner [7] reported an identifiable non-spiking neuron called _ inter- neuron I in the metathoracic ganglion of cockroach (see section III—A-1). It seems to be only possible way to cope with these difficulties that the input and output pathways of an interneuron should be examined for as many portions as possible in the nervous system and at the same time both its morphology and site of electrode penetration should be provided. Some theoretical approach would be of great help to interpret the data obtained in that way (see the following section). 3. Integrative Functions of Non-spiking Inter- neurons The only ultrastructural study on non-spiking interneurons has so far been carried out by Wilson and Phillips [31] in the locust mesothoracic ganglion. They showed that the main process, or the primary neurite, and the secondary ones leaving the primary one were completely wrapped in glial cells. Synaptic sites were always found to occur on glial-free, finer processes of usually less than 1 wm in diameter. Both the bar-type and discrete-type presynaptic configurations of Wood et al. [40] (referred to as 1:2 and 1:1 configuration respectively) were observed in the same interneuron. One of the postsynaptic cells in the bar-type structure was identified to be the motoneuron [31]. Most suggestive in their findings is that both the input and output synapses are mixed on the same fine branch. This is a feature common to synaptic connections among spiking neurons in the stomatogastric ganglion of lobster [41]. But the finding is especially important considering the non-spiking nature of the interneurons. Rall [42] has theoretically indicated by applying the cable analyses that voltage changes occurred at a fine branch become attenuated to less than 10% of the original value during the electrotonic spread to some other branches. In actual neurons, the input synapses from a particular neuron are not necessarily confined to any particular branches and so are the output synapses [41]. But, together with Rall’s indication, Wilson and Phillips’ finding strongly implies that some portion of the interneuron, if not a single branch, can operate as a functional unit inde- pendent of other portions of the interneuron since the output synapses would be affected most effectively by the neighboring input synapses. Burrows [33] showed that a single EPSP could evoke an IPSP in the postsynaptic motoneuron. This suggests that certain portions of the inter- neuron should have been outside the intracellular input-output pathway since the area where the single EPSP could spread electrotonically is restricted [42]. There seems to be no a priori reason to exclude the possibility that during this response the outside branches received some other input and their output synapses were activated to affect other neurons than that moto- neuron. Another important aspect in the integrative function of non-spiking interneurons is that they can operate in a similar way to the dendritic branches of ordinary spiking neurons. Although Rall [42] has suggested that the voltage attenuation during the electrotonic spread will be considerable from a certain branch to another, he also showed that it can still be lenient between two sites if the current experiences no serious increase in the input conductance of branches during the spread. Thus, depending on the relative positions of input and output synapses on the interneuron, the EPSP evoked at one particular site will spread Local Non-spiking Interneurons to the site of output synapses with still effective amplitude but with slower and less steeper time course as has been pointed out for the vertebrate spinal motoneurons by Rall [43]. By gathering many synaptic inputs from various branches, non-spiking interneurons could exert smooth, sustained output to their follower cells. Such sustained output might be suited for ‘“‘fine adjust- ment of central excitatory states’ [43] or for the control of relative threshold of the postsynaptic cells by changing their resting membrane potentials. In sum, non-spiking interneurons can operate as a multiple local information processor if we emphasize the function of individual branches and portions of the interneurons. They can also operate as a low-pass integrator if we place emphasis on the function as a whole neuron. However, experimental support is still lacking for both of them. To clarify the location of particular input and output synapses on the interneuron and the cable properties of the inter- neuron branches would be the first step for understanding how the non-spiking interneurons operate in an intact animal. Il, FUNCTIONAL ROLE OF NON-SPIKING INTERNEURONS Pearson [6], reviewing the reports available at that time, considered the functional advantages of non-spiking neurons. In addition to the local processing of information, i.e. the isolation of function within a single cell, as has been discussed above (section II-D-3), he pointed out several other important aspects for understanding the role of non-spiking interneurons in motor control. Firstly, he showed by computer simulation that in a simple system with one presynaptic and one postsynaptic spiking cells, discrete EPSPs could control the spike activity of the postsynaptic cell not in a continuous way but only discretely with several discontinuities in the input-output frequency relationship. The exceptions might occur when there is a 1:1 following or a sig- nificant reduction between the input and the output frequencies. For the presynaptic spiking neuron to continuously control the follower cell, many of them have to converge onto it as can be 689 seen in the invertebrate sensory systems. With the non-spiking property, only one presynaptic cell can control the follower cell continuously. A problem, however, would be how the graded membrane potential change of such a non-spiking neuron can be possible (see sections IV—B-2 and V). Secondly, non-spiking neurons would be advantageous if the activity of the follower cells is to be related to the net level of input rather than directly to its temporal and/or spatial pat- terns. The remote dendrites from the spike initiating site in spiking neurons would also be of great use for this purpose [42]. However, separation and assignment of the function of integrating the activity of many spiking inputs to a specific class of neurons would serve to enhance the capability of the nervous system for higher, more complex activity. Thirdly, graded potential can reflect very small change in the input in the form of analog signal. If that graded potential can lead to the graded release of transmitter as in the non-spiking inter- neurons, then that small change in the input signal can be transmitted with high fidelity. The argu- ment applies to many types of receptor cells both in vertebrate and invertebrate animals. For the pre-motor non-spiking interneurons, however, this point might first appear to be contradictory to the second point. But it is of significant importance if we consider the interaction between non-spiking interneurons reported by Burrows [44] (see section III-A-2). With these discussions in mind, we will review in the following sections the possible behavioral roles of non-spiking interneurons so far presented. A. Mediation of Specific Behavioral Acts Central nervous elements can be classified into those which mainly participate in the mediation of particular behavioral acts and those which mainly concern with the modulation of such mediating circuits. The classification is of tentative nature, and yet such an approach has been shown to be so powerful as a first but crucial step in the analysis of the nervous system function of invertebrate animals [45]. In this paper, we intentionally dichotomize the possible func- 690 M. HisADA, M. TAKAHATA AND T. NAGAYAMA tions of pre-motor non-spiking interneurons into the mediation and the modulation of be- havioral acts. They are to be re-examined in the synthetic process in the near future, but it would be necessary before that task to further provide precise analyses of the function of non-spiking interneurons in the control of a wide variety of behavioral acts. Both rhythmic and non-rhythmic, or episodic, behavioral acts have so far been reported where non-spiking interneurons are supposed to be involved in their control mechanisms. 1. Rhythmic Acts Mendelson [13] first showed in lobster and hermit crab that a pair of non-spiking neuron controlled the rhythmic activity of motoneurons innervating the second maxilla bearing the scaphognathite for gill ventilation. These non- spiking neurons showed an oscillation in the membrane potential (see section II-A) associated with rhythmic, reciprocal activation of the levator and depressor motoneurons. During the depo- larization phase of the non-spiking neuron, depressor motoneurons were excited while levators were suppressed. The oscillation frequency was about 1.7Hz. Artificial depolarization and hyperpolarization of its membrane potential caused the reciprocal activation of motoneurons which was observed during the depolarization and hyperpolarization phase respectively in the free-running oscillation. Brief current pulses injected into the non-spiking neuron could reset the rhythmic activity in motoneurons. Mendelson hypothesized that a single non-spiking oscillator neuron controls the scaphognathite beat rhythm. In 1980, Simmers and Bush [15] have found in crab an antagonistic pair of non-spiking oscillator neurons which control the rhythmic activity in the levator and depressor motoneurons. They showed that although current pulses injected into one of them could reset the timing of both the interneuron oscillation and the motoneuron activity, each one of them was by itself insufficient for controlling the rhythmic activity of the moto- neurons. They hypothesized a local interneuronal network as the rhythm generator. In the swimmeret beating system of crayfish, Heitler and Pearson {9] reported a similar com- plementary pair of non-spiking interneurons which showed membrane potential oscillation, which was out of phase with each other, with the rhythmic reciprocal activation of powerstroke and returnstroke motoneurons. They concluded that these interneurons are part of central pattern generator from the fact that they can reset the rhythm of motoneuron activation. In the walking system of cockroach, Pearson and Fourtner [7] identified in the metathoracic ganglion a non-spiking interneuron called inter- neuron I which showed oscillation during the rhythmic leg movements. During the depolar- ization and hyperpolarization phase, levator motoneurons showed an increase and a decrease in the spike discharge frequency respectively. Artificial depolarization and hyperpolarization caused an increase and a decrease in the moto- neuron activity. Brief current pulses injected into the interneuron could reset the timing of rhythmic motoneuron activity. But since the repolarization phase in the membrane potential oscillation of levator motoneurons seems to be generated by inhibitory inputs from neuron(s) other than interneuron I, they concluded that the motoneuron activity rhythm is generated not by interneuron I alone but by a network of non- spiking interneurons involving interneuron I. Other interneurons including those which are complementary to interneuron I have also been described [46]. Branches of interneuron I were entirely confined within the ganglion, some of them closely overlapping the dendritic branches of levator motoneurons. In the three motor systems mentioned above, non-spiking interneurons have been supposed to be involved in the pattern generating mechanism for the rhythmic movement. But the overall framework of such pattern generators is still to be examined. Is the pattern generation solely based on the network of non-spiking interneurons? Pearson and Fourtner [7] excluded the possibility that spiking neurons might be involved in the circuit because no spike activity could be correlated with the levator burst activity. Motoneurons, how- ever, can interact with each other without spikes. Heitler [47] suggested that motoneurons them- Local Non-spiking Interneurons 691 selves might be an integral part of the central pattern generator for the swimmeret beating system. Hence there still remains a possibility that other spiking neurons are also involved in the pattern generating circuit. Another, but related, question is how the slow oscillation in the membrane potential of non- spiking interneurons can be generated. If the assumption that the oscillation is endogenous is to be turned down, then it must be the result of the operation of some type of neuron networks (but see also section IV—B-2). Further identifi- cation of neural elements involved in the network is needed. The role of spiking neurons should also be studied at their subthreshold level (see section IV—B-1). 2. Episodic Acts Control mechanism of the locust hind leg posture has been most intensively analysed regarding the non-spiking local circuitry [31, 47-51]. Burrows [30] has reported a variety of local non-spiking interneurons which showed different outputs as revealed by intracellular current injection. The study was mainly concentrated on those which affected the motoneurons innervating the flexor and extensor tibiae muscles. Local non-spiking interneurons have been found which excite or inhibit one or more of motoneurons innervating either one of them. These interneurons had no significant output on the motoneurons innervating the other. Other local non-spiking interneurons have also been found which excite flexor (extensor) motoneurons and at the same time inhibit ex- tensor (flexor) motoneurons. One interneuron was found to excite both the flexor and extensor motoneurons. Many of them exerted motor effect On motoneurons innervating those muscles re- sponsible for moving other joints than the femoro- tibial one. A difficulty in interpreting the obtained results lies on the fact that the output connections shown above were studied based on intracellular current injection into each non-spiking interneuron. As has been discussed above (section II—D-3), the effect of current injection depends on the electrotonic distance between the site of output synapses and the site of electrode penetration. Even the same interneuron could show different motor outputs with different site of current in- jection. Furthermore, the interaction among non-spiking interneurons ({[44]; see below) makes the situation more complicated. The output of an interneuron is greatly affected by other pre- synaptic interneurons depending upon the external sensory input and the behavioral context of the animal. Nevertheless, the results clearly show that, with the current source at a certain site of the interneuron under certain internal and external conditions, non-spiking interneurons can exert a variety of motor outputs. Different motor outputs exhibited by local non-spiking interneurons can suggest some of their possible behavioral roles. For example, the interneuron which causes co-excitation of flexor and extensor tibiae motoneurons can be involved in the jump, producing the co-contraction of both muscles preceding the relaxation of flexor tibiae which triggers the rapid leg movement Pperast. In further considering the functional role of these interneurons in the motor control, following two points seem to be vital. Firstly, most local non-spiking interneurons seem to have no direct input from the peripheral sense organs [24, 48]. This may indicate that the interneurons mainly function as the member of central pattern generator for the leg movement. Jumping has been reported to be excited by either descending or ascending input. A clue to understand the relative position of non-spiking interneurons in the whole system of jump control would be hence to examine their central inputs. The second point is that non-spiking inter- neurons interact with each other in a graded way [44]. The mode of interaction is reported solely to be inhibitory: depolarization of one interneuron causes hyperpolarization of another. Thus interneurons which affected motoneurons _in- nervating the muscle of a certain joint exerted inhibitory effect on other interneurons which affected motoneurons innervating the muscles of other joints. The interaction among the non- spiking interneurons could serve for stabilizing and smoothing the movement of the leg as a whole in certain behavioral acts. 692 M. HisapA, M. TAKAHATA AND T. NAGAYAMA The interaction among the non-spiking inter- neurons also suggests that the operation of particular interneurons greatly depends on activity of other interneurons. As Pearson [6] pointed out, subtle change in the analog input can be precisely reflected in the output of non-spiking interneurons. Extrinsic modulation of specific reflex circuit would thus be most effectively carried out through their interconnections. This leads to a further possibility that non-spiking interneurons function not only in mediating specific behavioral acts but also in modulating them (see section III-B-2). Another case where non-spiking interneurons seem to mediate the episodic movement is the uropod steering system ((29, 54]; Takahata et al., in preparation). Non-spiking interneurons have been reported to be capable of controlling effectively the activity of motoneurons innervating uropod muscles in a variety of ways [10, 29]. Some of them control the antagonistic mo- toneurons in a reciprocal way. These inter- neurons thus might be involved in the reciprocal pattern formation during the steering act ((55]; Takahata et al., in preparation). A difference from the locust jump system is that some of the non-spiking interneurons receive sensory inputs from the tactile receptors on the tail-fan (Nagayama, unpublished observation). The connection seems to be monosynaptic but direct evidence is still lacking. Interneurons were also found which received recurrent input from the motoneurons which they affect (Takahata, un- published observation). These facts imply that some non-spiking interneurons in the crayfish terminal ganglion might function in integrating both the central and the peripheral inputs to generate various spatio-temporal patterns of motoneuron activity. In the steering system, however, basic reciprocal pattern in the antagonistic motoneuron activity has been shown to be produced by descending spiking interneurons (Takahata et al., in pre- paration). It may well be that the nervous system provides redundant mechanisms to generate particular acts for security against accidental injury and for compensatory function. But the situation strongly suggests that the non-spiking interneurons function in other ways than merely generating particular patterns of motoneuron activity. B. Modulation of Specific Reflex Pathways Several possible functions other than mediation of specific behavioral acts have been assigned to non-spiking interneurons in both rhythmic and episodic motor systems. In either case, the evi- dence is circumstantial. But reviewing these possibilities at the present time would serve to develop future experimental paradigms for the study of functions of non-spiking interneurons. 1. Rhythmic Acts Heitler and Pearson [9] reported a non-spiking interneuron which acts as a switching element in the swimmeret beating system of crayfish. When depolarizing current was injected into this inter- neuron, rhythmic reciprocal activation of power- stroke and returnstroke motoneurons was initiated in a quiescent animal. The rhythmic bursting of motoneurons lasted during the current injection (about 2sec). Other interneurons which have similar function have been reported in the crab ventilatory system [15] and in the cockroach walking system [56]. Their membrane potential is related to the oscillation cycle in the motoneuron burst activity. Injection of depolarizing current into a crab interneuron [15] increased the oscillation cycle and that of hyperpolarizing current decreased it. Excessive hyperpolarization (<—4nA) caused sustained spike discharge of motoneurons without oscillation. Heitler and Pearson [9] reported another type of non-spiking interneuron which acts as a balancing element in the swimmeret system. Artificial depolarization of this interneuron caused sustained reciprocal activation of antagonistic motoneurons: the powerstroke motoneurons were inhibited and the returnstroke motoneurons were excited. A possibility remains, however, that this interneuron itself might be an element of the rhythm generating circuit, although it showed no noticeable oscillation. Since the pattern generator for the rhythmic behavior remains still open, assessing the functional significance of these ‘“‘switching”’ and “balancing”? non-spiking interneurons is_ very Local Non-spiking Interneurons difficult at present. It should be first quantitatively analysed what factors influence the rhythmic behavior. The swimmeret beating system, for example, could be greatly influenced by operation of the walking system [57]. Here a possibility arises that the signals from the walking system are transmitted to those interneurons which have been reported by Heitler and Pearson [9] so that the beating rhythm can be modulated according to the activity level of the walking system. The most important step to elucidate the functional role of non-spiking interneurons, especially the modulatory ones, would be to identify each source of influence and to examine the mode of interaction since almost no behavioral act occurs regardless of behavioral context of the animal. 2. Episodic Acts The uropod steering response to body rolling has been shown to interact with a variety of locomotor acts [55, 58]. The interaction can be either facilitatory or inhibitory. The steering response is significantly facilitated, for example, by the abdominal posture movement (postural facili- tation, [55, 58]). The descending statocyst input evokes only subthreshold responses in the uropod motoneuron (Takahata ef al., in preparation). But during the abdominal posture movement, both the opener and the closer motoneurons of uropods show sustained depolarization and thus increase their excitability. The postural facili- tation of steering act could thus be accounted for by the summation of subthreshold EPSPs from the descending interneurons with the sustained depolarization (Takahata and Hisada, in pre- paration). This sustained depolarization of motoneurons has been shown to be generated by non-spiking interneurons [59]. Several lines of evidence indicate that the spike signals from the abdominal posture system are once transmitted to the local non-spiking interneurons and smoothed there as has been theoretically predicted by Rall [43] for remote EPSPs in vertebrate motoneuron dendrites (see section II-D-3) and finally transmitted to uropod motoneurons to form their sustained depolarization. Non-spiking interneurons which coactivate both the opener and closer motoneurons 693 [29] might be responsible for this extrinsic modu- lation of the motoneuron excitability. Some type of non-spiking interneurons are thus likely to function in gating of certain reflex pathways (Fig. 3). In the control of leg position in locust, Siegler [49, 50] reported that the membrane potential of some non-spiking interneurons which affected the flexor tibiae motoneuron was altered by the imposed change in the femoro-tibial joint angle, and that the output effect of the interneurons on the motoneuron activity also change with the joint angle. This could imply that the synaptic input to the interneuron causes different effects on the motoneuron depending on the leg position. Furthermore, she showed that the sustained depolarization of some interneurons could reverse the resistance reflex of the tibia and the com- pensatory reflex of the tarsus in response to imposed change in the femoro-tibial joint angle. LOSS OF Lec Contact LEG PROPRIOCEPTOR ETAT RNS ABDOMINAL STATOCYST INTERNEURON EXTENSION SPECIFIC INTERNEURON SUBTHRESHOLD EXTENSOR Uropoo MOTONEURON MOTONEURON ABDOMINAL EXTENSION Fic. 3. Modulatory role of non-spiking interneurons in the control of uropod steering response. Cen- tral pathway for the postural extension of the abdomen is summarized on the left based on Evoy and Kennedy [87], Larimer and Eggleston [88] and Page [89, 90]. Central pathway for the steering response is summarized on the right based on Takahata and Hisada [59, 91]. Facilitatory inter- action between these two motor systems has been suggested from the quantitative behavioral ob- servation [58]. Nerve signals which interact with the steering pathway have been shown to have central origin in the abdominal posture system [55]. Each rectangle indicates a population of neurons. NON-SPECIFIC SUBTHRESHOLD 694 M. HisADA, M. TAKAHATA AND T. NAGAYAMA When the tibia was extended from the femur, for example, flexor tibiae motoneurons showed maximal depolarization and slow extensor mo- toneurons did not spike. With the same stimulus but with the sustained depolarization of a non- spiking interneuron by current injection, the flexor motoneurons showed much smaller depolari- zation and the extensor motoneurons became excited, discharging spikes. Some other inter- neurons could enhance these reflexes when continuously depolarized with current injection. The non-spiking interneurons in the metathoracic ganglion thus can modulate the segmental reflexes in various fashion. For understanding the functional significance of non-spiking interneurons, it would be suggestive to ask a question: Can the supposed function of non-spiking interneurons be carried out by spiking neurons as proxy? Or, can the function not be possible without any type of non-spiking mode of neuron operation? Rhythm generation, for ex- ample, could be possible solely based on the network of spiking neurons (e.g. [60]). In a sense, non-spiking interneurons are not necessarily required to generate the rhythmic movement. And yet the fact is that non-spiking interneurons appear to crucially participate in the rhythm generating mechanism. A clue to this enigma seems to be in examining the function of spiking neurons in detail. Recent studies have revealed that ordinary spiking neurons also. communicate without spikes. In _ the following section, we will review the obtained results and further proceed to discuss about the biological significance of co-existence of the spiking and the non-spiking mode of neuron operation. IV. NON-SPIKING COMMUNICATION AMONG SPIKING NEURONS It has been widely accepted that the functional units in the nervous system are individual neurons which transmit their information to follower cells in the form of temporal patterns of spike trains. However, since Bullock [61] pointed out the possibility of intercellular communication in a graded manner without the intervention of all-or- none spikes, the importance of communication without spikes in the nervous function has slowly but steadily been appreciated by many investi- gators. The process received relatively little attention since, as Hodgkin [62] put it, “it has brought about by extensions of existing techniques, rather than by any striking invention”. This ‘“‘quiet revolution” in the traditional view on the neuron function has been summarized by Shepherd [63, 64]. Here we briefly sketch the classical view on the function of a single neuron and then point out some features newly found which the spiking neurons share with the non-spiking neurons. A. Traditional Views on the Single Neuron Function On the basis of anatomical studies, three distinct regions could be appreciated. These three regions differ from each other both anatomically and physiologically. The cell body contains various organelles including the nucleus. Axon is usually a single long process while dendrites show profuse arborization. Since Cajal [65], based on the gross morphology of neurons, different functions have been assigned to each of these three regions. Dendrites are the synaptic receptors, the cell body integrates synaptic inputs, and the axon generates and transmits spikes to the following neurons through the axon terminals which are the synaptic effectors. The single neuron is thus polarized functionally. Chains of these neurons as the unit of nervous system make up the reflex and central networks. The most serious objection to this view emerged from the electronmicroscopic study of the mam- malian olfactory bulb by Rall et al. [66]. They found presynaptic structures on the dendrites of the mitral cells, functionally suggesting that dendrites may have synaptic connections with other dendrites and that activation of dendritic synapses may occur by local graded synaptic potentials. Pre- synaptic structures on dendrites have thence been reported in many central neurons of vertebrate and invertebrate animals [4]. B. Novel Aspects: of Neuron Function Attempts to revise the classical neuron doctrine Local Non-spiking Interneurons 695 have been done by several investigators [61, 63, 64, 67]. Most important is the fact that spiking neurons are able to communicate with each other also without spikes. The direct consequence of this finding is the notion that the individual neurons are not the functional units in the nervous systems. Significance of this notion in under- standing the central nervous function has been discussed by Shepherd [63]. 1. Non-spiking Communication among Spiking Neurons Several types of neurons have recently been found to utilize both the spiking and the non- spiking mode of information processing. Dendro- dendritic synapses in a variety of spiking neurons could be the site of non-spiking communication. Amacrine cells of vertebrate retina also show both modes of operation [5]. A clear example where the spiking and non- spiking mode of operation seem to function in different ways has been provided by Graubard and her colleagues [14, 68, 69]. The stomatogastric ganglion of spiny lobster contains about 30 neurons. Their interconnections are responsible for the rhythmic contraction of stomach muscles. Subthreshold depolarization of a _ presynaptic lateral pyloric (LP) motoneuron caused a hyper- polarizing response in a postsynaptic pyloric dilator (PD) motoneruron. Spikes of LP motoneuron could also evoke hyperpolarizing potential change (IPSP) in PD motoneuron. The graded hyperpolarizing response had the reversal potential which was similar to that observed in the IPSPs evoked by LP spikes. Both responses were blocked in a graded fashion when the extracellular Ca** concentration was reduced. The graded hyperpolarization was also blocked by picrotoxin. All of these facts indicate that the non-spiking interaction between LP and PD motoneurons is mediated chemically. The postsynaptic effect was in proportion to the presynaptic membrane potential change. Hyper- polarization of the presynaptic neuron caused depolarization of the postsynaptic neuron, indi- cating that the presynaptic spiking neuron was continuously releasing transmitter at the resting potential level [69]. -With electronmicroscopy, King [41] have shown both input and output synapses on the dendrites of these motoneurons. This fact suggests that the graded interaction be- tween motoneurons occurs in dendrites. Similar interconnection between motoneurons has been reported among the motoneurons in the terminal abdominal ganglion innervating the uropod muscles of crayfish [70]. The interaction is excitatory as contrasted with that in the stomato- gastric ganglion. Subthreshold current injection into an identified motoneuron (Add) innervating the adductor muscle caused an increase in the spike discharge frequency in another identified moto- neuron (Red) innervating the reductor, a synergistic muscle of the adductor. The postsynaptic effect was proportional to the amount of current injected into the presynaptic Add motoneuron. Injection of hyperpolarizing current into the Add motoneuron caused a decrease of reductor moto- neuron activity. The site of interaction was deduced from the minimum current threshold for changing the Red motoneuron activity in the current injection test at various portions of the Add motoneuron. It was found that the interaction occurs within a certain confined portion of Add motoneuron. Evidence was shown that the interaction was mediated chemically, but further study is needed to clarify the nature of interaction. Presynaptic structure has been found on the motoneuron dendrites in locust [71] but still not in crustaceans PP aere le Especially interesting is the case of granule cells in the mammalian olfactory bulb. Granule cells have inhibitory output connections at their dendrites in the external plexiform layer to the secondary dendrites of mitral cells which receive direct inputs from the olfactory nerve and send outputs to olfactory cortical regions [64]. Granule cells not only mediate the recurrent and lateral inhibition of mitral cells but also exert central driving or biasing of them, gathering centrifugal inputs from various parts of the brain. Although the granule cells can generate spikes [74], their function has been thought to be based on the passive properties of their dendrites [75, 76]. The functional role of spikes in these cells, by contrast, is not known. 696 M. HisADA, M. TAKAHATA AND T. NAGAYAMA 2. Functional Difference between Spiking and Non-spiking Mode of Neuron Operation The functional significance of the excitatory graded interaction between motoneurons _in- nervating the synergistic uropod muscles [70] is not known. It could function in ensuring the co- contraction of two muscles. Similar excitatory interaction has also been observed between another pair of synergistic motoneurons, but has never been observed between motoneurons which are antagonistic to each other. It seems that the motoneurons utilize spikes to transmit their final output to target muscles and non-spiking com- munication to interact with each other to form the spatio-temporal patterns of spike discharges. The functional division of spiking and non- spiking modes of operation also exists in the stomatogastric system. The rhythmic spike activity in LP motoneuron and pyloric moto- neurons has been shown to be caused by endogenous oscillation in the activity of PD motoneurons and anterior burster (AB) cell [77]. Although these AB/PD cells show an oscillation in their membrane potential with spikes superimposed during the depolarization phase, Raper [78] showed that the spikes are not required to generate the rhythmic activity in the LP motoneuron. Localized application of tetrodotoxin (TTX) suppressed spike generation in AB/PD cells which still showed an endogeneous oscillation in the membrane potential. Under this condition, oscillation of the LP membrane potential could be also. observed. Injection of hyperpolarizing current into LP motoneuron showed reversal in the potential change, indicating the chemical transmission from AB/PD motoneurons to LP motoneuron. The results show that in the stomatogastric system, the rhythmic movement is generated by a neuron network operating in the non-spiking mode although most of the con- stituent elements of the network can generate spikes. Here the spikes are used to transmit final rhythmic output to stomach muscles. The granule cells of mammalian olfactory bulb which can generate spikes seem to Operate entirely in the non-spiking mode. It may be that the spiking mode is inappropriate for exerting the long-lasting inhibition of mitral cells. Another aspect of non-spiking mode of operation, however, is that non-spiking neurons can by their neuritic electrotonus integrate various spiking inputs to form a _ sustained output to follower cells representing the net level of these inputs (see section III). Thus Reese and Shepherd [79] suggested that the centrifugal inputs from the diagonal band and the anterior olfactory nucleus can control the overall sensitivity of the olfactory system through — tonic inhibition of mitral cells, the degree of which depends on the granule cell membrane potential. Centrifugal inputs have actually been reported not to be transmitted directly to mitral cells [64]. These examples indicate that the nervous system selects either one of the spiking and non-spiking modes of intercellular communication in indi- vidual cases, making the most of their advantages. Spikes are apparently suited for long-distance communication. The graded interaction among neurons has many advantages (see section III). It seems, however, that which type of communi- cation is finally adopted in particular cases depends not only on the fitness of each mode of interaction for the present purpose but also on the properties of the movement to be executed and on the interaction with other motor and/or sensory systems. In the rhythm generation, for example, graded interaction seems to be advantageous for continuously controlling the motor output but under certain conditions, spiking mode of inter- action could substitute the graded one or even become more advantageous as exemplified by many previous studies (e.g. [60)]). V. CONCLUSIONS We can now envisage a spectrum of neurons rather than discrete groups of neurons such as spiking and non-spiking neurons (Fig. 4). There could certainly exist those neurons which operate only with spikes and those which never generate spikes, communicating with graded interaction. But the non-spiking interaction among dendrites in several arthropod motor systems strongly suggests that the neurons in general communicate with both the spikes and the graded interaction. A large number of vertebrate neurons which have presynaptic dendrites also support the idea of the Local Non-spiking Interneurons 697 Non-SPIKER | INTRAGANGLIONIC INTER-) UNABLE TO SPIKE Stow GRADED CHANGE SPIKER WITH SPIKELESS COMMUNICATION [HII INTERGANGLIONIC (INTRA- ) ABLE TO SPIKE yt) TY | Ht i] INTERGANGLIONIC (INTRA- ) _—— ABLE TO SPIKE ALL oR None CHANGE IN POTENTIAL BOTH IN POTENTIAL Stow GRADED RELEASE Att or NONE RELEASE OF TRANSMITTER FROM BoTH OF TRANSMITTER FROM DISTRIBUTED SYNAPSES TERMINAL Less SPECIFIC INTERMEDIATE MorE SPECIFIC IN MODALITY IN MODALITY Fic. 4. A spectrum of central neurons with respect to their mode of information processing. The top row compares their structural feature (cf. section II-B). of information transmission are compared in middle rows. their inputs. spectral variation in the neuron function. One particular neuron thus resorts to spikes for some functional roles and to graded interaction for others. Non-spiking interneurons can be regarded as the specialized class of neurons sitting at one extreme and exclusively devoted to the nervous function where graded transmission plays the essential role. The mediation of specific behavioral acts in- cluding the motor pattern generation seems to be one major role of non-spiking interneurons. The precise and continuous regulation of motoneuron discharge over a wide range of frequencies would be most effectively attained by the non-spiking transmission as Pearson [6] pointed out. Many reports of non-spiking interneurons in the ar- thropod rhythm generating system support this conclusion. The same argument also applies to the control of episodic movements [48]. In both the rhythmic and episodic systems, non-spiking interneurons are functioning together with the Differences in their mode The bottom row compares spiking neurons which also communicate in a graded manner [9, 10, 14, 29, 47, 70, 80]. This co- operation might further enhance the system’s capability of attaining more subtle control of the motor output. In the vertebrate nervous system, there has been no report of purely non-spiking interneurons with the exception of retinal neurons. Spiking neurons, however, could function as the ‘“‘non- spiking neurons” with asynchronous convergence of many presynaptic spikes on the remote site from the output synapses. Actually many presynaptic dendrites have been reported in the vertebrate brain. It may well be that, in the vertebrate central nervous system with a vast number of neurons, no_ specialized class of non-spiking neurons has been needed during the evolution- ary process. The corollary of this argument is, as Simmers [81] pointed out, the fact that non- spiking interneurons have so far been reported only in arthropod species with far smaller number 698 M. HisapA, M. TAKAHATA AND T, NAGAYAMA of neurons. Functional isolation of neuritic regions in a single non-spiking interneuron based on the local processing of input-output signals also seems to favor the neuron economy. The modulation of specific neuronal pathways is another important role of non-spiking inter- neurons. Some local non-spiking interneurons which function in gating the uropod steering pathway provide an example of modulatory func- tion of graded communication [59]. Granule cells of mammalian olfactory bulb may also be included here. Overall responsiveness of the olfactory system could be at least partly deter- mined by granule cells which continuously regulate the excitability of mitral cells in a graded way despite their ability to generate all-or-none spikes. No such gating or biasing function in the non- spiking mode has been reported, however, in the motor control system of vertebrates. A developmental approach would further make a point in understanding the modulatory function of non-spiking interneurons. Goodman et al. [82] traced the electrical excitability of about 100 progeny cells of the dorsal unpaired median (DUM) neuroblast, which is one of more than 60 neuroblasts in each thoracic segment. The mor- phology and electrical excitability of the progeny cells were closely related to the order of birth in the proliferation showing a wide spectrum. Thus those with spiking cell bodies and spiking axons emerge earliest and those with locally confined non-spiking neurites, latest. They suggest that in the developmental process of the insect nervous system the motoneurons and _ interganglionic interneurons are produced first and local, non- spiking interneurons are added later. This model is consistent with our hypothesis that some of the non-spiking interneurons are the major element in the modulatory mechanism of various mediating circuits mainly comprising the interganglionic interneurons. The model also appears to apply to the development of vertebrate nervous system [3, 83, 84]. Non-spiking interneurons in the arthropod nervous system thus play crucial roles in the behavioral control. Phylogenetically, nervous systems of cnidarians utilize spikes for intercellular communication [85]. Even the unicellular or- ganisms can generate spikes [86]. It is hence unlikely that the non-spiking mode of neuron operation in the arthropod nervous system is a primitive form of intercellular communication. Rather the non-spiking interneurons are the products of evolutionary process during which the behavior to be executed has become more and more complicated. The fact that vertebrate brains have adopted a variety of dendrodendritic networks which operate in a graded manner also proves the importance of non-spiking communi- cation in the performance of subtle and complex nervous activity. The non-spiking interneurons and the dendrodendritic networks are thus the relevation of the behavioral strategy during the evolution of both the protostomic and the deutero- stomic animals. The separation of non-spiking interneurons as a specialized neuron class in arthropods might have come from the compromise between the neuron economy and the behavioral complexity. 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(1968) J. 102: ZOOLOGICAL SCIENCE 1: 701-709 (1984) © 1984 Zoological Society of Japan The Structure of Photoreceptor Cells in the Pineal Organ of the Japanese Common Newt, Cynops pyrrhogaster pyrrhogaster MISAO KIKUCHI and KIyosHI AOKI Life Science Institute, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102, Japan ABSTRACT — The pineal organ of the Japanese common newt, Cynops pyrrhogaster pyrrhogaster, is a flat organ situated on the dorsal surface of the diencephalon and consists of photoreceptor cells, supporting cells and neurons. protrude into the lumens. cones found in the retina. Within the pineal organ, the outer segments of the photoreceptor cells The outer segment is well-organized and resembles the lamellae of the The outer and inner segments are connected by a cilium and within the inner segment there are large numbers of mitochondria and a paraboloid-like concentration of glycogen granules. In the neuropile region, synapses between the basal processes of photoreceptor cells and neural dendrites characterized by synaptic ribbons are observed. The behavioral experiments have revealed that the pineal organ of the Japanese common newt could perceive light and was implicated in the entrainment of the locomotor activity rhythm. The present morphological studies also support the possibility that the pineal organ possesses these functions. INTRODUCTION Extensive studies on the pineal organs of various vertebrates show that the pineal organ of mammals is a secretory organ, but those of lower vertebrates such as fish, amphibians and reptiles have a direct photosensory function [1, 2]. Morphological studies have demonstrated photoreceptor cells in the frontal organ [3, 4] and the epiphysis [5-8] of anurans. The analysis of impulses recorded by the pineal nerve [9] or the pineal stalk [10, 11] using anurans proves that the pineal organ func- tions as a photoreceptor. Photoreceptor cells in the pineal organ of urodeles, Diemictylus viridescens viridescens [12, 13], Ensatina eschscholtzi [15], Cynops pyrrhogaster pyrrhogaster [16], have also been described in detail and development of the pineal organ was studied in Taricha torosa [17-20]. Behavioral experiments with amphibians [21, 22] have shown that eyeless newts and frogs can perceive light Ambystoma tigrinum [14], Accepted April 16, 1984 Received August 8, 1983 through extraocular photoreceptors [23, 24] and that the pineal organ is implicated in this response [25]. The tiger salamander, Ambystoma tigrinum, is able to perceive linearly polarized light used for orientation [26, 27]. On the other hand, there are only a few reports on the relationship between photoreception by the pineal organ and locomotor activity rhythms using urodeles [28, 29]. In birds, studies of the circadian rhythm of melatonin release [30] or N-acetyl-transferase activity in isolated pineal glands [31, 32] together with studies of activity rhythms after enucleation and trans- plantation of the pineal organ [33, 34] indicate the existence of a circadian oscillator within the pineal organ. We have found through behavioural experiments that the locomotor activity of the Japanese common newt, Cynops pyrrhogaster pyrrhogaster, was entrained to a light-dark cycle (LD 12: 12) and that entrainment was dependent upon the pineal organ (Chiba and Aoki, in preparation), suggest- ing that the pineal organ of Cynops had photo- receptive capability and was implicated in the entrainment of the locomotor activity rhythm. 702 M. KIKUCHI AND K. AOKI The purpose of this paper is to present evidence that the pineal organ of Cynops can perceive light on the basis of ultrastructural studies. The result will be discussed in relation to the circadian locomotor rhythm. MATERIALS AND METHODS Sexually mature adult Japanese common newts, Cynops pyrrhogaster pyrrhogaster, body length 10-12 cm, were killed by decapitation and the brains were quickly dissected and prefixed in 2.5% glutalaldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) at 0-4°C. After pre- fixation for 24 hr the brain was trimmed and postfixed in 1.5% osmium tetroxide in the same buffer for 2 hr. The tissue was block-stained with 0.3% uranyl acetate for 1 hr, dehydrated through a graded series of ethanol, infiltrated with pro- pyleneoxide and embedded in Epon 812. For light microscopic studies, blocks were cut with a Porter-Blum MT-1 ultramicrotome to give | wm sections and stained with 1% toluidine blue. For electron microscopic studies, ultrathin sections were mounted on grids. They were contrasted with uranyl acetate followed by lead citrate and examined with a Hitachi H-—300 elec- tron microscope. RESULTS Light microscopy The pineal organ of adult Cynops is situated just under the cranium at the roof of the dien- cephalon between the habenular commissure and the posterior commissure (Fig. 1). The pineal organ is flat, about 700 ~m in length and is tear- drop shaped with the narrow region directed anteriorly. The pineal organ of Cynops has neither the parapineal organ nor the frontal organ found in other lower vertebrates. It is enveloped in the basal lamina except at the central area of the ventral surface (proliferation zone, see Hendrickson and Kelly [18]) where it is directly connected to the third ventricle and the dien- cephalic roof. The pineal organ is mainly com- posed of photoreceptor cells and supporting cells. Oil droplets are also sometimes seen in the inter- cellular spaces. There are a number of lumens in the pineal organ and each is surrounded by the supporting cells and photoreceptor cells whose outer segments protude into the lumens. Electron microscopy The ultrastructure of the pineal organ of Cynops is similar to that of other urodeles. The pineal organ is mainly composed of photoreceptor cells, supporting cells and neurons. Near the basal lamina there are areas of neuropile (Fig. 2). The most abundant photoreceptor cells have well- developed outer segments, with a maximum length of 30 wm, protruding into the lumens (Fig. 3). The outer segment consists of a stack of lamellar discs similar to the cone-type lamellae of the photoreceptors found in the retina of vertebrates (Fig. 4). Although some outer segments are disorganized and present whorl-like profiles, most are long and well organized. The outer and inner WILL LLL LAAELILZAZAZzQQEEEAQEEEEZEE, LM EEL Fic. 1. posterior commissure; Sub. Com., Third Ventricle Schematic representation of a sagittal section through the newt brain showing the relationship between the pineal organ and surrounding tissues. go Thalamus , Hab. Com., habenular commissure; Pos. Com., subcommissural organ. Pineal Photoreceptor Cells in Newt 703 segments are connected by a cilium that has a 9+-0 centriolar apparatus (Figs. 5 and 6). In the inner segment, numerous stacks of mitochondria, Golgi complexes, rough endoplasmic reticulum, lysosomes and some vesicles are observed. Photo- receptors are connected to neighboring cells by desmosomes. The nucleus of the photoreceptor cell is large and irregularly shaped (Fig. 7a). Near the nucleus, a board-like structure, 0.1 “m thick, of unknown function, can sometimes be observed. At higher magnification, a_ highly ordered pattern is apparent (Fig. 7b). The pho- toreceptors are characterized by a large concentra- tion of glycogen granules similar to the paraboloid structure found in photoreceptors of the retina (Fig. 7a). There are synaptic ribbons in the photoreceptor basal process, and synaptic ribbon- like structures can also be observed in the inner segment near the outer segment at the junctions between photoreceptors. Ribbons generally ap- pear in twos and threes at the junction and are surrounded by clear vesicles of 50 nm in diameter. However, unlike the basal process there are no clear vesicles remote from ribbons (Fig. 10). Supporting cells found among the photorecep- tors contain much smooth endoplasmic reticulum. Compared to the photoreceptors, the mitochon- dria are fewer and more dispersed. The nucleus is more spherical and less electron dense. The Fic. 2. Schematic representation of the photoreceptor cells in the pineal organ. PC, photoreceptor cell; PA, concentration of glycogen granules; NR, neuropile region; BL, basal lamina; SC, supporting cell. Fic. 3. Low magnification electron micrograph showing the pineal lumen cotaining several outer segments(OS) Longitudinal section of the outer segment protruding into the lumen(L) is long M, mitochondria; BL, basal lamina. x 6,000. of photoreceptor cells. and well organized. IS, inner segment; Fic. 4. A photoreceptor cell showing a well-formed typical cone-like outer segment. 18,000. A portion of outer and inner segments connected by a cilium. The centriole(C) and the basal body(B) are oriented nearly perpendicularly. Fic. 6. Across section of the connecting cilium containing nine pairs of tubular filaments. 58,000. Longitudinal section of the photoreceptor cell containing mitochondria, Golgi complex, dense- cored vesicle (lysosomes) (DV), a nucleus and a concentration of glycogen granules(PA). BS, board-like D, desmosomes; OS, outer segment; SC, supporting cell. 6,800. 7b. High magnification electron microgragh of a portion of the board-like structure. It has a highly ordered pattern and seems composed of crystals of protein. x 100,000. M. KIKUCHI AND K. AOKI 704 Pineal Photoreceptor Cells in Newt 705 ‘ae a ie a a He 706 M. KIKUCHI AND K. AOKI Pineal Photoreceptor Cells in Newt 707 inner segments of the photoreceptors and support- ing cells contain dense-cored vesicles measuring about 0.2-1.0 “m in diameter. The neuropile regions are situated near the ventral basal lamina and are composed of the basal processes of photoreceptors filled with synaptic vesicles of 50 nm in diameter and neural dendrites (Fig. 8). Many synapses between photoreceptors and dendrites, indicated by synaptic ribbons, can be seen in the neuropile region. The synaptic ribbons are surrounded by clear synaptic vesicles and are either straight or curved and up to 2 “~m long. They show orientation to post-synaptic dendrites at synaptic junctions (Fig. 9). Some- times, several synaptic ribbons, up to a maximum of 10, can be seen (Fig. 11). Dendrites and synaptic terminals of the photoreceptors possess mitochondria. Dendrites contain microtubules and microfilaments (Fig. 9). Near the ventral base- ment lamina, there are electron dense vesicles of about 180 nm in diameter (Fig. 12). DISCUSSION The pineal organ of the Japanese common newt is situated immediately beneath the cranium and does not have a frontal organ like that of a frog. Light is easily accessible to the pineal organ through the epidermis and the translucent cra- nium. The photoreceptors in the pineal organ of Cynops are structurally identical to those found in the pineal organ of other lower vertebrates [35]. They have outer segments that protrude into several lumens existing within the pineal organ but the length of the outer segments is not uni- form. In general, the outer segments are long and well organized and consist of 200 to 400 lamellar disks. The number of lamellae may sometimes reach 600. Thus the structure of the pineal outer segment of Cynops is different from that of anurans, Rana esculenta L. and Rana temporaria L. [4, 5], or the salamander, Ambystoma tigrinum [14], which consists of only 60-120 lamellae. The outer segment of the mature newt, Taricha torosa, has been described as being disorganized and con- torted [1, 19]. From the number of photorecep- tors and overall structure of the pineal organ, it is clear that the pineal organ is not capable of form perception. In the case of the tiger salamander, Ambystoma tigrinum, it has been suggested that orientation may be mediated through the percep- tion of polarized light [27]. In the case of Cynops, there are many photoreceptors with outer segments horizontally protruding into lumens. However, these structural features alone do not allow infer- ences to be drawn about mechanisms for the perception of polarized light. The basal processes of the photoreceptors are connected with neural dendrites by ribbon syn- apses. Synaptic ribbons surrounded by numerous clear synaptic vesicles are possibly involved in neuro-chemical transmission. In Cynops, rib- bons are observed not only at the basal process, but at places in the inner segment where there are no synaptic connections with the neural dendrites. Such ribbons may be involved in the transfer of materials between photoreceptor cells. The daily locomotor activity rhythm of Cynops is clearly entrained to the light-dark cycle (LD 12: 12). Eyeless newts also show rhythms entrained to the light-dark cycle (Chiba and Aoki, in pre- paration). These results indicate that Cynops perceives light with the pineal organ and may use this information for the entrainment of the loco- motor activity rhythm. The pineal organ possesses secretory functions and in many animals, circadian variations in pineal melatonin synthesis have been observed [35]. The secretory pineal organ of mammals does not have photoreceptive capabilities, and while it is not Fic. 8. Neuropile region composing the basal processes of photoreceptor cells(PP) and neural dendrites(ND). SR, synaptic ribbon. 14,000. Fic. 9. A synaptic junction characterized by a synaptic ribbon surrounded by synaptic vesicles. process; ND, neural dendrite. 28,000. Fic. 10. Synaptic ribbons at the junction between photoreceptor cells. The basal process of the photoreceptor cell containing eight synaptic ribbons. Fic. 11. Fic. 12. Dense-cored vesicles situated near the ventral basal lamina. PP, basal PC, photoreceptor cell. » 18,000. * 14,000. x 27,000. 708 an independent oscillator, its melatonin or sero- tonin content and N-acetyl-transferase activity do exhibit circadian rhythms [36, 37]. When the pineal organ of chicken is isolated and cultured, serotonin N-acetyl-transferase activity follows the rhythm established before isolation [31, 32]. Fur- ther, when pineal organs are transplanted into pinealectomized sparrows, the phase or the period of the donor’s rhythm is transferred to the host [33, 34]. These facts indicate that there may be a circadian oscillator in the pineal organ. If rhythm formation is attributable to the pineal organ also in Cynops, melatonin may play an important role, although its existence within the pineal organ has not yet been confirmed in this species. Within the photoreceptor cells and supporting cells, there are electron dense particles with a diameter of 0.2-1.0 wm. At a position close to the basement lamina, particles with a diameter of about 180 nm are also present (Fig. 12). These particles are probably secretory. In order to elucidate the relationship between the pineal organ and the oscillator for rhythm forma- tion in Cynops, attention must be focused on the secretory function of the pineal organ. ACKNOWLEDGMENTS We thank Dr. A.D. 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Federa- tion Proc., 35: 2347-2352. ire J . J 7 al = ry af ree? > iy Bele pt As ne Laie Te) eae. S : « , ri . i fey a i me \ By : \ < i ¢ i c ‘i i i i ‘ 2 i ae ZOOLOGICAL SCIENCE 1: 711-717 (1984) Simultaneous Recording of Motile Responses of Light-Absorbing and Reflecting Chromatophores in Vitro NorIKo OsHIMA, Ryozo Fusit and HIROAKI KASUKAWA Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan ABSTRACT — A method for recording simultaneously the motile responses of both light-absorbing and reflecting chromatophores is described. A microscope in which specimens were observable both by usual transmission and by dark-field incidence illumination systems was put for use. By making use of a rotating photo-chopper disk, the paths of the transmission and the incidence illu- mination beams were opened alternately at more than 10 Hz. The resultant output currents of the photosensor in two trains, which were endowed with the changes due to activities of above-mentioned two sorts of cells, respectively, were first converted into voltages. They were then appropriately amplified, and led to two separate channels by making use of two sample-and-hold circuits, each of which was triggered synchronously with the opening of the corresponding illuminator path. In each channel, a certain DC component was appropriately subtracted from the output voltage of the sample- and-hold device, and the resultant voltage changes were widely registered on a dual channel electronic © 1984 Zoological Society of Japan chart recorder as a practically smooth trace. were presented, and its usability was discussed. INTRODUCTION It has now become rather popular to record the response of darkly pigmented chromatophores, the melanophores, automatically and continuously on an electronic paper-chart recorder by transduc- ing the intensity of light transmitted through a small area of skin within which the pigment cells lie into the electric current [1-4]. Although there are some difficult points to overcome, the motile activities of moderately light-absorbing chro- matophores, i.e., the xanthophores and _ the erythrophores, can also be registered now, if we adopt an appropriate skin preparation, employing appropriate apparatuses [5-7]. Meanwhile, a method to assess accurately the responses of light-reflecting chromatophores, i.e., the leucophores and the motile iridophores, has also been invented, in which an epi-illumination microscope was put to use in combination with a photomultiplier tube as a highly sensitive photo- sensor [6, 8]. No attempts have ever been made, Accepted April 25, 1984 Received April 13, 1984 Some examples of the application of the present method however, to record simultaneously the response of light-absorbing chromatophores and that of reflecting ones in the same portion of the skin. Recently, we have succeeded to do so by making good use of an assortment of electronic circuits coupled with a photo-chopper to switch over rapidly the light paths of transmission and inci- dence illumination beams. In this article, we describe the method and discuss the usefulness of its application to the chromatophore system of animals. MATERIAL Skin preparations in which light-absorbing and reflecting chromatophores exist contiguously or overlapped each other can be put to use. First, split dorsal-fin pieces of the damselfish, Chrysiptera cyanea, were adopted, in which dermal melano- phores were found just under the closely graveled monolayer of small motile iridophores [9]. Move- ments of a few melanophores and the overlying iridophores within a restricted area of the skin were tried to be recorded simultaneously. Another type of preparation used was the scale 712 N. Osuima, R. Fusm AND H. KASUKAWA isolated from dorsal trunk of the medaka, Oryzias latipes. In the wild-type forms of this species, a number of melanophores and a few leucophores were present in the dermis. Usually, each leu- cophore was closely associated with a melanophore. There, the perikaryon of the former lied just under that of the latter, constituting a concentric two-cell unit of chromatic effector. Further, the territory extended by both cells was about the same, when their chromatosomes were thoroughly dispersed throughout the cytoplasm. Thus, the responses of those two pigment cells of different sorts could be studied with the area restricted by circum- scribing their domain. CLS OL ———— a RRO, an OOIIORRIIII 1 MS SE 1E~ CL | CF it _ If | Cee Fee Fic. 1. measurement, (cf. Fig. 3), filter, CL: condenser lens, CVC: current-to-voltage converter, component cancel adapter, eye-piece, indifferent electrode, for experimental media, lens, AM: annular mirror, DCA: direct current amplifier, DER: dual channel electronic chart recorder, FRD: field-restricting diaphragm, IE: ILS: incidence illumination light source, M: motor, OP: outlet pipette for experimental media, ES: electronic stimulator, The skin preparations were excised in a physio- logical saline solution, which had the following composition (mM); NaCl 128, KCl 2.7, CaCl, 1.8, MgCl, 1.8, glucose 5.6, tris-HCl buffer 5.0 (pH 7.4). Being held under the cover slip on a small perfusion chamber, a skin piece was irrigated with the saline or one of the experimental solutions (Fig. 1). METHODS Stimulation Chemical stimulation was applied by perfusing Diagram of the apparatus for simultaneous photoelectric recording of light- absorbing and reflecting chromatophores. C-1: channel 1 (cf. Fig. 3), CD: circular diaphragm for iridophore measurement, CLS: controlling light source, AD: annular diaphragm for leucophore C2: channel 2 CF: color COMP: comparator, DCC: DC- EP: IP: inlet pipette OL: objective RD: MS: microscope stage, PC: perfusion chamber, rotating disk with sectoral openings for chopping illuminating and controlling light beams, SE: stimulating electrode, SHC: sample-and-hold circuit, SPD: silicon photodiode, TLS: transmission illumination light source. Chromatophore Responses on Two Channels 713 the skin piece with one of the experimental solu- tions containing stimulant substances. Such drugs used in this study included norepinephrine hydro- chloride (Sankyo, Tokyo) and melatonin (Nakarai Chemicals, Kyoto). To stimulate the nervous elements around the pigment cells, a K’*-rich saline was employed [10], which was prepared by substituting an equimolar KCl for NaCl in the primary saline. For this purpose, 50mM-K* saline was found to yield satisfactory results. In most experiments, therefore, we adopted the K*- rich solution of this type. The method for stimulating electrically the nerve fibers controlling chromatophores was essentially the same with that described elsewhere [2]. Optical system A microscope in which both the transmission and the dark-field epi-illumination systems are installed is employable (Fig. 1). In the present study, an industrial microscope (Nikon, Tokyo; Optiphot XF-BD with CF-BD lenses) was used. As a matter of course, light-absorbing chro- matophores were observed and measured of their motile responses by the ordinary transmission system, while the reflectance changes due to the light-reflecting cell activities were assessed under the dark-field incidence illumination [6, 8]. In order to minimize the complementary-light ab- sorbing effect of light-reflecting cells, the use of an appropriate sharp cut filter (CF in Fig. 1) is strongly recommended for the accurate assessment of the motile activities of light-absorbing cells. A Toshiba (Tokyo) O-—55 filter which cuts off light shorter than 55nm in wave-length was properly used for the study of melanophores coexisting with motile iridophores. On the other hand, one havy- ing a wider cut-off range of wave-length (e.g., Toshiba, R-60) brought about better results in eliminating the effects of leucophore movements. The filter for this purpose should of course be placed between the transmission illuminator and the condenser. Photosensor A semiconductor photoelectric transducer of high quality or a photomultiplier tube is employ- able. In the present study, a silicon photodiode (S1226-SBK or SBQ, Hamamatsu Photonics, Hamamatsu) or a reasonably priced photomul- tiplier of side-on type (931A, Hamamatsu Pho- tonics) was used. The former was put just above the field-restricting diaphragm inside the eyepiece in the photographic column of the trinocular assem- bly (SPD in Fig. 1; cf. also ref. 7). Being installed in a hand-made housing, the latter was also adapted on the photographic column [6, 8]. Photo-chopping and electronic processing Being incorporated in the diagram of the whole system, the newly invented portions in the present work are exhibited in Figure 1. The output current of the light-sensor was first conducted to a current-to-voltage converter, which has precisely been described lately elsewhere [7]. The use of such a high quality converter is also recommended, when we adopt a photomultiplier tube in place of the photodiode [6, 8]. In any case, the output voltage of the converter was led to two DC amplifiers, which could amplify the input signals independently. A black plastic rotating disk having two sets of Fic. 2. Rotating disk with two sets of sectoral openings employed to chop light beams for microscope illuminations and those for generating pulses triggering analog switches. CB,: path of controlling beam for incidence illumination, CB,: path of controlling beam for transmission illumi- nation, IIB: path of incidence illumination beam, MA: motor axle, RD: rotating disk, TIB: path of transmission illumination beam. 714 N. OsHima, R. Fusit AND H. KASUKAWA three sectoral openings was made and used as the photo-chopper (Fig. 2). The outer and wider sectors are for opening and shutting the light pathway of either transmission or incidence illu- minating beam. The interior angle of each sectoral opening is 60°. The inner set is just for generating trigger pulses in the controlling system, and has sectors of a smaller interior angle being incised to be about 20°. By rotating the disk, the light paths of the two illuminating systems can be opened alternately (Fig. 3C). Thus, the output of the current-to- voltage converter, and deservedly those of two DC amplifiers showed alternate and parallel changes corresponding with the alternation of the illumina- tion beams (Fig. 3B). Meanwhile, the narrower sectoral cuts allow a pair of photosensors in combination with the light sources beyond the disk plate to generate photo-current pulses of exactly the same frequency as that for the illu- mination chopping, but of shorter duration (Figs. Responses of light- |A absorbing and reflecting chromatophores (arbitrary unit) Qutput current of photo-sensor (arbitrary unit) Sequence of OPEN— i]lumination j close— chopping 2 and 3D). Through the comparator circuits those narrow pulses are transformed into accurate square pulses for triggering analog switches inside the sample-and-hold devices (Figs. 1 and 3D). When the trigger pulse is applied, the analog switch is turned on. During that period, the signal applied to the input terminal of the sample- and-hold circuit is directly conducted to its output as it is (Fig. 3E). During the period when the command pulse is off, the output voltage is held clamped at the value recorded when the pulse is just cut off (Fig. 3E). In such a way, the changing signal potential at the input is transferred to the recorder recurrently, renewing the former value. In order to apply the trigger pulses around the middle one-third within the term when the light path of either transmission or incidence illu- mination is open (Fig. 3C, D), the narrower sectoral cuts as well as the controlling light source- sensor assemblies are so positioned as shown in Figure 2. In this way, two kinds of output C-1 close— Angle of rotation of ————> ———_0@__ 1 photo-chopper 9 ee 240 360 480 600 : sample — — disk (degree) P D C-1 hold— ) | Sequence of | les | analog switch- tone | | | | ) | : : | C-2 trigger pulses hold— Output voltage of sample-hold circuits (arbitrary unit) Fic. 3. Schematic representation of the changes in representative parameters in the device due to the photo-chopping and the responses of both light- absorbing and reflecting chromatophores. C-1: channel 1, e.g., for re- flectance measurements, C-2: channel 2, e.g., for transmission measure- ments. Chromatophore Responses on Two Channels 715 signals alternately generated in the main photo- sensor can be picked up alternately, leading finally to the registration of two traces on the recorder. If the frequency of the chopping is sufficiently high in comparison with the changing rate of cellular responses, a practically smooth trace can justly be registered. For pursuing the motile response of common chromatophores, the chop- ping at about 10 Hz had naturally been estimated to be sufficient enough, and actually revealed to be so. Usually, therefore, we made use of the ap- paratus at the chopping frequency of approximately 15 Hz. Since the chopper disk had three open- ings, about five revolutions per second could bring about the above-mentioned frequency of opera- tion, although faster chopping at least up to 100 Hz could have been performed in the trial manu- facture. By way of parenthesis, we can use the apparatus just for assessing activities of either the light-ab- sorbing or reflecting cells exclusively, by keeping the transmission or the incidence light path open, respectively, without rotating the chopper disk. Recorder For registering continuously two separate events, i.e., the responses of both light-absorbing and reflecting chromatophores, an electronic chart recorder having two or more channels or a poly- graph has to be employed. In the present inves- tigation, a dual channels recorder (EPR-221A; Toa Electronics, Tokyo, or SS—250F; Sekonic, Tokyo) was put to use. The output of the sample-and-hold circuit mentioned before was led to the recorder usually through a DC-component cancelling adapter [6, 7). In the channel for detecting light reflection (C-1 in Figs. 1 and 3), the use of such a device may not be necessary, since the pen positioner, usually equipped already in the recorder, would simply substitute for the device. For a more large voltage cancellation, inevitable in the other channel, the device has been disclosed to be very convenient for positioning the trace. RESULTS As an example of the application of the present method, an actual record registering the responses of both a few melanophores and the overlying motile iridophores within a small area (130 “m in diameter) of a split dorsal-fin preparation of the damselfish, Chrysiptera cyanea, is exhibited in Figure 4. In this particular recording, the pre- paration was stimulated nervously to elicit either the melanosome aggregation in the melanophores or the color response of the motile iridophores. Incidentally, the color response of such iridophores has lately been defined as the shift of the spectral peak of the reflecting light from the cells concerned from near ultraviolet towards the longer wave- length region of up to greenish yellow [9]. The summation of small unit responses to a low frequency nervous volley can clearly be observed on both traces exhibiting the responses of these two sorts of pigment cells. Working on the same material, we have already obtained some interesting results, including the i ee aM le su a oe ee : . $a 7. i, : sates “cus a ih a Eee! aoe gear ee I. ; Be ‘ Sor 5 EMCEE | AAR re eit, CSS, PERT, oO : 4 SS a ee eee ae Color response, \ “- ee eee aes See H Aggregation, ee eee te ne te a Fic. 4. Example of actual simultaneous recording of the responses of melanophores (lower trace) and overlying motile iridophores (upper trace) of the damselfish, Chrysiptera cyanea, to electrical nervous stimulation. N.S.: nervous stimulation. — Bi- phasic square pulses of 10 V in strength and 1.0 msec in duration were applied at 0.14 Hz on the proximal part of a split dorsal-fin preparation. Maximal level of chromatophore responses (100%) was attained by applying 5*10°°M _ nor- epinephrine for 5 min, following the recording shown here. 716 N. OsuimaA, R. Fusm AND H. KASUKAWA fact that the responses of these two kinds of cells generally proceeded side by side, although the iridophores were rather refractory to hormonal stimuli. Part of the outcomes will appear shortly [9], and further details will be dealt with in a series of future publications. The simple chromatophore unit of wild-type Oryzias, composed of a-melanophore and a leu- cophore, also provided an attracting material to be studied by the present method. In the actual measurements, the isolated scale was held epider- mal side down under the cover slip. The leu- cophore could thus be observed on the top of the melanophore, being able to be assessed of its light reflectance more precisely. In this study, further- more, we adopted a photomultiplier tube as the photosensor, since the light reflection from the leucophore was so weak as to be undetectable quantitatively and without noise by the semicon- ductor detector. Another point to be noted here is that an annular diaphragm (AD in Fig. 1, cf. ref. 8) was adopted, since we already knew it to be more adequate for the present purpose than the usual circular one (CD in Fig. 1) commonly used in the iridophore study. Figure 5 illustrates an instance of measurements in which K*-rich saline gave rise to an aggregation of melanosomes within the melanophore, and at the same time a dispersion of leucosomes in the leucophore. It should be pointed out here that Fic. 5. Another example of actual recording which shows the responses of a single simultaneous melanophore (lower trace) and its associated leucophore (upper trace) on a scale of the medaka, Oryzias latipes, to 50-mM K* saline solution. The explanation given in the legend for Fig. 4 regarding the ordinate also applies to this figure. the directions of the responses were completely reversed in terms of the movement of the pigmen- tary inclusions. Although not shown in the figures, the same apparatus has also revealed to be extremely useful in examining the effects of chemical substances, such as norepinephrine or melatonin, on those compound chromatic systems. DISCUSSION Light-absorbing and reflecting chromatophores are frequently found to lie one upon another in the skin, forming a structural and functional unit for color change. In the medaka, Oryzias latipes [11, 12], and the killifish, Fundulus heteroclitus [13], for instance, melanophores and leucophores have been shown to move oppositely: When melanosomes in the melanophore disperse, leu- cosomes in the leucophore usually aggregate, and vice versa. With respect to the integumentary coloration, however, such a phenomenon was quite reasonable in producing additive chromatic effects, thus leading to efficient changes of tint from dark to light and the other way. In damselfish, on the other hand, coordinated movements of melanophores and motile irido- phores have been shown to be closely implicated with their normal cobalt-blue coloration, excite- ment darkening, and so forth {9]. Similar in- stances in which two or more sorts of chromato- phores comprizing light-absorbing as well as reflecting ones constitute a chromatic unit are believed to exist abundantly in the animal kingdom. Those entities may play an essential role in produc- ing characteristic and subtle hues and also in changing the tone delicately. In any case, the recording simultaneously the responses of those chromatophores must be of greater use than the conventional methods in which the responses of chromatophores of differ- ent kinds had to be separately assessed. It is a matter of course, furthermore, that we can accom- plish more than twice the work within the same period of time. Until the present, attempts have never been made to record quantitatively the responses of two different kinds of pigment cells at the same time. Chromatophore Responses on Two Channels Only an exclusive was the recent trial by Iwata et al. [14], in which the responses of both melano- phores and leucophores on the same portion of the Oryzias scale were endeavored to be quantified. In their study, the transmission and the incidence illumination lights of the microscope were turned on and off alternately every 20 seconds, and two discontinuous curves corresponding to the re- sponses of two kinds of cells were registered on one channel of the polygraph. In consideration of the actual rate of chromatophore responses, much faster switchover of the lights stands to reason. In the apparatus invented this time, the switching at more than 10 Hz could have easily been realized. Since the time constant of the response of common chromatophores is not less than a few seconds, the recurrent period less than 100 msec is sufficient enough to register the trace practically smooth and continuous. If we employ electronic shutters which are now available commercially, extraordinary rapid chop- ping may be achieved. As mentioned above, however, the switchover frequency used in this study was well enough to perfection for quantifying the responses of the effector cells of this category. Therefore, it is unnecessary to get and employ such expensive devices as a substitute for the photo- chopper. Utilizing the apparatus described here, we are now able to perform more detailed investigations on the motile as well as regulatory mechanisms of chromatophore units in the animal skin. A more profound understanding of fine and delicate color changes including pattern formation and vanish- ment seen in case of environmental adaptations and ethological encounters would thus be obtain- able hereafter. ACKNOWLEDGMENTS This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, and also by a grant from the Ito Foundation for the Promotion of Ichthyological Research. REFERENCES 1 Fujii,R. and Novales,R.R. (1968) Tetrodotoxin: 11 13 717 Effects on fish and frog melanophores. Science, 160: 1123-1124. 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. Iga, T. (1975) Effects of sulfhydryl inhibitors on migration of pigment granules in the melano- phore of Oryzias latipes. Mem. Fac. Lit. & Sci. Shimane Univ., Nat. Sci., 8: 75-84. Nagahama, H. and Katayama, H. (1982) Ba-pulsa- tions of innervated and denervated melanophores of the teleost fish in the presence of adrenaline. J. Sci. Hiroshima Univ., Ser. B, Div. 1, 30: 159- AFi. Fujii, R. and Oshima, N. (1984) Biological assay using chromatophore responses. In ‘Bioassay of Physiologically Active Substances”. Ed. by N. Ikekawa, S. Marumo and M. Hoshi, Kodansha Scientific, Tokyo, pp. 262-267. (In Japanese) Fujii, R. and Oshima, N. (1984) Movement of chromatophores. “Jikken Seibutsugaku K6za”, 10: “Undo Seibutsugaku” (Series in Experimental Biology, Vol. 10: Biology of Movement). Ed. by H. Sugi and Y. Hiramoto, Maruzen, Tokyo, pp. 255-270. (In Japanese) Oshima, N. and Fujii, R. (1984) A _ precision photoelectric method for recording chromatophore responses in vitro. Zool. Sci., 1: 545-552. Fujii, R. and Miyashita, Y. (1979) Photoelectric recording of motile responses of fish leucophores. Annotationes Zool. Japon., 52: 87-94. Oshima, N., Sato, M., Kumazawa, T., Okeda, N., Kasukawa, H. and _ Fujii, R. (1984) Motile iridophores play the leading role in damselfish coloration. Pigment Cell, 7: in press. Fujii, R. (1959) Mechanism of ionic action in the melanophore system of fish I. Melanophore- concentrating action of potassium and some other ions. Annot. Zool. Japon., 32: 47-59. Miyoshi, S. (1952) Responses of iridocytes in isolated scale of the medaka (Oryzias latipes) to chlorides. Annot. Zool. Japon., 25: 21-29. Kinosita, H. (1953) Studies on the mechanism of pigment migration within fish melanophores with special reference to their electric potentials. Annot. Zool. Japon., 26: 115-127. Menter, D.G., Obika,M., Tchen, T.T. and Taylor, J. D. (1979) Leucophores and iridophores of Fundulus heteroclitus: Biophysical and ultra- structural properties. J. Morphol., 160: 103-120. Iwata, K. S., Takahashi, T. and Okada, Y. (1981) Nervous control in chromatophores of the medaka. In “Pigment Cell 1981". Ed. by M. Seiji, Univ. Tokyo Press, Tokyo, pp. 433-438. ZOOLOGICAL SCIENCE 1: 719-723 (1984) © 1984 Zoological Society of Japan Nucleolus Organizer Region Lateral Asymmetry Induced by BUdR Incorporation in Mouse Chromosomes SUSUMU TAKAYAMA and KyoMU MATSUMOTO Biological Laboratory, Faculty of Science, Kwansei Gakuin University, Nishinomiya 662, Japan ABSTRACT — Mouse lung cells from a primary culture were incubated with BUdR to obtain chromosomes with BT-TB, BB-TB and TB-TT chromatid constitution. After the silver staining, all of these differently BUdR-substituted chromosomes showed a marked increase of nucleolus organizer region lateral asymmetry (NLA) in which no silver dot or a smaller dot was present in the chromatid which contained more BUGR than did its sister chromatid. The NLA found in the BB-TB and TB—TT chromosomes mainly belonged to an extreme type of NLA in which only one silver dot was found almost exclusively in the TB- and TT-chromatid, respectively. The similarity in the extent of NLA occurred in the BB-TB and TB-TT chromosomes seemed to suggest that there may be some compensatory mechanism against the inhibitory effect of BUdR on the NOR activity. INTRODUCTION The thymidine analog, 5-bromo-2’-deoxyuridine (BUdR), seems to have diverse effects on the various differentiating systems. BUdR suppresses the synthesis of a number of proteins particularly those which characterize a given differentiated cells, without greatly influencing over-all cellular vitality [1-4]. Some recent studies have suggested that an alteration in the activity of nucleolus organizer regions (NORs) was induced by BUdR incorporation. NORs where the rRNA genes are active can be specifically stained by the Ag—AS method [5]. Lau and Arrighi [6] reported that the bifilarly BUdR substituted chromatids of human chromosomes showed little or no silver deposit while unifilarly substituted chromatids exhibited normal amounts of silver. Such a lateral asym- metry in NOR silver staining was also observed by Strobel et al. [7] on human lymphocytes and Vogel et al. [8] on both human lymphocytes and Chinese hamster strain cells. The present study was conducted to investigate the phenomenon of NOR lateral asymmetry in detail using a mouse primary culture. Accepted May 22, 1984 Received April 5, 1984 MATERIAL AND METHODS Lung cells of a primary culture from a ddy male mouse were incubated in the medium con- taining various concentrations of BUdR for one and two rounds of replication as well as during last but one S phase to obtain chromosomes with BI-TB, BB-TB and TB-TT chromatid constitution, respectively. Colchicine-arrested metaphases were treated with 0.075 M KCl, fixed in methanol: acetic acid (3: 1) and air-dried. Silver staining was carried out according to the 1-step method of Howell and Black [9]. NOR- lateral asymmetry (NLA) observed was classified into the following three grades; 3+-, the complete absence of silver grain on one sister NOR, 2-+-, the difference in size between two silver dots of sister NORs is roughly more than twofold, and 1+, the difference is less than twofold. Chromo- somes with ambiguous silver positive NORs, i.e., those with a large silver mass, or those sharing silver-stained material, were separately scored as “‘unclassifiable’’. To confirm the inhibitory effect of BUdR- substitution on NOR activity, another set of BUdR-substituted chromosome preparations was first subjected to the 4Na~-EDTA Giemsa method for Giemsa SCD [10] and photographed. The ~~] S. TAKAYAMA AND K. MATSUMOTO 0 * : t * | o*, ) a a a ee e f Fic. 1. Partial metaphase spreads of mouse cells incubated with 20 »M BUGR in the three dif- ferent ways. a: TB—BT chromosomes stained with the 4Na-EDTA Giemsa method, showing B-bands. b: The same chromosomes as in a, restained with the silver method, some of them showing clear silver dots. Arrowhead and small arrows indicate 2+ and 1+ grade of NAL, respectively. c: TB-ITT chromosomes showing B-dark type Giemsa SCD. d: The same chromosomes as in c, silver stained, large arrows indicating 3+ NLA. e: BB-TB chromo- somes showing B-dark type Giemsa SCD. f: The same chromosomes as in e, silver stained, NLA seen here all belonging to 3+ grade. Bar represents 10 um. NOR Lateral Asymmetry Induced by BUdR 721 slides were then completely destained by immersing in methanol-acetic acid (3: 1) and treated with the silver staining method. The resulting silver dots were located on the same chromosomes as photographed after Giemsa SCD. The Giemsa staining used has been known to result in the dark staining of BUdR-substituted chromosomes or chromosome regions compared to those less substituted or unsubstituted ones. RESULTS As seen in Figure 1, BUdR-substituted and silver stained metaphase chromosomes revealed obvious asymmetry in the NOR dots between their sister chromatids. Three grades of NLA images described in Material and Methods with symbols of 3+, 2+ and 1+ are exemplified here and indicated with arrows, arrowheads and small arrows, respectively. Since there was the possibility that the Giemsa SCD procedure might alter the results of the silver staining, scoring of NLA was first carried out on the slides which had been subjected to the silver staining only. The results obtained were summarized in Table 1 and clearly suggested that BUdR-substitution led to induce NLA. TABLE 1. However, the direct evidence for this was obtained by comparing the Giemsa SCD images with the distribution of asymmetric silver dots in the same metaphase chromosomes. The TB-BT chromosomes shown in Figure la exhibited Giemsa positive bands which we had called B-band [11], and some of the chromosomes revealed obviously asymmetric silver dots (Fig. 1b). Differentially BUdR substituted TB-TT chromo- somes showed clear B-dark type of Giemsa SCD (Fig. lc), in which the TB-chromatids stained dark and the TT-ones light. As seen in Figure 1d, silver stained TB-TT chromosomes revealed marked NLA in which no silver dot or smaller one was usually located in the BUdR-substituted (TB) chromatid. The Giemsa staining of BB~TB chromosomes also resulted in B-dark SCD (Fig. le). The silver staining of them showed an_ increased number of asymmetric dots, and the absence of dot or the presence of less conspicuous dot was usually restricted to the BB-chromatid and not to the TB-one (Fig. If). These results are summarized in Table 2. In 30 cells substituted with BUdR to have TB-TT chromosomes, 92 chromosomes were found to have asymmetric silver dots. In this case 82 Frequency of NLA in chromosomes substituted with different concentrations of BUdR in three different ways, and in unsubstituted controls NOR-asymmetry NOR- Unclassi- Chromosomes Chromosomes Number symmetry _fiable with silver with dot of cells 3+ 2+ 1+ = Total (%)* dot per cell scored Control 0 Las 14 ( 7.8) 166 8 188 3.8 50 BT-TB 20uM S120) - 63 88 (39.5) 135 45 268 5.4 50 50 34 41 30 = 105 (53.6) 91 20 238 4.8 45 100 30 29 65 #146 (63.2) 85 39 270 5.4 50 TB-TT 20uM Ss de Se) | 2 Oo) 53 37 349 4.3 81 50 144 16 ili 171 (86.8) 26 8 205 4.1 50 100 68 23 16 107 (94.7) 6 15 128 3.9 50 BB-TB 20uM 189 61 6 256 (91.8) 23 14 293 3.5 84 50 147 9 2 158 (95.8) 7 10 175 3.5 50 100 150 16 4 170 (96.6) 6 16 192 3.2 60 * Percentage of total chromosomes with symmetric and asymmetric NORs. 722 S. TAKAYAMA AND K. MATSUMOTO TABLE 2. Frequency of NLA in 20 ~M BUdR-substituted chromosomes which have been stained first for the Giemsa SCD and then subjected to the silver staining Asymmetric dot: smaller dots or no dots on Symmietnie Uncles Ne rar ot able Dark chromatid Light chromatid Total scored TB-TT TB TT 82 (89.1)* 10 (10.9) 92 (100) oD 12 30 BB-TB BB TB 82 (84.5) IS CS.5)) 97 (100) 4 13 30 * Percentage of total chromosomes having smaller or no dots either in their dark or light chromatids. out of the 92 chromosomes (89.1%) were seen to have either no silver dot or less conspicuous dot on the dark (TB) chromatid, whereas only 10 chromosomes (10.9%) revealed such a minor dot on their light (TT) chromatid. Analysis in other 30 cells containing BB-TB chromosomes showed similar results. DISCUSSION In the study of satellite association frequency in human lymphocyte cultures, Sigmund et al. [12] incubated human lymphocytes with BUdR (10 wg/ml) for 2 cycles and found that BUdR- labeled acrocentric chromosomes exhibited NOR lateral asymmetry. Compared to these data from human lymphocytes, the frequency of NLA obtained in our study using mouse cells was extremely high, reaching more than a ten-fold increase over control level. It is interesting to note that such a high rate of NLA induction was obtained in the mouse cells using even a lower concentration of BUdR than that used in the human materials. This may suggest that the BUdR effect to induce NLA varies considerably according to materials to be examined. The good coincidence between the scoring of NLA from the preparations subjected to the silver staining alone (Table 1) and that from the preparations stained with silver after Giemsa SCD (Table 2) indicates that the prior Giemsa SCD procedure does not exert any significant effect on the result of subsequent silver staining. It has been reported that in man-mouse hybrid cell lines, only those NORs are silver-stainable in mitotic chromosomes that were actively trans- cribing during the preceding interphase [13, 14]. The BI-TB chromosomes showed an obvious increase of NLA over control level, and the extent of NLA observed in the TB-TT chromosomes appeared almost identical to that seen in the BB-TB chromosomes not only quantitatively but also qualitatively. Therefore, the inhibitory effect of BUdR on the silver staining does not seem to be simply due to the reduction of silver stainability of BUdR-substituted chromatin, but seems to be in some ways involved in the reduction of NOR activity. It is well accepted that in eukaryotes genetic codes are restricted to only one strand of a DNA duplex and that one chromatid of a metaphase chromosome consists of a single DNA duplex. This must be applicable to the situation of genes involving the NOR activity. Therefore, if BUdR-substitution can reduce the NOR activity, all the BI-TB chromosomes are theoretically expected to exhibit NLA, and the BUdR effect should appear much more severely in the BB-TB chromosomes compared to the TB-TT ones. However, the present results were not at all reconcilable with such simple expectations, but seem to indicate that there may be some com- pensatory mechanisms to act in such a way that they make the NOR activity in the TB-chroamtid of BB-TB chromosome as high as that in the TT of TB-TT. Although it seems at least possible to say that the inhibition effect is a direct result of BUdR incorporation into DNA, the mechanism underlying the NLA induction seems highly complicated and remains to be solved with some other suitable approaches. NOR Lateral Asymmetry Induced by BUdR 723 REFERENCES Holtzer, H. and Abbott, J. (1968) Oscilation of the chondriogenic phenotype in vitro. In “The Stability of the Differentiated State”. Ed. by H. Ursprung, Springer-Verlag, New York, pp. 1-16. Stellwagen, R.H. and Tomkins,G.M. (1971) Preferential inhibition by 5-bromodeoxyuridine of the synthesis of tyrosine aminotransferase in hepatoma cell cultures. J. Mol. Biol., 56: 167-182. Weintraub, H., Campbell, G. L-M. and Holtzer, H. (1972) Identification of a developmental program using bromodeoxyuridine. J. Mol. Biol., 70: 337- 350. Walther, B.T., Pictet,R.L, David, J.D. and Rutter, W. (1974) On the mechanism of 5- bromodeoxyuridine inhibition of exocrine pancreas differentiation. J. Biol. Chem., 249: 1953-1964. Goodpasture, C. and Bloom, S.E. (1975) Vis- ualization of nucleolar regions in mammalian chromosomes using silver staining. Chromosoma, 53: 37-S0. Lau, Y-F. and Arrighi, F. E. (1977) Comparative studies of N-banding and silver staining of NORs in human chromosomes. In “Joint Seminar and Workshop — Aspects of the Chromosome Or- ganization and Function”. Ed. by M.E. Drets, M. Brum-Zirrukka and G.A. Folle, Latin American Congress of Genetics, Montevideo, Uruguay, pp. 49-53. Strobel, R. J., Pathak, S. and Hsu, T.C. (1981) NOR lateral asymmetry and its effect on satellite association in BUdR-labeled human lymphocyte cultures. Hum. Genet., 59: 259-262. Vogel, W, Schempp, W. and Puel, V. (1978) Silver-staining specificity in metaphases after incorporation of 5-bromodeoxyuridine (BUdR). Hum. Genet., 40: 199-203. Howell, W.M. and Black, D. A. (1980) Con- trolled silver-staining of nucleolus organizer regions with a_ protective colloidal developer: a l-step method. Experientia, 36: 1014-1015. Takayama, S. and Tachibana, K. (1980) Two opposite types of sister chromatid differential staining in BUdR-substituted chromosomes using tetrasodium salt of EDTA. Exp. Cell Res., 126: 498—SO1. Takayama, S. and Matsumoto, K. (1982) G- band-like structures and centromeric asymmetry in the BrdU containing mouse chromosomes. Chromosoma, 85: 583-590. Sigmund, J., Schwarzacher, H. G. and Mikelsaar, A-V. (1979) Satellite association frequency and number of nucleoli depend on cell cycle duration and NOR-activity. Hum. Genet., 50: 81-91. Miller, D. A., Dev, V.G, Tantravahi, R. and Miller, O. J. (1976) Suppression of human nucleolus organizer activity in mouse-human somatic hybrid cells. 243. Miller, O.J., Miller, D. A., Dev, V.G., Tantravahi, R. and Croce, C.M. (1976) Expression of human and suppression of mouse nucleolus acti- vity in mouse-human somatic cell hybrids. Proc. Natl. Acad. Sci. USA, 73: 4531-4535. Exp. Cell Res., 101: 235- ZOOLOGICAL SCIENCE 1: 725-736 (1984) Effect of Several Redox Dyes on the Respiration of Unfertilized Eggs of Sea Urchin IkUO YASUMASU!, AKIKO FUJIWARA!, AKIYA HINo!3 and KouicH! ASAMI? ‘Department of Biology, School of Education, Waseda University, Nishiwaseda, Shinjuku-ku, Tokyo 160, and *Division of Biology, National Institute of Radiological Sciences, 4—9-] Anagawa, Chiba-shi 283, Japan ABSTRACT — The respiration of unfertilized sea urchin eggs became as high in its rate as that of fertilized eggs by adding N, N, N’, N’-tetramethyl-p-phenylenediamine (TMPD), phenazine metho- sulfate (PMS) and dichlorophenol indophenol (DCPIP). The TMPD-stimulated respiration was inhibited by cyanide. The respiration, observed about 10 min after adding PMS, DCPIP or sperm, was inhibited by antimycin A (AMA) and cyanide. During 10 min from adding PMS, DCPIP or sperm, the respiration was insensitive to these inhibitors and became sensitive to them following another addition of TMPD. Cyanide and AMA prolonged the duration of cyanide insensitive respiration. These suggest that electron transport is inhibited in unfertilized eggs in two spans of mitochondrial respiratory chain between flavoprotein and cytochrome 6 and between cytochrome band cytochrome c, making the respiratory rate quite low; the inhibition in the former span is released by adding PMS, DCPIP and sperm, and the inhibition in the latter is gradually eliminated. Following the release of inhibition in the former span, a leakage of electron equivalent from a site between flavoprotein and cytochrome 5 probably occurs, resulting in cyanide insensitive respiration, which is turned off by the release of inhibition in the latter span. Fertilization also induced another cyanide- insensitive respiration accompanied by peroxide release. © 1984 Zoological Society of Japan INTRODUCTION Pioneer works by Warburg [1, 2] have demon- strated that the respiratory rate is suddenly enhanced in sea urchin eggs following fertilization. Thereafter, many investigators have studied on the metabolic systems in sea urchin eggs in order to understand the cellular mechanism for the fertilization-caused stimulation of respiration. However, the mechanism has not satisfactorily been understood yet. ADP control of respiration was once supposed to be a reason as to why the respiratory rate in unfertilized eggs was quite low. ATP utilization is expected to increase in its rate following fertilization, because of fertilization- Accepted June 5, 1984 Received March 19, 1984 3 Present address: Department of Biology, Nagoya University, Nagoya 464, Japan. induced activation of cell functions. However, the levels of ATP, ADP, AMP and inorganic phosphate hardly change before and after fertili- zation [3,4]. It has been demonstrated that mitochondria are in condensed conformation in unfertilized eggs and in orthodox conformation in fertilized eggs [5]. It is well known that orthodox conformation indicates an inactive state observed in the absence of ADP and condensed one shows an active state in its presence [6]. Thus, these observations suggest that the low respiratory rate in unfertilized eggs is not due to acceptor control of respiration. On the other hand, the respiratory rate of unfertilized eggs has been reported to be enhanced by several redox dyes, such as_ dimethyl p-phenylenediamine, pyocyanine, methylene blue, toluidin blue, menadione, naphthoquinone and so on [7-10]. These redox dyes may act as artificial 726 I. YASUMASU, A. FusIwARA, A. HINO AND K. ASAMI electron carriers, resulting in a bypass of electron transport in a span of mitochondrial respiratory chain, in which the electron transport is inhibited in unfertilized eggs. This assumption has been postulated by Runnstrom [11], prior to the works concerning acceptor control of respiration. Indeed, it has been reported that an inhibitor of cytochrome oxidase presents in unfertilized eggs and decreases in its level following fertilization fi}. As has been postulated by Runnstrom [11], these findings lead us to an assumption that the low rate of respiration in unfertilized eggs of sea urchin is ascribed to the inhibition of electron transport in mitochondrial respiratory chain and fertilization- induced release of the inhibition enhances the respiratory rate. However, the spans of mito- chondrial respiratory chain, in which electron transport is inhibited, are not clearly known at present, although the presence of a, b and c type of cytochromes is shown [13, 14]. Furthermore, it has been found that peroxide production occurs [15, 16] and the respiration is somewhat insensitive to cyanide during initial several minutes after fertilization [17]. Peroxide production and cyanide insensitive respiration do not seem to be due to electron transport through whole span of mitochondrial respiratory chain. Thus, we decided to investigate the effect of several redox dyes on the respiration of unfertilized eggs to find out the spans of mitochondrial respiratory chain, where electron transport is inhibited. Insemination in the presence of these redox dyes was also performed to find out possible respiratory systems other than mitochondrial one, which might be stimulated by fertilization. MATERIALS AND METHODS The eggs and sperm of the sea urchin, Hemicen- trotus pulcherrimus, were obtained by an injection of 0.5 M KCI into the body cavity. The eggs were washed three times with artificial sea water (Jamalin Lab., Osaka) and 5% egg suspension (V/V) was made. The dry sperm was stored in an ice bath until use. Estimation of respiratory rate: Three ml of 5% egg suspension was transferred to a closed vessel, to which an oxygen electrode (Yellow Springs Co.) was attached. The oxygen concentration in the egg suspension was monitored at 20°C by a recorder (QPD 36, Hitachi Co., Tokyo). Through a small hole in the stopper of the closed vessel, an addition of 5 wl dry sperm was performed to fertilize the eggs in 3 ml of the suspension, and 3-30 yl of solutions of redox dyes and inhibitors were also added, respectively. Fertilization rate was checked after the estimation of the respiratory rate had been finished. Unless fertilization rate was higher than 90%, the data obtained were omitted. Phenazine methosulfate (PMS), dichlorophenol indophenol (DCPIP), menadione and antimycin A (AMA) were dissolved in 95% ethanol. Meth- ylene blue, NaCN and N, N, N’, N’-tetramethyl- p-phenylenediamine (TMPD) were dissolved in distilled water. The concentrations of these substances are shown in the figure legends. Solvents used to make the solutions of these redox dyes and inhibitors did not exert any effect on the respiratory rate of the eggs. Estimation of peroxide: Peroxide produced by the eggs was estimated by luminescence photometer (Luminometer 1250, LKB), equipped with a recorder (QPD 36, Hitachi Co., Tokyo), in 3 ml of the egg suspension containing 10 g/ml of lumi- nol and 5 pg/ml of peroxidase. Luminescence by the oxidation of luminol with peroxidase reaction depending on peroxide produced in the eggs was estimated, and the amount of peroxide was obtained using H,O, as the standard. Chemicals: PMS, DCPIP, TMPD and meth- ylene blue were the products of Kanto Chem. Co., Tokyo. AMA and peroxidase were obtained from Sigma Chemical Co., Mo. Luminol and menadione were from Tokyo Kasei Co., Tokyo. All other chemicals were of analytical grade. RESULTS Figure 1 shows the change in oxygen con- sumption of the eggs. In unfertilized eggs, the respiratory rate was quite low. An addition of dry sperm to the suspension of unfertilized eggs caused a sudden increase in the respiratory rate with about 30sec delay from the insemination. The respiratory rate became maximum within Respiration of Sea Urchin Eggs 9.8 (10.6: 2.1) 100 nmole O 186 | 2 /1 o°eggs (179 +11.2) <-> min 60.0 n> 526-4 a) Soleg ei aoe 60.9. --.+ oe sP (65,429.2) 30, 492 GNC i as Awe Tngligisiellen oll? oe : TMPD| CN AOA RTE (52.1+4.2) 7 Sein a AM AMA TMPD 1 o.2> | 8.2 Fic. 1. Change in the respiratory rate of sea urchin eggs following fertilization and sensitivities of respiration of the eggs to antimycin A and cyanide. Tracing records of change in oxygen concentration in the egg suspension, obtained by an oxygen electrode, areshown. The values in the figure indicate the respiratory rates expressed as nmole O,/min/10° eggs. In parentheses, mean values + SEM (standard error of mean) obtained on 10 experiments are shown. Arrows show the times of additions of sperm (sp), antimycin A (AMA: 50 7M), cyanide (CN~: 4 mM) and tetramethy|-p- phenylenediamine (TMPD: 80 M), respectively. The solid lines marked with A and B, show the tracing records obtained following addition of spermatozoa. Dotted lines indicate those obtained by adding AMA or cyanide. The additions of these inhibitors were performed 1, 5, 9 and 12 min after sperm addition. Steady state respiration, which is CN~ sensitive, was obtained about 10 min after fertilization. The tracing records marked with C and D show the effects of AMA and cyanide on the respiration of unfertilized eggs. 728 I. YAsuMAsu, A. FUJIWARA, A. HINO AND K. ASAMI 2 min after insemination, then gradually decreased until a constant level of the respiratory rate at about 10 min. The constant rate of respiration in fertilized eggs was markedly higher than that in unfertilized eggs. This profile of the change in the respiratory rate in Hemicentrotus eggs following fertilization is similar to that observed in the eggs of the other species [18, 19]. This sudden increase in the respiratory rate has been reported to be not due to the respiration of added sperm [20]. For the convenience, the sudden increase in the respiration is nominated as “burst of respiration” and the following a constant respiration as the “‘steady state’’ respiration. As shown in Figure 1, the respiration during the burst was somewhat insensitive to AMA and cyanide, but the steady state respiration was inhibited by these inhibitors. The sensitivity of respiration to AMA and cyanide increased gradually during the burst of respiration. By adding TMPD, the inhibition of respiration by AMA was reversed. The response of respiration in unfertilized eggs to these compounds was the same as that cf the steady state respiration in fertilized eggs. These indicate that the steady state respiration in fertilized eggs, as well as in unfertilized ones, results from electron transport through whole span of mito- chondrial respiratory chain. By adding TMPD to the suspension of unferti- lized eggs, the respiration was instantly enhanced to a level similar to that of the steady state re- spiration in fertilized eggs (Fig. 2). The TMPD- induced respiration was inhibited by cyanide but was, of course, insensitive to AMA. Even in the presence of AMA, TMPD induced the respiration in unfertilized eggs with a rate as high as in the steady state respiration of fertilized eggs (Fig. 1). Cyanide sensitivity of the TMPD-induced respiration in unfertilized eggs suggests that the electron equivalent accepted by cytochrome c by the aid of TMPD [21] is transported to molecular oxygen through a span of mitochondrial respiratory chain between cytochrome c and cytochrome a, a3. The rate of TMPD stimulated respiration in unfertilized eggs was similar to the rate of steady state respiration in fertilized eggs and TMPD enhanced only slightly the rate in fertilized eggs (Fig. 2). Thus, the capacity of electron transport TMPD A 9.8 J z CN B ~14.2 yTMPD ee | [0 nmole 0,/10°eggs pan min Fic. 2. The effect of TMPD on the respiration of unfertilized eggs. Tracing records of oxygen concentration in the suspension of unfertilized egg are shown in A, B, C, D and E. F shows the tracing record obtained on fertilized eggs 30 min after insemination. Values in the figure show the respiratory rates expressed as nmole O,/min/10° eggs. Arrow’ shows addition of TMPD, AMA (50 4M) and CN7 (5 mM), respectively. Concentrations of TMPD are 10 »M for A, 200M for B, 70 uM for C arid D, 100 uM for E and F. to molecular oxygen through a span between cytochrome c and cytochrome a, a; in unfertilized eggs is almost the same as in fertilized eggs. Figure 3 shows the effect of PMS and DCPIP, which are known to accept. electron equivalent from flavoprotein, on the respiration of unfertilized eggs. Following the addition of these compounds, the respiratory rate increased in a similar manner as Respiration of Sea Urchin Eggs 729 /10°eggs o— 2 min Fic. 3. Effect of PMS and DCPIP on the respiration of unfertilized eggs of sea urchin. Arrows show the times of additions of PMS, DCPIP, AMA (50 4M) and cyanide (5mM). The values shown are the rates of respiration expressed as nmole O,/min/ 10° eggs. Concentrations of PMS added to unfertilized egg suspension are 1.0 «»M for A, 5 uM for B and C, and 15 ~M for D and E. E shows tracing records in the egg suspension obtained 30 min after PMS addition. Concentrations of DCPIP were 100 #M for F, 300 »M for G and H, and 1.0mM forlandJ. J shows tracing records in the suspension of unfertilized eggs obtained 30 min after DCPIP addition. 730 I. YAsuMAsu, A. FusIWARA, A. HINO AND K. ASAMI observed following fertilization (Fig. 1). After the burst of respiration induced by these redox dyes, the steady state respiration, which was almost the same level as observed in fertilized eggs, occurred. The amount of molecular oxygen utilized during the burst was somewhat correlated to the concentrations of these redox dyes but the steady state respiration was almost similar in its rate irrespective of their concentrations. The same occurred in unfertilized eggs by adding methylene blue and menadione in place of PMS and DCPIP (data not shown). Menadione was unstable and oxidized in sea water at alkaline pH’s where the respiration of the eggs was estimated. During the burst induced by these redox dyes, the respiration, which was hardly inhibited by AMA and cyanide during initial period of the burst, became gradually sensitive to these inhibitors (Fig. 3) in a similar manner as observed during fertilization induced burst of respiration (Fig. 1). The steady state respiration, following the burst DCPIP TMPD 100 nmole O05 /10°eggs a 2 min induced by PMS and DCPIP, was also inhibited by AMA and cyanide, and AMA-caused inhibition of respiration was reversed by TMPD. These suggest that, in the case of the steady state re- Spiration, the electron equivalent accepted by PMS and DCPIP is transported to molecular oxygen through a span of respiratory chain between cytochrome 5 and cytochrome a, a;. PMS and DCPIP probably cause a bypass of electron transport in a span between flavoprotein and cytochrome b. The rate of steady state respiration induced by these compounds (Fig. 3) was as high as that of the steady state one in fertilized eggs (Fig. 1). An addition of TMPD at the initial period of the respiratory burst induced by PMS and DCPIP, as well as by sperm, made the respiration sensitivie to cyanide, as shown in Figure 4. PMS and DCPIP enhanced, even though slightly, the re- Spiratory rate of the TMPD-treated unfertilized eggs, but they failed to induce cyanide insensitive burst of respiration in the TMPD treated ones. Fic. 4. The effect of TMPD on the respiration just after adding PMS, DCPIP and sperm. Arrows show the times of addition of PMS (15 »M), DCPIP (300 1M), sperm, TMPD (70 »M) and cyanide (5 mM). expressed as nmole O,/min/10° eggs. The values in the figure indicate the respiratory rates Solid lines show the changes in the respiratory rates following addition of PMS, DCPIP and sperm into the unfertilized egg suspension. The tracing records obtained after adding TMPD are shown with dotted lines. A, B and C indicate effects of cyanide 5 min after addition of PMS, DCPIP and sperm, re- spectively. D, E and F show the effect of cyanide on the respiration of the eggs induced by adding TMPD 2 min after the addition of PMS, DCPIP and sperm, respectively. Respiration of Sea Urchin Eggs 731 Fia. 5. sp AMA ‘ | 100 nmole 0 /10° eggs Effects of cyanide and AMA added just after stimulation of the eggs by PMS, DCPIP and sperm on the respiration of the eggs. Arrows show the times of additions of PMS (15 7M), DCPIP (300 M), sperm (sp), CN~ (5 mM) and AMA (50M). The values in the figure indicate the respiratory rates expressed as nmole O,/10° eggs. Solid lines show the oxygen decrease due to the respiration of the eggs stimulated by PMS, DCPIP and sperm, respectively. The effects of cyanide added 2 min and 10 min after the addition of PMS, DCPIP and sperm, on the respiration in the eggs kept in the absence and presence of AMA are shown with dotted lines, respectively. By the addition of AMA or cyanide during the burst, the duration, where cyanide insensitive respiration occurs, was prolonged, as shown in Figure 5. The same occurred when AMA or cyanide was added during the fertilization induced burst of réspiration. Inhibition by AMA of electron transport in a span between cytochrome b and cytochrome c elongated the duration where cyanide-insensitive respiration occurred, and the bypass of electron transport caused by TMPD at this span turned off cyanide insensitive respiration. Cyanide, which inhibits electron transport in a span between cytochrome a, a; and molecular oxygen, also prolonged the duration of the burst, probably resulting from a limitation of electron transport by cyanide in a span between cytochrome 6 and cytochrome c, practically in a similar manner as in the case of the inhibition by AMA. TMPD failed to turn off cyanide insensitive respiration when cyanide was added just after and before TMPD addition, as also shown in Figure 6. The insemination, performed in the presence of TMPD, PMS or DCPIP, caused fertilization membrane elevation in the eggs. In the presence of these redox dyes, the burst of respiration, also occurred following fertilization, though it was smaller than in the absence of these redox dyes (Fig. 7). In the eggs, which had been kept in the presence of these redox dyes for several minutes, cyanide-sensitive steady state respiration was observed (Figs. 2 and 3), and fertilization of thus treated eggs induced another burst of respiration, 732 I. YAsuMASU, A. FusrwarRA, A. HINO AND K. ASAMI CN 489 100 nmole O02 /10°eggs = 2 min Fic. 6. Effect of TMPD on the cyanide-insensitive respiration in unfetilized eggs obtained after adding PMS. Arrows show the times of additions of TMPD (70 4M), PMS (15 4M), and cyanide (5S mM). Additions of cyanide were performed at various times before and after TMPD addition, respectively. which was smaller than in their absence (Fig. 7). This burst of respiration was not inhibited by AMA and cyanide (data not shown). Figure 8 shows the rate of peroxide production caused by insemination. By adding PMS or DCPIP, very low rate of peroxide production, if any, was observed in unfertilized eggs. TMPD failed to cause peroxide production. Only by adding spermatozoa, peroxide production was evidently observed during the period where cyanide insenitive respiration occurred. Even in the presence of these redox dyes, the rate of peroxide production by adding spermatozoa was almost the same as observed in the absence of these redox dyes. An addition of sperm to the suspension of the unfertilized eggs fixed with glutaraldehyde [20] did not cause any production of peroxide. This indicates that peroxide production does not occur in spermatozoa following the interaction between sperm and eggs, but in the eggs by fertilization. DISCUSSION In the present study, it was observed that the respiratory rate of unfertilized eggs was instantly enhanced by TMPD and the stimulated respiration was completely inhibited by cyanide. It has been demonstrated that TMPD accepts electron equi- valent from ascorbate and cytochrome 5b and transport it to cytochrome c [21]. Thus, electron equivalent is probably transported from ascorbate or cytochrome b to cytochrome c by the aid of TMPD and finally to molecular oxygen in the reaction catalyzed by terminal oxidase, which is sensitive to cyanide. Cyanide-sensitive respiration induced by TMPD, therefore, probably results from electron transport to molecular oxygen through a span of mitochondrial respiratory chain between cytochrome c and cytochrome a, 43. The rate of TMPD-induced respiration of unferti- lized eggs was almost the same as the rate of steady state respiration of fertilized eggs. Hence, the capacity of electron transport through a span between cytochrome c and molecular oxygen in unfertilized eggs does not seem to be lower than in fertilized eggs. Low respiratory rate in unfertilized eggs is probably ascribed to the deficiency of electron transport in a span between flavoprotein and cytochrome c. The inhibitor of cytochrome oxidase found in unfertilized eggs [13] may not contribute to keeping the respiratory rate quite low in unfertilized eggs of the sea urchin used in the present study. PMS and DCPIP, known to accept electron Respiration of Sea Urchin Eggs 733 200 100 100 Respiratory rate (nmole O2/min/10° eggs) 0 100 8 | DCPIP 0 -10 0 TMPD (154M), PMS (154M) and DCPIP (300 uM). 10 20 Time after insemination (min) Fic. 7. The change in the respiratory rate caused by fertilization of the eggs kept in Changes in the respiratory rate of the eggs caused by fertilization in the presence of TMPD (A), PMS (B) and DCPIP (C). In Fig. 7A, the changes in the rate of respiration fol- lowing fertilization in the absence of these redox dyes is superimposed with thin line. equivalent from flavoprotein enhanced respiratory rate of unfertilized eggs with a burst of respiration. The profile of change in the respiratory rate ob- served following the addition of these redox dyes was similar to that observed following insemi- nation. The steady state respiration in unfertilized eggs, following the respiratory burst induced by these redox dyes, was inhibited by AMA and cyanide, as well as the steady state respiration of fertilized eggs. In the steady state respiration, electron equivalent accepted by PMS and DCPIP from flavoprotein is probably transported to mo- lecular oxygen through a span between cytochrome b and cytochrome a, a;. This AMA-sensitive steady state respiration induced by PMS or DCPIP was as high in its rate as that of fertilized eggs. Thus, it seems probable that electron transport in unfertilized eggs is inhibited between flavoproteins and cytochrome b, and that PMS or DCPIP establishes a bypass skipping the inhibited site. Insemination probably releases the inhibition of electron transport in this span of mitochondrial respiratory chain, resulting in an elevation of respiratory rate. 734 I. YAsumasu, A. FustwaraA, A. HINO AND K. ASAMI 20 10 20 10 3 20 10} DCPIP 30 20 Peroxide production (nmole H,O,equ/min/10 eggs) -10 0) ll | A 10 20 Time after adding sperm (min) Fic. 8. Peroxide production foltowing fertilization. Peroxide released from the eggs was estimated by luminesence caused by oxidation of luminol by peroxidase which was added in the surrounding sea water. Arrow shows the times of ad- ditions of TMPD (15 uM), PMS (15 uM), DCPIP (300 »M) and sperm (sp). On the other hand, the initial burst of respiration induced by PMS and DCPIP, as well as by ferti- lization, was somewhat insensitive to AMA and cyanide. The sensitivity of the respiration to AMA and cyanide gradually increases during the respiratory burst. Furthermore, the addition of AMA or cyanide soon after adding PMS, DCPIP or sperm elongated the duration of the respiratory burst. These suggest that respiratory burst occurs due to an inhibition of electron trans- port to molecular oxygen through a span between cytochrome b and cytochrome a, a3. Since no inhibition of electron transport occurs in a span between cytochrome c and cytochrome a, a; in unfertilized eggs, as described above, this inhibition is assumed to be only in the span between cyto- chrome b and cytochrome c. Indeed, the re- spiration during the burst induced by these redox dyes and sperm became sensitive to cyanide for a while after another addition of TMPD, which establishes a bypass of electron transport in a span between cytochrome b and cytochrome c Respiration of Sea Urchin Eggs 735 [21]. Thus, electron transport in unfertilized eggs is probably inhibited not only in a span between flavoprotein and cytochrome 6 but also in a span between cytochrome 6 and cytochrome c. It is also probable that the inhibition of electron transport in the latter span is gradually released following the elimination of the inhibition in the former span. The inhibition of electron trasnsport in the latter span probably causes, when the inhibition in the former span is released, a burst of respiration. The gradual increase in the sensitivity of the respiration to AMA and cyanide may occur during the burst keeping in pace with the release of the latter span. The same is pro- bably true in the case of addition of methylene blue, pyocyanine, naphthoquinone and so on, which have been demonstrated to _ elevate respiratory rate of unfertilized sea urchin eggs [7-10]. In isolated rat heart mitochondria, hydrogen peroxide is found to be produced in the presence of AMA [22, 23]. This indicates that an electron leakage in a span between flavoprotein and cytochrome 4 occurs in mammalian mitochondria, when electron transport is inhibited in a span between cytochrome 6 and cytochrome c. The situation in sea urchin eggs just after fertilization is very similar to that in mammalian mitochondria in the presence of AMA. The release of inhi- bition between flavoprotein and cytochrome b by the dyes or by fertilization probably allows electron flow to reach the second inhibited site between cytochrome 6b and c. The increased electron flow will cause a leak of electron around the second inhibited site. In sea urchin eggs, however, the leakage of electron equivalent in this span seems to be extraordinarily high in its rate during the burst. In the steady state respiration in unfertilized eggs stimulated by PMS and DCPIP or in fertilized eggs, cyanide insensitive respiration was too low to be detected as in mammalian mitochondria even in the presence of AMA, but was markedly high during the burst. Thus, it is probable that the defects in a span of mitochondrial respiratory chain between flavoprotein and cyto- chrome 6, which occur temporally in unfertilized eggs and in just fertilized eggs, allow the re- spiratory chain to leak an enoumous amount of electron equivalent, resulting in cyanide sensitive respiration, when the supply of electron equivalent increases in this span. The burst of respiration in the eggs stimulated by PMS, DCPIP or sperm became cyanide sensitive for a while after the TMPD addition, but an failure of turning off this cyanide-insensitive respiration occurred by adding cyanide just after or prior to TMPD addition. This indicates that elevated electron flow caused by TMPD for a considerable duration is indispensable for turning off the cyanide insensitive respiration. This also suggests a possibility that a forced increase in flow rate of electron transport by TMPD through the span, where the defect of electron transport occurred, might cure the defect itself. Also fol- lowing fertilization, this defect might be cured by increasing rate of electron flow through this span and in consequence cyanide insensitive respiration due to this defect would be turned off and .altered to the respiration resulting from electron transport through whole span of mito- chondrial respiratory chain. Fertilization probably induces cyanide insensitive respiration not only resulting from this leakage of electron equivalent but also from the other re- actions. Addition of sperm to unfertilized eggs incubated with these redox dyes for at least 10 min, when the burst of cyanide-insensitive respiration had been turned off and the respiration became sensitive to AMA and cyanide, resulted in another cyanide-insensitive burst of respiration, although it was smaller than that observed following normal fertilization. This indicates that the eggs contain another cyanide-insensitive respiratory system than the mitochondrial electron transport system. The production of peroxide occurred following fertilization at almost the same rate irrespective of the presence or absence of these redox dyes but was quite low in its rate following the addition of these redox dyes. In the present experiment, we probably estimated the peroxide released from the eggs to the surrounding medium, because the peroxidase added to estimate peroxide might not penetrate into the eggs. It is probable that hydrogen peroxide produced inside the eggs is hardly detectable, since most of the produced peroxide can be broken down by cytoplasmic 736 I. YAsuMASU, A. FustwaARA, A. HINO AND K. ASAMI enzymes such as catalase [9]. Thus, fertilization- Pubbl. Staz. Zool. Napoli, 28: 315-340. induced release of peroxide during the burst of 12 Magio, R. and Monroy, A. (1956) An inhibitor respiration is probably due to the other system(s) of cytochrome oxidase activity in the sea urchin : : : : egg. Nature (Lond.), 184: 68-69. than mitochondrial one. The peroxide releasing 13 Maggio, R. and’ Ghiretti: Mapaldi ae Mees) system(s) in the eggs is not known at present and The cytochrome system in mitochondria of now under investigation. unfertilized sea urchin eggs. Exp. Cell Res., 15: 95-102. 14 Okabayashi, K. and Nakano, E. (1983) The REFERENCES cytochrome system of sea urchin eggs and embryos. Arch. Biochem. Biophys., 225: 271-278. 15 Foerder, C. A. and Shapiro, B. M. (1977) Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. Proc. Natl. Acad. Sci. USA, 74: 4214-4218. 16 Foerder, C. A., Klebanoff, S.J. and Shapiro, B. M. (1978) Hydrogen peroxide production, chemilu- tae f hi 2 -E minescence, and the respiratory burst of fertiliza- noHoWine (eritlizalion tot sca gurchingceesie se. tion: Interrelated events in early sea urchin SoU SSS SESE development. Proc. Natl. Acad. Sci. USA, 75: 4 Yasumasu,I., Asami, K., Shoger, R. L. and 3183-3187. Fujiwara, A. (1973) Glycolysis of sea urchin eggs. Rate-limiting steps and activation at fertilization. Exp. Cell Res., 80: 361-371. 5 Innis, M. A., Beers, T. R. and Craig, S. P. (1976) Mitochondrial regulation in sea _ urchins. 1. Mitochondrial ultrastructure transformations and changes in the ADP: ATP ratio at fertilization. Exp. Cell Res., 98: 47-56. 6 Hackenblock, C. R. (1966) Ultrastructural basis for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural 1 Warburg, O. (1908) Beobachtungen iiber die Oxydationsprozesse im Seeigelei. Hoppe-Seyler’s Z. Physiol. Chem., 57: 1-16. 2 Warburg, O. (1910) Uber die Oxydationen in lebenden Zellen nach Versuchen am Seeigelei. Hoppe-Seyler’s Z. Phisiol. Chem., 66: 305-340. 3 Epel, D. (1969) Does ADP regulate respiration 17 Shimada, H., Terayama, H., Fujiwara, A. and Yasumasu, I. (1982) Méelittin, a component of bee venom, activates unfertilized sea urchin eggs. Develop. Growth Differ., 24: 7-16. 18 Ohnishi, T. and Sugiyama, M. (1963) Polaro- graphic studies of oxygen uptake of sea urchin egg. Embryologia, 8: 79-86. 19 Epel,D. (1965) Some aspects of metabolic control in the fertilization transition of sea urchin eggs. In “Control of Energy Metabolism’. Ed. by : 3 ‘ B. Chance, R. W. Estabrook and J. R. Williamson, change with changes in metabolic steady state : in isolated mitochondria. J. Cell Biol., 30: PERSIE EES ING WON hy BO 269-297. 20 Hino, A., Fujiwara, A. and Yasumasu, I. (1980) 7 Rumirm, . (932) Die Besinfussing der [biden of espaton in es urchin peat Atmung und Spaltung im Seeigelei durch Dimethyl- ; oc oiecicadiemin tad ledhedkinen, Brcto: I. Change in the respiratory rate of spermatozoa plasma, 15: 532-565 : in the presence of fixed eggs. Develop. Growth , ‘ Differ., 22: 421-428. 8.) -Runnsizém, J. (1935). On the infinence Of” 511) peet@ a PJandEmisten 1S 5m eee eee nem pyocyanine on the respiration of the sea urchin ege. Biol. Bull., 68: 327-334. electron acceptors for abbreviated phosphorylaing . 36 Nm electron transport: flavin-cytochrome c. In 9 Krahl, M.E. (1950) Metabolic activities and ee . es Z Method E . Vol. 10, Ed. b cleavage of eggs of the sea urchin, Arbacia ethod, in, Enzymology, . . y ; 2 R. W. Estabrook and M.E. Pullman, Academic bees A review, 1932-1949. Biol. Bull., Press, New York, pp. 33-37. | tes i vee. 22 Boveris, A. and Chance, B. (1973) The mito- 10 De Vincentis,M., Fucci, L. and Lancieri, M. ; : ; ; chondrial generation of hydrogen peroxide. (1970) Stato funzionale della catena_ respi- G . : E ; : eneral properties and effect of hyperbaric ratoria, prima e dopo la fecondazione, nelle oxygen. Biochem. J., 134: 707-716 uova di riccio di mare. Atti Soc. Peloritana Sci. ; wa Mischa é : ; 23 Cadenas, E. and Boveris, A. (1980) Enhancement Fis. Mat. Nat., 16: 237-255. ; ; of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mito- chondria. Biochem. J., 188: 31-37. | 11 Runnstr6m, J. (1956) Some considerations on metabolic changes occurring at fertilization and during early development of the sea urchin egg. ZOOLOGICAL SCIENCE 1: 737-742 (1984) Influence of Nerve Cord Removal on Pharynx Induction by Head Piece Transplanted in Postpharyngeal Body Region of the Planarian, Dugesia japonica japonica YOSHIKAZU KISHIDA and ETsuo AsaAI! Department of Biology, School of Education, Okayama University, Tsushima, Okayama 700, and ‘Department of Biology, Kanazawa Medical University, Uchinada-machi, Ishikawa 920-02, Japan ABSTRACT — The effect of nerve cord removal on the inductive formation of new pharynges in freshwater planarian, Dugesia japonica japonica, was studied. The head piece was previously trans- planted in the postpharyngeal region of the animals. Both left and right nerve cords of the host animals were extirpated in the region anterior to the given head piece at varying periods of time after transplantation. New pharynges were found to be induced in these animals and their number and developmental stages were examined for respective specimens. Plural induced pharynges were found only in specimens in which the nerve cords were extirpated on the second day after trans- plantation of the head piece. Some of these pharynges showed the incomplete rudimentary structure and their polarity was distorted. These results show that the nerve cords have inhibitory action for the apparition of the pharynx and that they dominate the pharyngeal polarity, probably through deter- © 1984 Zoological Society of Japan mining the body polarity. In addition, it was suggested that the nerve cords might secrete a substance which would be utilized as nutrient and promote the growth of the developing pharynx. INTRODUCTION Some evidences have been given by several authors that the presence of nerve cords has an important meaning in regeneration of the body fragments of the freshwater planarians. If the body fragments are made without the nerve cords, they can not pursue a course of normal regenera- tion. For example, the fragment taken from the lateral body margin containing no nerve cords abnormally regenerates to form ‘head-hump syndrome’, termed by Sperry ef al. [1, 2], and the prepharyngeal body fragment from which the nerve cords were extirpated develops into bipolar head regenerate [3]. Further, our previous ex- periments that dealt with the removal and/or transplantation of nerve cords and demecolchine treatment have showed that the nerve cord is a significant factor in determining the regeneration Accepted June 5, 1984 Received February 15, 1984 blastema to form either head or tail [4—6]. On the other hand, inductive effect of the nerv- ous tissue has been shown for some other organs. Lender [7-9] ascertained that the neurosecretory substances induced the formation of the eyes in Polycelis nigra. Schilt [10] found in Dugesia lugubris that the repeated incisions of nerve cords could evoke the formation of additional pharynx. We obtained also the similar results in Dugesia japonica japonica. However, what role nerve cords actually play in the formation of the pharynx still remains to be known. Present study was concerned with this question. MATERIALS AND METHODS The sexual forms of Dugesia japonica japonica collected from a stream in the suburbs of Kanazawa City were exclusively used as materials for this study. Animals of about 20mm in length were kept under starvation for 10 days prior to experi- ments. The worms were anesthetized in an 738 Y. KISHIDA AND E. ASAI aqueous solution of chloretone and placed on a piece of wet filter paper. The graft was obtained as_a rectangular piece by cutting the head region of the animal. The host animal was operated to make a hole in the postpharyngeal region of which the size is about the same with a grafting piece. Thus a grafting piece was transplanted into this hole. Some operated animals were reared for control, and some were given for further operation. On the 2nd, 4th, 6th, 8th and 10th days after the transplantation, we chosed such animals that showed complete fusion between host and graft tissues. Each of them was placed on a wet filter paper put on small ice-block, but with its ventral surface upwards. Both left and right nerve cords of the set animals were extirpated through the region anterior to the graft. The operated-on animals were reared in decalcified and aerated tap water at 18°C. The worms were fixed in Helly’s solution on the 14th day after the transplantation. The serial Paraplast sections of 54m were made, stained by Mayer’s haematoxylin and eosin B. Histology of the induced pharynges was re- ferred to the following three stages according to the previous criteria [11]. Stage I shows a primitive pharyngeal cavity lined with the single layered epithelial cells and only a few basophilic mesenchymal cells. Stage II has somewhat enlarged pharyngeal cavity having the mass of basophilic mesenchymal cells located in one side but the tiny pharyngeal lumen does not pierce throughout the cavity yet. Stage III shows small in size but alomost com- plete pharyngeal structures. TABLE 1. RESULTS The results were summarized in Table 1 in which the number and the developmental stages of the induced pharynges were given according to the difference in time until the nerve cord removal after the head piece transplantation. In the control animals all 11 cases showed only one induced pharynx at the region posterior to the graft, and 9 cases (82%) had it at the region anterior to it. In the experimental worms, pharyngeal induc- tion was also recognized, regardless of the differ- ence in time when the nerve cords were extirpated. But, it should be noted that occurrence of plural number of induced pharynges and the develop- mental degree of them showed some relation to the time of nerve cord removal. When the nerve cord removal was done on the 2nd day after head piece transplantation, 4 out of 14 cases gave rise to plural pharynges at both regions or either one, anterior or posterior to the graft. As the result, 39 pharynges were recognized in postpharyngeal region of the 14 operated animals. But when it was given on the 4th day or later after that, only one pharynx was induced in either region of almost all cases and no plural formation of pharynges occurred. As to the degree of histological differentiation of the induced pharynges, it could be pointed out that when the nerve cord removal was done in the more later days after the head piece transplanta- tion, the more numbers of the operated animals would show the advanced degree in pharyngeal Numbers and developmental stages of the induced pharynges in the postpharyngeal region deprived of nerve cords after transplantation of the head piece Number of Developmental stages Day* worms Total number operated I II Iil Control** 11 ) 3 L7/ 20 2 14 7 122 20 39 4 14 6 6 13 PS) 6 12 DD i 13 Dd 8 12 0 6 16 22 10 12 0 6 15 72) * Time in days until nerve cord removal after head piece transplantation. ** No nerve cords were extirpated. Nerve Cord and Pharynx Induction 739 Fic. 1. Photomicrographs showing the induced pharynges in Specimen-1 in the experimental group, in which the nerve cords were extirpated two days after transplantation. a, b, c, d, e, f, g, h: the sections through the induced pharynges, ph-1, ph-2, ph—3, ph-4, ph-5, ph—6, ph—7 and ph-8 (arrowheads) respectively. i: the section through the induced pharynges, ph-9 (upper arrowhead) and ph-10 (lower arrowhead). G: grafted region. Anterior is upward and right is dorsal. Magnification is all x 800. 740 Y. KiIsHIDA AND E. ASAI differentiation. Namely, the rudimentary pharyn- ges (Stage I) were noted only in the cases where the nerve cords were extirpated within 6 days after the head piece transplantation, while the well- developed pharynges (Stage IIJ) appeared more frequently in the cases where they were removed on the 8th and 10th days after it (Table 1). Since occurrence of the plural number of induced pharynges seems to give us some clue to see the role of the nerve cord for the inductive formation of pharynx, Table 2 was prepared in order to show the number and the developmental stages of induced pharynges in respective specimens belong- ing to the experimental group in which the nerve cord removal was done on the 2nd day after head piece transplantation. Specimen-1, -2, -4 and -6 had more than three pharynges induced by the head graft. Specimen-1 was the most conspicuous case of them. It had three pharynges in anterior region and seven in the posterior one (ph—1 through ph-10 in total). Most of them, however, did not well developed. Three of them (ph-3, —6 and ~7) showed the rudimentary structure at Stage I and 6 (ph-1, —2, -4, -5, -9 and —10) at Stage II. Only one (ph—8) developed enough to reach Stage Ill (Fig. 1). Two of the first 3 pharynges (ph-3 TABLE 2. Numbers and developmental stages of induced pharynges in the worms deprived of the nerve cords on the 2nd day after trans- plantation of the head piece Developmental stages Pen Total number I II Ill 1 3 6 1 10 2 0 2 2 4 3 0 0 2 Z 4 0 1 2 3 5 0 0 Z 2 6 0 1 2 3 7 1 0 1 2 8 0 0 2 2 9 0 1 1 2 10 0 0 2 Z 11 2 0 0 22 12 0 0 1 1 13 1 0 1 2 14 0 1 1 2) anterior GC) .. 6 1g 0 ? posterior Fic. 2. Schematic diagram to illustrate the situation of the induced pharynges in Specimen-1. G: graft, Numbers (1-10): numbers of the induced pharynges, ph—1 through ph-10. ) 7 =x 4 Qe s~€ & and —6) appeared at the dorsal side of the specimen, and one (ph—1) was found to be reversed its polarity. The situation is diagrammatically shown in Figure De DISCUSSION The present result showed that when the nerve cords were extirpated in the postpharyngeal region after transplanting the head piece, the plural num- ber of pharynges were induced in the host tissue. This may insist that the nerve cords of the host worm are not only unessential but may have inhibitory effect for the inductive formation of pharynges. This situation could be seen most clearly in such cases that the nerve cords were extirpated on the 2nd day after the transplantation of the head piece. That is, the determination of the pharynx formation by head graft in the host tissue seems to occur within 2 days after transplanta- tion of the head piece, though the nerve cord removal in less than two days after head piece transplantation was not carried out in the present study because of the high motility. This result coincides with that of the earlier worker’s experi- ment [12] in which the effect inducing the multi- Nerve Cord and Pharynx Induction 741 pharyngeal regenerates was noted only when the actinomycin D treatment was given to the body sections at the time during the first 48 hr. While, the present result has showed that most of these plural induced pharynges remained in lower degree of developmental stage and some of them were reversed their polarity. Our previous works have given some related phenomena that the isola- ted fragments deprived of nerve cords have the pharynx with distorted polarity [13], and that the polarity of the fragments with the reversely ori- ented nerve cord was determined in accordance with an original polarity of the nerve cord [4]. Thus it may be convincing that the nerve cord can determine the pharynx polarity through predomi- nation of the body polarity. Next, a tendency was noted that the longer the time until the nerve cord removal after the head piece transplantation passed, the more ad- vanced the degree of differentiation of the induced pharynges came to be. This result suggests that existence of the nerve cords promotes the growth of the induced pharynx. It may be true that the secreting substances from the nerve cords are utilized as nutrient for developing pharyngeal cells as in amphibian limb regeneration, in which it is well known that the neurosecretory substances from the cut ends of the nerves promote the blastema growth [14]. We may offer an interpretation as to the pharynx formation in Dugesia japonica japonica from above consideration. When the head piece is trans- planted into the postpharyngeal region, the new pharyngeal zone is induced by reorganization of the host tissue contacted with the graft [5, 15]. Induced pharyngeal zone is provided with the latent condition which should perhaps develop several pharynges. The formation of such multi- pharyngeal condition in postpharyngeal region was suggested from the experiment by Ansevin and Wimberly [12] in which the body pieces were treated with actinomycin D in Dugesia tigrina. In their study, it was assumed that the multiple pharynges came to be induced in random orienta- tion in the isolated postpharyngeal sections by pre-existing ‘pharynx-mRNA’ for coding proteins which transform undifferentiated neoblasts into the tissue of the pharynx. In that time, the nerve cords may act to inhibit the apparition of the latent pharyngeal buds with exception of one of normal oriented pharynx and regulate the tissue to force to construct ‘single pharynx system’. After that, the nerve cords exude the substance which is utilized as nutrient for the developing pharynx to promote their growth. Further investigation as to some substances related to each step of this process is required. REFERENCES 1 Sperry, P.J., Ansevin, K.D. and Tittle, F. K. (1973) Inductive role of the nerve cord in regeneration of isolated postpharyngeal body sections of Dugesia dorotocephala. J. Exp. Zool., 186: 159-174. 2 wsperry, P: J. and Ansevin, K. D. (1975) Determination in regenerating tissues of Dugesia dorotocephala: the influence of nerve cord grafts. J. Embryol. Exp. Morphol., 33: 85-93. 3 Kishida, Y. and Kurabuchi, S. (1978) The role of the nervous system in the planarian re- generation I. Regeneration of body fragments deprived of ventral nerve cords. Annot. Zool. Japon., 51: 90-99. 4 Kurabuchi, S. and Kishida, Y (1978) The role of the nervous system in the planarian re- generation II. Regeneration of the body fragments with the reversed nerve cord. Sci. Rep. Kanazawa Univ., 23: 57-63. 5 Kurabuchi, S. and Kishida, Y. (1979) The role of the nervous system in the planarian regeneration III. The influence of the head and the nerve cords on the blastema regeneration. Annot. Zool. Japon., 52: 179-187. 6 Kishida, Y. and Mimura, M. (1982) Effects of the nerve cords and demecolchine on the polarity of the freshwater planarian, Dugesia japonica japonica. Bull. Sch. Educ. Okayama _ Upniv., 61: 1-8. 7 Lender, Th. (1952) Le role inducteur du cerveau dans la régénération des yeux d’une planaire d’eau douce. Ann. Biol., 28: 191-198. 8 Lender, Th. (1955) Mise en évidence et propriétés de l’organisine de la régénération des yeux chez la planaire Polycelis nigra. Rev. Suiss. Zool., 62: 268-275. 9 Lender, Th. (1956) Recherches expérimentales sur la nature et les propriétés de |’inducteur de la régénération des Yeux de la planaire Polycelis nigra. J. Embryol. Exp. Morphol., 4: 196-216. 10 Schilt, J. (1972) Induction d’un pharynx sup- plémentaire par des incisions répétées chez la 742 japonica japonica. planaire Dugesia lugubris O. Schm. J. Embryol. Exp. Morphol., 27: 15-24. Asai, E. (1980) Influence of X-irradiation on pharyngeal induction in the planarian Dugesia Zool. Mag., 89: 220-226. Ansevin, K.D. and Wimberly, M.A. (1970) Modification of regeneration in Dugesia tigrina by actinomycin D. J. Exp. Zool., 172: 349-362. Kishida, Y. and Nakagiri, K. (1982) Effect of the nerve cord on the pharynx formation in freshwater planarian. Bull. Sch. Educ. Okayama 14 3) Y. KISHIDA AND E. ASAI Univ., 60: 19-31. Singer, M. and Ilan, J. (1977) Nerve-dependent regulation of absolute rate of protein synthesis in the newt limb regeneration: Measurement of methionine specific activity in peptidyle-t-RNA of the growing polypeptide chain. Devel. Biol., 57: 174-187. Sengel, P. (1953) Sur l’induction d’une zone pharyngienne chez la Planaire d’eau douce Dugesia lugubrisO.Schm. Arch. Anat. Microsc. Morphol. Exp., 42: 57-66. . ZOOLOGICAL SCIENCE 1: 743-750 (1984) © 1984 Zoological Society of Japan Comparison of Zoeal Development between the Two Carcinoplacid Crabs, Carcinoplax longimana (De Haan) and Eucrate crenata De Haan, Reared in Laboratory MASAYUKI TERADA Yokosuka High School, Osuka-cho, Shizuoka 437-13, Japan ABSTRACT — Larvae of the two crabs, Carcinoplax longimana (De Haan) and Eucrate crenata De Haan, were reared on Artemia nauplii in the laboratory from hatching throughout zoeal stages. The entire course of the zoeal development are described in this paper. The general features of C. /ongimana zoeae agree well with Kurata’s descriptions of zoeae of the same species in respective stages. Zoeae of C. longimana are different from those of E. crenata by the number of zoeal stages, types of the antenna II and telson, setation of the endopodial maxilla I, the presence or absence of chromatophores on the base of dorsal carapace spine and the lateral surfaces of the carapace, and mid-lateral projections on the abdominal segments IV and V. INTRODUCTION Although fifteen species of the carcinoplacid crabs have been recorded from Japan [1], few studies have been published on the larval develop- ment of these crabs. So far as the author is aware, only a single species, Carcinoplax longimana (as C. longimanus), has been reared in the labora- tory from zoeal stage I to megalopa stage [2]. The purpose of this paper is to give a detailed description of the zoeal development of the two carcinoplacids, C. longimana and Eucrate crenata. MATERIALS AND METHODS An ovigerous female of Carcinoplax longimana was obtained with trawl from the bottom con- sisting of mud, sand or broken shells, 150 meter deep, of the Sea of Enshunada, on July 10, 1983, and an ovigerous female of Fucrate crenata was caught with trawl from the muddy-sandy bottom, 30 meter deep, of the Mikawa Bay on August 27, 1983. The specimens were kept in a plastic vessel (70 x40 x 30cm) equipped with a bubbler until Accepted May 21, 1984 Received March 21, 1984 the larvae hatched out. Immediately after hatching, the stage I zoeae were isolated from the females and transferred to separate glass vessels (15cm in diameter, 10cm in depth), with filtered sea water about 7 cm deep, approxi- mately 200 larvae being placed in each vessel. Newly hatched Artemia nauplii were provided as food for the larvae and culture water was changed every day. Water temperature at 11 a.m. during the period of observations ranged from 25.0 to 29.3°C for C. longimana and from 24.0 to 27.5°C for E. crenata. The larvae were checked every day for moulting and death. scopic observations, at least eight larvae and five exuviae at each zoeal stage were preserved in 6% buffered sea water formalin. The ap- pendages were studied mostly at 200 magni- fication and details of setations at * 400 or more. Of the appendages, mandible and rudimentary maxilliped III were not examined. In this paper, C length of larvae denotes the length from the anterior edge of the eye to the middle of the posterior margin of the carapace, and R-D length that from the tip of the rostral spine to the tip of the dorsal carapace spine. For micro- OBSERVATIONS Zoeae of C. longimana hatched out on July 18 / and those of E. crenata on September 5. The former species has four zoeal stages and the latter five. Unfortunately, megalopae of the two species could not be obtained. In C. longimana, the minimum duration of each zoeal stage was five days for stage I, three days for II and five days for III, and in E. crenata, was three days for stages I and II, four days for III, and three days for IV. Gross morphology of zoeal stages Carcinoplax longimana Stage I (Fig. 1, AI, HI) — C length, 0.64 mm; R-D length, 1.80mm. Cephalothorax is spherical in shape in lateral view, with a long rostral and dorsal spines, and a pair of short lateral ones. Rostral spine is approximately equal in length to antenna II, but slightly shorter than dorsal spine. Chromatophores are absent on the base of dorsal carapace spine and the lateral surface of carapace. These characteristics remain un- altered throughout zoeal stages. Abdomen consists of five segments and telson. During the whole zoeal period, abdominal segment II has a mid-lateral projection pointing forwards on each side and segments IJI-V bear a small spine, curving backward on each side. Posterior margin of abdominal segments IIJI-V with a short spine extending a little over the following segment. Telson is bifurcated into two long processes to make a folk, each furca bearing one lateral and one dorsal spines, but no hair in all zoeal stages, and three pairs of spines on the inner margin. According to Terada’s criteria [3], the telson falls into type A240. Both pereiopods and pleopods have not been developed as yet. Stage II (Fig. 1, AII, HIT) — C length, 0.85 mm; R-D length, 2.58mm. Zoea is approximately the same in gross morphology as that of stage I. Postero-lateral spines on abdominal segments II-V are slightly longer than in the previous stage. Pereiopods have made their appearance as rudi- ments, although pleopods are still absent. Stage III (Fig. 1, AIII, MHIII)—C length, Fic. 1. antenna lI; D, maxillaI; E, maxilla II; panying scales represent 0.1 mm. Zoeal stages of Carcinoplax longimana (De Haan). F, maxilliped I; Numerals associated with respective alphabets indicate the ordinal numbers of zoeal stages. 744 M. TERADA 0.96 mm; R-D length, 3.08 mm. Postero-lateral spines of abdominal segments III-V have increased in length, reaching about half the length of the next abdominal segment. A pair of short hair-like processes has appeared between furcae of telson (setation 6+2). Abdominal segment VI is articu- lated from telson. Thus, abdomen is now com- posed of six segments and telson. Pereiopod buds are longer than those in the previous stage. Pleopods appear on the ventral surface of abdominal segments II-VI as blunt processes. Stage IV (Fig.1, AIV, HIV)—C length, 1.08 mm; R-D length, 3.50mm. _ Postero-lateral spines of abdominal segments III-V are longer than the next segment. Inner surface of telson bears five pairs of spines (setation 6+4). The number of abdominal segments is six-+telson as in stage III. Rudiments of pereiopods are con- siderably enlarged and protrude from the carapace. Pleopods are well developed, biramous with minute endopods and unarmed exopods in segments IJ-V. Those of segment VI are uniramous. Eucrate crenata Stage I (Fig. 2, AI; Fig. 3, HI) —C length, 0.58 mm; R-D length, 1.45mm. All zoeal stages of E. crenata and C. longimana are approximately similar in structure, except for size, chromatophore patterns and abdominal armature. Chromato- phores are present in the base of dorsal spine and on the lateral surface of carapace. Abdominal segments II and III are armed with a pair of mid- lateral projections. In segment II, projections are larger and directed anteriorly, while in segment III, they are very small and directed posteriorly. Telson is bifurcated into two long furcae, each having only one lateral spine. Therefore, it falls into type Ay, [3]. On inner margin of furcae, there are three pairs of equal-sized spines (setation 6+0). Postero-lateral margins of ab- dominal segments II-V bear a pair of short blunt processes. Stage II (Fig. 2, AII; Fig. 3, HII) — C length, 0.60mm; R-D length, 2.21 mm. Except for a A, total view (lateral); B, antenna I; C, G, maxilliped II; H, abdomen (dorsal). Accom- Zoeal Development of the Carcinoplacid Crabs 745 Fic. 2. Zoeal stages of Eucrate crenata De Haan. A, total view (lateral); B, antenna I; C, antenna II; D, maxillaI; E, maxillaII. Numerals associated with respective alphabets indicate the ordinal numbers of zoeal stages. Accompanying scales represent 0.1 mm. Zoeal Development of the Carcinoplacid Crabs . Hl Gia. Gl H| Fic. 3. Zoeal stages of Eucrate crenata De Haan. (dorsal). Accompanying scales represent 0.1 mm. slightly larger size of stage II zoeae, these are no differences in structure between stages I and II. Stage III (Fig. 2, AIII; Fig. 3, HIII) — C length, 0.75mm; R-D length, 2.88mm. Abdominal segments have increased to six. Pereiopod rudi- ments first appear within carapace. Stage IV (Fig. 2, AIV; Fig. 3, HIV) — C length, 1.00mm; R-D length, 3.88mm. Pereiopods are visible as small bulbous projections. Small rudiments of pleopods have appeared on the ventral side of all abdominal segments except for the first and the last. Stage V (Fig. 2, AV; Fig. 3, HV) —C length, 1.30mm; R-D length, 4.96 mm. Rudimentary pereiopods as well as pleopods are considerably enlarged in stage V than in stage IV. Cephalothoracic appendages of zoeae Carcinoplax longimana Antennal (Fig. 1, BI-BIV)— Antennal is conical in shape, non-segmented and terminates in a group of aesthetes and short setae. The total numbers of aesthetes and setae are four in stage I, five in stage II, six in stage III, and eight in stage IV. Basal portion of antenna I is thickened and a small knob-like endopod is visible in the last stage zoeae. Antenna Il (Fig. 1, CI-CIV)—Exopod of antenna II is about 2/3 as long as spinous process, 747 Hil F, maxilliped I; G, maxilliped If; H, abdomen Numerals associated with respective alphabets indicate the ordinal numbers of zoeal stages. and armed with two or more spines in its middle region in all zoeal stages. According to Aikawa’s criteria [4, 5], this antenna II seems to fall into type B;. Endopod appears as a minute bud in the basal region of spinous process in stage II, growing up to about 1/5 as long as spinous process in the next stage, and to 1/2 in the last stage. MaxillaI (Fig. 1, DI-DIV)—‘Setation of endopod is 4-2-1 (i.e. endopod carries four apical setae and two mid-inner setae on distal segment and one on proximal). The setation remains unaltered throughout four zoeal stages. Basal endite possesses five setae in stage I, eight in II, nine in III, and 13 or 14 in IV. Coxal setae in stages I to IV count seven, seven, nine or ten, and 12 or 13, respectively. A long plumose seta appears on the outer side of basal endite in stage II, and is retained through the subsequent stages. MaxillaIl (Fig. 1, EI-EIV) — Endopodous setation of maxilla II is 3: 2-3 (i.e. bilobed endopod with three terminal and two subterminal setae on outer lobe and three setae on inner). Basal endite is also bilobed with setation of 4-5 (i.e. basal endite has four setae on outer lobe and five on inner) in stages I and II, 5-5 in III, and 7-6 or 6-7 in IV. Setation of inner lobe of coxal endite is 1:3 (i.e. inner lobe with one apical and three lateral setae) in stages I and II, 1:4 in stage III, and 2:5 in the final stage. ee — 748 M. TERADA Scaphognathite in stage I bears four plumose setae along its anterior and lateral margins and a process somewhat like a plumose seta in ap- pearance extends posteriorly from its proximal end (setation being 4:1). In the following stages, plumose setae occur along the whole margin, the total numbers being 12, 20-22, and 29-33 in stages II to IV, respectively. Maxilliped I (Fig. 1, FI, FID) — Chromato- phores are distributed on protopod. Protopodial setation of maxilliped I is 3-3-2-2, setae being distributed along distal to proximo-medial margin. Setation of five-segmented endopod is 4- 1—2—1-2-3 (i.e. endopod bears four apical and one outer lateral setae on segment V, two on IV and II, one on III, and three on J) in stages I and II. But addition of a second, inner seta to segment V in stage III gives a setation of 4-2-2-1—2-3 which remains unchanged in the following zoeal stage. Exopod is composed of two segments, with four natatory setae at its end (cut short in the figure) in stage I, six in II, eight or nine in III, and nine or ten in IV. Maxilliped II (Fig. 1, GIV)—As in makxil- liped I, no structural changes occur throughout zoeal development in maxilliped II. Three- segmented endopod has setation of 3-3-1-1 (i.e. endopod with three terminal and three sub- terminal setae on distal segment, one seta on middle, and one on proximal) in all zoeal stages. Protopodial setae are arranged along distal to proximo-medial margin setation being 1-1-1-1. Chromatophores are associated with protopod. Exopod is two-segmented, with four, six, eight or nine and nine or ten terminal natatory setae in stages I to IV, respectively. Eucrate crenata Antenna I (Fig. 2, BI, BV) — This appendage is an unsegmented process bearing a group of aesthetes and simple setae at its tip, as that of C. longimana. The number of aesthetes and setae varies with advance of development: 2-2 (i.e. with two termina! aesthetes and two terminal setae), 4-1, 3-1, 6-1 and 8-1 in stages I-V, res- pectively. An unsegmented rudiment of endopod makes its appearance in the last stage as in the previous species. Antenna II (Fig. 2, CI-CV)—Exopod of antenna II with two similar accessorial hairs in the middle part, is nearly as long as spinous process and edged by two rows of spiny hairs from the middle to the tip. The antenna II falls into type A; [4,5]. Endopod makes its first appearance in stage III as minute bud arising from the basal region of spinous process, and grows up to about 1/4 the length of spinous process in stage IV, and 3/5 in stage V. Maxilla I (Fig. 2, DI-DV) — Lamellar maxilla I is small and composed of three parts, basal and coxal endites, and two-segmented endopod, with setation of 4-2-0. Basal endite possesses five setae in stage I, nine, 12 and 13 in subsequent stages. Setae on coxal endite are seven in number in the first three stages, nine in stage IV, and ten in stage V. One developed plumose seta appears on the outer edge of basal endite in stage II. This is retained throughout the subsequent stages. Maxilla II (Fig. 2, EI-EV) — Endopodous setation is 3:2-3 in all zoeal stages as in C. longimana. Endites are feebly bilobed, each lobe with several setae. Setation of basal endite in 4-5 in stages I and II, 5-5 in IH, 5-6 in IV, and 7-7 in V. Setation of inner lobe of coxal endite is 2:3 in the first three stages, 2:4 in stage IV and 3:5 in stage V. Large plate-like scaphognathite in stage I bears four plumose setae along anterior and lateral ‘margins, its proximal end is produced posteriorly to a long triangular extension. In the succeeding stages, the proximal tip of scaphognathite is rounded, bearing plumose setae along its entire margin, setation being 11-12, ca. 19, 26-30 and ca. 37. Maxilliped I (Fig. 3, FI, FIV) — Unsegmented protopod always possesses a 3-3-2+2 setation, like that found in C. longimana zoeae. Setation of endopod is 4-1—2-1-2-3 in the first three stages, but an additional seta appears on inner margin of distal segment and outer margin of segment III in stages IV and V (4-2-2-2-3). Exopod has four, six, eight, ten and 11 natatory setae at its tip in stages I to V, respectively. Maxilliped II (Fig. 3, GI, GIII) — Setation of endopod is 3-2-1-1 in stages I and II, whereas in the following three stages 3-3—-1-1 as in the previous Zoeal Development of the Carcinoplacid Crabs 749 species. Protopod has 1-1-1-1 setation in all stages. Exopod is slightly constricted in its middle with four long natatory plumose setae in stage I, six or seven in stage II, nine in stage III, ten or 11 in stage IV, and 13 in the last stage. DISCUSSION Kurata [2] described all larval stages of C. longimana on the basis of specimens reared in the laboratory. The present results are in good agreement with his findings with respect to the number of zoeal stages, the carapace spines, types of the antenna II and telson, mid-lateral projections of the abdomen, endopodial setations of the maxilla I, maxilla I], and maxilliped II. Kurata’s descriptions also accord well with the present findings that the endopodial rudiment of the antennal appears as a rod-like process in its basal region in the last stage; setation of the telson is 6+0 in the first two stages, but changes into 6+2 in III, and 6+4 in IV; the telson be- comes distinctly articulated in stage III; and rudiments of four pairs of pleopods on the abdominal segments II to V and a pair of uropods are visible as small buds in stage III, increasing in length in stage IV. However, Kurata made no mention on many other characters of zoeae. Feature common to C. longimana and E. crenata are as follows. The cephalothorax is spherical in shape, with single long rostral and dorsal spines and a pair of short lateral ones. The endopodous setation of the maxilla II is 3: 2-3. The protopodial setation of the maxil- lipeds I and II are 3-3-2-2 and 1-1-1-1, re- spectively. Chromatophores are distributed on the protopods of the two maxillipeds. The endopodial rudiment of the antenna I first appears in its basal region in the last stage. A plumose seta comes in sight on the outer side of the basal endite of the maxilla I in stage II, and persists throughout the subsequent stages. The pereiopods are visible in the antepenultimate stage as small rudiments, growing longer in the following two stages. Five pairs of the pleopods are first recogni- zable in the penultimate stage as small papillae, developing into rod-like limbs in the ultimate stage. The abdomen is composed of five segments and telson in stages I and II, but the sixth segment becomes separate from the telson in stage III. In stage I, the scaphognathite of the maxilla II bears four plumose setae along the anterior and lateral margins, its proximal end is produced posteriorly to a broad triangular extension. In the following Stages, the proximal tip of the scaphognathite is rounded, bearing many plumose setae along its entire margin. C. longimana zoeae are a little larger than stage-matched E. crenata larvae. However, 1) the number of zoeal stages is four in C. /ongimana and five in E. crenata. 2) Zoeae of C. longimana have chromatophores neither on the lateral surface of the carapace nor in the base of the dorsal carapace spine, while in E. crenata zoeae chromato- phores occur in both of the two locations. 3) In C. longimana, setation of endopod of the maxilla I is 4-2-1 throughout zoeal period, but in E. crenata it is 4-2-0. 4) In the former species, types of the antennalII and telson are B; and Ano, respectively, while in the latter A; and Ayy4o, respectively. 5) Zoeae of C. longimana bear a pair of the mid-lateral projections on the abdominal segments II to V, whereas those of E. crenata have similar projections only on segments II and III. Moreover, setation of endopod of the maxil- liped II is always 3-3-1-1 in C. longimana zoeae, while in E. crenata zoeae it is 3-2-1-1 in stages I and II. In zoeae of C. longimana, the inner lobe of the coxal endite of the maxilla II in stages I and II shows a setation 1: 3, while E. crenata zoeae of 2: 3 in the first three stages. ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. Kiyoshi Takewaki, Professor Emeritus of the University of Tokyo, for his valuable advice and kind looking over of the manuscript. Thanks are also due to the staff of the Thermal Excellent Uti- lization Research Center, Shizuoka Pref., for their valuable assistance in obtaining filtered sea water. REFERENCES 1 Miyake, S. (1983) Japanese Crustacean Decapods and Stomatopods in Color. II, Brachyura (Crabs). Hoikusha, Osaka, pp. 1-277. 2 Kurata, H. (1968) Carcinoplax longimanus (De 750 M. TERADA Haan) (Goneplacidae). Bull. Tokai Reg. Fish. Res. Lab., 56: 167-171. 3 Terada, M. (1980) Zoea larvae of four crabs in the subfamily Xanthinae. Zool. Mag., 89: 138-148. 4 Aikawa, H. (1929) On larval forms of some 5 Brachyura. Rec. Oceanogr. Works. Jap., 2: 17-55, pls. 2-5. Aikawa, H. (1933) On larval forms of some Brachyura. II; A note on indeterminable zoeas. Rec. Oceanogr. Works. Jap., 5: 124-254. ZOOLOGICAL SCIENCE 1: 751-754 (1984) © 1984 Zoological Society of Japan Termination of Adult Diapause by a Juvenile Hormone Analogue in the Bean Bug, Riptortus clavatus HIDEHARU NUMATA! and TosHITAKA HIDAKA Department of Zoology, Faculty of Science, Kyoto University, Sakyo, Kyoto 606, Japan ABSTRACT — Diapausing adults of both sexes of Riptortus clavatus Thunberg were treated topically with a juvenile hormone analogue (JHA). behavior in most of the males at the same dosage. in decapitated females at a dose of 6 ug/insect. JHA administered at 0.6 or 6 wg/insect induced yolk deposition in all females and complete ovarian development in some of them. It also evoked sexual JHA was even effective in inducing yolk deposition Therefore, we concluded that adult diapause in R. clavatus is due to the cessation of JH secretion, and JHA can terminate diapause without intervention of the protocerebral neurosecretion. INTRODUCTION Diapause in adult insects is characterized by reproductive arrest and its hormonal mechanism has been demonstrated in many cases [1, 2]. The application of exogenous juvenile hormone (JH) or its analogue (JHA) terminates adult diapause and induces yolk deposition or oviposition in many species [3-11]. A few authors have reported that the frequency of mating increases in diapaus- ing pairs when both sexes are treated with JH or JHA [3, 6, 8, 11]. The bean bug, Riptortus clavatus Thunberg (Heteroptera: Coreidae), exhibits a facultative adult diapause which is controlled by photoperiod [12]. The present paper describes the effect of a JHA on diapause termination in both sexes. MATERIALS AND METHODS Adults of R. clavatus were collected from legume fields in Kyoto. Their eggs were used for experi- ments performed at 25+1.5°C. Nymphs were reared by a method previously reported under a Accepted May 24, 1984 Received March 6, 1984 1 Present address: Department of Biology, Faculty of Science, Osaka City University, Sumiyoshi, Osaka 558, Japan. photoperiod with a 10-hr photophase and a 14-hr scotophase (10L-14D), which induced and main- tained diapause in this species, or a diapause- preventing photoperiod of 16L-8D [12]. Two adults of the same sex were reared in a 200 ml plastic cup with soybeans and water. JHA was obtained commercially as Manta® (Otsuka Chemicals Co.), an ethanol solution of methoprene (isopropyl [2E, 4E] 11-methoxy-3, 7, 11-trimethyl-2, 4-dodecadienoate). Seven days after adult energence, experimental groups of diapausing insects were treated topically on the abdomen with various doses of JHA in 5 ul ethanol. Control groups were either treated with ethanol or left untreated. Seven days after JHA treatment, the diapause status, whether maintained or terminated, was examined. Statistical analyses were carried out between each experimental group and the control group, ethanol-treated and untreated groups combined, by Fisher’s exact probability test. Females were dissected and the developmental stages of their ovaries were classified as follows: (—) no yolk was deposited in oocytes; (+-) light- blue colored yolk was deposited in oocytes; (++) mature eggs were ovulated into the oviduct. In- dividuals which had ovaries in stage (+) or (++) were considered to be diapause-terminated because light-blue yolk deposition never occurred in 752 H. NUMATA AND T. HIDAKA diapausing females [12]. JHA was also applied to some decapitated individuals in order to exclude the effects of the brain. In R. clavatus, spermatozoa were found in the testes of the diapausing males, which were dis- tinguished from the nondiapausing ones only by the lack of mating activity [12]. Assessment of diapause in males was therefore carried out upon induction of sexual behavior. Each experimental male was placed in an 80 ml plastic cup with a virgin nondiapausing female of the same age (14 days qld) and the behavior of the male was ob- served for 30 min. In the sexual behavior of this species, the male invariably quivers while facing the female before mounting it. Therefore, quiver- ing and copulation were employed as indices for mating activity in the male. RESULTS Females Diapause was maintained in the control groups. Although application of JHA at 0.06 wg/insect was ineffective for diapause termination, it was effective at 0.2 wg/insect. JHA induced yolk deposition in all females and induced complete ovarian development in some of them at 0.6 or 6 pg/insect (Table 1). Eight male/female pairs treated with JHA at 6 yg/insect were reared. .The females began to lay eggs 6-8 days after the JHA application. JHA was effective in inducing yolk deposition and exhibited a lethal effect at 6 yg/ insect in diapausing females which were decapi- TABLE 2. Effect of JHA on ovarian development in decapitated diapausing adults of Riptortus clavatus (10L-14D, 25°C) Stage of ovaries* Treatment No. No. ———_ treated died ~ 4: ir Ethanol 20 1 19 0O 0 6ngJHA 20 gt 4 FFF + Seven days after treatment. For —, +, +, see text. * 0.01 ‘Department of Parasitology, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-11, and *Department of Biology, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236, Japan ABSTRACT — Estrogen-independent persistent proliferation and cornification of the vaginal epi- thelium occurred in ovariectomized (OVX) adult mice which had received 7 daily injections of 20 ng 178-estradiol (E,) starting on the day of birth. The permanent vaginal changes were suppressed by neonatal injections of 400 IU retinol acetate (VA) given concurrently with E,. The suppression by VA was also found in adult OVX, neonatally E,-treated mice receiving VA injections for 7 days beginning on the 2nd, 4th or 6th postnatal day. The histological features and mitotic rate of the vaginal epithelium in neonatally E, plus VA-treated mice (E, plus VA mice) were similar to those © 1984 Zoological Society of Japan of E,-treated mice until 30 days of age. However, the mitotic rate in the vaginal epithelium of E, plus VA mice began to decrease after 60 days of age when clear cells appeared in the basal layer. By 120 days, the epithelium became atrophic, indicating a markedly lowered mitotic rate. In addition, mitotic rate of the vaginal epithelium was significantly lowered in adult OVX offspring of mothers given 3 daily injections of 4,000 IU VA from days 16 to 18 of gestation. INTRODUCTION Perinatal treatments of female mice with estrogen or androgen induce estrogen-independent persistent proliferation and cornification of the vaginal epithelium. These vaginal changes are irreversible, frequently resulting in precancerous or cancerous lesions at an advanced age [1-11]. The occurrence of permanent vaginal changes induced by neo- natally administered estrogen or androgen is prevented by injections of vitamin A given sim- ultaneously with the sex hormones [12-17]. The vaginal epithelium of mice treated neonatally with estrogen or androgen concurrently with vitamin A initially undergoes proliferation and cornification and then degenerates gradually, requring more than 3-4 months for the complete Accepted May 24, 1984 Received March 30, 1984 > To whom reprints should be requested. abolition of the persistent vaginal proliferation [14, 16]. In addition, the previous studies have shown that the occurrence of permanent vaginal changes is never blocked by vitamin A when given after the sex hormone treatment [13, 16]. However, it has been unelucidated whether the permanent vaginal changes of neonatally sex hormone-treated mice are prevented by vitamin A treatment starting at different perinatal periods. The present study, therefore, was aimed at determining the starting time for vitamin A administration to be effective in the prevention of estrogen-induced permanent proliferation of the vaginal epithelium and also at examining sequential changes in structure and mitotic rate of the vaginal epithelium of mice treated perinatally with estrogen and vitamin A. MATERIALS AND METHODS Female mice of the C57BL/Tw strain were 778 H. TACHIBANA, T. IGUCHI AND N. TAKASUGI used in the present experiments. A group of female mice were injected subcutaneously with a daily dose of 20 ug 178-estradiol (E,) dissolved in 0.02 ml sesame oil for 7 days starting within 24 hr after birth (0 day of age). Four groups of the neonatally E,-treated mice were given 7 daily injections of 400 IU retinol acetate (VA) beginning at 0, 1, 3 or 5 days of age. E, and VA used in the present experiments were purchased from Sigma Chemical Co., St. Louis, MO, USA. Two other groups of neonatally E,-treated mice received 7 or 14 daily injections of VA starting at 7 days of age. Another group of mice was given daily injections of 400 IU VA alone from day 0 of age. Eleven female mice delivered from the pregnant mothers which had received daily intraperitoneal injections of 4,000 IU VA for 3 days from day 16 to day 18 of gestation (day 0 of gestation is the day on which the vaginal plug was found) were given E, injections for 7 days from day 0 of age. Seven mice receiving injections of oil vehicle only for 7 postnatal days served as controls. All mice were ovariectomized at 30 days of age and killed at 120 days. For the study of sequential changes in mitotic rate of the vaginal epithelium in E, plus VA- treated mice, two groups of 30 mice each were given daily subcutaneous injections of either 20 wg E, dissolved in oil, or of 20 wg E, plus 400 IU VA in oil for 7 days starting on the day of birth, respectively. Seven daily injections of oil vehicle alone were given to 30 neonatal mice as controls. In 5 intact female mice, mitotic rate of the vaginal epithelium was also examined on the day of birth. The treated mice were separated into 18 groups consisting of 5 mice each and killed at 7, 10, 15, 30, 60 or 120 days of age. Animals scheduled for sacrifice at 60 and 120 days of age were Ovariectomized at 30 days of age. On the day following the first, third and fifth injections, 5 mice each of the E,-, E. plus VA- and oil-treated groups were sacrificed for examining mitotic rate of the vaginal epithelium. All animals were given a single subcutaneous injection of colchicine, dissolved in a 0.9% NaCl solution 5 hr before sacrifice. The colchicine dose was altered with the age of the animals at sacrifice, being 0.5-1 yg at 0-7 days, 50 wg/20g body weight at 10 and 15 days and 100 wg/20g body weight at 30, 60 and 120 days. Vaginae were fixed in Bouin’s solution, sectioned longitudinally in paraffin at 7 wm, and stained with Delafield’s hematoxylin and eosin. In 5 sections from each vagina, dividing cells at metaphase were counted in the cranial part (upper 3/5) and the caudal part (lower 2/5) of the vaginal epithelium. In addition to these experiments, mitotic figures were also counted in the middle part of the epithelium corresponding to the same part of the vagina in the preceding experiments [18]. Mitotic rate per hr (%) was estimated by counting the dividing cells in 1,000 basal cells. The thickness of 5 flat epithelial portions was measured in the middle part of vagina. The number of non-cornified layers of the mid-vaginal epithelium was also counted in 5 flat portions. RESULTS Effects of vitamin A given in different perinatal periods on the occurrence of permanent vaginal changes induced by neonatal estrogen treatment The vaginal epithelium of ovariectomized mice given neonatal injections of vitamin A acetate (VA) alone was atrophic, consisting of 2-3 layers of cells as was seen in the controls. In 7 of 8 ovariectomized mice injected neonatally with 178-estradiol (E,), the vagina had proliferated epithelium composed of 5-10 cell layers with superficial cornified (n=4) or parakeratotic layers (n=3). The vaginal epithelium of the remaining mouse was composed of 3-5 layers showing neither cornification nor parakeratosis. However, in 4 groups of neonatally E,-injected mice which had received 7 daily injections of VA given either starting at 0 day of age (E, plus 0-6 d VA) or starting at 1, 3 or 5 days of age (E, plus 1-7, 3-9, 5-11 d VA), the vagina had an atrophic epithelium consisting of 2-4 layers without superficial cornification except one mouse of E, plus 3-9 d VA group, which showed a proliferated epithelium consisting of 5—7 layers with cornifi- cation. Superficial parakeratosis was found in some mice of groups of E, plus 0-6 d VA (n=2), E, plus 1-7 d VA(n=1), E, plus 3-9 d VA(n=2) Suppression by VA of Permanent Vaginal Changes and E, plus 5-11 d VA (n=2). Mucification was also observed in the proximal part of the epithelium in one individual each of E, plus 1-7 d VA and E, plus 5-11 d VA groups. In neonatally E,-treated mice receiving daily VA injections from the 8th to the 14th postnatal day (E, plus 7-13 d VA), the vaginal epithelium was composed of 4-8 layers with superficial cornifi- cation. Four of 6 mice given 14 daily injections of VA starting on the day after neonatal E,-treatment (E, plus 7-20 d VA) had vaginae lined by 2-5 epithelial cell layers. The superficial epithelial layer was parakeratotic in one of the four animals. In the remaining two, the vaginal epithelium consisted of 8-12 layers with cornification. Eleven mice delivered from the mothers which had received 3 daily intraperitoneal injections of 4,000 IU VA were given 7 daily injections of E, starting at 0 day of age (Pre VA plus E,). Seven of them had their vaginae lined with the epithelium consisting of 2-5 layers without cornification. In the remaining four, the vaginal epithelium TABLE 1. 119 consisted of 5-11 layers. Superficial epithelial cornification was observed in 2 of the 4 animals. In one or 2 mice of each group of E, plus VA (from 0-6 d to 5-11 d), E, plus 7-20 d VA and Pre VA plus E,, clear cells were found in the basal layer of the cranial part of the epithelium, whereas the mice of the E, plus 7-13 d VA group showed no such clear cells (Table 1). In all mice having 4- to 7-layered vaginal epithelium without cornification, the vaginal stroma was often invaded by numerous lymphocytes. The superficial layer of the epithelium of these mice also showed an occasional infiltration of a large number of leucocytes. Mice possessing vaginae with less than 4 epithelial cell layers on the average accounted for 40-100% of all groups of E, plus VA-treated mice, being high in the groups of E, plus 0-6 d_ VA and E, plus 5-11 d_ VA, and low in the group of E, plus 7-13 d_ VA (Table 1). Mitotic rate in the vaginal epithelium was markedly higher in neonatally E,.-treated mice than that in oil controls. However, the rate decreased in mice when given VA _ injections Number of mice having a vaginal epithelium consisting of less than 4 cell layers and of those showing “‘clear cells” in the basal layer of the epithelium : No. of mice having No. of mice Group (postnatal days, Oday of birth) mice 168 than 4 epithelial showing | A Oil (0-6)* 7 7 (100) 0 B VA (0-6) 5 5 (100) 0 & E, (0-6) 8 1 (3) 0 D E, (0-6)+VA (0-6) > 5 (100) Z E E, (0-6)+VA (1-7) 6 5 (83) Z F E, (0-6)+VA (3-9) 7 6 (86) 2 G E, (06)+VA (5-11) 7 7 (100) 2 H E, (06)+VA (7-13) 5 2 (40) 0 I E, (0-6)+VA (7-20) 6 4 (67) 2 J VA* (Pre 16—-18)+E, (0-6) 1] 7 (64) I CvsD P=0.0047 CvsE P=0.0160 Fisher’s exact probability test CvsF P=0.0089 CvsG P=0.0012 CvsJ P=0.0370 E,: 204g 17f-estradiol/day, VA: 400 IU retinol acetate/day, VA*: 4,000 IU VA/day. a: Numbers in parentheses indicate the postnatal period of treatment. Pre 16-18: Period of days 16~-18 of gestation. 780 H. TACHIBANA, T. IGUCHI AND N. TAKASUGI TABLE 2. Number of cell layers, thickness and mitotic rate of the vaginal epithelium in ovariectomized mice treated perinatally with estrogen and vitamin A Gis Tr “(pasnatal day No es Ney a co) NG Mitons ta A Oil (0-6) 7 2.1+0.1? 11.0+ 0.6 0.10.02 B VA (0-6) 5 2.0-+0.0 10.4+ 1.2 0.10.01 6; E, (0-6) 8 Voll seL7 VWeDSEUNS) 2.3+0.34 D E, (0-6)+VA (0-6) 5 3.4+0.2 27.8+ 1.7 1.10.23 E E, (0-6)+VA (1-7) 6 3.9+0.6 SUSE D7 0.8+0.22 F E, (0-6)+VA (3-9) 7 3.5+0.6 28.2+ 6.0 1.0+0.21 G E, (0-6)+VA (5-11) 7 3.4+0.2 Alsae Zell 0.8 +0.10 H E, (0-6)+VA (7-13) 5) 6.3+0.6 51.7 4.7 1.7+0.25 I E, (0-6)+VA (7-20) 6 S36 Ill 46.5+11.7 135-023 J VA* (Pre 16-18)+E, (0-6) 11 5.3+0.8 52.6+11.2 1.2+0.17 Cvs D P<0.005 (0.01)” P<0O.01 (0.05) P<0.05 (0.05) CvsE P<0.01 (0.01) P<0.01 (0.05) P<0.01 (0.01) Cvs F P<0.005 (0.01) P<0.005 (0.01) P<0.01 (0.05) Student’s t-test CvsG P<0.001 (0.01) P<0.002 (0.01) P<0.002 (0.01) Cvs H NS Cvs I NS° NS P<0.05 (Fi,=4.42)? Cvs J P<0.01 (0.01) D vs H P<0.02 (0.01) P<0.02 (0.01) N Bet be ee a E,: 20 vg 178-estradiol/day, Sars. non-significant, d: A boundary value. concurrently with E, treatment (P<0.05, Student’s t-test). In E, plus 1-7 d VA, E, plus 3-9 d VA and E, plus 5-11 d VA mice, the vaginal epithelium showed significantly lower mitotic rate than that in E, mice (Table 2). In mice of E, plus 7-20 d VA and Pre VA plus E, groups, mitotic rate in the vaginal epithelium was also lower as compared with that in E, mice, though the number of layers as well as the epithelial thickness were not significantly less than those in E, mice. On the other hand, no difference was found in the epithelium between E, mice and E, plus 7-13 d VA mice. The number of layers and the epithelial thickness were higher in E, plus 7-13 d VA mice as compared with those of E, plus 0-6 d VA mice. Sequential changes in structure of the vaginal epithelium following neonatal injections of estrogen with or without vitamin A VA: 400 IU retionl acetate/day, b: Values in parentheses indicate a significant level by ANOVA except d, VA*: 4,000 IU VA/day. a: Mean+ c: Statistically a. Vaginal epithelium of control mice In normal oil-injected control mice at 0-3 days of age, the cranial part of the vaginal epithelium consisted of a single layer of pseudostratified columnar cells lining a narrow lumen, whereas the epithelium in the distal part was composed of cuboidal cells without a lumen. At 5—15 days of age, the vaginal epithelium consisted of 2-3 layers of cuboidal cells, though the vagina still remained imperforate. Vaginae of 30-day-old mice showed a 2- to 4-layered epithelium with superficial mucification; the distal vagina formed a canal with orifice. In 60- and 120-day-old ovariectomized mice, vaginae had an atrophic epithelium consisting of 2—3 layers of cuboidal cells. b. Vaginal epithelium in mice treated neonatally with estrogen The vaginal epithelium of 1-day-old mice receiving a single injection of E, consisted of pseudostratified columnar cells as did the controls. Suppression by VA of Permanent Vaginal Changes 781 However, enlarged polygonal cells (B-cells) [19] frequently appeared in the basal layer of the middle part. The distal vagina was connected with the urethra to form a common canal. In 3-day-old mice given 3 daily E, injections, the cranial part of the vaginal epithelium contained either small nodules of B cells or a sheet of fused B-cell nodules with superficial cornification. In mice receiving 5 daily injections of E,, B-cell nodules increased in number, fused into sheets consisting of 5-7 layers of cells. The outermost layer of the sheet underlying the original columnar epithelium underwent cornification, resulting in degeneration and exfoliation of the original epithelium. In mice given 7 daily injections of E,, all parts of the vaginal epithelium showed cornification except for the fornical region where B-cell nodules were occasionally found under the columnar epithelium. On the 3rd day after 7 daily E, injections, the epithelium was composed of the superficial layer of cornified cells, intermediate layers of prickle cells and a _ basal-cell layer. In the fornico-cervical region, however, the superficial layer was not cornified. In 15-day-old E, mice, the epithelium consisted of 4~7 layers with a superficial parakeratotic layer except in the fornical region where the superficial layer was mucified. The vaginal epithelium in 4 of 5 30-day-old E, mice consisted of 5—9 layers of cells with superficial cornification; the epithelium of the remaining mouse was stratified. In 60- and 120-day-old E, mice which had been ovari- ectomized at 30 days of age, the vaginal epithelium was markedly proliferated, consisting of 5—10 layers with superficial cornification or parakeratosis. In the present observation, however, adenosis-like lesions were not detected during the observation of longitudinally sectioned vaginal epithelium in adult ovariectomized E, mice, whereas such lesions were found in the transversely sectioned epithelium of neonatally estrogenized mice [20, 21). c. Vaginal epithelium in mice treated neonatally with estrogen plus vitamin A The vaginal epithelium of mice aged 1—30 days was not different in E, and E, plus VA groups. In 60-day-old ovariectomized E, plus VA mice, however, the vagina had an epithelium consisting of 47 layers without cornification. Neither parakeratosis nor mucification was found except for one. Vaginae of ovariectomized E, plus VA mice aged 120 days showed an epithelium consisting of 24 layers with (n=2) or without parakeratosis (n=3). Clear cells appeared in the: basal layer of the cranial part of 60-day-old (n=4) and 120-day-old (n=2) ovariectomized E, plus VA mice. In 60- and 120-day-old E, plus VA mice showing parakeratosis, numerous lymphocytes were aggregated in the stroma along with many leucocytes infiltrating into the superficial layer of the epithelium. Changes in mitotic activity in the vaginal epithelium of neonatally estrogen-and estrogen plus vitamin A-treated mice a. Cranial part of vagina Mitotic rate in the basal layer of the vaginal epithelium of control mice declined gradually until 120 days of age, though the rate was elevated slightly at 7 days. In 1-day-old mice receiving a singel injection of E,, the rate rose to a higher level than in the controls (P<0.05, Fig. la). In contrast, 3 daily E, injections caused an abrupt fall of the rate, resulting in low mitotic activity similar to that in the controls. On the day fol- lowing the last E, injection, mitotic rate showed a second peak, but declined at 15 days. Then, the rate again rose at 30 days, persisting at a high level until 120 days of age. The mitotic activity pattern observed in groups of E, plus VA mice was similar to that in E, mice until 30 days of age. However, the activity in E, plus VA mice declined at 60 days, reaching a significantly lower level than that in E, mice at 120 days of age (P<0.05, Fig. 1a). b. Caudal part of vagina Mitotic rate in the vaginal epithelium of the controls increased gradually until 7 days, but it decreased thereafter, persisting at a low level after 15 days of age. In E, mice, mitotic rate slightly rose between the first and the Sth postnatal day. At 7 days of age, however, a sharp increase in mitotic rate was found, but the rate declined strikingly between 10 and 15 days of age. The rate rose again, reaching the high level at 30 days. This high mitotic activity persisted until the 782 H. TACHIBANA, T. IGUCHI AND N. TAKASUGI a) Cranial part Mitotic rate (%) Onl Se On ian nO 15 30 60 120 Pann b) Caudal part Aye i: ; 4 2 2 24 —a>— oie io siti aa OFS 5. 7: 30 60 ; i} i | t t i Days after birth Fic. 1. Mitotic rate of the cranial (a) and caudal (b) parts of the vaginal epithelium. Normal newborn (C1); mice treated neonatally with 20 wg 17f-estradiol (E2) (O); 20 vg E, plus 400 IU retinol acetate (@); oil vehicle (A). The vertical bars indicate S. E. of the mean. Each point represents the mean mitotic rate of 5 mice. Arrows indicate daily subcutaneous injections. sacrifice at 120 days. The vaginal epithelium of E, plus VA mice showed a mitotic activity pattern similar to that in E, mice until 15 days of age. However, in E, plus VA mice mitotic rate was significantly lower at 30 days compared with that in E, mice. The low level of mitotic activity persisted thereafter until sacrifice (Fig. 1b). Suppression by VA of Permanent Vaginal Changes 783 DISCUSSION Female mice treated perinatally with large doses of natural and synthetic estrogens or androgens exhibit estrogen-independent, persistent proliferation and cornification of the vaginal epithelium. The permanent vaginal changes are irreversible, frequently resulting in precancerous or cancerous lesions in old mice (for reviews, see [22-24]). On the other hand, it has been demon- strated that the occurrence of permanent vaginal changes is prevented by neonatal injections of retinol acetate (VA) given concurrently with 178-estradiol (E,) [12-15, 17], diethylstilbestrol (DES) (Iguchi ert a/., submitted) or Sa-dihydro- testosterone (DHT) [16]. Previous studies revealed that the permanent vaginal changes induced by neonatal injections of E, or DHT were not suppressed by 5 daily injec- tions of VA given starting on the day after the neonatal treatment with sex steroids [13, 16]. In the present study, approximately the same degree of suppression of the permanent vaginal changes was found in adult mice given VA injections starting on the 2nd, 4th or 6th postnatal day as well as on the day of birth. However, 7 daily injections of VA failed to prevent the occurrence of permanent vaginal changes when started on the 8th postnatal day, whereas 14 daily VA injections beginning on the same postnatal day resulted in some decline in mitotic rate of the vaginal epithelium. This finding suggests that the permanent vaginal changes induced by neo- natally administered E, are suppressed by a longer period (14 days) of VA injections even when started on the day after the E, treatment, though VA given for a shorter period (5-7 postnatal days) is incapable of preventing the occurrence of permanent changes in E, mice. These results suggest that the period of VA treatment for the prevention of permanent vaginal changes must be prolonged when the treatment started later after birth. However, the possibility cannot be excluded that the prolongation of treatment period is not required if a higher dose of VA is administered to neonatally estrogenized mice. In addition, neonatally E,-treated mice (E, mice) delivered from the pregnant mothers which had received VA injections from days 16 to 18 of gestation showed a lower mitotic rate in the vaginal epithelium than did E, mice. Ac- cordingly, it appears that the perinatal period (3 prenatal or 7 postnatal days) of VA treatment is at least within the range of time when the VA treatment must be started for the prevention of permanent vaginal changes, though effects of VA given before day 16 of gestation on the vagina were not examined in the present study. In the previous studies, it was demonstrated that nodules of relatively large cells (B cell) ap- pearing under the original columnar epithelium fuse together and form a sheet of B cells, which sheds the original epithelium by cornification of the superficial layer of B cell-derived sheet [18, 19, 25]. Histological observation of sequential changes in the vaginal epithelium of E, plus VA mice indicated no appreciable difference in this developmental process as compared with that in E, mice until 30 days of age. At one and 7 days of age, mitotic rates in the vaginal epithelium of E, mice showed 2 peaks in the cranial part as reported previously [18]. The present study of mitotic activity in the vaginal epithelium of E, plus VA mice also demonstrated a fluctuation pattern similar to that observed in E, mice until 30 days of age. At 60 and 120 days of age, how- ever, differences were found in histology and mitotic activity between E, and E, plus VA mice. In E, plus VA mice, the number of epithelial layers as well as mitotic rate began to decrease starting at 60 days, resulting in a level at 120 days sig- nificantly lower than those in E, mice. Clear cells with pale cytoplasm frequently appear in the basal layer of degenerating vaginal epithelium of E, plus VA, DES plus VA and DHT plus VA mice [14-16]. In the present study, clear cells were found in the degenerating epithelium of E, plus VA mice aged 60 and 120 days, but not in the same age groups of E, mice. The present histological findings are in accordance with previous observations of vaginal epithelium in mice treated neonatally with a combination of sex hormone and VA. The vaginal epithelium of neonatally E, plus VA, DES plus VA and DHT plus VA mice once underwent proliferation with or without cornifi- 784 cation, and then degenerated gradually. Eventu- ally, more than 3-4 months were required for complete prevention of the occurrence of permanently changed vaginal epithelium in mice following neonatal treatment with sex hormone and VA as demonstrated by the present study. The atrophic vaginal epithelium of adult ovari- ectomized, E, plus VA mice responded normally to estrogen given postpubertally [17]. Accordingly, the present study suggests that vitamin A, when given starting within 7 days after birth, can alter the estrogen-independent type of the vaginal epithelium induced by neonatally administered estrogen to an estrogen-dependent type as seen in normal female mice. ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 Dunn, T.B. and Green, A. W. (1963) Cysts of the epididymis, cancer of the cervix, glandular cell myoblastoma, and other lesions after estrogen injection innewborn mice. J. Natl. Cancer Inst., 31: 425-455. 2 Takasugi, N. (1963) Vaginal cornification in persistent-estrous mice. Endocrinology, 72: 607— 619. 3 Takasugi, N. and Bern,H.A. (1964) Tissue changes in mice with persistent vaginal cornifica- tion induced by early postnatal treatment with estrogen. J. Natl. Cancer Inst., 33: 855-865. 4 Kimura, T. and Nandi,S. (1967) Nature of induced persistent vaginal cornification in mice. IV. Changes in the vaginal epithelium of old mice treated neonatally with estradiol or testosterone. J. Natl. Cancer Inst., 39: 75-93. 5 Takasugi, N. (1972) Carcinogenesis by vaginal transplants from ovariectomized, neonatally estro- genized mice into ovariectomized normal hosts. Gann, 63: 73-77. 6 Kimura, T. (1975) Persistent vaginal cornifica- tion in mice treated with estrogen prenatally. Endocr. Japon., 22: 497-502. 7 Iguchi, T. and Takasugi, N. (1976) Occurrence of permanent changes in vaginal and uterine epithe- lia in mice treated neonatally with progestin, estrogen and aromatizable or nonaromatizable androgens. Endocr. Japon., 23: 327-332. 8 10 11 12 13 15 16 17 18 ID) H. TACHIBANA, T. IGUCHI AND N. TAKASUGI Taguchi, O., Takasugi, N. and Nishizuka, Y. (1977) Irreversible lesions in female reproductive tracts of mice after prenatal exposure to testos- terone propionate. Endocr. Japon., 24: 385-391. Jones, L. A. and Bern, H. A. (1977) Longterm effects of neonatal treatment with progesterone, alone and in combination with estrogen, on the mammary gland and reproductive tract of female BALB/cfC3H mice. Cancer Res., 37: 67-75. Jones, L.A. and Bern, H. A. (1979) Cervico- vaginal and mammary gland abnormalities in BALB/cCrgl mice treated neonatally with proges- terone and estrogen, alone or in combination. Cancer Res., 39: 2560-2567. Jones, L.A. and _ Pacillas-Verjan, R. (1979) Transplantability and sex steroid hormone respon- siveness of cervicovaginal tumors derived from female BALB/cCrgl mice neonatally treated with ovarian steroids. Cancer Res., 39: 2591-2594. Mori, T. (1968) Effects of neonatal injections of estrogen in combination with vitamin A on the vaginal epithelium of adult mice. Annot. Zool. Japon., 41: 113-118. Mori, T. (1969) Further studies on the inhibitory effect of vitamin A on the development of ovary- independent vaginal cornification in neonatally estrogenized mice. Proc. Japan Acad., 45: 115— 120. Yasui, T. and Takasugi, N. (1977) Prevention by vitamin A of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice. An electron microscopic study. Cell Tiss. Res., 179: 475-482. Yasui, T., Iguchi, T. and Takasugi, N. (1977) Blockage of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice by vitamin A; Parabiosis and transplantation studies. Endocr. Japon., 24: 393-398. Iguchi, T. and Takasugi, N. (1979) Blockade by vitamin A of the occurrence of permanent vaginal changes in mice treated neonatally with 5a- dihydrotestosterone. Anat. Embryol., 155: 127- 134. Tachibana, H. and Takasugi, N. (1980) Resto- ration of normal responsiveness of vaginal and uterine epithelia to estrogen in neonatally estro- genized, A-vitaminized adult mice. Proc. Japan Acad., 56: 162-166. Iguchi, T., Ohta, Y. and Takasugi, N. (1976) Mitotic activity of vaginal epithelial cells following neonatal injections of different doses of estrogen in mice. Develop. Growth Differ., 18: 69-78. Takasugi, N. and Kamishima, Y. (1973) Devel- opment of vaginal epithelium showing irreversible proliferation and cornification in neonatally estro- genized mice: An electron microscopic study. 20 21 22 23 Suppression by VA of Permanent Vaginal Changes 785 Develop. Growth Differ., 15: 127-140. Forsberg, J.-G. (1972) Estrogen, vaginal cancer and vaginal development. Am. J. Obstet. Gyne- col., 133: 139-151. Plapinger, L. and Bern, H. A. (1979) Adenosis- like and other cervicovaginal abnormalities in mice treated perinatally with estrogen. J. Natl. Cancer Inst., 63: 507-518. Takasugi, N. (1976) Cytological basis for per- manent vaginal changes in mice treated neonatally with steroid hormones. Int. Rev. Cytol., 44: 193-224. Takasugi, N. (1979) Development of permanent- ly proliferated and cornified vaginal epithelium in 24 25 mice treated neonatally with steroid hromones and the implication in tumorigenesis. Natl. Cancer Inst. Monogr., 51: 57-66. Bern, H. A. and Talamantes, F. J. (1981) Neonatal mouse models and their relation to disease in the human female. In ‘‘Developmental Effects of Diethylstilbestrol (DES) in Pregnancy”’. Ed. by A.L. Herbst and H.A. Bern, Thieme Stratton, New York, pp. 129-147. Takasugi, N. (1971) Morphogenesis of estrogen- independent proliferation and cornification of the vaginal epithelium in neonatally estrogenized mice. Proc. Japan Acad., 47: 193-198. exnryutnd Lome, monn be cxgey few viLastiberciont ‘ DATs lsh a 7 j t att an riuks fo alfa, oy ; ry 4 kA bee) mh yout * Beth. ; cer he papas KOSS «9, = Noone hl O43 fin ,. 1 ae 4 ; ey { J my 2 ivi: Al YNEIGAI h, io) esl “ae $ arte fo ince | m8) luau} Pi y i ; : | - et | I : F ™ ( | ii ad } i ; Ke j | t : ; | i N | a or , ste WD = : S 4 ‘ yh in Go toa 43 f ’ % Ki VItY 20 Velen epithet ; Pe in] (Reo Cee cae p ; a = “ ane pn any is DT iy Ae ¥ i Nag a : le Poth ti li le bes | hick they Pkt pueuithe ROHN i 1¢ Glsite ictal ic 3 whe An Genie ainie = i ot? i Gy ZOOLOGICAL SCIENCE 1: 787-793 (1984) © 1984 Zoological Society of Japan Changes in Eye Color during Aggressive Interaction in the Chocolate Cichlid, Cichlasoma coryphaenoides MUNETAKA WATANABE, TOMOMICHI KOBAYASHI and HARUE TERAMI Department of Biology, College of Liberal Arts and Sciences, Okayama University, 2-I-1 Tsushima, Okayama 700, Japan ABSTRACT — Changes in the eye color and body color of the chocolate cichlid Cichlasoma cory- phaenoides were recorded by means of an iris tone index and body mark index during aggressive interaction. Under isolated conditions, the cichlids exhibited a lightened iris and a body color pattern consisting of a longitudinal stripe, a dorso-lateral and a peduncular spots. When they were attacked or chased, the iris darkened clearly and instantly. The iris darkening was so sensitive to motivational shift that the iris tone sometimes darkened even when the fish saw another fish approach. When the fish attacked others, the iris tone either scarcely changed or lightened a little. The body marks hardly shifted in the few seconds during which the fish attacked or were under attack. INTRODUCTION Changes in eye color during encounters between fish has been reported by several investigators [1-5]. In the desert pupfish, Cyprinodon macu- larius, Barlow [1] and Cox [2] and in Pomacentrus jenkinsi, Rasa [3] stated that dominant males kept exhibiting darkened eyes, and supposedly this darkening was due to a rise in aggressive motiva- tion. On the contrary, Kingston [6] reported in 17 species of the goodeid Cyprinodontiformes, that the eye darkening of these fishes was related to fright and the loss of fights in encounters. Body color changes in fish have been studied as an index of motivational condition and its signal function has been actively researched [7, 8]. How- ever, the biological meaning of eye color change has not yet been thoroughly analyzed, probably because eye color changes are not as clear as those of the body surface, and the changes often appear and disappear in a few seconds. We recently noticed in the chocolate cichlid [10] that its large eyes change color tone very quickly and distinctly from pale under normal conditions Accepted April 17, 1984 Received January 6, 1984 to extreme dark, corresponding to emotional shifts due to disturbances in the environment and motivational changes during encounters with conspecifics. Here we studied changes in iris tone and body marks of the cichlids during aggressive interaction using our newly developed method for recording and assessing such quick changes. MATERIALS AND METHODS Chocolate cichlids, Cichlasoma coryphaenoides, freshwater fish native to South America were used in the experiment. Individuals of 6 to 8cm in total length were purchased from a tropical fish dealer in Okayama, Japan. They were reared in groups of four in white-graveled tanks 90 « 27 x 30cm deep, with aquatic plants and stones and the water temperature adjusted to 28°C. They were fed pellets every morning. The cichlids wear a dorso-lateral spot, a pedun- cular spot and a longitudinal stripe on each side of body (Fig. 1). When they are heavily fear- motivated, they display a pattern of black vertical stripes on the brown ground coloration, excepting for small light areas at the bases of the dorsal fin rays. They have relatively large eyes and their 788 M. WATANABE, T. KOBAYASHI AND H. TERAMI Fic. 1. The chocolate cichlid fish Cichlasoma coryphaenoides in an aggressive bout. Note the difference in the tone of the iris between the chaser and the chased fish. Index Iris pattern body mark ——Y \Y, INO V/ Y) Y @ 4 3 © & Fic. 2. The iris tone index (/;) and body mark index (/,) for assessing the coloration of the iris and body. Changes in Eye Color in a Cichlid Fish 789 irises change tone quickly. The fish rest in suspension in the water, main- taining their bodies sometimes horizontally and sometimes at an angle of 40—S0° upward to the horizon with minute motions of pectoral and dorsal fins. They often settle on the leaves and stems of aquatic plants, where the fear-motivated body color can act as cryptic coloration. The iris tone of the cichlid darkens very clearly and quickly when it is attacked or chased by stronger fish (Fig. 1). The iris darkening is so sensitive to motivational shift that the iris tone sometimes darkens even when the fish sees another fish approach. For assessing the iris color, the iris tone index (/;) was defined as follows (Fig. 2): J;=1— white, J,=2— uniformly light grey or partially black, J; =3 — grey with a golden ring around the pupil, I; =4 — all black. The body marks, too, wax and wane with changes in environmental and motivational con- ditions. The cichlids wear a ground coloration of body from silver white to dark brown. The body mark index (/,) was also defined according to the Mm ASW & mem NM WW (continued) number and tone of the marks: /,=1— no body mark, /,=2-— dorsal spot only, /,=3 — dorsal and peduncular spot, /,—4—the spots plus a longitudinal stripe, with half steps of 1.5, 2.5 and Sy. When a fish oriented itself and approached another fish, or two fish approached each other, the situation was assumed to be an attacking interaction, regardless of whether or not their bodies came into contact with each other. Ob- servations and recordings were conducted on the behavior of the fish in the rearing tank in situ, in order that a transfer to a new tank for observation would not affect the fish. Switch devices were interposed in the circuit of a dual channel pen-recorder (Nihon Kohden RM-20) at a chart speed of 0.75 mm per second. The indices were simultaneously recorded by a pair of observers (J; by one observer and /, by the other) by pushing the switches when the indices changed (Fig. 3). One of the observers also noted the time that a particular fish attacked or was attacked by another fish by pushing another switch. The identity of the opponent each time Fic. 3. cichlid during aggressive interaction, retouched by inking. attacked; |, the fish was attacked. Simultaneous oscillographic records of the shifts of 7; (upper) and /, (bottom) of a chocolate Signs in the interzone: ff, the fish 790 M. WATANABE, T. KOBAYASHI AND H. TERAMI was not recorded. The values of the shifts, from the index at 5 sec before a bout to the index at 5 sec after it, were recorded for six 10-minute sessions, and the total of the values divided by the number of bouts was named the mean index shift (MIS). This value describes the average amount by which a fish changed its index during 10 sec. TABLE |. other fish GROUP I Fish A (2nd ranker) After bouts Iris ind TiS Index 1 ) 3 4 a 1 13 6 4 oe 5 i D) 6 17 8 = oO m 3 ee 3 6 5 a 4 en on 2 D Bones After bouts O index Z iO 1S 20 28 29 35 20 hkO S (tet oes Mires teenie ean ee 2 Stueail 5 Dt ae ee eee Sip )0/ snl Di gems 5 [i ela 0 je Aen en eee ee Ory 53.0 Fol) = | Ee ee ele 4 5 Pee ete oe 9 Giana aes eres 40 S22 0 = 2 Se Fish B (4th ranker) Ris After bouts Iris index 1 9) 3 4 g ! —- = a = } iS D ae 1 = 11 oO = 3 ae ee 3 21 D aa) 4 es me 3 18 After bout Body index ses se LO 15° 20 2:5. 310° 355metO Oe 2s es ee eS ee g S50 ee ee a | Se ae ee 3 ON eS OS a) | ee » hat ae i 4 Oy, 4 yet ee 0 aa) CG. Mee RAPED E 2 8 Gs TOIT 1G 1 AO SEs BERS RY Since we can not relate M/S to aggressive bouts for solitary fish, their MIS was derived as follows: I, and J, were recorded at 30-second intervals, and the shift values during the 10 sec from before until after the marks were obtained; then M/S was calculated in the same manner as described above. The shifts of 7; and J, of chocolate cichlids from 5 sec before to 5 sec after being attacked by GROUP II Fish C (1st ranker) After bouts Iris index 1 2 3 4 i) 1 13 12 3 = 5 Ss 2) 2 10 13 i iD) i 3 = 4 12 5 oO ara) 4 BS » mal Fish D (3rd ranker) After bouts Iris index 2 3 4 a) 1 — — 3 1 3 £ 2 ee Be 3 5 3 2 6 S ee i ~ 4 == 1 1 1D Fish E (4th ranker) ae After bouts Iris index 1 2 3 4 iz) 1 Dy — — D4 =>) 5 2 — — 2 — 5 3 2 2 10 E ee a 4 ia 1 1 45 Changes in Eye Color in a Cichlid Fish 791 TaBLeE 2. The shifts of 7; and 7, of chocolate cichlids from 5 sec before to 5 sec after attacking other fish GROUP I Fish A (2nd ranker) After bouts Iris index 1 2 3 4 2 1 21 4 I a =. 3 2 10 5 2 =e 2 5 3 = 3 I I vo faa) 4 os zea wae a After bout Body index i hares fas 20 25 30 35 40 0 ee ee eee er pee 2 - 2 (ee eee 8 2 —- —~ 4 4 —~ —~ — 2 Pee eae _ on 8 5 BRIE OE 2 ueereeat 214 ferey reel pt oigiw: 2h ea ES eee i ddteive = 0 ee RESULTS Table 1 represents the shifts of J; and/or J, of the 2nd (fish A) and 4th (B) rankers in Group I and the Ist (C), 3rd (D) and 4th (E) rankers in Group II when they were attacked by other fish. The higher rankers A and C exhibited lighter irises (J;=1 or 2) in most cases, while the lower rankers B, D and E had darker irises (/;=3 or 4) until the onset of bouts. An increase of /; from before until after the bout was seen in every fish tested, particularly in lower rankers. The M/S of J, of fish E does not appear to be as large as one might expect (Table 4). This is due to the fact that the M/S could no longer be increased in 45 out of the total of 67 bouts, since J; was already at the maximum value of 4 just before the bouts. If the M/S is calculated on 22 (67 minus 45) bouts, we can obtain a fairly high value of 0.591. The MIS of J, of either fish A or B when it was attacked, was near zero or 0.1 at most, indicating that body marks scarcely changed. When attacking other fish, the M/S of /; of all tested fish A, C and D was near zero or had a minus value, that is, the iris remained unchanged or lightened (Tables 2 and 4). When put in solitude, both the higher ranker GROUP II Fish C (1st ranker) After bouts Iris index 1 2 3 4 Mod l | be 2 =e Sk =) 5 2 5 7 1 ae = S 3 — 6 2 a4 faa) 4 2 5 1 — Fish D (3rd ranker) — Aft t Iris index aoa 1 2 3 4 2 1 1] 6 — _ =) £ 2) 4 4 I — 2 oS 3 — | | — oO jaa) 4 — = — es TABLE 3. The shifts of J; of isolated chocolate cichlids Fish A (2nd ranker) After bouts Iris index | 2 3 4 2 1 12 2 — — 3 = 2 4 24 = _ vo 5 3 — I — — D fae 4 er fe Be Aah Fish B (4th ranker) After bouts Iris index | 2 3 4 2 l 12 6 ] — Ss 2 6 16 = P S 3 2 | — Vv fa) 4 ns hans — For measurement details, see text. (fish A) and lower ranker (fish B) exhibited light- ened irises (J;,=1 or 2; Table 3). They always exhibited distinct body marks (/,=3.5 or 4.0) without exception. 792 M. WATANABE, T. KOBAYASHI AND H. TERAMI TABLE 4. The mean index shifts (WIS) of J; and J, of the chocolate cichlid under three conditions: being attacked, attacking and being in isolation Attacking In isolation I; I, I; I, Condition Attacked Index qT; I, A 0.222 (72) —0.014 (72) Group I B 0.702 (57) 0.097 (57) C 0.338 (77) — Group H D 0.838 (37) — E 0.194 (67) — —0.083 (48) 0.104 (48) —0.070 (43) 0.000 (43) 0.044 (45) 0.011 (45) 0.071 (28) — se yi —0.460 (50) — ie ee The numbers in parentheses represent the frequency of bouts. DISCUSSION The present study shows that the iris of the chocolate cichlid rapidly darkens when the fish is attacked, while it hardly changes or lightens somewhat when the fish attacks others. The body marks shift little in the matter of seconds it takes to either attack or be attacked. The MIS of J; of the attacked fish gives the appearance of being fairly small, from near 0.2 to 0.8. But these values seem to be large enough to indicate that the eye color darkens when the fish is attacked, because in the limited space of our tanks, slight contact (without bodily touch) very often occurred, although they would be nominally included as ‘attacking interaction (p. 789)’, which stimulated the fish little. The darkening of the eye color in defeated fish in this cichlid as well as in goodeid fish [6] is in contrast to the eye darkening of winning fish in Cyprinodon [1] and Pomacentrus [3]. Such a diversity in the mode of eye color change suggests that the change might have some signal function which developed correspondingly to the life form of each species. In the chocolate cichlid, espe- cially, the eye darkening is very quick and occurs all over the iris, while a change in body color is hardly seen. Therefore, it is quite possible that the eye color change acts as an important signal for communication in this fish. Some fishes display body colorations and pat- terns corresponding to the order of dominance. In the Japanese freshwater serranid Coreoperca kawamebari, the top-ranking fish wears a yellow- brown body color and the lowest ranker, dark stripes and a white vertex band [9]. The chocolate cichlids also display iris tones according to their social ranking when they are put together in a limited space. The higher ranker has a white iris, while the lower rankers display a fairly dark iris which further darkens in tone when the higher fish approaches them. The top-ranker is apt to attack the fish having a white iris more intensely. This suggests that an effect of the darkened eyes is to inhibit attack by conspecifics. On the other hand, we often observed that when a fish which had darkened eyes after being chased by a stronger fish, approached another fish, it was chased again. This observation favors the attack-releasing effect of darkened eyes. In order to elucidate the significance of eye color changes in the chocolate cichlid it is neces- sary to perform more quantitative observations on their behavior in various social relationships, using the present method. ACKNOWLEDGMENT We wish to express our gratitude to Dr. P. H. Greenwood of the British Museum (Natural History) for identifying the fish. REFERENCES 1 Barlow, G. W. (1961) Social behavior of the desert pupfish, Cyprinodon macularius, in the field and in the aquarium. Amer. Midland Naturalist, 65: 339-359. 2 Cox, T.J. (1966) A behavioral and ecological study of the desert pupfish (Cyprinodon macularius) in Quitobaquito Springs, Organ Pipe Cactus Na- tional Monument, Arizona. Ph. D. thesis, Univ. Changes in Eye Color in a Cichlid Fish Arizona, Tucson. Rasa, O.A.E. (1969) Territoriality and _ the establishment of dominance by means of visual cues in Pomacentrus jenkinsi (Pisces: Pomacentridae). Z. Tierpsychol., 26: 825-845. Echelle, A. A. (1981) Behavior of the pupfish, Cyprinodon rubrofluviatilis. Copeia, 1981: 68-76. Martin, F. D. and Hengstebeck, M. F. (1981) Eye color and aggression in juvenile guppies, Poecilia reticulata Peters (Pisces: Poeciliidae). Anim. Behav., 29: 325-331. Kingston, D. I. (1980) Eye-color changes during aggressive displays in the goodeid fishes. Copeia, 1980: 169-171. 793 7 Baerends,G.P. and Baerends-van Roon, J. M. 10 (1950) An introduction to the study of the etho- logy of cichlid fishes. Behaviour, Suppl. 1: 1-242. Kohda, Y. and Watanabe, M. (1982) Agonistic behavior and color pattern in a Japanese fresh- water serranid fish, Coreoperca kawamebari. Zool. Mag. 91: 61-69. Kohda, Y. and Watanabe, M. (1982) Relationship of color pattern to dominance order in a fresh- water serranid fish, Coreoperca kawamebari. Zool. Mag., 91: 140-145. Terami, H. and Watanabe, M. (1982) On the darkening of iris in a tropical fish Cichlasoma coryphaenoides. Zool. Mag. 91: 644. , \ eA ie = 4 ai NM 7 yanip ola . é 2. 4 . 7 - _ 4 rT s000." Ari s ; cf Jai z,: ¢ & Z. % = a oe 2 9 4 14 — I - i J re = ¢ ’ es = “I ) = . 2 = ee 4 4 | z: | s = { 1 { { | ZOOLOGICAL SCIENCE 1: 795-800 (1984) © 1984 Zoological Society of Japan Rigid Isolation between the Northern Population and the Southern Population of the Medaka, Oryzias latipes MITSURU SAKAIZUMI! Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan ABSTRACT — The genetic characters of the wild populations of the medaka, Oryzias latipes, were studied in the boundary between the Northern Population and the Southern Population in Japan. The two major populations were found to be isolated from each other rigidly, and the boundary was well correlated with mountain barrier. The populations with a genotype different from both major populations were found in the quite limited area around the western end of the boundary. INTRODUCTION In the course of the allozyme studies of Oryzias latipes, it has been shown that the Japanese wild populations of this species contain two genetically different groups, the Northern Population and the Southern Population. Of 21 protein loci ex- amined four loci are nearly fixed for different alleles between these two groups, suggesting that these groups were isolated from each other in considerably ancient times. Little clinal distribu- tion of alleles is observed at these loci, but the boundary is very distinct [1, 2]. In Japan the medaka is found in three major islands (Honshu, Shikoku, and Kyushu) and many small islands around them (Fig. 1). Thus, Aomori Prefecture seems the northernmost limit of the natural distribution of this species. The North- ern Population inhabits the region along the Sea of Japan coast of northern half of Honshu. On the other hand, the Southern Population is dis- tributed along the Pacific coast and along the western part of the Sea of Japan coast. The boundary of these two groups is well correlated with the backbone mountains of Honshu for the Accepted May 18, 1984 Received April 17, 1984 1 Present Address: Department of Medicinal Chemi- stry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199- 01, Japan. most part. This species has not been found in the northern half of Iwate Prefecture (Takada, personal communication). As the northernmost limit of the Southern Population is the southern part of this prefecture, and as the medaka with the genotype of the Northern Population inhabits the Pacific coast of Aomori Prefecture, the two genetically different populations are separated by mountain barrier in the northern half of Iwate Prefecture. In this paper I report the distribution and the genetic characters of this species in the boundary region between the Northern Population and the Southern Population, mainly around the western end of the boundary where a continuous distribution of this species is observed. The object of the present study is to examine the extent of genetic isolation between the two populations and the correlation between genetic difference and geographic features in a limited area. MATERIALS AND METHODS In 1983 specimens were collected in the region around the Wakasa Bay (Fig. 1); fish were then brought alive to the laboratory. Collection sites are listed below with the number of electrophore- tically examined samples in parentheses: 1. Iburihashi, Kaga (12); 2. Urushibara, Sabae (10); 3. Kikuyama, Tsuruga (10); 4. Kohgasaki, Obama (10); 5. Ichiba, Maizuru (10); 6. Shimazu, Amino (10); 7. Miyajima, Toyooka (10); 8. Ohto, 796 M. SAKAIZUMI HOKKAIDO Northern Begin: Adh? SEA OF JAPAN Wakasa Bay HONSHU.’ Southern Population SHIKOKU Fic. 1. Kinomoto (10); 9. Nakasuji-cho, Ayabe (11); 10. Koyama, Tottori (13). The procedures of tissue preparation and elec- trophoresis were those described in Sakaizumi et al. [1, 2]. Electrophoretic patterns were observed for 8 enzyme systems; alcohol dehydrogenase (Adh), L-iditol dehydrogenase (/dh), superoxide Distribution ranges of the Japanese two major populations of Oryzias latipes. Adh* Ian" Pgm? Sod” PACIFIC OCEAN Closed circles show the collection sites of samples electrophoretically examined. The rectangular area is the subject of the present study, which is enlarged in Fig. 4. Populations are numbered as in the text. dismutase (Sod), phosphoglucomutase (Pgm), acid phosphatase (Acp), amylase (Amy), muscle lactate dehydrogenase (Ldh), and liver esterases. Adh, Idh, Sod and Pgm are the diagnostic enzymes of the Northern Population. Acp, Amy, and Ldh-A are specific markers of the Inland Sea Subpopula- tion and the San’in Subpopulation in the Southern Genetic Isolation of Medaka Populations Population. As for the liver esterases, it has been known that all individuals of the Northern Popula- tion show the same pattern with two major bands, while many variants, with 1-7 bands, are found in the Southern Population [2]. Although the ge- netic control has not been interpreted yet, this enzyme system was used as a diagnostic marker of the Northern Population. RESULTS in Table 1. at each locus are as follows: 797 The allele frequencies at 7 enzyme loci are shown Characteristics of allelic distribution (1) Adh: This locus TABLE |. Allele frequencies at 7 enzyme loci from 10 populations of Oryzias latipes Population Locus 1 2 3 4 5 6 rt 8 9 10 Adh a .08 —_ 45 — — 1.0 1.0 1.0 95 1.0 Br «92 1.0 55 1.0 1.0 —- —_ — 05 — Idh a — — a _ a - — 1.0 1.0 .96 b 1 1.0 1 1 1.0 1.0 — — .04 Sod a 1.0 1.0 1.0 1 1 95 .90 — — — b — — — — — .05 10 1.0 1.0 .96 d = a —- = a .04 Pgm a” 1.0 1.0 1.0 1.0 1.0 35 — — — — b — — — — — 05 — 1.0 1.0 1.0 d — — 60 1.0 — — — Acp 443.0 1.0 1.0 1.0 1.0 1.0 85 20 20 92 b — — — -— a — _ os —- .08 c — a — — — — 15 .80 80 — Amy Acne hO 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 a2, b — a — — _- — — — as 88 Ldh-A a — a — — .05 — 10 —- — Coe 1.0 1.0 1.0 95 95 90 Sls) — .08 d — -- — — — — — 85 1.0 — e — = — — 05 — — — .92 Populations are numbered as in the text. a oraa Fic. 2. Phosphoglucomutase allozyme phenotypes from liver of three pupulations of medaka. Lanes 1—5, Amino; lane 6, Kaga; lane 7, Tottori. 798 O 1. 2. Se 4S 6 Fic. 3. 1-5, Maizuru; lane 6, Kaga; Esterase allozyme phenotypes from liver of four populations of medaka. lane 7, Tottori; M. SAKAIZUMI T & oe 39 10 11 12 Lanes lanes 8-12, Toyooka. TABLE 2. Pattern frequency of the liver-esterases from 10 populations of Oryzias latipes Population Pattern 1 2, 3 4 5 6 7 8 9 10 ‘northern’ -type 1.0 .90 .60 .20 1.0 — ae os ee ey ‘southern’ -type —— 10 .40 .80 — 1.0 1.0 1.0 1.0 1.0 Populations are numbered as in the text. was nearly fixed for Adh* or Adh’. Tsuruga population showed polymorphism. The popu- lations from Kaga, Sabae, Obama, and Maizuru had ‘northern’ allele, Adh’. (2) Idh and (3) Sod: These loci showed similar distribution pattern of the ‘northern’ and the ‘southern’ alleles. They were essentially monomorphic in each population. The ‘southern’ alleles were predominant in three populations, Kinomoto, Ayabe, and Tottori. (4) Pgm: This locus was fixed for ‘northern’ allele in the populations from Kaga, Sabae, Tsuruga, Obama, and Maizuru. It was fixed for ‘southern’ allele in the populations from Kinomoto, Ayabe, and Tottori. An unique allele, Pgm*, was pre- dominant at Amino and Toyooka (Fig. 2). (5) Acp: Except for three populations this locus was essentially fixed for Acp*. Acp* was frequently observed in the populations from Kinomoto and Ayabe. (6) Amy: Amy’ was found only at Tottori. This locus was fixed for Amy’ in other populations. (7) Ldh-A: Kinomoto and Ayabe populations showed high frequency of Ldh-A*. San’in District specific Ldh-A° was exclusively observed at Tottori. The ‘northern’ pattern of liver-esterase system was predominant at Kaga, Sabae, Tsuruga, and Maizuru (Fig. 3). The frequencies of the ‘north- ern’ and the ‘southern’ (=non-northern) patterns are shown in Table 2. DISCUSSION Based on 8 enzyme markers, 10 populations examined in the present study can be classified Genetic Isolation of Medaka Populations 799 Sea of Japan Baan AMINO OTOYOOKA TOTTORI Wakasa Bay Lake Biwa 50 km Fic. 4. Allele frequencies at four variable loci and pattern frequencies of electromorph of liver-esterases in the populations around the Wakasa Bay. N-type, ‘northern’-type allele (pattern); S-type, “southern’- type allele (patterns). into three groups; 1) the ‘northern’-type popula- tions (Kaga, Sabae, Tsuruga, Obama, Maizuru), 2) the ‘southern’-type populations (Kinomoto, Ayabe, Tottori), and 3) the boundary region populations (Amino, Toyooka). The genetic characters and distribution of these groups are illustrated in Figure 4. The populations from Kaga, Sabae, and Maizuru show a typical genotype of the Northern Popula- tion. Although the ‘southern’ allele at Adh locus and the ‘southern’ patterns of liver-esterases are observed at Tsuruga and Obama, these popula- tions can be regarded as the southernmost popu- lations of the Northern Population. Polymor- phism of Adh and esterases seem to be due to the secondary gene flow of the ‘southern’ characters into the Northern Population. The ‘southern’-type group is composed of two different populations, the Inland Sea-type (Kinomoto, Ayabe) and the San’in-type (Tottori) populations [2]. They show typical genotypes of the Southern Population. The population from Kinomoto has quite different genotype from Tsuruga population in spite of the short geographic distance (ca.20km). This result suggests that the mountain barrier between these two sites has been effective to maintain the genetic isolation between these two populations. Although Ayabe and Maizuru belong to the same water system, the genotypes of populations are considerably different. This phenomenon seems to reflect the history of this drainage system (e.g. stream capture) after the establishment of the distribution of the North- ern Population and the Southern Population [3]. The third group shows an unique genotype different from any of the Northern Population, the Inland Sea Subpopulation, and the San’in Sub- population. This type of population is found in a quite limited area where is the western end of the boundary between two major populations in Japan. They have ‘northern’ alleles at Jdh and Sod loci, while Adh and liver esterases are fixed 800 M. SAKAIZUMI for ‘southern’ types. It should be emphasized that they have an unique allele, Pgm*. Because this allele has not been observed elsewhere, it is likely that Pgm* has originated and propagated recently, probably after the formation of populations in this area. The results obtained in the present study indi- cate that the Northern Population and the South- ern Population are isolated rigidly and that the isolation has been maintained chiefly by mountain barrier. It should be mentioned that both ends of the boundary between the two major popula- tions are the regions where mountains sink into the sea and little flat area exists along the sea coast. The unique genotype of the populations from the boundary region suggests that these popula- tions were formed by an introgression between the Northern Population and the Southern Population followed by random drift at each locus. At the same time, it suggests that two major populations are not back-crossing in spite of full fertility of the laboratory reared F, hybrid of these two populations (unpublished data). In order to speculate the time when the populations of the boundary area occurred, I can find a clue that Adh, Idh, and Pgm loci are fixed for specific alleles, respectively, in Toyooka population. As the effective population size seems not so small, it is unpublished data. possible that the origin of the boundary-region populations goes back to fairly old times, for instance, to the last glacial period or so. ACKNOWLEDGMENTS I am grateful to Professor Nobuo Egami of the University of Tokyo for his useful commentary on the early draft of the manuscript. Kind help in the collec- tion of materials by Kiyoshi Naruse is greatly appre- ciated. J thank Mr. Muneo Takada of Iwate Educa- tion Center for Sciences for allowing me to use his This work was supported in part by a Grant-in-Aid for Fundamental Scientific Research from the Ministry of Education, Science and Culture to Professor N. Egami. REFERENCES 1 Sakaizumi, M., Egami, N. and Moriwaki, K. (1980) Allozymic variation in wild populations of the fish, Oryzias latipes. Proc. Japan Acad., 56 (B): 448- 451. 2 Sakaizumi, M., Moriwaki, K. and Egami, N. (1983) Allozymic variation and regional differentiation in the wild populations of the fish Oryzias latipes. Copeia, 1983: 311-318. 3 Okada, A. and Takahashi, K. (1969) Geomorphic development of the drainage basin of the River Yura. J. Geography, 78: 19-37 (In Japanese with English abstract). ZOOLOGICAL SCIENCE 1: 801-807 (1984) Systematics of the Drosophila montium Species Subgroup: a Biochemical Approach SEIDO OHNISHI! and TAKAO K. WATANABE? National Institute of Genetics, 1111 Yata, Mishima 411, Japan ABSTRACT — Genetic relationships among 29 species in the Drosophila montium species subgroup were investigated using two electrophoretic techniques, O’Farrell’s two-dimensional electrophoresis (2DE) and conventional starch gel electrophoresis (SGE). Based on data of genetic distances estimated by protein differences detected by 2DE, a dendrogram was constructed for this subgroup. In addition, species specific allozymes (diagnostic enzymes) were surveyed by SGE in order to dis- © 1984 Zoological Society of Japan tinguish species easily. helpful for identifying species. It was found that six enzymes (Aldox, G6pdh, Men, 6Pgdh, Idh and Est-6) are For example, when two enzymes, A/dox and G6pdh run in a starch gel, we could distinguish 26 of 29 species (about 90%). Thus, it is clear that these two biochemical techniques are useful tools for studying Drosophila systematics. INTRODUCTION The Drosophila montium species subgroup, involving more than fifty species, is the largest in the D. melanogaster species group and provides interesting material for the study of Drosophila systematics. Classification has been primarily made by morphological characters such as sex combs, male genitalia [1-3] and karyotypes [4, 5]. However, there is less information available con- cerning quantitative investigations of genetic differentiation between species in this subgroup. We report here genetic relationships proven by protein differences detected by 2DE for 29 species in this subgroup, of which 14 species reported previously [6] are included. This article also demonstrates that species specific allozymes (diagnostic enzymes) are useful for species identi- fication. MATERIALS AND METHODS Twenty nine species of the Drosophila montium Accepted May 7, 1984 Received February 10, 1984 1 Present address: Research Laboratories, Nippon Merck-Banyu Co., Ltd., 810 Nishijo, Menuma- machi, Saitama 360—02, Japan. ? To whom correspondence should be sent. species subgroup were used. They are listed in Table 1. Each of these species is originated from one fertilized female which was sampled in natural populations (isofemale line). Of the 29 species analyzed here, 14 species (marked by an asterisk in the table) are the same as described in Ohnishi et al. [6, 7]. The others were collected in different years and in different localities by the members of a scientific expedition from Tokyo Metropolitan University (see Table 1 for more details). Species numbered 1-7 and 11-15 are classified as the D. kikkawai complex and D. auraria complex, re- spectively. The taxonomical positions of the other species are not clear (except for a group which includes three species, D. jambulina, D. punjabiensis- like and D. punjabiensis [6, 8). The procedures for 2DE and SGE are the same as previously reported [6]. The 2DE is, in principle, the same as O’Farrell’s method [9] in which proteins sampled from six male adults for each species are separated by isoelectric points (pl) in the first dimension and by molecular weight (mw) in the second dimension. An example of a 2DE gel is shown in Figure 1. Based on the data of protein differences between two species detected by 2DE, genetic distances (D) were calculated by the Aquadro and Avise equation [10]: D=1—2n,,/ (n,-+n,), where n,, is the number of shared spots in both species and n, or n, is the total number of 802 S. OHNISHI AND T. K. WATANABE burlai seguyl birchii serrata baimaii WS). khaoyana 26. khaoyana-like Die . lacteicornis 28 parvula L111, Nairobi (Kenya), 1979 K2, Mombasa (Kenya), 1979 WAUI67, Wau (Papua New Guinia), 1981 POMS03, Port Moresby (Papua New Guinia), 1981 Y150, Penang (Malaysia), 1979 Chi-ton (Taiwan), 1982 A-strain, Wulai (Taiwan), 1979 IRO78, Iriomote Island, 1978 SHL26, Shillong (India), 1981 TABLE 1. Flies of the Drosophila montium species subgroup used in this study Species Source 1. D. pennae* Texas stock no. 3028.1 25 ‘Dubocki= AO-1, sent by Dr. V. Baimai 3. D. kikkawai* Okinawa-strain 4. D. leontia* AO-2, sent by Dr. V. Baimai Sy JD ial Texas stock no. 3146.1 6. D. lini-like MM Y326, Maymyo (Burma), 1981 7. D. barbarae* Texas stock no. 3033.1 8. D. jambulina* TMU, India, 1979 9. D. punjabiensis-like* Texas stock no. 3116.11, Thailand 10. D. punjabiensis* TMU, India, 1979 11. D. quadraria* Texas stock no. 3075.1, Taiwan 12. D. triauraria* Tsukuba, 1976 13. D. auraria* Mishima, 1978 14. D. biauraria* Tsukuba, 1976 15. D. subauraria KT-4, Kitagami, 1982, sent by Dr. M. T. Kimura 16. DP raja Mishima, 1978 17. D. mayri WAUI153, Wau (Papua New Guinia), 1981 18. D. bicornuta T238, Kota Kinabalu (Malaysia), 1979 19. D. truncata RGN-210-17, Rangoon (Burma), 1982 D. D. D. D. D. D. D. D D. D . thopaloa-like CM71, Chiangmai (Thailand), 1977 The 14 species noted with an asterisk (*) are the same as previously reported by Ohnishi e¢ a/. [6] and the remaining 15 species are newly added in this study. scored spots in each species. Based on the matrix of genetic distances, a dendrogram was constructed following the UPGM (unweighted pair group method) of Sokal and Sneath [11]. Standard starch gel electrophoresis (SGE) was also carried out to analyze eight allozymes (Aldox, G6pdh, Men, 6Pgdh, Idh, mMdh, cMdh and aGpdh) for all 29 species. The procedure for SGE is the same as that in previous reports [6, 7, 12]. Four flies in every species were run separately (i.e., four single fly analyses were carried out) and the allozyme patterns were scored. RESULTS A dendrogram of 29 species in the Drosophila montium species subgroup is illustrated in Figure 2. Of 15 species analyzed here, 13 species are distantly related to 14 species analyzed in a previous report [6]. The genetic distances are more than 0.3, with two exceptions, D. lini-like of the kikkawai com- plex and D. subauraria of the auraria comples. The largest value (about 0.5) of genetic distance found in the D. montium subgroup exceeds the largest one (0.36) estimated by 2DE in the D. Systematics of Drosophila 803 peng el 100K Saas 70K 40K a * 25K SDS Fic. 1. lacteicornis). focusing in the first dimension. ao 4.5 An example of a two-dimensional electrophoretic gel (a sample from six male adults of Drosophila The numbers in the abscissa show isoelectric points (pl) with IEF denoting isoelectric Numbers in the ordinate show molecular weight (mw) expressed as K (x 10°) separated by SDS slab gel electrophoresis in the second dimension. virilis (or D. montana) subgroup [7]. This may suggest that the speciation level in the D. montium subgroup is more advanced than in D. virilis subgroup. A new species, D. subauraria of the auraria complex, recently found by Kimura [13], was found to be more closely related to D. biauraria than to the others by means of 2DE analysis in this complex. This tendency is also confirmed by a morphological study on male genitalia [13] and by allozyme analysis with Jdh and G6 pdh (Table 2). The 2DE analysis shows that D. lini-like seems to belong to the D. kikkawai complex, although it is not morphologically so closely related to D. Jini. This is consistent with the data from allozymes (see Table 2) and from_ interspecific crosses (Ohnishi, unpublished data). A species of D. rhopaloa-like has an apical black patch on the male wings which is never seen in the other members of this subgroup, except for the D. suzukii subgroup. Judging from morphological traits, including this patch, it is expected that D. rhopaloa-like would differ most from all other members in this sub- group. The results by 2DE analysis also supports this expectation, i.e., the genetic distance found between D. rhopaloa-like and the others was 804 S. OHNISHI AND T. K. WATANABE D. pennae D. bocki D. kikkawai D. leontia D. lini D.lini-like D. barbarae 2DE D.jambulina . punjabiensis-like . punsjabiensis .guadraria _triauraria _auraria _bjauraria _Subauraria _ rufa mayri . bicornuta . truncata _burlai .seguy!l . birch serrata baimatt _khaoyana khaoyana-like lacteicornis . parvula SIS SUS es oe SoS SS 5 © s & . thopaloa-like a aaa mac ai (ae ) ei a| 0.6 0.4 0.2 0 Genetic Distance (D) Fic. 2. A dendrogram of 29 species of the Drosophila montium species subgroup, constructed from data showing protein differences detected by two-dimensional electrophoresis (2ED). The genetic distances (D) between species are calculated by the Aquadro and Avise (10) equation: D=1—F= 1—2n,,/(n,-+n,), in which n,, is the number of shared spots in both species and n, or ny, is the total number of scored spots in each species. approximately 0.5, which indicates the most subgroup. distant relationship within the subgroup, sug- Enzymes which reveal no variation among gesting that this species may belong to another species are of no practical use for species identi- Systematics of Drosophila 805 TABLE 2. Relative mobilities of common alleles at eight allozyme loci in the Drosophila montium species subgroup Species Aldox G6pdh Men 6Pgdh Idh cMdh mMdh_ aGpdh pennae 10 — 4 3 8 4 4 4 bocki 5 — 4 3 8 4 4 4 kikkawai 10 9 4 3 8 4 4 4 leontia 10 8 4 3 8 4 4 4 lini 8 8 4 3 8 4 4 4 lini-like 8 11 4 3 8 4 4 4 barbarae 8 14 3 3 6 4 4 4 jambulina 13 10 4 aS 2 4/6* 4 4 punjabiensis-like 4 11 8 6 7 4 4 4 punjabiensis 1 10.5 10 6 8 4 4 4 quadraria 2 4 6 4.5 7 4 4 4 triauraria BS) 4 6 4.5 8 4 4 4 auraria 3.3 4 6 4.5 8 4 4 4 biauraria 35 7 6 4 6 4 4 4 subauraria 3.5 f | 8 GI 6 4 4 4 rufa 8 3 Ea) 5 8 4 4 4 mayri 3B 6 3 a0 6 4 4 4 bicornuta 10 — Z 8 5 4 4 4 truncata 42 12 2 8 6 6 4 4 burlai 14 11 4.5 4 6 4 4 4 seguyi 10 4 4 3 4 4 4 4 birchii 12 2 3 3 6 4 4 4 serrata 3 — l 3 5 4 4 4 baimaii 3.5 10 4.5 1 8 6 4 4 khaoyana 6 8 3 8 8’ 4 4 4 khaoyana-like 5 7 3 8 8 4 4 4 lacteicornis 4 7 3 5 8 4 4 4 parvula 3.5 8 10 4 6’ 2 4 4 rhopaloa-like 12 3 4.5 4 5 = 4 4 No. of alleles (12) (12) (9) (9) (8) (4) (1) (1) The relative mobility of each allele was determined by four flies for each species. The numbers showing different alleles were arbitrarily assigned, although fastermigrating alleles in a more cathodal system were indicated by a larger number at that locus. * Including heterozygous flies. — Not detectable. fication, as in mMdh or aGpdh (see Table 2). On the other hand, enzyme loci at which different alleles have become mutually fixed for each species are applicable to species identification. We discovered that five enzymes, Aldox, Men, Idh, G6pdh and 6Pegdh are all useful (see Tables 2 and 3). These enzymes are called ‘“‘diagnostic enzymes” (species specific enzymes), because if we run a single enzyme, Aldox, about 40° (12/29) of the species are distinguishable; when two enzymes, Aldox and G6pdh are run together, about 90% (26/29) are distinguishable. The results described above indicate that a survey of these enzymes facilitates the identification of almost all of the species. However, D. auraria and D. triauraria could not be distinguished by means of this survey 806 S. OHNISHI AND T. K. WATANABE TABLE 3. Percentage of species which can be distinguished by using different combi- nations of two or three allozyme loci Aldox G6pdh Men 6Pgdh_ Idh Aldox 41.4* G6pdh 89.7 44.8* Men 193) B82-8" —3ik0F 6Pgdh 82.8) 875.9) 655) Si1kOs Idh 1S 1D) CW BD ~ Zo" Aldox +Gé6pdh Vo Vor” Lol * Percentage of species distinguished by only one allozyme locus. ** If Est-6 or Est—C is analyzed in addition to these three allozyme loci, all 29 species can be distinguished by only using allozymatic data. even including Men and 6Pgdh (Table 2). Ac- cording to Ohnishi et al. [6], they are dis- tinguishable by using an additional enzyme, Est-6 or Est-C. Therefore, if we performed a survey using four enzyme systems, Aldox, Gé pdh, Men and Est-6 (or Est-C), the 29 species concerned are wholly distinguishable. DISCUSSION Aquadro and Avise [10] and Ohnishi ef al. [6, 7] pointed out that two-dimensional electrophoresis (2DE) is a valuable tool in the study of systematics. A dendrogram based on the data obtained by 2DE appeared to be the most reliable for systematic considerations and is supported by data from interspecific hybridization and allozymes [6, 7]. The construction of a reliable dendrogram (Fig. 2) for 29 species of the D. montium species subgroup is meaningful because it is the first application of the 2DE technique to quantitative estimation of genetic relationships among several members of this subgroup. A survey of allozyme variability showed that four or five allozyme loci were useful for the identi- fication of species in this subgroup. The diagnostic enzymes detected by starch gel electro- phoresis (SGE) as well as proteins analyzed by 2DE are also important to the taxonomy of this subgroup in addition to conventional taxonomical traits (morphology). The importance of diagnostic enzymes has also been proposed by Ayala and Powell [14]. However, it is necessary for us to note one qualification concerning species identi- fication by diagnostic enzymes. If the detected allozyme locus is monomorphic within a species, there is no problem in making an identification. On the other hand, if the loci are polymorphic, the utility of this tool is subject to certain restrictions. Actually, we discovered one such case in cMdh of D. jambulina (see Table 2), even though there was a small sample (four flies for each species) for an investigation of enzymatic variation. Of course, it is necessary for us to carry out a more extensive survey in order to say whether the locus is polymorphic or not. In conclusion, the two biochemical techniques (2DE and SGE) were very helpful for studying the systematics of the D. montium species subgroup, along with morphological and karyological clas- sifications. In particular, 2DE is useful for the quantitative estimation of genetic distances between species, and the SGE for qualitative identification of species. ACKNOWLEDGMENTS We would like to thank Dr. R. A. Voelker of NIEHS, Research Triangle Park, NC, USA and an anonymous referee for their critical reading of this manuscript. Contribution No. 1540 from the National Institute of Genetics, Mishima, Japan. REFERENCES 1 Parshad, R. and Paika, I. J. (1964) Drosophilid survey of India. II. Taxonomy and cytology of the subgenus Sophophora (Drosophiia). Res. Bull. Panjab Univ., 15: 225-252. 2 Bock, I. R. and Wheeler, M. R. (1972) The Dro- sophila melanogaster species group. Univ. Texas Publ., No. 7213: 1-102. 3 Tsacas, L. and David, J. (1977) Systematics and biogeography of the Drosophila kikkawai-complex, with descriptions of new species (Diptera, Dro- sophilidae). Annls. Soc. ent. Fr. (N.S.), 13: 675-693. 4 Baimai, V. (1979) A new species of the Dro- sophila kikkawai complex from Thailand (Diptera: Drosophilidae). Pacific Insects, 21: 235-240. 5 Baimai, V. (1980) Metaphase karyotypes of cer- tain species of the Drosophila montium subgroup. Jpn. J. Genet., 55: 165-175. Systematics of Drosophila Ohnishi, S., Kim, K.W. and Watanabe, T. K. (1983) Biochemical phylogeny of the Drosophila montium species subgroup. Jpn. J. Genet., 58: 141-151. Ohnishi, S., Kawanishi, M. and Watanabe, T. K. (1983) Biochemical phylogenies of Drosophila: Protein differences detected by two-dimensional electrophoresis. Genetica, 61: 55-63. Watanabe, T. K., Matsuda, M., Ohnishi, S. and Hihara, F. (1982) Notes on systematics of Dro- sophila jambulina. Jpn. J. Genet., 57: 561-567. O'Farrell, P.H. (1975) High resolution two- dimensional electrophoresis of proteins. J. Biol. Chem., 250: 4007-4021. Aquadro, C. F. and Avise, J.C. (1981) Genetic divergence between rodent species assessed by using two-dimensional electrophoresis. Proc. Natl. Acad. Sci. U. S. A., 78: 3784-3788. Il 807 Sokal, R. R. and Sneath, P. H. A. (1963) ciples of Numerical Taxonomy. Francisco and London. Voelker, R. A., Langley, C. H., Leigh Brown, A. J., Ohnishi, S., Dickson, B., Montgomery, E. and Smith,S.C. (1980) Enzyme null alleles in natural populations of Drosophila melanogaster: Fre- quencies in a North Carolina population. Proc. Natl. Acad. Sci. U.S. A., 77: 1091-1095. Kimura, M. T. (1983) A new species of the Dro- sophila auraria complex, D. subauraria, from northern Japan (Diptera, Drosophilidae). Kontyd, 51: 593-595. Ayala, F. J. and Powell, J. R. (1972) Allozymes as diagnostic characters of sibling species of Dro- sophila. Proc. Natl. Acad. Sci. U.S.A., 69: 1094-1096. Prin- Freeman, San | —s deat abcinet caspase ve ao erie “l ‘Sth a fe 4 A = s ” ip ' ee 1 hi, ER | = a , iitihe Re 1 wid = ha veer edieed F i . | mi dte sail 4 “ f ' & wii 7 ue t { } i ' r ( | (e; ~~ & " a j i i fi % é ¥ ~~ —_" E i : angel a ‘ ‘e a ' f [ ” sie iis ie. 41ieeehs th ih \ ; 7 a, ‘a i a9 ‘ay soghetha em Bt | one ‘v2 ie . ; r > I — , = Ep hae io A a. } S a _ 7 ° aoe ? ZOOLOGICAL SCIENCE 1: 809-817 (1984) Paralepidapedon g.n. (Trematoda: Lepocreadiidae), with Descriptions of Metacercariae of Paralepidapedon hoplognathi (Yamaguti, 1938) comb. n. and of Two Other Species from Sea Urchins TAKESHI SHIMAZU and SHIGERU SHIMURA! Nagano-ken Junior College, 49-7 Miwa 8-chome, Nagano 380, and ‘Department of Fisheries, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT — Paralepidapedon g. n. (Trematoda: Lepocreadiidae) is erected to receive P. hoplognathi (Yamaguti, 1938) comb. n. (type species), P. antarcticum (Prudhoe et Bray, 1973) comb. n., P. dubium (Prudhoe et Bray, 1973) comb. n. and P. sebastisci (Yamaguti, 1938) comb. n. This genus is most closely related to Neolepidapedon Manter, 1954, but different from it in having the uroproct. Meta- cercariae of P. hoplognathi were found encysted in the sea urchins, Anthocidaris crassispina, Hemi- centrotus pulcherrimus and Pseudocentrotus depressus, at Misaki, Kanagawa Prefecture, and developed to sexual maturity in the fish, Oplegnathus fasciatus, in feeding experiments. The metacercarial and adult worms are described. The development, structure and phylogenetic importance of the cirrus pouch in the genus are discussed. In addition, simultaneously obtained metacercariae of Proctoeces maculatus (Looss, 1901) Odhner, 1911 (Fellodistomidae), from A. crassispina, Diadema setosum and H. pulcherrimus, and of an unidentified trematode from A. crassispina are briefly © 1984 Zoological Society of Japan described. Metacercariae of a lepocreadiid trematode were found in sea urchins. They were fed to fish, from which juvenile and adult worms were recovered. This trematode was identified as Neolepidapedon hoplognathi (Yamaguti, 1938) Manter, 1954. Close examination of these metacercarial and adult flukes and Yamaguti’s [1, 2] specimens of this species showed that the species represents a new lepocreadiid genus, which is proposed with it as the type species in this paper. MATERIALS AND METHODS Sea urchins, Anthocidaris crassispina, were collected from around the pier of the Misaki Marine Biological Station, University of Tokyo, Misaki, Kanagawa Prefecture, on several oc- casions from May 1980 to July 1983. They were crushed and examined organ by organ for parasites. Accepted April 16, 1984 Received February 3, 1984 In July 1983, a small collection of Diadema setosum, Hemicentrotus pulcherrimus and Pseudocentrotus depressus taken at the same locality was also examined. In order to obtain adult flukes, two experiments were conducted to feed metacercariae isolated from A. crassispina to fish, Oplegnathus fasciatus, by means of a gastric tube. In the first experiment, about 30 metacercariae each were given to three fish. One of them was autopsied 1 day later and the rest were examined 3 days later. In the second experiment, about 110 metacercariae were fed to one fish, followed by about 130 metacercariae 2 days later, and it was autopsied 3 days after the last feeding. The fish used were commercially supplied and had been kept in an aquarium before use for a few weeks after capture in the sea. Parasites were flattened, fixed in Schaudinn’s solution, stained with Heidenhain’s iron hematox- ylin or alum carmine and mounted in Canada balsam. Infected organs of hosts were made into serial paraffin sections and stained with hema- 810 T. SHIMAZU AND S. SHIMURA toxylin and eosin or with the PAS. Repre- sentatives of the specimens studied are deposited in the collection of the National Science Museum (Natural History), Tokyo. All measurements are given in millimeters. Specimens including the holotypes and paratypes of species hitherto assigned to the genus Neolepi- dapedon Manter, 1954, were restudied, whenever possible, on loan from the Meguro Parasitological Museum, Tokyo; the U.S. National Museum Helminthological Collection, Beltsville, Maryland, U.S. A.; and the Harold W. Manter Laboratory, University of Nebraska State Museum, Lincoln, Nebraska, U.S. A. RESULTS Results of surveys Encysted metacercariae of the trematode (NSMT-PI1 2625-2636) were found mostly in the gonads and rarely in the muscles of Aristotle’s lantern and ampullae of A. crassispina, H. pulcher- rimus and Ps. depressus. The first was much more heavily infected than the two others. On July 10, 1980, 29 individuals (43 to 62 mm in testa diameter) of A. crassispina were examined, and 22 or 76% of them harbored 1 to 66 (mean, 14.9) parasites in the gonads. Besides these metacer- cariae, those of two other species of trematodes were obtained, and they will be dealt with later (see pp. 815-816). Metacercaria (Figs. 1 and 2) Measurements based on 10 individuals. Cyst single-layered, globular to ellipsoidal, increasing in size to more than 0.9 in diameter with growth of worm in it, about 0.02 in thickness. Body fili- form, spinose, 1.14-4.80 long by 0.20—0.66 wide. Eye-spot pigment absent. Oral sucker funnel- shaped, subterminal, 0.09-0.30 long by 0.07-0.32 wide. Prepharynx long, 0.24-0.60 long. Pharynx 0.06-0.18 long by 0.05—0.14 wide. E- sophagus very short, 0.02-0.12 long, one-fifth to -fourth of prepharynx length. Intestinal bifur- cation near posterior one-third of forebody. Intestinal ceca a little undulating, containing red- dish brown material, opening into excretory vesicle to form uroproct near posterior end of body. Ventral sucker 0.08—0.26 long by 0.09-0.25 wide, equatorial or slightly anterior to it; sucker width ratio 1:0.81-1.22. Reproductive organs fairly well differentiated, similar to but less developed than those of sexually immature specimens found in the fish in the second experiment as will be described later. Testes postovarian, tandem, separated, less than 0.20 in diameter. Ovary 3- to 4-lobed, small, near middle of hindbody, less than 0.06 in diameter. Vitellaria not developed. Excretory vesicle tubular, reaching to ovary; main collecting canals extending laterally to near pharynx; flame cells numerous, formula not worked out; pore terminal. Results of feeding experiments (Figs. 3 and 4) In the first experiment, three juvenile worms (NSMT-PI 2637) were found in the small intestine of the fish 1 day after feeding. Morphologically, they agreed well with the foregoing metacercaria. From the small intestine of one of the two fish examined 3 days after feeding, two gravid worm with several eggs in the uterus (NSMT-PI 2638) were recovered. No worm was detected in the other fish. The morphology (Fig. 3) and measure- ments of these two adults were as follows. Fics. 1-5. Fic. 1. Paralepidapedon hoplognathi (Yamaguti, 1938) comb. n. Encysted metacercaria from Anthocidaris crassispina. Fic. 2. Excysted metacercaria from A. crassispina, entire body (a) and posterior part of body (b), ventral view. Fic. 3. terminal genitalia, ventral view. Fic. 4. lia and ovarian complex, dorsal view. Fic. 5. Gravid worm recovered from Oplegnathus fasciatus 3 days after feeding of metacercariae, male Juvenile worm recovered from O. fasciatus (3 days after feeding of metacercariae?), terminal genita- Yamaguti’s specimen (MPM Coll. No. 22111) from O. fasciatus, posterior part of body, ventral view. Fic. 6. Yamaguti’s specimen (MPM Coll. No. 22113) of P. sebastisci (Yamaguti, 1938) comb. n. from Sebastes inermis, male terminal genitalia, ventral view. Scale bars: 1=0.3mm; 2a=1mm; 2b=0.1 mm; 3, 4, 6=0.2 mm; 5=0.1 mm. 811 Metacercaria of Paralepidapedon hoplognathi A ks a 812 T. SHIMAZU AND S. SHIMURA Body slender, armed with scalelike spines, 2.64-3.80 long by 0.52-0.60 wide. Oral sucker 0.10-0.16 long by 0.14 wide. Prepharynx 0.22- 0.38 long. Pharynx 0.12-0.14 long by 0.14 wide. Esophagus very short, 0.04-0.06 long. Intestinal ceca thick, bifurcated about posterior third of forebody. Uroproct present. Ventral sucker located about anteror two-fifth of body, 0.19—0.20 long by 0.20-0.21 wide; sucker width ratio 1: 1.39-1.59. Testes globular, tandem, separate, postovarian, 0.22-0.32 long by 0.22-0.28 wide. Vasa efferentia running forward at first apart from each other and then contiguously in preovarian region. Seminal vesicle large, sinuous, lying free in parenchyma but leaving its distal parts in cirrus pouch, in about anterior one-fourth of hindbody, with well-developed inner circular and outer longitudinal, probably sphincter muscles in its distalmost part. Pars prostatica ovoid, large, in cirrus pouch. Prostatic cells present both in cirrus pouch and around seminal vesicle outside cirrus pouch. Ejaculatory duct very shori, in cirrus pouch. Cirrus practically absent. Only anterior thick-walled portion of cirrus pouch seen, claviform, slightly longer than ventral sucker. Genital atrium small, shallow. Genital pore just anterosinistral to ventral sucker, with small gland cells around it. Ovary 4-lobed, median, in middle of hindbody or a little anterior to it, 0.14-0.24 long by 0.19-0.24 wide. Seminal receptacle elongate- oval, posterodorsal to ovary, 0.04-0.05 long by 0.02-0.04 wide. Laurer’s canal postovarian, submedian. Ootype-complex lateral to ovary. Uterus intercecal, between ovary and ventral sucker; metraterm weakly developed, smooth, without a valvelike muscular structure just in front of its aperture at base of genital atrium. Eggs operculate, not embryonated, 0.056-0.060 by 0.040. Vitelline follicles distributed from posterior end of body to seminal vesicle. Excretory vesicle tubular, extending to ovary; pore terminal. In the second experiment, seven worms (NSMT-— P] 2639) were found in the small intestine of the fish. They were 2.46 to 3.20 mm long by 0.50 to 0.70 mm wide and almost mature but not gravid, with fairly well-developed genital organs. The cirrus pouch was longer than the ventral sucker, composed of the anterior thick-walled and the posterior thin-walled portion, and still enclosed a long tubular seminal vesicle, the pars prostatica, prostatic cells and ejaculatory duct; sometimes a weak constriction was seen on the border between the two portions (Fig. 4). Neither vas deferens nor external seminal vesicle was observed. The vitelline glands were differentiated from the posterior end of the body to the midlevel between the ovary and the ventral sucker. DISCUSSION Neolepidapedon hoplognathi was described first as Lepidapedon hoplognathi from the small intestine of Hoplognathus punctatus [=Oplegnathus punctatus] taken in Suruga Bay [1]. It was also recorded from H. fasciatus [=O. fasciatus] at Hamazima, Mie Prefecture [2]. Later, it was transferred from the genus Lepidapedon Stafford, 1904, to a new genus, Neolepidapedon Manter, 1954 [3]. The flukes recovered in the present experiments grew and developed to sexual maturity with day after feeding. Their morphology was closely paralleled to that of the present metacercaria. The fish used had been held in captivity for a few weeks before use. Therefore, the flukes are considered to have been of experimental infection. The present trematode agrees well with N. hoplognathi [1,2] in all morphological respects except in the presence of a uroproct and structure of the male terminal genitalia. Yamaguti [1] observed that the intestinal ceca terminated at the extreme posterior end of the body and that both the internal and the external seminal vesicle were present. However, restudies of the type specimens (MPM Coll. No. 22376) and his [2] ones (MPM Coll. No. 22111) showed that a uroproct was present in them (Fig. 5) and that their male terminal genitalia were identical in structure with those in the present fluke. What he called the oval vesicula seminalis interna proved to be the muscular dis- talmost part of the seminal vesicle, with an expanded lumen, located in the anterior portion of the cirrus pouch. Therefore, the present trematode is identified as N. hoplognathi. This species most closely resembles Neo- lepidapedon but differs from it in having the uro- Metacercaria of Paralepidapedon hoplognathi 813 proct. This difference is considered of generic significance. Consequently, a new genus, Para- lepidapedon, is created to accomodate the species as type species, along with three others at present. The diagonosis of this genus is given below. Paralepidapedon g. n. Generic diagnosis Lepocreadiidae: Lepidapedinae. Body elon- gate, spinose. Eye-spot pigment not seen even in metacercariae. Prepharynx usually long. Phar- ynx present. Esophagus short or not. Intestinal ceca opening into excretory vesicle to form uro- proct with terminal pore. Ventral sucker com- paratively small, in anterior half of body. Testes tandem, in posterior half of body. Greater part of seminal vesicle convoluted, free in parenchyma in the adult stage, surrounded by prostatic cells, with well-developed, probably sphincter muscles in distal end. No external seminal vesicle present. Cirrus pouch consisting of anterior thick-walled and posterior thin-walled portions and including seminal vesicle, pars prostatica, prostatic cells and ejaculatory duct in young specimens; only the former portion seen in adults, containing distal parts of seminal vesicle, pars prostatica, a number of prostatic cells and ejaculatory duct. Cirrus practically absent. Genital atrium small. Genital pore anterolateral to ventral sucker. Ovary median, pretesticular. Seminal receptacle and Laurer’s canal present. Uterus _preovarian, intercecal; metraterm not spinose, with or without valvelike muscular structure just in front of its aperture at base of genital atrium. Vitelline follicles extensive, confined to hindbody. Eggs operculate, not embryonated. Excretory vesicle tubular. Intestinal parasites of marine teleosts. Encysted metacrecariae known from sea urchins. Type species: P. hoplognathi (Yamaguti, 1938) comb. n. (synonyms: Lepidapedon hoplognathi Yamaguti, 1938; Neolepidapedon hoplognathi (Yamaguti, 1938) Manter, 1954). Other species: P. antarcticum (Prudhoe et Bray, 1973) comb. n. (synonym: N. antarcticum Prudhoe et Bray, 1973). P. dubium (Prudhoe et Bray, 1973) comb. n. (synonym: N. dubium Prudhoe et Bray, 1973). P. sebastisci (Yamaguti, 1938) comb. n. (synonyms: L. sebastisci Yamaguti, 1938; N. sebastisci (Yamaguti, 1938) Manter, 1954). Manter [3] did not mention the nature of the intestinal ceca either in the description of N. polyprioni Manter, 1954, the type species of Neolepidapedon, or in the diagnosis of this genus. However, his Figure 7 shows the ceca terminating blindly in this species. Restudy of a paratype of the species (MPM Coll. No. 22385) failed to confirm it because the posterior parts of the ceca were poorly stained and obscured by the vitellaria. Professor Mary Hanson Pritchard kindly re- examined on behalf of us two other paratypes deposited in the collection of the Harold W. Manter Laboratory and informed us that the ceca ended blindly in them. Yamaguti [4] listed 14 species under WNeo- lepidapedon. Since then, seven species have been named in the genus: antarcticum, dubium, helicoleni and trematomi Prudhoe et Bray, 1973 [5]; Aisti- opteri Korotaeva, 1975 [6]; belizense Fischthal, 1977 [7]; and israelense Fischthal, 1980 [8]. Among these 21 species, the following three besides hoplognathi (now P. hoplognathi) possess the uro- proct: antarcticum [5], dubium [5] and _ sebastisci Yamaguti, 1938 [2]. The first two were found in the pyloric ceca of the same host, Coryphaenoides whitsoni, from the Antarctic Sea [5]. Their de- scriptions [5] suggest that the structure of their male terminal genitalia is similar to that in P. Consequently, they also should be was hoplognathi. placed in Paralepidapedon. N. described first as Lepidapedon Yamaguti [1] from the small intestine and pyloric ceca of “Sebastiscus albofasciatus from Nagasaki and Suruga Bay.”” The type material (three slides) of this species was restudied. A slide (the holo- type and a paratype; MPM Coll. No. 22377) was labelled ‘“‘Opechona sebastisci n. sp.; small intestine; kasago [=S. marmoratus]; Nagasaki; sebastisci sebastisci by 814 T. SHIMAZU AND S. SHIMURA 26/4 35.” Another slide (four paratypes and a specimen of Plagioporus isaitschikowi identified by him; MPM Coll. No. 22112) was _ labelled ““Opechona sebastisci; upper part of small intestine and pyloric ceca; ayamekasago [=S. albofasciatus]; Numazu; 13/4 35.” A third slide (a paratype; MPM Coll. No. 22112) was labelled ““Opechona sebastisci; upper part of small intestine; ayame- kasago; Numazu; 13/4 35.” The generic name Opechona was corrected to Lepidapedon on their labels by Shunya Kamegai on March 15, 1972. It seems likely that the type host is not S. albofasciatus but S. marmoratus and the type locality is Nagasaki. He [2] also recorded the species from ‘“‘the intestine of Sebastiscus marmoratus Cuv. et Valenc. at Hamazima, Mie Prefecture.’ His slide (six specimens; MPM Coll. No. 22113) was labelled “‘Lepidapedon sebastisci; stomach and small intestine; mebaru [=Sebastes inermis]; Hamazima; 12/4 39.”’> He may have made a mistake in description of the host name. Later, Manter [3] transferred the species from Lepidapedon to Neolepidapedon, presumably with- out knowing the presence of the uroproct in the species [2]. Reexamination of Yamaguti’s [1, 2] specimens confirmed it and revealed that the male terminal genitalia in them (Fig. 6) was identical in anatomy with those in P. hoplognathi. The valvelike muscular structure in the genital atrium, which Yamaguti [1, 2] had not referred to, was observed. Therefore, the species also should be allocated to Paralepidapedon. P. hoplognathi is distinguishable from the three other species by having the prepharynx being much longer than the very short esophagus and by lacking the valvelike muscular structure in the genital atrium. The latter three are very like. P. sebastisci may be separated from the two others by a larger sucker width ratio, which was 1: 1.28 to 1.68 in nine better prepared ones of Yamaguti’s specimens re- examined. P. dubium is said to differ from P. antarcticum in having the somewhat smaller size of body, oral sucker being distinctly smaller than the ventral sucker and definitely larger eggs [5]. As for the remaining 16 species in Neolepidapedon the exact nature of the intestinal ceca in them is not necessarily clear. The descriptions or figures indicate the blindly ending ceca in cablei Manter, 1954 [3]; dollfusi Durio et Manter, 1968 [9]; epinepheli Siddiqi et Cable, 1960 [10]; helicoleni [5]; histiopteri [6]; hypoplectri Nahhas et Cable, 1964 [11]; israelense [8]; macrum Overstreet, 1969 [12]; pugetense (Acena, 1947) Yamaguti, 1971 [13]; retrusum (Linton, 1940) Sogandares-Bernal et Hutton, 1960 [14]; and trematomi [5]. The descriptions and figures omit details of the ceca in belizense [7]; equilatum Siddiqi et Cable, 1960 [10]; medialunae Montgomery, 1957 [15]; mycteropercae and trachinoti Siddiqi et Cable, 1960 [10]. Restudy of four paratypes of cablei (HWML 0814), a paratype of dollfusi (HWML 0608), the holotype and a paratype (?) of epinepheli (USNM Helm. Coll. No. 39381), a paratype of hypoplectri (HWML 21631), and the holotypes of israelense, macrum, mycteropercae and _ trachinoti (USNM Helm. Coll. Nos. 75002, 71305, 39383 and 39380, respectively) ascertained that the ceca ended blindly in these species. Therefore, they are retained in Neolepidapedon. The species macrum is very like mycteropercae and appears to be synonymous with it. The holotypes of belizense, equilatum, medialunae and pugetense (USNM Helm. Coll. Nos. 74164, 39382, 38194 and 59587, respectively) and three paratypes of retrusum (USNM Helm. Coll. No. 8274) were reexamined, but it was impossible to trace the full extent of the ceca in them because the posterior parts of the ceca were hidden by the vitellaria. The species, helicoleni, histiopteri, pugetense, retrusum and trematomi, may also be preserved in the genus. The position of belizense, equilatum and medialunae remains indeterminate. The present study demonstrates that the cirrus pouch in P. hoplognathi changes in structure as worms develop. During stages of development earlier than the adult stage, it was elongated, consisted of the anterior thick-walled and the posterior thin-walled portion, and included the seminal vesicle, pars prostatica, prostatic cells and ejaculatory duct. In the adult stage, on the other hand, only its anterior portion was seen containing the distal parts of the seminal vesicle, pars pro- statica, a number of the prostatic cells and the ejaculatory duct; the greater part of the seminal vesicle and most of the prostatic cells were situated free in the parenchyma. It is suggested that, about Metacercaria ‘of Paralepidapedon hoplognathi 815 the time when worms attain sexual maturity, the posterior portion atrophies and eventually disap- pears, or bursts under too great pressure of the developing and enlarging seminal vesicle from the inside and wrinkles to become practically invisible, and that, as a result, the greater part of the seminal vesicle and most of the prostatic cells come to lie free in the parenchyma. It is as if this part of the seminal vesicle were a true external seminal vesicle. The same may be said of the other members of Paralepidapedon and of those of Neolepidapedon because the present reexamination of many of them showed that the cirrus pouch was thick and lacked a well-defined posterior end in them. The unde- veloped cirrus pouch in the earlier stages in Paralepidapedon recalls Manter’s [3] statement, “It [a membraneous sac surrounding the gland cells and the posterior portion of the seminal vesicle in Lepidapedon] is separated by a deep constriction or by a narrow isthmus from a more anterior, typical cirrus sac containing a seminal vesicle, prostatic cells, and cirrus. Its membrane is apparently continuous with the wall of the cirrus sac, and it is probably more correct to describe the cirrus sac as divided into two distinct portions.” This cirrus pouch is identical with the undeveloped one in Paralepidapedon. As to the cirrus pouch, this type is regarded as primitive, and that in the adult stage of Paralepidapedon and Neolepidapedon secondary. These three genera are very like in all features except in the structure of the cirrus pouch and in the presence or absence of the uroproct. It is possible that Neolepidapedon was first de- scended from Lepidapedon and then Paralepi- dapedon originated from the former with acqui- sition of the uroproct. The family Lepocreadiidae (Odhner, 1905) Nicoll, 1935, comprises a large number of described genera [4]. It has long been said that an external seminal vesicle with or with- out the prostatic cells around it is present in some of them but absent in others. Evidently, however, reexamination of the structure and formation of the so-called external seminal vesicle in them is needed. This is the first paper to report the metacercaria of P. hoplognathi from the sea urchins. At Misaki, metacercariae of a trematode were re- corded from the ovary of about 25% of Mespilia globulus taken in 1930 to 1931 [16]. Since this record is without any morphological description or figure, it is quite impossible now to identify this trematode. No individuals of this sea urchin could be collected around the Station in the present study. Other helminth parasites (1) Metacercariae of Proctoeces maculatus (Looss, 1901) Odhner, 1911 (Trematoda: Fellodistomidae) Unencysted metacercariae were found in the gonads of A. crassispina (two specimens, NSMT-— P|] 2640), D. setosum (two specimens, NSMT-PIl 2641) and H. pulcherrimus (one specimen, NSMT-— P] 2642). The incidence of infection of each of these sea urchins was not recorded. The mor- phology and measurements of the five meta- cercariae were: body immature, 1.80-4.20 long by 0.44-1.00 wide; oral sucker 0.26—-0.56 long by 0.26-0.54 wide; pharynx 0.16—0.32 long by 0.12- 0.34 wide; ventral sucker 0.36—0.76 long by 0.40— 0.86 wide; sucker width ratio 1: 1.40-1.81; testes 0.10—0.44 in diameter; cirrus pouch 0.28—1.00 long; ovary rounded or trilobed, 0.06—0.30 in diameter. (2) Metacercariae of an unidentified trematode (Figs. 7 and 8) Encysted metacercariae were found in the gonads of A. crassipsina (NSMT-PI 2643-2645). The incidence and intensity of infection were not recorded. The morphology (Figs. 7 and 8) and measurements (based on five specimens) were: cyst globular, single-layered, 0.16-0.25 by 0.18— 0.25 in size, 0.004—0.008 in thickness; body delicate, oval, smooth, 0.28-0.34 long by 0.12-0.16 wide; eye-spot pigment absent; oral sucker subterminal, 0.05—0.07 long by 0.06—0.08 wide; prepharynx 0.02 long; pharynx 0.03-0.05 long by 0.03-0.04 wide; esophagus 0.02-0.04 long; intestinal ceca bifurcated about halfway between two suckers, ending blindly at some distance from posterior end of body; ventral sucker large, a little postequatorial, 0.13- 0.17 long by 0.13-0.20 wide; sucker width ratio 1: 1.97-2.45; testes symmetrical, small, located near posterior ends of ceca; other gential organs (ovarian complex, uterus and terminal genitalia) located median between excretory vesicle and 816 AW Ce = 5 100 Hymenolepis Kesennuma-shi, Miyagi Pref. rashomonensis 5) Mase-d6 Dec. 4 2, 1 50 H. rashomonensis 6) Ddnomoto-d6 May 29 3 y 67 H. rashomonensis Toéei-ché, Aichi Pref. 7) Jigoku-ana May 29 1 1 100 HA. rashomonensis Shidara-cho, Aichi Pref. 8) Disused air raid shelter, No. 1 Nov. 19 y, 2 100 HA. rashomonensis Obama-shi, Fukui Pref. 9) Disused air raid shelter, No. 2 Nov. 19 2 0 0 10) Shiraishi-d6 Nov. 19 5 4 80 H. rashomonensis 13) Abandoned mine, No. 2 Nov. 19 1 1 100 H. rashomonensis 15) Koyo abandoned mine May 21 2 1 50 A. rashomonensis Nishiyoshino-mura, Nara Pref. 16) Shinsen-d6 Dec. 14 D 2 100 H. rashomonensis Tenkawa-mura, Nara Pref. 17) Komori-no-kutsu Dec. 14 1 0 0 Tenkawa-mura, Nara Pref. 18) To6r6-no-kutsu Dec. 14 D, 0 0 Tenkawa-mura, Nara Pref. 20) Muso6-d6 Jan. 16 2 0 0 Kamikitayama-mura, Nara Pref. 21) Akakura abandoned mine Apr. 29 1 1 100 H. rashomonensis 23) Kawamata abandoned mine Nov. 8 1 1 100 A. rashomonensis 24) Ryujin-d6 Nov. 5 2 1 50 H. rashomonensis 25) Itadani gongen-d6 Nov. 5 2 1 50 H. rashomonensis Fukue-mura, Tottori Pref. 26) K6jiro-no-ana July 28 5 y 67 H. rashomonensis 27) Sanmy6dji old mound Nov. 6 1 0 0 Sanmy6ji-ch6, Tottori Pref. 28) Oo-ana July 27 7 3 43 H. rashomonensis Shimane-ch6, Shimane Pref. 30) Inome-ana Mar. 29 2D 2 100 H. rashomonensis Inome-ch6o, Shimane Pref. 31) Sea eroded cave Mar. 29 4 4 100 H. rashomonensis Inome-ché, Shimane Pref. 32) Sea eroded cave Aug. 30 yy yy 100 H. rashomonensis 33) Mikomori-ana Mar. 29 D, 2 100 A. rashomonensis Nima-cho, Shimane Pref. Cestodes of Bats from Japan TABLE 1. (Continued) 823 Host species Cave and locality 35) Underground raceway 39) Disused tunnel Kamiyaku-ché, Kagoshima Pref. 40) Abandoned charcoal kiln Yaku-ch6, Kagoshima Pref. Vespertilionidae (5) (6) (7) (8) (9) (10) Miniopterus schreibersii fuliginosus 11) Water tunnel Kaminaka-cho, Fukui Pref. 29) Komori-ana Shimane-ch6é, Shimane Pref. 34) Hiradokogawa abandoned mine Miniopterus schreibersii blepotis 45) Otomi-daini-d6 Myotis macrodactylus 11) Water tunnel 19) Suish6-no-kutsu 21) Akakura abandoned mine 28) Oo-ana 35) Underground raceway 36) Underground raceway Kotonami-cho, Kagawa Pref. 38) Tunnel Nakatane-ch6, Kagoshima Pref. Plecotus auritus sacrimontis 15) Koyo abandoned mine 16) Shinsen-d6o 19) Suishé-no-kutsu 21) Akakura abandoned mine 22) Kose abandoned mine Tenkawa-mura, Nara Pref. Vespertilio namiyei 37) Sea eroded cave Otsukue-island, Fukuoka Pref. Vespertilio orientalis 1) Temmadate shrine Temmabayashi-mura, Aomori Pref. * Serial No. of localities shown in Fig. 1. Date of collection Mar. 28 Aug. 29 Aug. 29 Aug. 7 Aug. 21 Number of bats examined infected 10 IZ A Ano — — NN S| SS SS eS (Sh ere Si S&S wee GG OG & Cestode species 50. H. nishidai 100 H. nishidai 40 H. rashomonensis 10 V. hidaensis 25 V. hidaensis 20 V. wakasensis Spi i: (SP (SP (a=) & \o V. tanegashimensis sp. n. V. sp. \o Zi [O76 S&S ‘© 5 unidentified (larva) 100 V. multihamata 824 I. SAWADA Fia. 2. b: Rostellar hooks. a: Scolex. gashima), Kagoshima Prefecture; August 27, 1983. Type specimens: Holotype NUE Lab. Coll. No. 8301; paratype NUE Lab. Coll. No. 8302. Remarks: The present new species most closely resembles V. multihamata Sawada, 1967 [1] from the Oriental frosted bat Vespertilio orientalis in the number and the length of rostellar hooks. However, it differs from V. multihamata in that the strobila is longer (42-46 vs. 20-25); the scolex is larger (0.277 by 0.457 vs. 0.105-0.119 by 0.246— 0.280); the neck is slender (2.5 vs. absent); and the testes are larger (0.207—0.235 by 0.235-0.305 vs. 0.046-0.049 by 0.077). This is the first cestode to be reported from M. macrodactylus in Japan [2-5]. Vampirolepis wakasensis sp. n. (Fig. 3) Five bats, Myotis macrodactylus, were collected in a disused water tunnel at Kaminaka-ch6é, Fukui Prefecture, on November 20, 1983. One of them Vampirolepis tanegashimensis sp. n. c: Mature proglottid. Scales in mm. was found to be infected with two gravid speci- mens of this cestode. Description: | Medium-sized hymenolepidid; mature strobila 91-96 in length and 1.6-1.8 in maximum width. Strobila margins slightly serrate. All proglottides wider than long. Scolex round when the rostellum is invaginated, 0.280-0.315 by 0.385—-0.399, not sharply demarcated from neck. Rostellum 0.105—0.112 by 0.133-0.140, armed with a crown of 42 Y-shaped hooks, each measuring 0.035 in length. Hook handle long; guard bluntly round at its end, shorter than blade. Suckers discoidal, unarmed 0.112—0.126 in diameter. Neck region behind scolex 1.5-1.8 long by 0.36—0.41 wide. Genital pores unilateral, located a little anterior to middle of proglottid. Testes three in number, spherical, 0.112-0.133 by 0.119-0.140, situated in posterior field of proglottid, arranged in a trans- verse row or in triangular position, one poral and two aporal. Cirrus sac pyriform, 0.147—-0.189 Cestodes of Bats from Japan 825 b 0.02 - . ” * 5 Ye fea 2 . Fic. 3. Vampirolepis wakasensis sp. n. a: Scolex. b: Rostellar hooks. c: Mature proglottid. d: Egg. Scales in mm. long and 0.042 wide, occupied by internal seminal vesicle measuring 0.070—-0.084 by 0.034—0.040. Duct from external seminal vesicle to cirrus sac forming a loop. External seminal vesicle, ellip- soidal, 0.105—0.140 by 0.049-0.056. Ovary trans- versely elongated and digitate in mature proglottid, 0.490-0.518 wide. Vitelline gland distinctly trilobated, 0.196-0.224 by 0.084-0.105, situated near midline in space between first and second testes in posterior field of proglottid. Vagina opening in genital atrium, extending to median field, posterior to cirrus sac. Seminal receptacle 0.154-0.180 by 0.084—-0.098, situated anterior to poral testis. Uterus arising directly from ovarian lobes as a lobe sac, gradually enlarging, filling all available space in proglottid. Numerous eggs present in uterus, spherical or oval, 0.053—0.056 in diameter, surrounded by four envelopes, outermost chorion thick, with smooth surface. Onchosphere spherical, 0.032 in diameter; embryonic hooks 0.014 long. Type host: Myotis macrodactylus. Site of infection: Small intestine. Type locality and date: Kaminaka-ché, Fukui Prefecture; November 20, 1983. Type specimens: Holotype NUE Lab. Coll. No. 8303, paratype NUE Lab. Coll. No. 8304. Remarks: The present species most closely 826 I. SAWADA Kl Ss Wy > E ~= baw, | ‘a ive ORE lee et of, Ga eee a ee ik Eh see * a ' i p>) z : ; ‘ 5 F 7s) x 7 . a a : . < Pye :. ‘ ie ; Ae oo ‘o gaurd? iaiinish : ‘ it Perse om 4 | ; a ; . | : | : | ; : J 3 J ; ; ; 3 ; NG gs ~ x u: Yh ; ee - < aaa r : . : Scar TR 7 = i fie j ii nf ; \ } : J a4 o . : a * ’ ay f: Tf ; n ‘ = : j i Ge = e { 7 , ; , \ P xy t , i i this \ re ; i By 4 ‘ vA i J a le " % ZOOLOGICAL SCIENCE 1: 829-832 (1984) [COMMUNICATION] © 1984 Zoological Society of Japan Neuronal Pathways from the Tectal “Snapping-Evoking Area” to the Tongue-Muscle-Controlling Motoneurons in the Japanese Toad: Evidence of the Intervention of Excitatory Interneurons MASAHIKO SATOU, TOSHIYA MATSUSHIMA and KAzUoO UEDA Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan ABSTRACT — Using intracellular recording tech- niques in paralyzed Japanese toads, tongue-muscle- controlling motoneurons were identified antidromically and neuronal pathways from the tectal “snapping- evoking area’ (the ventrolateral part of the optic tectum) to these motoneurons were searched. The tongue-muscle-controlling motoneurons _ generally showed hyperpolarizing potentials in response to a single electrical stimulus applied to either side of the “snapping-evoking area.’ On the other hand, large and long-lasting excitatory postsynaptic potentials (EPSPs) which prevailed over the hyperpolarizing potentials were evoked in response to repeated elec- trical stimuli at short intervals, i.e., a temporal facilita- tion of EPSPs was observed. In some motoneurons tested, these EPSPs were further spatially facilitated; i.e., the EPSPs were facilitated when electrical stimuli were applied bilaterally to the “‘snapping-evoking area’. These results strongly suggest (/) that exci- tatory interneurons intervene in the _ tecto-bulbar pathways from the “‘snapping-evoking area” to the tongue-muscle-controlling motoneurons, (2) that the bilateral descending volleys from this area converge on these interneurons, and (3) that temporal and spatial facilitation of spike discharges occurs in these interneurons. From these results, it is proposed that temporal and spatial integration of synaptic inputs in premotor interneurons plays an important role in motor-pattern generation for prey-catching. It has been hypothesized that the anuran optic tectum carries ““command signals” for visually- Accepted May 18, 1984 Received April 12, 1984 guided prey-catching behavior such as “‘turning’’, ““fixating’’, and “‘snapping” (for reviews see refs. [2, 4]). These tectal commands are supposed to activate, through descending tectal efferents, the ‘““motor-pattern generators” for prey-catching in the bulbo-spinal regions [13]. In agreement with this supposition, various types of tectal neurons linked to visual prey/non-prey analysis have been activated antidromically by electrical stimuli applied to the tecto-bulbar/spinal pathways in the caudal medulla [9]. Although there have been attempts to elucidate neural circuits of the prey-catching motor-pattern generators [3, 4, 12, 13], detailed neurophysiological analyses are scarce. As a first step in analysis, we have paid special attention to the tongue-flipping movement of the prey-catching sequence in the Japanese toad [7] and have searched in the present experiments for neuronal pathways activating the tongue- muscle-controlling motoneurons from the tectal ““snapping-evoking area’. It will be shown that interneurons, on which descending tectal volleys of both sides converge, mediate excitatory synaptic transmission from the tectal “‘snapping-evoking area” to the tongue-muscle-controlling moto- neurons. MATERIALS AND METHODS Twelve female Japanese toads (Bufo japonicus) 830 (body weight, 160-335 g) were used as experi- mental materials. They were anesthetized with ethyl m-aminobenzoate methanesulfonate (25 mg/ 100 g of body weight) and later paralyzed with d-tubocurarine chloride (6mg/100 g of body weight) or succinylcholine (0.8-1.2 mg/100 g of body weight). Electrical stimuli (duration: 0.1 msec; supramaximal intensity) were applied to the branches of the hypoglossal nerve innervating the tongue-protracting and tongue-retracting muscles, respectively, through bipolar silver-wire electrodes. In addition, both sides of the ventrolateral part of the optic tectum (the ‘“‘snapping-evoking area’’ [1]), where we could often elicit a ““snapping”’ response by repeated electrical stimuli in freely moving Japanese toads [7], was stimulated through bipolar stainless steel electrodes. The parameters of electrical stimuli (duration: 0.5 msec; intensity: 90-160 A; interval: 10 msec) were of the same range as those used in the above-mentioned M. SATou, T. MATSUSHIMA AND K. UEDA experiments on freely moving toads. Glass micropipettes filled with 2M of K-citrate were used for intracellular recording. Recorded po- tentials were displayed on an oscilloscope by the use of a conventional preamplifier designed for recording and passing currents through single microelectrodes. RESULTS Thirty-three tongue-protractor motoneurons (PMNs) and 15 tongue-retractor motoneurons (RMNs) - were analyzed intracellularly. They were identified antidromically by stimulating the nerve branches innervating respective muscles. Figure 1A shows a typical example of the responses of a PMN to electrical stimuli applied to the tectal “‘snapping-evoking area”. The antidromic spike response is shown in Da. Aa-—Ad show the responses to 1-4 ipsilateral tectal GOT) stimuli, ior © iOT D | iOT : cOT a eR Ee ne ao ON nee a ee ail a a ‘ (ee a a: CSS A eS at , OT cOT b ietous va 62) ee ate b a ie Th ameneciaie caneail b Ty ne, CU eae see aoe iOT ¢ 5 KITT g fr jl rae s elas: asi ‘é ei ; cOT aoe cOT 5 mv (AC) Wr " f | 50 mV (D) c € f — ee b 40ms (A-C) 4 MM 10ms (p) d/; is hr = it Fic. 1. Intracellular responses of tongue-muscle-controlling motoneurons to electrical stimuli applied to the tectal “‘snapping-evoking area’’ (ventrolateral part of the optic tectum). Upper traces in A-C show intracellular responses of the motoneurons; lower traces show extracellular field potentials just outside the impaled cells. Aa-—Ad show, respectively, the responses of a PMN to 1, 2, 3, and 4 successive electrical stimuli at 10-msec intervals applied to the iOT; Ae—Ah show, respectively, the responses to 1, 2, 3, and 4 successive stimuli (10-msec intervals) applied to the cOT. Ba and Bb show the responses of a PMN to 2 iOT and 2 cOT electrical stimuli, respectively, at 10- msec intervals. Bc shows the response when iOT and cOT stimuli were simultaneously applied. The arrowheads in Ba and Bb show EPSPs superimposed on large hyperpolarizing potentials. Ca and Cb show the responses of an RMN to 4 iOT and 5 cOT electrical stimuli, respectively, at 10-msec intervals. Cc shows the responses when the iOT and cOT stimuli were simultaneously applied. Da-De show the antidromic action potentials of cells A-—-C, respectively. The dots indicate the time of electrical stimulation. Toad’s Snapping Pathway 831 respectively. The response to a single iOT stimulus was mainly hyperpolarizing in direction and lasted for about 160 msec (Aa). In response to 2 successive stimuli of the same intensity and of a short interval (10 msec), depolarizing potentials appeared during the peak of hyper- polarization (Ab). These depolarizing potentials were much larger and lasted for a longer period of time when 3 or 4 successive stimuli were ap- plied (Ac, Ad). Bursts of discharges were often superimposed on the facilitated depolarizing potentials. Thus, temporal facilitation of the depolarizing potentials was seen in this cell. Basically similar results were obtained in response to contralateral tectal (COT) stimuli (Ae—Ah). Such temporal facilitation of the depolarizing potentials (presumably EPSPs; see below) was a common observation in all of the PMNs and RMNs so far tested. In order to determine the properties of facilitated depolarizing potentials, the effect of a polarizing current injection on these potentials was examined. The amplitude of the facilitated depolarizing potentials became larger as the hyperpolarizing currents increased, and became smaller as the depolarizing currents increased. These results suggest that facilitated depolarizing potentials are facilitated EPSPs. In some PMNs, as well as RMNs, tested, “spatial” facilitation of the EPSPs, in addition to the temporal facilitation mentioned above, was observed. Figure 1B and Figure 1C show typical examples of responses of a PMN and an RMN, respectively. The antidromic responses are shown in Db and Dc, respectively. In the B cell (PMN), the intensity of iOT(Ba) and cOT (Bb) stimuli was adjusted so that EPSPs of a small amplitude (indicated by the arrowheads in Ba and Bb), which were superimposed on the large hyper- polarizing potentials, were evoked in response to 2 successive stimuli at 10-msec intervals. EPSPs of a much larger amplitude were evoked when iOT and cOT stimuli were simultaneously applied (Bc). The amplitude of the EPSPs exceeded the sum of those evoked solely by iOT (Ba) and cOT (Bb) stimuli. Thus, “spatial” facilitation of EPSPs was observed in this cell in addition to temporal facilitation (not illustrated). In the C cell (RMN), 4 or 5 successive stimuli of 10-msec intervals were applied to iOT (Ca) and cOT (Cb), respectively. Spatial facilitation of EPSPs was also observed in this cell when iOT and cOT stimuli were simultaneously applied (Cc). DISCUSSION Since the descending tectofugal efferents pass mainly through the “‘snapping-evoking area’”’ (the ventrolateral part of the optic tectum) [8, 11], it is likely that the present electrical stimuli applied to this area mainly activated these tectofugal efferents. However, the possibility that fibers from other regions (e.g., the subtectum) were activated in addition to the descending tectofugal fibers must be considered (cf., refs. [5, 13]). The present experiments have shown that EPSPs evoked in the tongue-muscle-controlling moto- neurons in response to descending volleys from the tectal “‘snapping-evoking area’’ were facilitated temporally as well as spatially. These results can be explained by supposing (/) that excitatory interneurons, on which the bilateral tectofugal inputs converge, intervene at a certain point of the tecto-bulbar pathway from the tectal “‘snapping- evoking area’ to the tongue-muscle-controlling motoneurons and (2) that temporal and spatial facilitation of spike discharges occurs in these interneurons (cf., refs. [6,10]). It has been supposed that interneuronal circuitry interposed between descending tectofugal efferents and moto- neurons concerning prey-catching is involved in the generation of the motor pattern for this behavior [3, 12, 13]. The present study has provided the first direct evidence for this hypothesis. Further- more, we have shown that both temporal and spatial interactions of excitatory synaptic inputs to the presumed interneurons are important for activation of the tongue-muscle-controlling moto- neurons. Therefore, temporal and spatial integra- tion of synaptic inputs in premotor interneurons may play a critical role in motor-pattern genera- tion for prey-catching. REFERENCES 1 Ewert, J.-P. (1967) Z. Vergl. Physiol., 54: 455- 832 481. Ewert, J. -P., Burghagen, H. and Schiirg-Pfeiffer, E. (1983) In ‘“‘Advances in Vertebrate Neuro- ethology”. Ed. by J.-P. Ewert, R. R. Capranica and D. J. Ingle, Plenum Press, London, New York, pp. 413-475. Grobstein, P., Comer, C. and Kostyk, S. K. (1983) In “Advances in Vertebrate Neuroethology’’. Ed. by J.-P. Ewert, R.R.Capranica and D. J. Ingle, Plenum Press, London, New York, pp. 331-347. Ingle, D. J. (1983) In “Advances in Vertebrate Neuroethology”. Ed. by J.-P. Ewert, R.R. Capranica and D. J. Ingle, Plenum Press, London, New York, pp. 177-226. Lazar, G. Y., Totk, P., Csank, G. Y. and Kicliter, E. (1983) J. Comp. Neurol., 215: 108-120. Lundberg, A. (1970) In “Excitatory Synaptic Mechanisms”. Ed. by P. Andersen and J. K. S. 10 11 13 M. SATou, T. MATSUSHIMA AND K. UEDA Jansen, Universitetesforlaget, Oslo, pp. 333-340. Matsushima, T., Satou, M. and Ueda, K. (1982) Zool. Mag., 91: 462. Rubinson, K. (1968) Brain Behav. 529-561. Satou, M. and Ewert, J.-P. (1984) Naturwiss., 71: 52-53. Satou, M., Mori, K., Tazawa, Y. and Takagi, S. F. (1983) J. Neurophysiol., 50: 74-88. Székely, G. (1973) In “‘Handbook of Sensory Physiology, Vol. 7/3B’”. Ed. by R. Jung, Springer, Berlin, Heidelberg, New York, pp. 1-26. Weerasuriya, A. (1983) In ‘“‘Advances in Verte- brate Neuroethology’. Ed. by J. -P. Ewert, R. R. Capranica and D. J. Ingle, Plenum Press, London, New York, pp. 613-627. Weerasuriya, A. and Ewert, J. -P. Comp. Physiol., 144: 429-434. Evoly 1: 98h) J: ZOOLOGICAL SCIENCE 1: 833-836 (1984) [COMMUNICATION] © 1984 Zoological Society of Japan Saturation Density of Tetrahymena with or without Agitation at Various Surface-to-Volume Ratios of Culture Test Tubes TADAO SAITOH and HirRosuHI ASAI Department of Physics, School of Science and Engineering, Waseda University, 3-4-1] Okubo, Shinjuku-ku, Tokyo 160, Japan ABSTRACT — Saturation density of Tetrahymena pyriformis in a test tube without shaking can be greatly enhanced, if a small amount of culture medium is layered on top of solid agar containing the same culture medium in the test tube so that the ratio of area of air- medium interface to volume is a large one. Growth of Tetrahymena pyriformis in such cultivating tubes with and without shaking was investigated under various conditions of the surface-to-volume ratio. The saturation density of the cells reached 1 x 10® cells/ml in the tube with the largest surface-to-volume ratio (39 cm~') without shaking. When such a tube was shaken during cultivation, the saturation density of the cells was significantly lower than that without shaking. 7. pyriformis was also cultivated in a test tube with a small surface-to-volume ratio such that the oxygen supply was insufficient. The saturation density of the cells in such a tube of small surface-to-volume ratio with shaking was slightly higher than that with- out shaking. These results suggest that the saturation density of the cells in a tube of large surface-to-volume ratio is markedly restricted by slight mechanical agita- tion (shaking). We found previously that the lag time of growth of Tetrahymena pyrviformis was changed by end products from the cells [1]. On the other hand, the end products had no effect on the saturation density of the cells. Namely, the saturation density obtained by cell growth after inoculation in a conditioned medium already containing the end products was almost the same as that obtained by cell growth after inoculation in a fresh medium [1]. Further, in order to elucidate factor(s) re- stricting the saturation density in a conditioned Accepted May 2, 1984 Received November 18, 1983 medium or in a fresh medium, a ‘“‘crowding”’ effect involved in the population growth of 7. pyriformis was investigated by the use of hollow spherical glass beads with a size and weight similar to those of the cells [2]. It was suggested that growth inhibition of Tetrahymena under such conditions may be a result of cell-to-cell and cell-to-bead collision. Formerly, it was suggested by some investigators [3-5] that saturation density was dependent upon the amount of oxygen supplied to the culture medium. We had tentatively con- cluded that the supply of oxygen to the culture medium in culture tubes used in our earlier studies [1,2] was ample, because the culture tubes were shaken during the culture period in an incubator. However, Dobra et al. [6] reported recently that growth of Tetrahymena on an agar surface was facilitated by an optimal surface-to-volume ratio, yielding a high density of cells. Furthermore, they mertioned that as cells reached higher den- sities, their motility decreased. A_ surface-to- volume ratio of about 1.3cm™' in the culture tubes was used in our previous studies. Thus, their results could be interpreted as indicating that the culture medium in our culture tubes might not have obtained enough oxygen from air. On the other hand, it could be considered that the reason why the saturation density of Tetrahymena cells was very high in their work might have been that the motility of Tetrahymena was re- stricted by the presence of agar. Accordingly, their results could be compatible with our previous proposal with regard to “cell-to-cell collision” as described above. However, it is certain that oxygen supply to the culture medium 834 T. SAITOH AND H. AsaAI is one of the factors which can restrict the satura- tion density of growth of Tetrahymena. Thus, in this experiment, the test tubes of various surface-to-volume ratios were used for cultivating Tetrahymena cells and measuring their saturation densities at various degree of oxygen supply. We confirmed that the saturation density of Tetrahymena cells in a culture tube with a large surface-to-volume ratio is decreased by mechanical agitation even though the oxygen supply is more efficient than it is without mechanical agitation. MATERIALS AND METHODS Tetrahymena pyriformis strain W [7] was used in this investigation. The culture medium was composed of 2% proteose peptone (Difco), 0.7% dextrose, 0.5% yeast extract (Wako), 0.5mM Mg?’*, 0.05 mM Ca’*, 25 g/ml penicillin and 40 ug/ml streptomycin (Kaken) and was adjusted to pH 6.8 with Tris-maleate buffer. The presence of 0.5 mM Mg”* and 0.05 mM Ca’** was required for effective growth, as described in our previous paper [8]. Solution containing the culture medium with 1.5% agar (Shuzui) was boiled and delivered in 5 ml aliquots to 40 culture tubes (39cm, 6.16 cm’ section area). All the culture tubes con- taining the solution were stoppered with cotton and autoclaved for 15 min at 120°C and 2 atm. The culture medium in a cotton-stoppered 250 ml Erlenmeyer flask and some pipettes were also autoclaved under the same conditions. For inoculation, 1 ml of stock culture of Tetrahymena was transferred to 100 ml of culture medium in the 250 ml Erlenmeyer flask. Aliquots of 0.2 to 10.0 ml of culture medium containing stock cells in the 250ml Erlenmeyer flask were pipetted aseptically into culture tubes containing solid agar which had previously been formed at room tem- perature (Fig. 1). This procedure resulted in an initial inoculum of about 10 cells/ml. Tetrahymena cells in 0.2-10.0 ml of medium were grown in cotton-stoppered upright culture tubes. The solid agar was helpful in preventing the culture medium from evaporating. Twenty of the culture tubes, in which the surface-to-volume ratios were variable, were agitated through an cotton stopper culture medium (0.2—10,0 ml ) solid agar Fic. 1. A test tube (30<90mm) containing solid agar and culture medium with cotton-stopper for cul- tivating Tetrahymena cells. excursion of 4cm at a rate of 40 oscillations/min in an incubator (Taiyo M-100) at 27°C. The other 20 tubes, in which the surface-to-volume ratios were also variable, were not agitated, but were kept in an incubator (Taiyo Mini-80) at 27°C. Each of the incubators was covered with a vinyl bag to prevent the culture medium in the culture tubes from evaporating. When Tetrahymena cells were grown in the culture medium, the generation time at the loga- rithmic growth phase was about 3.5hr. The stationary growth phase of the culture having the largest (39cm) surface-to-volume ratio was reached about 6 days after inoculation. The stationary growth phase was maintained for at least 5 more days. For quantitative measure- ments of the saturation density, a culture medium at 7 days after inoculation was diluted 200—10,000 times with fresh culture medium and then 5 yl of the diluted solution was placed on a glass slide of hematocyte counting chamber. The number of Tetrahymena cells on the glass slide was counted under a dark-field microscope. Such dilution and counting was repeated 4 times for each culture medium. After the measurements of the satura- tion density, the culture medium was transferred to a test tube as a small beaker and then the pH Saturation Density of Tetrahymena 835 and volume were measured. The pH and volume of the medium before and after cultivation were constant within 5%. RESULTS AND DISCUSSION Culture medium on solid agar was used to cultivate Tetrahymena cells as shown in Figure 1. When the culture medium had a large surface-to- volume ratio, the saturation density of the cells reached 1 x 10° cells/ml without shaking. Dobra et al. [6] reported that as cells reach higher densities on solid agar, their motility decreases. We also observed under a microscope that the higher the population density of cells in the culture medium was, the lower was their swimming speed, when the culture medium had a large surface-to-volume ratio. When the cultivation of Tetrahymena cells was x10? 10 co mo Saturation density (cells/ml ) done in culture tubes with a surface-to-volume ratio of less than 3 cm™', the saturation density of the cells in a culture tube which had been shaken for 7 days in the incubator was higher than that in a culture tube which had not been shaken. For example, when the cultivation was done at a sur- face-to-volume ratio of 1.3cm™', the saturation densities of the cells in shaken and unshaken culture tubes were 1.2 x 10° cells/ml and 0.7 x 10° cells/ml, respectively (see the inset in Fig. 2). This result suggests that the amount of oxygen supplied to the culture medium in the culture tube was increased by shaking the culture tube and that growth of Tetrahymena was promoted. When the cultivation of Tetrahymena cells was done at a surface-to-volume ratio of more than 3 cm’, the saturation density of the cells in a culture tube which had been shaken for 7 days in incubator was lower than that of the cells in a culture tube 4 ° o/ Surface/ volume (cm-' ) Fic. 2. Saturation densities of T. pyriformis grown for 7 days postinoculation in culture tubes containing solid agar, at various surface-to-volume ratios. Saturation densities of 7. pyriformis in culture tubes with shaking at a rate of 40 oscillations/min () and without shaking (O). When Tefrahy- mena cells reached the saturation density under shaking conditions, as shown by an arrow in Fig. 2, the culture tube with a surface-to-volume ratio of 16cm™! was transferred to an incubator at the same incubating temperature of 27°C for 4 further days without shaking. Its saturation density of T. pyriformis reached more than twice that with shaking. Saturation densities of 7. pyriformis in culture tubes with shaking (x) and without shaking (O) at surface-to-volume ratios of less than 4 cm™~ are shown in the inset in enlarged scale. 836 T. SAITOH AND H. ASAI which had not been shaken. For example, when the cultivation was done at a surface-to-volume ratio of 16cm", the saturation densities of the cells in shaken and unshaken culture tubes were 1.2 x 10’ cells/ml and 3.1 x 10’ cells/ml, respectively (see Fig. 2). Furthermore, the density of 1.2 x 10’ cells/ml in a shaken tube could be increased to 2.8 x10’ cells/ml if the culture tube was further kept without shaking for 4 days in the incubator at 27°C. This suggests that growth of Tetrahymena in culture tubes was inhibited by shaking, even though a sufficient amount of oxygen was supplied to the culture medium. The fact that growth of Tetrahymena was inhibited by shaking supports the result of our previous investigation [2]. Namely, in our previous paper it was suggested that growth inhibition is a result of cell-to-cell collision, and is not due to the production of waste materials or to exhaustion of nutrients in the medium. When the cultivation of the cells was done at a surface-to-volume ratio of more than 20cm’, the saturation density of the cells reached more than 4x10’ cells/ml. By slanting, we observed that the culture medium containing the cells had high viscosity. Hjelm [9] reported that the maximum popula- tion density of Tetrahymena can reach 1.5 x 10° cells/ml if culture is done in a rotating bottle which provides a large surface-to-volume ratio. This value seems to be at least twice as high as reported for other methods. Hjelm ascribed this to the high oxygen concentration available, though he also mentioned that other factors, such as the accompanying lower CO, concentration, may be of importance. Our results indicate that he might have obtained an even higher population density maximum than 210° cells/ml if the culture bottle had not been rotated, since the oxygen supply should have been ample. In a quite separate study of Tetrahymena, Elliott et al. [10] observed that the number and the contents of mitochondria, lysosomes and lipids in Tetrahymena were markedly influenced by agitation. Our studies also suggest that physical activity such as cell-to-cell collision has a profound effect on the metabolism of Tetrahymena and restricts cell division. ACKNOWLEDGMENT We wish to express our thanks to Professor I. Yasumasu of Waseda University for his interest on this work. REFERENCES 1 Saitoh, T. and Asai, H. (1980) Experientia., 36: 1375-1376. 2 Saitoh, T. and Asai, H. (1982) Experientia., 38: 248-249. 3 Pace, D.M. and Ireland, R.L. (1945) J. Gen. Physiol., 28: 547-557. 4 Levy,M.R. and Scherbaum, O. H. Gen. Microbiol., 38: 221-230. 5 Malecki, M. T., Licko, V. and Eiler, J. J. (1971) Curr. Mod. Biol., 3: 291-298. 6 Dobra, K.W., Mcardle, E.W. and Ehret, C. F. (1980) J. Protozool., 27: 226-230. 7 Nanney, D. L. and McCoy, J. W. (1976) Trans. Am. Microsc. Soc., 95: 664-682. 8 Saitoh, T. and Asai, H. (1979) J. Protozool., 26: 286-290. 9 Hyjelm, K. K. (1970) Exp. Cell Res., 60: 191-198. 10 Elliott, A.M., Travis, D.M. and Work, J. A. (1966) J. Exp. Zool., 161: 177-192. (1965) eel ZOOLOGICAL SCIENCE 1: 837-839 (1984) [COMMUNICATION ] © 1984 Zoological Society of Japan Direct Visualization of the Effect of Mg** on the Elongation at the Pointed Ends of Limulus Acrosomal Actin Bundles KoscAK MARUYAMA, KIYOMI TSUKAGOSHI and Issg1 MABUCHI! Department of Biology, Faculty of Science, Chiba University, Chiba 260, Department of Cell Biology, National Institute for Basic Biology,Okazaki, Aichi 444, and ‘Department of Biology, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan ABSTRACT — Elongation at the pointed ends of Limulus sperm actin bundles was to a smaller extent in the presence of 0.05 mM MgCl, than in its absence in 50mM KCl and 5mM Tris-HCl, pH 8.0 at 25°C. This tendency was more evident in the presence of 4 uM cytochalasin D in addition to Mg’*. It appears that Mg** enhances dissociation of monomers from the pointed end of an actin filament at steady state. Actin filaments have structural polarity, and the rate of elongation is much faster at one end (barbed end) than the other end (pointed end) (for a review, see Pollard and Craig ([I)). Cytochalasins preferentially inhibit the growth at the barbed end of an actin filament (cf. [1]). Recently, using fluorescent dye-labelled actin, we have observed that the extent of actin polymeri- zation in the presence of cytochalasin D at steady state is markedly affected by Mg’*, and suggested that the equilibrium at the pointed end is shifted to the monomer side in the presence of 0.01-1 mM MgCl, [2]. In fact, Bonder ef al. [3] have already reported that the critical concentration at the pointed end becomes larger in the presence of 2mM MgCl, and 50 mM KCI than in 50 mM KCl alone. . Actin bundles of sperm acrosomal process of the horseshoe crab Limulus polyphemus consist of oriented actin filaments of the same polarity and Accepted April 13, 1984 Received March 30, 1984 can be used for effective seeds on addition of rabbit skeletal muscle actin [3-6]. In the present investigation, the effect of Mg** on the elongation of the pointed ends of the actin filaments was directly visualized using Limulus sperm actin bundles. MATERIALS AND METHODS Rabbit skeletal muscle actin was prepared by a routine procedure [2]. Limulus polyphemus acrosomal process was isolated according to Tilney [7] and stored in 50% glycerine containing 15mM Tris-HCl, pH7.5 and 15mM MgCl, at —20°C. The actin bundles were sonicated for 10 sec in 10mM Tris-HCl, pH 8.0 in a Tomy sonifier (20 kc). Actin samples negatively stained with 2% uranyl acetate were observed under a JEM 100S electron microscope at 80 kv. RESULTS AND DISCUSSION As shown in Figure la and b, short Limulus actin bundles served as nuclei at the both ends on addition of rabbit monomeric actin. In Figure la and b, elongated filaments from the barbed ends were partly shown. In the presence of cyto- chalasin D, only one end (pointed end) elongated (Fig. lcandd). The polarity of the actin filaments was checked by adding heavy meromyosin and also by morphological differences of the both ends 838 K. MARUYAMA, K. TSUKAGOSHI AND I. MABUCHI Elongation of Limulus Actin Bundle 839 TABLE 1. Lengths of actin filaments elongated from the pointed ends of Limulus acrosomal actin bundles under the influence of Mg?* and cytochalasin D Lengths (7m) Additions io 30’ «180 (min) 50 mM KCl Control RIS: 143-746 4 uM cytochalasin D 1.14 1.20 1.28 50 mM KCI+0.05 mM MgCl, Control 0.80 0.93 0.73 4 uM cytochalasin D 0.44 0.79 0.54 G-actin, 0.13 mg/ml; 0.2 mM ATP; 5 mM Tris-HCl, pH 8.0; Zs. Conditions: of Limulus bundles [4, 6]. It is clearly seen that the extent of growth at the pointed end during incu- bation with rabbit actin was much smaller in the presence of 0.05mM MgCl, (Fig. 1d) than in its absence (50 mM KCI and 4M cytochalasin D) (Fig. Ic). . The lengths of elongated actin filaments at the pointed ends of Limulus actin bundles were measured on the electron micrographs, and the average lengths of some ten bundles (40-100 filaments) are listed in Table 1. There were not much differences in the elongated lengths in the presence and absence of cytochalasin D in 50 mM KCI (cf. Fig. la and c). On the other hand, when 0.05 mM MgCl, was added with 50 mM KCl, there was a marked decrease in the lengths (cf. Fig. 1b). This tendency was more remarkable in the presence of cytochalasin D (cf. Fig. 1d). It should be mentioned that in the presence of Mg’* the lengths became larger between 10 and 30 min of incubation, and then became smaller at 180 min. This was evidently due to a transient growth of nuclei reaching eventually the equilibrium state (cf. [3]). However, this was not the case with 50mM KCI alone. Therefore, it is thought that Mg’* accelerates dissociation of actin monomers from the pointed ends of actin filaments at steady state irrespective of the presence and absence of cytochalasin D (cf. [8]). It should be noted here that capping of the barbed end with cytochalasin D appears to decrease both the rate and extent at the other free end (pointed end) (see Table 1). The decreased rate of growth at the pointed end by cytochalasins as compared to intact one has been already described under different conditions using brush border microvilli as seeds [9, 10]. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science and Culture, and from the Yamada Science Foundation. REFERENCES 1 Pollard, T.D. and Craig, S.W. (1982) Trends in Biochem. Sci., 7: 55-58. 2 Maruyama, K. and Tsukagoshi, K. Biochem., 96: 605-616. 3. Bonder, E.M., Fishkind, D.J. and Mooseker, M.S. (1983) Cell, 34: 491-501. 4 Tilney, L.G., Bonder, E. M. and DeRosier, D. J. (1981) J. Cell Biol., 90: 485-494. 5 Bonder, E.M. and Mooseker, M.S. (1983) J. Cell Biol., 96: 1097-1107. 6 Mabuchi, I. (1983) J. Biochem., 94: 1897-1900. Tilney, L. G. (1975) J. Cell Biol., 64: 289-310. 8 Coluccio, L. M. and Tilny, L. G. (1983) J. Cell Biol., 97: 1629-1634. 9 Pollard, T.D. and Mooseker, M.S. (1981) J. Cell Biol., 88: 654-659. 10 Yokota, E. and Maruyama, K. (1983) J. Bio- chem., 94: 1897-1900. (1984) J. Fic. 1. Electron micrographs of elongated actin bundles of Limulus acrosomal process under the in- fluence of Mg?* and cytochalasin D. a, 5SO0mM KCl; b, 50mM KCI+0.05mM MgCl; c, 50 mM KCI+4 uM cytochalasin D; d, 50mM KCI+0.05mM MgCl,+4 4M cytochalasin D. Incubated for 10 min in the presence of rabbit skeletal muscle G-actin, 0.13 mg/ml, 0.2 mM ATP and 5 mM Tris-HCl, pH 8.0 at 25°C. Scale, 0.5 um. , 8 y hay, F His ap : D Aenean 7 ' | 9 Soren adn! iat OF 16F boladgeml } a YS os He SOReRT Ment. se y ZOOLOGICAL SCIENCE 1: 841-843 (1984) [COMMUNICATION ] © 1984 Zoological Society of Japan Occurrence of a Physiologically Active Calcitonin in the Brain of the Bullfrog, Rana catesheiana CHITARU OGURO, HIROYUKI NoGAwa! and YUICHI SASAYAMA Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan ABSTRACT — Administration of a brain extract from the bullfrog caused responses typical of calcitonin, hypocalcemia accompanied with hypophosphatemia, in rats. Neither muscle extract, acetylcholine, dopamine, noradrenaline nor serotonin had any effects on the serum calcium and inorganic phosphorus con- centrations when administered to rats. These results suggest that immunohistochemically detected calcitonin in the bullfrog brain is a calcitonin which is physiologi- cally active. Calcitonin (CT), which is known to be a hypo- calcemic hormone in mammals, was _ recently detected in the brain of the bullfrog by means of immunohistochemistry and radioimmunoassay for human CT [1]. The question arises as to whether this immunoreactive CT (iCT) is physiologically active (with hypocalcemic potency) and what its function in the brain is. The present study was performed to see if the iCT in the bullfrog brain has hypocalcemic potency using a rat bioassay. MATERIALS AND METHODS Adult male bullfrogs, Rana catesbeiana, 300-400 g bw, were purchased from a commercial source. The brain and pituitary gland were removed after each animal was pithed. These organs were homogenized in 1 N acetic acid then boiled for a Accepted May 22, 1984 Received April 18, 1984 1 Present address: Research Laboratory, Otsuka Pharmaceutical Factory Inc., Naruto, Tokushima 772, Japan few minutes, chilled rapidly and _ centrifuged (5,000 rpm, 15 min). The supernatant was lyo- phylized and stored until use. The stock material was dissolved in 0.99% NaCl solution before use. The method of CT extraction employed in the present study noted above was the same to that used by Yui ef a/. [1] for CT radioimmunoassay. Male Wistar rats 4-5 weeks of age were fasted overnight before the experiment. While anes- thetized with ether they were cannulated with polyethylene tubing (PE 10) in the iliac artery and iliac vein. The former was used for the blood sampling and the latter for administration. Each rat received extract of 2 brains (average wt 400 mg) or 2 pituitary glands. Synthetic salmon CT (SCT) (10 mU per rat) was used as a standard of com- parison for the hypocalcemic potency. Some neuroregulators were examined as well as the brain extract for their hypocalcemic activity. Acetylcholine chloride (Nakarai), dopamine hy- drochloric acid (Nakarai), noradrenaline (Nakarai) and serotonin creatinine sulfate (Merk) were dissolved in 0.9°% NaCl solution at concentrations of 1.2, 0.4, 0.6 and 0.4 wg per 400 wl, respectively. For the control, saline (0.9% NaCl solution) and muscle (400 mg) extract, prepared in the same manner as the brain extract, were used. Every dose was adjusted to 400 wl per rat. The numbers of rats given each agent were: brain, 8; pituitary gland, 11; muscle, 6; acetylcholine, 5; dopamine, 5; noradrenaline, 6; serotonin, 5; SCT, 8; control saline, 10. Before the administration and 30, 60, 120 and 180 min after administration blood was sampled. 842 C. OcuRO, H. NOGAWA AND Y. SASAYAMA Serum was obtained by centrifuging the blood at 12,000 rpm for 3 min. Serum Ca and Na concen- trations were determined by atomic absorption spectrophotometry. Serum inorganic phosphorus (Pi) was measured according to a modified method of Fiske and Subbarow [2]. The Student’s f-test was used for the statistical treatment. Significancy was set at P<0.05. RESULTS The brain extract caused a significant decrease in serum Ca concentration 30 and 60min after administration (Fig. 1). Two hours after admini- stration, however, the serum Ca recovered to a nearly normal level. The serum Pi concentration was also decreased significantly by the administra- tion of the brain extract (Fig. 2). These changes were very similar to the typical changes caused by SCT (Figs. 1 and 2). The pituitary extract brought about hypo- calcemia 1 hr after it was administered (Fig. 1). However, the change in the serum Pi concentration caused by the pituitary extract was not statistically significant (Fig. 2). The serum Ca and Pi con- centrations were not changed by either the saline or muscle extract used as controls (Figs. 1 and 2). None of the neuroregulators examined had any effect on the serum Ca and Pi concentrations at the dose used. The serum Na concentration was not changed by any administration. oo E 10 SS 010 OF oO eg 2 Lal's tat @ : pees iss) * O 90 = = z Ye Lu Y i 1 2 HOURS Z Fic. 1. Time course of serum Ca changes in rats following the administration of extracts of bullfrog brain (O), muscle (©) or of pituitary gland (A), SCT (@) and control saline (#). Each point represents the average value. The SE is not shown to avoid complicating the figure. Differs from the control saline; * P<0.05, ** P<0.01. 110 mee ie ¢——_=3 A A C2 = 6° a ma \ fe = oO (=) fo) (2) * * O SERUM Pi mg/100mI © ° \ \ Oo fo) — NO 3 HOURS Fic. 2. Time course of serum Pi changes in rats fol- lowing the administration of extracts of bullfrog brain (©), muscle (©), or of pituitary gland (A), SCT (@) and control saline (#). Each point represents the average value. The SE is not shown to avoid complicating the figure. Differs from the control saline; *« P<0.05, *« P<0.01. DISCUSSION The occurrence of what appears to be calcitonin in the ultimobranchial gland of amphibians has been revealed in earlier studies by a rat bioassay [3-5] and by immunohistochemistry [6, 7]. In mammals extra-thyroid CT has been reported in a number of tissues and organs [8-10]. However, knowledge of the localization of CT in extra- ultimobranchial tissues in lower vertebrates is scanty [10-12]. Recently, the occurrence of iCT was reported in the bullfrog brain [1]. The main aim of the present study was to deter- mine whether or not this iCT has hypocalcemic potency. The results show that the bullfrog brain extract caused a significant hypocalcemia and hypophosphatemia resembling that caused by CT. This suggests that the iCT in the bullfrog brain is a CT with hypocalcemic potency. However, it is unlikely that the main function of this CT is the regulation of serum Ca concentration. At present the function of CT in the bullfrog brain is not known, although it has been suggested that in mammals CT is an agent affecting the control of the central nervous system [13-16]. The present study was supported in part by a Grant- in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (no. 75480024). Calcitonin in Bullfrog Brain REFERENCES Yui, R., Yamada, Y., Kawamori, R. and Fujita, T. (1981) Biomed. Res., 2: 208-216. Fiske, C. H. and Subbarow, Y. (1925) J. Biol. Chem., 66: 375-400. Oguro, C. and Uchiyama, M. (1980) In “Hor- mones, Adaptation and Evolution’. Ed. by S. Ishii, T. Hirano and M. Wada, Japan Sci. Soc. Press, Tokyo/Springer Verlag, Berlin. pp. 113-121. Oguro, C., Nagai, K-I., Tarui, H. and Sasayama, Y. (1981) Comp. Biochem. Physiol., 68A: 95-97. Oguro, C., Tarui, H. and Sasayama, Y. (1983) Gen. Comp. Endocrinol., 51: 272-277. Van Noorden,S. and Pearse, A.G.E. (1971) His- tochemie, 26: 95-97. Treilhou-Lahille, F., Jullienne, A., Aziz, M., Beaumont, A. and Moukhtar, M.S. (1984) Gen. Comp. Endocrinol., 53: 241-251. Becker, K. L., Snider, R. H., Moore, C. F., Monaghan, K.G. and Silva, O. L. (1979) Acta Endocrinol., 92: 746-751. 9 15 16 843 Cooper CW... Pens, TC... Obie, J.P. and Garner, S.C. (1980) Endocrinology, 107: 98- 107. Deftos, L. J., Burton, D. W., Watkins, W. B. and Catherwood, B. D. (1980) Gen. Comp. Endo- crinol., 42: 9-18. Galan Galan, F., Rogers, R.M., Girgis, S.1., Arnett, T. R., Ravazzola,M., Orci, L. and MacIntyre, I. (1981) 432. Ravazzola, M., Orci, L., Girgis, S.I., Galan Galan, F. and MacIntyre, I. (1981) Cell Biol., 5: 937-944. Pecile, A., Serri, S., Braga, P.C. and Olgiati, V.R. (1975) Experientia, 31: 332-333. Nakhla, A.M. and Majumdar, A. P. N. Biochem. J., 170: 445-448. Carman, J.S. and Wyatt, R. J. Gen. Psych., 36: 72-75. Freed, W.J., Perlow, M. J. (1979) Science, 206: 850-852. Acta Endocrinol., 97: 427-— (1978) (1979) Arch. and Wyatt, R. J. ee ee ews: vil sYNieo® oct gD ealgheales $e hod (Te A rere yu neo yee ne. oO f I F ; pl iba | toe a y i it - rat L Labi ee 4 ot [ te naly vied. hee ine . yeu baad Pobeai . ee na ie a ' [ g \ : | SP gra rec See rar ey SE : < f a y a Fee Bh Ot ies MiG & skis RENN ¢ Mi , : ’ a ' ¥ a # be lig one P2 = Q ‘ J i al p f & y = ty i & Be) f a ‘ i : is aN. i vue ritcs re « » ; ¥ - VM pects TPG: Shia \ ,* uy we \ nwa “ * ‘ . eights a 5 r tad ’ j ™ : ice 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 patho- genesis, 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. 26, No. 5. 1. H. MercHANtT-Larios, L. Porova and M. R. Brion: Early morphogenesis of chick gonad in the absence of mesonephros 2. R. MuRAKAMI and T. Mizuno: Histogenesis of the Os penis and Os clitoridis in rats 3. N. Castro, F. D. Barsiert, D.C. Micevt and C. D. PASQUALINI: Teratoid tumors derived from mouse embryos grown in an immunologically privileged site 4. K. I. Hirano, M. Masupba and H. Sato: Ultrastructural study of asters induced by microinjection with sperm centriolar fraction in sea urchin eggs 5. S. Lovtrup, A. ReHNHOLM and R, Perris: Induction of the synthesis of melanin and pteridine in cells isolated from the axolotl embryo 6. K. Nukura, A. Fustwara and I. Yasumasu: Effect of ami- nopterin and deoxyribonucleosides on the cleavage and embryogenesis of the sea urchin, Hemicentrotus pulcherrimus Information Six issues yearly in one volume. Annual sub- scription rate; $90.00.* Address all corre- spondence regarding new subscriptions and renewals to the following sole agents: JAPAN PUBLICATIONS TRADING Co., Ltd., P.O. Box 5030 Tokyo International, Tokyo, Japan. MARUZEN Co., Ltd., P.O. Box 605, Tokyo, Japan. | JAPAN PUBLICATIONS TRADING iInc., (U. S. A.) 300 Industrial Way, Brisbane, Cali- fornia, 94005, U.S.A. NAUKA Co., Ltd., Jinbo-cho 212, Kanda, Chiyoda-ku, Tokyo. 7. K. Kumar and A. K. 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GEE ty bs ove s ¥ iz: % ir : EA a di by : ee . / ae ’ wt i ; tat 1 \ 7 ; | nigger t 7 =f “a B 2 bs : = aie hey ort) Aer yi 2 bees ; ' | Wey moa rear AU at Mraeat ih " hits Wort ata ‘ sosge clos. gaiwollgt oat Gr VIGART OF ‘ONT Mevatin. @ | eg eal evlet DEOR x08 3G ; ; i Ra Ae OU, seid pd ’ i° OMMLAST BVT: Suite wae { } hase a Tt oy OO CA ; ABOU st00HR . , ee ‘prio: adaty ed Pages at Besides Analytical and Prepara- : : : ; Speed (rpm) Description tive ’ Ultracentrifuges, Hitachi SCR20BB > | Automatic with built-in Koki, a comprehensive centrifuge SCR20BA | 300 ~ 20,000 3 L microprocessor control, Digital version manufacturer, brings you a com- = an x00] 2 | 2 < | Automatic control, plete line of High Speed Refrig- 20PR520 | 300 ~20,000] =| § | = | Analog version : : D p+ = erated Centrifuges, Table-Top SCR7B S|] x : eS SCR7BC 7,000 “u | ¢ | $ | Large capacity, multi-place type Centrifuges and Miniature Cen- SCR7BA = trifuges with wide-ranging et & | Compact type for biochemistry speeds, capacities and _ tubes/ seTsa ee seats $= S Oe Eo tyes - oge — —————$ For clinical Automatic control bottles versatility for your acre 4,000 8 | laboratory test . . . Coda t ae oe 2 es u mati 1 icr r 25 r clinical laboratory routine as well = ~scrise [2,000 = 15,000] & | Miniature | control ‘ - basic research. cas 12,000 r Hematoerit | scapula Dil a es Fe ieee ____| Dual purpose a MC-300 430 ~ 2800| Cell washing se a ae. HITACHI Refrigerated/ anit Table-Top/Miniature Centrifuges Manufacturer @ Nissei SangyoCo..td. © Hitachi Koki Co.,Ltd. Instrument International Dept. Mori 17th Bldg., 26-5 Toranomon 1-chome, Minato-ku, Tokyo 105, Japan C.P.0O. Box: 1316, Tokyo 100-91, Japan Phone: 03-504-7282 Telex: J 22412 NISSEICOA ee ZOOLOGICAL SCIENCE VOLUME 1 NUMBER 5 OCTOBER 1984 CONTENTS REVIEW Hisada, M., M. Takahata and T. Nagayama: Local non-spiking interneurons in the arthro- pod motor control systems: Characteriza- tion and their functional significance ......... 681 ORIGINAL PAPERS Physiology Kikuchi, M. and K. Aoki: The structure of photoreceptor cells in the pineal organ of the Japanese common newt, Cynops pyrrhogaster PYTRNOLASTCH «SPs Rocce. stein nc eee oe ree 701 Oshima, N., R. Fujii and H. Kasukawa: Simultaneous recording of motile responses of light-absorbing and reflecting chromato- PINOLES THEVIMIO! Leas eck Bel enc sece actos oa es lel Satou, M., T. Matsushima and_ K. Ueda: Neuronal pathways from the tectal “‘snap- ping-evoking area”’ to the tongue-muscle-con- trolling motoneurons in the Japanese toad: Evidence of the intervention of excitatory interneurons (COMMUNICATION) ......... 829 Cell Biology Takayama, S. and K. Matsumoto: Nucleolus organizer region lateral asymmetry induced by BUdR incorporation in mouse chromo- Saitoh, T. and H. Asai: Saturation density of Tetrahymena with or without agitation at various surface-to-volume ratios of culture test tubes (COMMUNICATION) Maruyama, K., K. Tsukagoshi and I. Mabuchi: Direct visualization of the effect of Mg’** on the elongation at the pointed ends of Limulus acrosomal actin bundles (COMMUNICA- TION) Developmental Biology Yasumasu, I., A. Fujiwara, A. Hino and K. Asami: Effect of several redox dyes on the respiration of unfertilized eggs of sea PAR MMU es oo scd ont cnet. c' a ener ee 125 Kishida, Y. and E. Asai: Influence of nerve cord removal on pharynx induction by head piece transplanted in postpharyngeal body region of the planarian, Dugesia japonica YAU OINCA Ares eet ae ORD, Cee h oo roaet aire ee VSG. Terada, M.: Comparison of zoeal develop- ment between the two carcinoplacid crabs, Carcinoplax longimana (De Haan) and Eucra- te crenata De Haan, reared in laboratory ...743 Endocrinology Numata, H. and T. Hidaka: Termination of adult diapause by a juvenile hormone analogue in the bean bug, Ripftortus clavatus Sasayama, Y., C.Oguro, R. Yui and A. Kambegawa: Immunohistochemical de- monstration of calcitonin in ultimobranchial glands of some lower vertebrates ............... WDD Takami, S., Y. Jokura and A. Urano: Sub- nuclear organization of the preoptic nucleus in the toad, Bufo japonicus. =a eee 759 Hutz, R. J., M. Ghosh and W.R. Dukelow: Steroid uptake and *H-uridine incorporation by early hamster and squirrel monkey (in vitro fertilized) embryos: Effects of ovulatory regimen . Tachibana, H., T. Iguchi and N. Takasugi: Different perinatal periods of vitamin A administration for prevention of the oc- currence of permanent vaginal changes in mice treated neonatally with estrogen ......... TA. Oguro, C., H.Nogawa and _ Y. Sasayama: Occurrence of a physiologically active calcitonin in the brain of the bullfrog, Rana catesbeiana (COMMUNICATION) _......... 841 | Behavior Biology Watanabe, M., T. Kobayashi and H. Terami: Changes in eye color during aggressive interaction in the chocolate cichlid, Cichlasoma coryphaenOides.........00cccccreeeeees 787 Genetics ‘Sakaizumi, M.: Rigid isolation between the northern population and the southern population of the medaka, Oryzias latipes asi besesdeeibedeasleorece sls nebecheetCeteee = === 195 Taxonomy Ohnishi, S. and T. K. Watanabe: Systematics of the Drosophila montium species subgroup: A biochemical approach Shimazu, T. and S. Shimura: Paralepidapedon g. n. (Trematoda: Lepocreadiidae), with descriptions of metacercariae of Para- lepidapedon hoplognathi (Yamaguti, 1938) comb. n. and of two other species from sea UPCHINS © ..66.4.0054.0gdesceos de ceuke desde eee 809 Sawada, I.: Cestodes of bats from Japan, with descriptions of new species of the genus Vampirolepis (Cestoda: Hymenolepididae) ...819 Printed by Daigaku Letterpress Co., Ltd. Hiroshima, Japan ZOOLOGICAL SCIENCE tinuation of the “Zoological Magazine” and the “Annotationes Zoologicae Japonenses ZOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Editor-in-Chief: The Zoological Society of Japan: Nobuo Egami (Tokyo) Toshin-building, Hongo 2—27-2, Bunkyo-ku, Associate Editors: Tokyo 113, Japan. (03)-814-5675 Susumu Ishii (Tokyo) Officers: Seiichiro Kawashima (Hiroshima) President: Hideshi Kobayashi (Funabashi) Koscak Maruyama (Chiba) Secretary: Makoto Asashima (Yokohama) Assistant Editors: Treasurer: Kiyoaki Kuwasawa (Tokyo) Takeo Machida (Hiroshima) Librarian: Shun-Ichi Uéno (Tokyo) Kazuyoshi Tsutsui (Hiroshima) Editorial Board: Howard A. Bern (Berkeley, endocrinology) Aubrey Gorbman (Seattle, endocrinology) Robert B. Hill (Kingston, physiology) Yukio Hiramoto (Tokyo, cell physiology) Yukiaki Kuroda (Mishima, cell genetics) Hiromichi Morita (Fukuoka, sensory physiology) Tokindo S. Okada (Kyoto, developmental biology) Andreas Oksche (Giessen, neuroanatomy) Hidemi Sato (Nagoya, cell biology) Koichi Sekiguchi (Tsukuba, phylogeny) Mayumi Yamada (Sapporo, systematic zoology) Ryuzo Yanagimachi (Honolulu, reproductive biology) 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, Associate 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, Park Voorn 4, 3454 JR De Meern, P. O. Box 2073, 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 volumes. 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 1984, 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 1: 845-846 (1984) © 1984 Zoological Society of Japan OBITUARY Soichi Fukuda (1907-1984) Soichi Fukuda, an eminent insect endocrinologist, died on March 10, 1984, at the age of 76. He was born at Shirahama, Wakayama Prefecture, on November 4, 1907. Influenced by his teacher of biology at Osaka High School, he became interested in zoology and attended Tokyo Imperial University (now the University of Tokyo), enrolling in the zoology course. After gradua- tion from the university in March 1933, he continued his studies in the post-graduate course. In 1936, he was appointed Research Assistant and later he became the Head of the Katakura Seri- cultural Experiment Station in Matsumoto. In 1962, he was called to the professorship of Nagoya University. After retirement from Nagoya University, he successively served at Aichi Medical University and Aichi Gakuin University as professor of biology. Following is an abbreviated resume of his successful scientific career. While enrolled in the graduate course, he collaborated in research work with his colleague, Kiyoshi Takewaki (later Professor at the University of Tokyo), in the laboratory of Professor Naohide Yatsu. One of their joint research projects concerned the effect of gonadectomy and testicular transplantation on the secondary sex-characters of the lizard, Takydromus takydromoides. This work introduced him to the philosophy and methodology of modern endocrinology. Before his appointment to the staff of the Katakura Sericultural Experiment Station, he had practically no experience in sericulture or insect physiology. One day, in late May of 1936, h entered in a room where a large culture of silkworm cocoons was maintained. The cocoons | been brought there from a farmer in the Izu Peninsula. He saw several maggots crawling he floor. The maggots were larvae of the parasitic fly, Sturmia sericariae, liberated from the s «worm cocoons and they were seeking for places to pupate. Looking at this scene, his attention ‘ocussed on a maggot, which had failed to leave the shell of the cocoon and was immobilized ha uring the escape process. It was constricted at a midpoint of the body, which divided ted half from the residually larval half. This observation inspired in him the idea to |), ects with threads. The procedure is now routinely used in insect endocrinology, but ( that time. 846 OBITUARY Experiments with Sturmia eventually led him to his life-long research field: insect endocrinology. Then he chose the more convenient laboratory tool, the domestic silkworm, Bombyx mori, for his experimental investigative material. By experiments with this insect, he discovered that the prothoracic glands are the source of the moulting hormone. The morphology of this gland in Bombyx had been described by K. Toyama, but its function had remained unclarified. A full compilation of Prof. Fukuda’s work on the endocrine mechanisms of the silkworm was published in the midst of the Second World War. These studies provided the basis for the general under- standing of the fundamental endocrine mechanisms of insect metamorphosis. He then turned his interest to another phenomenon which also is important in the insect life cycle: diapause. These studies, again with Bombyx, revealed that a factor causing the production of diapausing embryos is secreted from a pair of neurosecretory cells located in the subesophageal ganglion. His interest further extended to the hormonal control of cycle seasonal forms of several lepidop- teran species. He loved to do experiments with his own hands and frequently he sat until midnight in front of his binocular microscope. Although he devoted almost all of his time to research at his laboratory, he nevertheless was attached to his family and received the warm sympathy and assistance of his wife, Sagako. All of us who know his contributions to biology well, and were so fortunate as to be influenced by him, will continue to honor him, in memory. E1J1 OHNISHI Biological Institute Faculty of Science Nagoya University ZOOLOGICAL SCIENCE 1: 847-849 (1984) © 1984 Zoological Society of Japan OBITUARY Haruo Kanatani (1930-1984) Professor Haruo Kanatani, Director of the National Institute for Basic Biology, Okazaki, died of pancreatic cancer on February 13, 1984. He was 54 years old. Dr. Kanatani’s untimely death left much grief and sadness among those who knew him and those who worked with him, and experienced as a great loss to the world of biological sciences both in Japan and abroad. Haruo Kanatani was born in Ashiya near Kobe on February 3, 1930. As a student at Konan High School he was inspired by the late Professor Hiroshi Takaya, a distinguished embryologist. It was at this time that he began his life’s work which was destined to become a life-long research in the field of cellular differentiation. He graduated from the Department of Zoology, Faculty of Science, the University of Tokyo in 1952. During the initial research period he concentrated his efforts on the problem of polarity in planarian regeneration and worked under the direction of Professor Takashi Fujii who profoundly influenced Kanatani’s academic carrier. He continued work on this classical problem and was awarded the degree of D. Sc. in 1961. From 1957 to 1965 he served as an instructor at the Misaki Marine Biological Station of the University of Tokyo, where he became closely acquainted with a number of leading biologists including Katsuma and Jean Dan. It was at this time that Kanatani initiated his fascinating studies on starfish oocyte spawning and maturation that were to lead to future discoveries on the molecular mechanism of gametogenesis. In 1965 he was promoted to an associate professor at the Ocean Research Insti- tute, the University of Tokyo where he worked in the laboratory of Professor Seiichiro Utida. Kanatani was then also deeply involved in planning new national institutes for science to be set up in Okazaki. In 1976 he was appointed a professor of the newly founded Institute for Basic Biology there and in early 1983 he was nominated as Director of the Institute. In starfish the fully grown ovary is filled with immature oocytes each with a prominent germinal vesicle and each surrounded by an envelope consisting of follicle cells. In 1959, Chaet and McConnaughy observed that injections of hot water-extracts of starfish radial nerves into the coelomic cavity of the ripe females could induce shedding of the immature eggs. Stimulated by 848 OBITUARY this early work, Kanatani in 1966 began his search for the active factor in these extracts. This factor later called gonad-stimulating substance or GSS was isolated by him in 1971. From 70g of an acetone-dried powder of nerves collected from 7360 (!) starfish, he was able to isolate 1.3 mg of purified GSS. This substance was found to induce discharge of eggs from isolated ovaries at 0.001 g/ml. This represents a high degree of purification, over 6000-fold, since the original extract was only active at 6.6 mg/ml. It was then shown that GSS is a peptide consisting of 22 amino acids (MW, 2066). In the course of the above work, Kanatani noticed that oocytes shed under the stimulus of GSS failed to mature in the presence of GSS-sea water. Maturation would occur however if the same eggs were incubated in the presence of ovarian fragments or in the medium conditioned by such fragments. He put forward a hypothesis that induction of maturation be caused by a second factor which Kanatani termed maturation-inducing substance or MIS. He predicted that MIS would be produced in the ovary under the stimulus of the neural peptide hormone, GSS. Purification of MIS was carried out starting with 20 kg of ovaries obtained from 3000 starfish. The ovaries were incubated for 6 hours in sea water containing GSS derived from 20g of lyphophilized radial nerves. At the end of tedious isolation procedure the system yielded 8.5 mg of pure MIS. This was in 1969. The MIS was effective at the level of 0.02 ug/ml in inducing maturation of isolated oocytes. The chemical structure of MIS was worked out by Professor Koji Nakanishi then at Tohoku University (currently Columbia University), and MIS was identified as 1-methyl adenine. In accordance with Kanatani’s earlier hypothesis it was concluded that GSS stimulates the produc- tion of MIS in the follicle cells surrounding the oocytes. The mechanisms for this stimulation very probably involve an activation of a methyltransferase according to Kanatani (1979). When MIS was microinjected directly into immature oocytes however, there was no induced maturation. This observation suggested that MIS works at the level of the cell membrane through a receptor-mediated production of a third substance named maturation-promoting factor or MPF. MPF then initiates a cytoplasmic process leading eventually to maturation. Indeed, a MIS receptor has been isolated from oocyte cell membranes. Kanatani and his group became extremely active at the National Institute for Basic Biology in attempts to isolate MPF. This material is quite unstable and has not yet been separated in a pure form. It has been identified however as a protein. The activity of MPF is, interestingly, effective on amphibian as well as starfish eggs. Furthermore, MPF obtained from cultured cells of hamster ovary origin induces maturation in starfish oocytes (1982). Hence it appears now that MPF is common to vertebrates as well as invertebrates. In 1983, MPF was shown to induce both germinal vesicle breakdown and follicle envelope breakdown in immature ovarian tissues. At the time of his death, Kanatani had reached the conclusion that MPF is involved not only in meiosis but also mitosis of ovarian cells. The purification of MPF is now being carried out by Kanatani’s reliable collaborators, especially Drs. Hiroko Shirai, Takeo Kishimoto, and Yoshitaka Nagahama. We all may hope for an early completion of this exciting project. When I was a freshman (Zenki) student in the Department of Zoology, the University of Tokyo in 1950, Kanatani was in the sophomore (Chuki) class. He was unique; always cheerful and humorous. He was of a poetic turn and wrote poems himself. Kanatani always spoke loudly in a very shrill voice with Kansaiben (west Japan accent). His nickname was ‘‘Rokusuke” or the even friendlier “‘Roku-san” named after the hero in the then popular movie entitled ““Aoi Sanmyaku (Blue Mountains)”. Kanatani often astonished his friends with his deep knowledge of French literature and philosophy. Haruo Chino (currently professor of insect biochemistry, Hokkaido University), Hideo Mohri (currently professor of biology, the University of Tokyo) and myself often joined Kanatani to talk about our researches and about how to improve the organization of biological research in our country. We all agreed on the necessity of encouraging molecular OBITUARY 849 studies of any biological phenomena, and Kanatani was quick to emphasize the necessity of providing the explanation in its simplest form. Note that even a spectrophotometer was not equipped in the laboratories of the Department of Zoology at that time. These discussions con- tinued over a thirty year period, the last one taking place at Chino’s home in Sapporo in the fall of 1982 on the occasion of the annual meeting of the Zoological Society of Japan. All of us enjoyed drinking sake (Japanese liquor) until late. Kanatani’s concern for better science never waived. Kanatani’s ultimate aim was to understand the principle underlying the cellular differentiation at the molecular level. He once told me his desire to return to the classical problem of planarian polarity and to isolate the “‘polarity-determing substance’’ or PDS. He even knew of several secret places where he could collect a large number of planaria that would be needed for such an attempt. Unfortunately, death intervened. Kanatani worked very hard and effectively: he used to show himself in his laboratory in the afternoon but would not leave until early in the morning. He was rigorous in evaluating the limits of a technique and always scrutinized carefully the results of his experiments. He combined this tough mindness with imagination and could be quite unconventional in designing new experi- ments. He was graceful in his personal interactions and maintained good working relationships with a great variety of colleagues both in Japan and abroad. He brought all of these skills plus per- sonal warmth and humanity to his role in planning the new national institute. Without Kanatani the road to the creation of the Okazaki Institutes would not have been as smooth as it was. On December 16, 1983, I received a last letter from Kanatani, then hospitalized at the Aichi Cancer Center: “I feel severe bellyaches. But I want to recover as soon as possible. For younger generation we have to commit ourselves to numerous administrative jobs, remaining at the same time active in research. I hope I can go out for a drink with you soon.” I felt relieved to have heard that he was optimistic right up to the end of his life. Dr. Kanatani published 137 articles from 1957 to 1984. Despite his relatively short life, he was honored by numerous academic awards: the Gold Medal of Pius XI from the Pontifical Academy of Sciences (1970), the Zoological Society of Japan Prize (1971), the Matsunaga Prize (1974), Foreign Membership of the French Academy of Sciences (1981), and Person of Cultural Merits of Japan (1983). To improve the research system of science in Japan, he served as a scientific adviser to the Bureau of Science and International Affairs, the Ministry of Education, Science, and Culture (1982-84). Dr. Kanatani also contributed much to the Zoological Society of Japan as an active member of the Board of Council. Dr. Haruo Kanatani is survived by his beloved wife Kazuko, his son Kentaro, and his daughter Kiyoko. KoscAK MARUYAMA Associate Editor Zoological Science eo : | re " ae, aah . q ea 7, | _ yume Soiogretseare lie a cone burt watt oe i i 1h OS At, aN dae hy. Vd tala sane : /* | THOR oe eevee Ore h ye anual uel et aeeyinals } Whe PhS Date cae ee we aoa gery! ne 7 Ge bay “ii a i abe - 4 t 4 i] et j : ‘e: \ F : 2! 4 eats we hey é Reeky reid) 2A 2 a y i i aig is i lig: Ou OTS F be Mb lelet wiieas iy ae aad ay = \, Kone x re < ' = 5 ww. \ mi s \ h a 4 = t vi gl = 1 % Vet iB i} ; Proceedings of the Fifty-Fifth Annual Meeting of the Zoological Society of Japan September 27-29, 1984 Morioka 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 EC : ecology TS : taxonomy and systematics MI: miscellaneous ZOOLOGICAL SCIENCE 1: 852-853 (1984) © 1984 Zoological Society of Japan President’s Message The 55th Annual Meeting of the Zoological Society of Japan held in Morioka, Iwate Prefecture, was a resounding success. On behalf of the Society, I would like to thank Professor Ketichiro Miya, Iwate University and Chairman of the Meeting, Professor Kagehide Shiraishi, Iwate Medical University, Professor Zen’ichiro Hoshino, Iwate University, and other members of the Organizing Committee for their unstinting efforts which produced such a fruitful meeting. At the past two Annual Meetings of the Zoological Society, in 1982 and 1983, I have outlined the goals of the Society for the coming few years. At this time I would like to restate these goals and enlarge upon them. (1) To increase the membership in order to broaden the Society’s base. This would assure meeting the financial needs of the Society and to increase its already important impact on public policy. The Society should cerainly try to prevent any possible decrease in .the number of its members owing to the reorganization of the structure of the Society (see Zool. Sci. 1 (1), 1984). (2) To improve communication between itself and other agencies, societies concerned with biology, and departments of biology at universities and colleges not only in Japan but also abroad. Improved communications can be expected to result in enhanced research progress through the exchange of ideas and techniques. (3) To minimize fragmentation and polarization of the biological sciences despite their di- versity. Increased contact among biologists working in different areas is necessary to foster interdisciplinary interaction. The Society should include investigators, especially younger people, representing the full spectrum of animal biology. (4) To improve presentation of research results. As the number of members increases, the number of presentations at the annual meeting naturally increases. Almost one dozen halls and rooms were needed for oral presentations at recent meetings. It is impossible for participants to hear interesting presentations scheduled at the same time in different rooms; to minimize this problem at the present meeting we are experimenting with the use of poster sessions. In addi- tion, we should consider Symposia, Plenary Lectures and Special Interest Dinners or Lunches. The Society’s meetings must be more than a pleasant place at which to meet old friends. A high quality of scientific presentation is essential. (5) To support the new journal ‘Zoological Science’, which is off to a good start. I hope that it continues to publish good papers and that it attains a world-wide distribution. (6) To foster awareness of humanitarian issues and bioethics at a time when biotechniques, such as human gene manipulation, embryo transfer, and artificial and in vitro fertilization with human gametes, have made great strides, and when natural environments are being menaced. Weneed always to be concerned about the nature and quality of life of the individual —the ultimate social organismal unit — in the biosphere. Changing human ecology in terms of a logarithmically increasing human population, which is dependent upon diminishing environmental resources of the earth, is a biologists’ problem and it cannot be left entirely to the social scientist. To under- stand the individual in his social and physical environment, we, with our broad and basic under- standing of biological principles, must respond to our social as well as technical responsibilities. The Zoological Society has experienced a profound metamorphosis through its reorganization during the past two years, and this has made progress in moving toward attainment of our goals rather slow. We may compare our small short term progress to the statement of the first lunar explorer, then he set foot onto the moon “‘one small step for this man, one giant step for mankind”’. President’s Message 853 Further, we know that advances of tomorrow will grow out of what we accomplish today. The Science Council of Japan now is also under reorganization, and the Zoological Society will have a much more intimate relationship with it than before. The activity of the Society is now becoming increasingly important inside and outside Japan. Finally, I must say that I feel highly honored to have been chosen by my colleagues to serve as President of The Zoological Society of Japan for 1985 and 1986. IL look forward to the coming two years as president, working with our members toward achievement of our goals. September 29, 1984 HIDESHI KOBAYASHI The President of the Zoological Society of Japan ZOOLOGICAL SCIENCE 1: 854 (1984) [THE ZOOLOGICAL SOCIETY PRIZE] Sexual Differentiation of Neural Circuits: © 1984 Zoological Society of Japan Implications for Sexually Dimorphic Neuroendocrine and Behavioral Brain Functions YASUMASA ARAI Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113 Male-female differences in morphologic parameters of the brain function have been demonstrated in certain regions. Recently, evidence is accumulating that sex steroid plays a significant role in modulating postnatal neuronal maturation and neurite formation. Synapto- genesis can be facilitated by neonatal treatment with estrogen in the arcuate and medial amyg- daloid nuclei [1]. Furthermore, the synaptic organization in these nuclear groups was found to be sexually dimorphic [2, 3]. Recently we found sexual difference in synaptic pattern in the ventromedical nucleus_ which seems to be responsible for sexually dimorphic behavioral functions such as feeding and sexual behaviors. There is evidence suggesting a regional difference in sex steroid-concentrating neurons within this nucleus. Receptors for sex Steroid are most abundant in the ventromedial part of the nucleus, whereas they are rare in the dorsomedial part. At the electron microscopic level, synaptic organization also showed a regional difference associated with sexually dimorphic pattern. In the ventrolateral part of the nucleus, for example, the number of synapses was sig- nificantly greater in male rats than that of females, while no sex difference in synaptic number was seen in the dorsomedial part. The synaptic pattern of the dorsomedial part in both males and females was similar to that of the ventro- lateral part in females. Furthermore, sex reversal of the synaptic pattern by the manipulation of the neonatal sex steroid environment was only seen in the ventrolateral part of the nucleus. This provides clear evidence indicating that synaptic sexual differentiation occurs specifically in the sex steroid concentrating neuronal system. Neural components such as the dorsomedial part of the ventromedial nucleus which lacks in sex steroid receptors cannot be modified by the organizational action of sex steroid during the neonatal period. Available morphological evidence for sexual dimorphism is still limited in the hypothalamic, preoptic and limbic structures [1]. The neuropil environment of these neuronal substrates in neo- natal rats possesses considerable plasticity to the organizational action of sex steroids. The major neural circuits driving postpubertal neuroendocrine and/or behavioral functions can be formed under these circumstances during postnatal development. Therefore, the sum of the sexually dimorphic ‘wiring program’ in the various parts of the neuroendocrine brain plays a critical role in the psychoneuroendocrinological differences between males and females. 1 Arai, Y. (1981) Trend. Neurosci., 4: 291-293. Matsumoto, A. and Arai, Y. (1980) Brain Res., 190: 238-242. 3 Nishizuka, M. and Arai, Y. (1981) Brain Res., 212: 31-38. ZOOLOGICAL SCIENCE 1: 855 (1984) [THE ZOOLOGICAL SOCIETY PRIZE] © 1984 Zoological Society of Japan Transitions of Myofibrillar Protein Isoforms and Their Assembly in Developing Muscle Cells TAKASHI OBINATA Department of Biology, Faculty of Science, Chiba University, Yayoi-cho, Chiba 260 Contractile apparatus (myofibril) of vertebrate striated muscles has a highly organized cross- striated structure and both contractile and regulatory proteins are localized in the particular regions of this structure. How cross-striated myofibrillar structure is established during striated muscle development has been of our particular interest. In this research, we examined the dif- ferentiation process of contractile and regulatory proteins specific to striated muscles during myogenesis, characterized protein isoforms ex- pressed in the embryonic and neonatal tissues and then their assembly to myofibrillar structure. 1. Transition of muscle protein isoforms during development Most of myofibrillar proteins exhibit character- istic transitions of isoform expression during embryonic and postnatal development of chicken skeletal muscle. Our general conclusion are as follows. i) The differentiation process of various proteins, myosin, actin, troponin, tropomyosin and C-protein, are not simultaneous. ti) The changes in each isoform proceed in the order: embryonic-type—neonatal-type—adult-type. For example, during embryonic development, cardiac- type C-protein is first expressed in chicken breast muscle and followed by the expression of slow- and fast-type C-protein. During postnatal de- velopment of this muscle, expression of slow C-protein is shut off and therefore only fast-type C-protein exists in the adult muscle. iii) The protein isoforms expressed in cultured muscle cells resemble those in embryonic muscles. iv) One of the factors regulating the conversion of the neonatal type isoforms to adult ones is related to nerve. In the denervated chicken breast muscle, leg troponin T, 3-tropomyosin and _ slow-type C-protein are expressed as in the neonatal muscle. v) In dystrophic muscle, the isoform transitions are depressed and as a result the neonatal type isoforms are generally expressed for a long period. 2. Assembly of contractile and regulatory proteins into myofibril Since the process of myofibril fromation is complicated, we focused our investigation on several steps of myofibril assembly. First, the amounts of monomeric and polymeric actins in embryonic chicken skeletal muscle were examined by the DNase I inhibition assay method so as to understand the polymerization of nascent actin molecules into filamentous forms in the developing muscle. We found that the embryonic chicken skeletal muscle at an early developmental stage contains a large amount of G-actin in comparison with well-developed muscle of adult. We suggests that actin polymerization may be suppressed by some factor in embryonic muscle tissue. Next, the polarity of thin filaments in relation to thick filaments was investigated at early phase of myofibrillogenesis so as to understand the process of the formation of thin and thick filaments bundles. The majority of thin filaments exhibited the right polarity and spatial position similar to that seen in mature mayofibrils. interaction may play an important role in the process of ordered assembly of two different filaments. Thirdly, we examined the assembly of troponin in myofibrilar structures. by an im- Myosin-actin munoelectron microscopy. We obtained evidences which show that three different troponin com- ponents are expressed simultaneously and assembled along actin filaments at early phase of myofibrillogenesis. 856 Physiology eli dl ACTION OF MELANIN-CONCENTRATING HORMONE (MCH) ON EISH MELANOPHORES. H.Kasukawa, M. agai ;- N.Oshima,! R.Fujiil and M.E.Hadley. Dept. Git WIOGl., PAG. Ot Sci., Toho Univ., Funabashi, and Dept. of Anat., Unsaivo Or Amazonia, LLucsonp musa Synthetic salmon MCH was shown to aggregate the pigment granules within melanophores of some fishes (Wilkes et al., 1984). In the present study, we have pharmacologically and physiologically examined the effect of MCH on the nerve- melanophore system iim Gis ikimels Ol teleost species. In the melanoohores of all fishes used, MCH showed the potent melanosome-aggregating action, but had no dispersing effect. The effect was dose- dependent, and the, threshold concentra- tion was about 10 M. Alpha- and beta- adrenergic antagonists did not affect MCH action on adrenergically-innervated mel- anophores. Muscarinic cholinolytics also did not inhibit the action of the hormone on cholinergically-innervated cells of Siluroids. Ca ions were not indispensable for the pigment-aggregating action. Furthermore, MCH aggregated melanosomes in the cells which were insensitive to melatonin. The conclusion was that the effects of MCH are direct on che target cells through its specific receptors. PH 2 ACTION OF MELANIN-CONCENTRATING HORMONE (MCH) ON NON-MELANOPHORAL CHROMATOPHORES IN TELEOSTS. N.Oshimal, H.Kasukawal, Y.Shigematsu! , R. Fujii! and M.E.Hadley?. !1Dept. of Biol., Fac. of Sci., Toho Univ., Funabashi, and Gunz Gi Meshes, Winshy, Gie ANelvoma, Wiese; USA. ey ES a a oe Le The effects of synthetic salmon melanin-concentrating hormone (MCH) on the chromatophores other than the melanophores were examined. In the erythrophores on the split-fin preparations of the platyfish Xiphophorus maculatus and the swordtail xX. helleri, and further in the xanthophores and amelanotic melanophores on the scale of the medaka Oryzias latipes, the pigment aggregation was potently induced by MCH (100M) even in the absence” of Cal vonis- The results obtained by the pharmacologi- cal analyses using the blocking agents such as phentolamine or propranolol suggest that the action of the peptide is probably mediated through its specific receptors on the target cells. in con- trast, the leucophores of the medaka responded by the pigment dispersion only when Ca ions were present. This response is Similar to that induced by alpha-MSH. On the other hand, the motile iridophores of the blue damselfish, Chrysiptera cyanea, which play a major role in coloration and its rapid changes, were not affected by the hormone. PH 3 CONTROL OF GRANULAR MOVEMENTS OF THE AMELANOTIC MELANOPHORES IN THE LIGHT- COLORED VARIETIES OF THE MEDAKA (ORYZIAS LATIPES) . M.Sugimoto, N.Yamaji, N.Oshima and R. IMUG|alaL 5 IDEINes GOie wWalOls, WAC. OF SEs, Woino© Univ., Funabashi. It is known that so-called "colorless" or "amelanotic" melanophores are present in the skin of the orange-red and white varieties of the medaka, Oryzias latipes, which lack the fully pigmented melano- phores. ite is considered that these melanophores possess only inactive tyrosi- nase precursors and are deficient in tyrosine, and therefore have only a small amount of melanin (Hishida et al., 1961). In this study, we have investigated the control mechanisms of motile responses of them. The granules in these cells aggre- gated in response to K ions, catechol- amines, MCH and melatonin. Beta adrenergic agonists, adenosine, atropine and alpha- MSH aroused the granular dispersion in the Cells « These pharmacological analyses indicate that these cells are controlled both neurally through alpha adrenoceptors and adenosine receptors, and hormonally through beta adrenoceptors and receptors for hormones from the pineal and the pituitary. The conclusion was, therefore, that the controlling mechanisms of these cells are just the same as those of the normal melanophores. PH 4 AUTORADIOGRAPHIC DEMONSTRATION OF ADRENER- GIC INNERVATION TO FISH ERYTHROPHORE. S.Miyata and K.Yamada. Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. The pattern of innervation to erythro- phores and melanophores in reddish scales of the swordtail, Xiphophorus helleri ('neon' and 'tuxedo' strains), was deter- mined using 3-norepinephrine (7H-NE) by light microscopic autoradiography. When isolated scales of 'neon' fish were incu- bated with 3H-NE, autoradiograms revealed that dense plexuses of varicose fibers labeled with 3y-NE enclosed the dendrites and cell bodies of melanophores, but no exact plexus that corresponds to erythro- phores could be observed. In '‘'tuxedo' scales, in which only erythrophores exist, a fairly dense varicose plexus was seen to enclose each erythrophore. These labeled fibers were never observed in scales incubated in the presence of excess cold NE or in denervated ones. Potassium ions induced a marked decrease in the labeling of varicose fibers in addition to the aggregation of pigment granules within both chromatophores. These findings suggest that the observed labeled fibers are adrenergic nerves which control the pigment-aggregation response of chromato- phores and further that erythrophores and melanophores of the present fish receive the same adrenergic innervation. Physiology 857 PH 5 PIGMENT AGGREGATION RESPONSE OF MELANO- PHORES OF THE GOBY, TRIDENTIGER OBSCURUS, TO ACIDS. H.Katayama and K.Yamada. Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. Physiological solution acidified with inorganic or organic acid, when applied externally, induced a reversible pigment aggregation within denervated melanophores in isolated caudal fins of the goby. The effective pH which caused the reversible aggregation was 3.0 to 4.2 for HCl, H,SO, and citric acid solutions and 3.2 to 4.8 for ascorbic acid solution, respectively. Monensin (10 pM) and nigericin (2-10 pM), H -ionophores, in isotonic KCl or choline chloride solution also induced a similar aggregation of pigment within melano- phores, respectively. From the response of melanophores to H -ionophores in iso- tonic KCl under various pH, the threshold of intracelular pH to cause aggregation of pigment was estimated and determined to be 6.4. Na waS indispensable for the redispersion of pigment from the aggregat- ed states which were brought about b treatment with acidic solutions and H -ionophores. In this case, Li could be substituted for Na , but K and choline could not. These findings suggest that increase in intracellular H causes aggre- gation of pigment and that H -Na exchange is involved in the process of pigment redispersion. PH 6 BETA ADRENERGIC RECEPTORS IN MELANOPHORE AND LEUCOPHORE OF THE MEDAKA, ORYZIAS LATIPES. F.Morishita and K.Yamada. Zool. Inst., Pac. of Sci., Hiroshima Univ., Hiroshima. The mode of pigment-dispersion re- sponse to beta adrenergic agonists differs considerably between melanophores and leu- cophores of the medaka. To determine the precise nature of the receptors, the responses of chromatophores to specific agonists and antagonist were examined in the present study. Both salbutamol and terbutaline, known as beta-2 agonists, reduced the pigment-aggregation response of melanophores to norepinephrine, causing dispersion of pigment. Propranolol, a beta blocker, blocked the action of these agonists. The agonists did not have any direct pigment-dispersing effect on leuco- phores, but inhibited the dispersion response of cells to isoproterenol. Both atenolol and metoprolol, beta-l antago- nists, inhibited the dispersion response of leucophores to isoproterenol, but did not interfere with the dispersion response of melanophores to isoproterenol. The results suggest that receptors involved in the pigment-dispersion response of the present melanophores are beta-2 in nature, while those of leucophores are beta-l in nature. PH 7 PATTERN OF INNERVATION TO MELANOPHORES IN TAIL FINS OF TELEOSTS. K.Yamada and S.Miyata. Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. The pattern of adrenergic innervation to melanophores in tail fins of the goby, Tridentiger obscurus, and the - guppy, Lebistes reticulatus, was determined using tritiated norepinephrine by light micro- scopic autoradiography. In. taille Gins! 208 the goby, several thick nerve bundles ran approximately parallel to fin rays from central to peripheral. A few thin fibers which branched out from the thick fibers passed along each melanophore to form varicose terminals en passant. No thick bundle was observed in the case of guppy, but numerous thin fibers enclosed the dendritic processes and cell bodies of melanophores, establishing fine nerve plexuses with dense varicose terminals. Repetitive administration of isotonic KCl to fresh fins of the guppy invariably induced a full aggregation of melanosomes within melanophores. By contrast, the degree of melanosome aggregation within the goby melanophores decreased consider- ably with progress in the treatment. The difference in melanophore response between the two fish species may depend on the difference between the pattern of innerva- tion observed. PH 8 EFFECTS OF DIVALENT CATIONS ON PIGMENT AGGREGATION RESPONSE OF FISH MELANOPHORE TON Tit S.Namoto. Zool. iInst.., Fach | 2054 -SGia, Hiroshima Univ., Hiroshima. Li’ induces aggregation of melanosomes within scale melanophores of the medaka, Oryzias latipes. Since Li is known to inhibit the activity of adenylate cyclase, effects of sugh enzyme activator ions as Mg and Mn and an adrenergic beta agonist, isoproterenol, were examined with regard to the pigment aggregating action- of Li . Normally, 65 mM Li causes about 70% of maximum aggregation of pigment within melangphores, but in the presence of 49 mM Mg or 2 mM Mn’ , the level of aggregation decreased to 20 to 30%. When A23187, an ionophore, was added, the level further decreased and melanopohores as7; sumed a nearly fully dispersed state. Li action was also antagonized considerably in the presence of isoproterenol. This effect of amine waS augmented in the presence of Mg , but completely abolished when propranolol, a beta antagonist, was added. These findings suggest that Li causes aggregation of pigment within the present melanophore, reducing the level of cellular cyclic AMP through bition of adenylate cyclase activity. CO ‘n CO PH 9 ADRENERGIC INNERVATION TO SCALE XANTHO- PHORES OF TELEOSTS. I.Kawail, S.Miyata2, K.Yamada2, and kK. Nakane’. Biola Lab. seAnchieP neces College of Nuring, Nagayal, cio! Zo@ils Uimsts, Irec. Of SEk eH ros malsUmdeviens Hiroshima2. The pattern of adrenergic innervation to xanthophores in scales of the medaka, Oryzias latipes, the minnow, Pseudorasbora parva, the molly, Molliensia velifera, and the platy, Xiphophorus maculatus, was in- vestigated using tritiated norepinephrine by light microscopic autoradiography. In scales of the medaka and minnow, in which melanophores are present with xanthophores, dense plexuses of varicose fibers were seen to enclose melanophores. However, such plexus which corresponds to xantho- phores could not observed, but a few fi- bers, which interconnect the plexuses of individual melanophores, were seen to pass along each xanthophore. In the molly scales, where amelanotic melanophores co- exist, a Similar pattern of innervation was observed as in the case of medaka. In the platy scales, in which only xantho- phores exist, dense plexus could not be seen but sparse networks of varicose fi- bers were observed around the individual xanthophores. Based on the observed differ- ences in pattern of innervation, the dif- ference in the mode of pigment aggregation response of xanthophores to Kt, among the present fish species, was considered. An) due ADAPTATION OF A FRESHWATER GOBY Rhino- gobius brunneus TO BACKGROUNDS. T.Naitoh and H.Yamanaka. Dept. of Biol., Shimane Univ., Matsue. In an attempt to understand the mecha- nism of adaptive patterning and de-pattern- ing of fish, color change of the striped type of Rhinogobius brunneus was observed. The fish was nearly pale on the solidly white background, gray on the gray, dark on the black, and striped on the 5 mm black-and-white checkerboard. When one of the colorations of the black-and-white checkerboard was altered to light-gray or dark-gray, leaving the opposite coloration unchanged, melanophores in the dark stripe regions of the skin reacted variedly in response to both the degree of contrast between the background figures and general shades of the background. Melanophores in the pale stripe regions of the skin, on the other hand, were concentrated irre- spective of the strength of contrast and general shade of backgrounds except for 2 of 7 specimens on the dark-gray and black checkerboard pattern in which much of the melanophores dispersed. Melanophores in the dark stripe regions and pale stripe regions of the skin were influenced in different manner by the contrast between background figures. It is likely that melanophores in respective regions basically form a separate set of melanophore groups in their reactions. Physiology Pin} lit IRIDOPHORES OF ODONTOBUTIS OBSCURA ARE MOTILE. Te) gal and yA. Matsuno. Depry Bole mioer Sci., Shimane Univ., Matsue. 2 De Ls ee Light reflecting chromatophores of the scale of a freshwater goby, Odontobutis obscura, are of an iridophore type: These iridophores contain lots of reflecting platelets that are arranged in highly oriented stacks in such a state as these organelles are aggregated in the perinucle- ar regions of the cells. It was found that these iridophores are motile, that is, these cells respond with translocations of the platelets within the cells to various stimuli. The isotonic KCl solution induced dispersion of the platelets in innervated scale preparations, but not in denervated ones. Norepinephrine induced the disper- sion response of the iridophores in both innervated and denervated preparations. Propranolol and dichloroisoproterenol, beta adrenergic antagonists, could not block the dispersion response caused by K* and norepinephrine. Melatonin, also, caused platelet dispersion of the iridophores. a-MSH was effective in aggregating the platelets into the centrospheres of the cells. The conclusions reached are; I) iradophores "Of T= Obsicunaganemmotimncr 2) The movement of the iridophores is under the nervous and hormonal control. PH 12 EFFECTS OF RETINALS ON REGULATION SYSTEMS OF C-GMP PHOSPHODIESTERASE ACTIVITY IN ROD OUTER SEGMENTS OF FROG RETINA. H.Takahashi and T.Yoshizawa. Dept. of Biophys., Fac. of Sci., Kyoto Univ., Kyoto The rod outer segments of vertebrate visual cell contain an enzyme cascade system which plays the important role in visual transduction. The phototrans- formation of rhodopsin to metarhodopsin II activates cGMP phosphodiesterase (PDEase). Metarhodopsin II thermally decomposes all- trans retinal and opsin, the latter of which needs 11-cis retinal for the regeneration of rhodopsin. We have studied effects of retinal isomers on PDEase activation systems. It was observed that 11-cis retinal and all-trans retinal inhibited inactivation of light-induced PDEase activity in frog rod outer segments, but gave no effect on soluble proteins, namely G-protein, PDEase inhibitor, and others. Then the effect of retinal on metarhodopsin II was examined. The amount of metarhodopsin II was measured by circular dichroism (CD) at 360 nm. The results obtained showed that metarhodopsin II in sonicated vesicles of rod outer segments displayed no difference in thermal decay between the presence and absence of all-trans retinal. This results may suggest the presence of some component which may preserve the light induced PDEase activity. Physiology 859 PH 13 WATER SOLUBLE PHOSPHOPROTEINS IN ROD OUTER SEGMENTS OF FROG RETINA. T.Yoshizawa and T.Shinozawa. Dept. of Biophys., Fac. of Sci., Kyoto Univ., Kyoto Wee Sel eS 2 ee ee ee eee The addition of cGMP or cAMP in the pre- sence of Mg or Mn ion together with7- 34 P-ATP to dark adapted disk membranes of rod outer segments (ROS) enhanced the phosphorylation of ROS proteins with 10.5K (Band 1) and 8.5K (Band 2) daltons accord- ing to estimations by a SDS PAGE. But GMP, GDP, GTP and ADP gave no enhancement. The cGMP activated protein kinase showed almost full activity between pH 6.5 and 8.0. The phosphorylated Bands (P-Bands) 1&2 were water soluble and exhibited the Same isoelectric point (5.2). P-Bands 1&2 in a native preparation exhibited to be 19.5K dalton. The phosphorylated sites of P-Bands 1&2 were serine resi- due (s). DEAE-Sephadex A-50 column chromatography could separate P-Bands 1&2 from other phosphorylated ROS pro- teins. Dephosphorylation of the P-Bands 1&2 in dark adapted disk membrane suspen- sion required Mn or Mg ion; the former was more effective than the latter. These data indicate the presence of a Mn or Mg ion activated phosphoprotein phospka- tase in ROS. The phosphorylation ant dephosphorylation of Bands 1&2 were inhibited by the addition of Zn ion. The possible significance of these data was discussed in terms of regulation of Na ion channels in the ROS plasma Membrane. PH 14 DEFECTIVE PHOSPHATIDIC ACID METABOLISM IN THE RETINULAR CELL MEMBRANE OF VISUAL TRANSDUCTION MUTANTS OF DROSOPHILA. H.Inoue, T.Yoshioka and Y.Hotta* Yokohama Ciby:~ Urav. , Sch. of Med., Yokohama, *Paculty of Sci., Univ. of Tokyo, Tokyo. The phosphorylation of photoreceptor phospholipids in the three alleles of Drosophila visual mutant (norpA:no receptor potential A gene) was studied. In the normal strain, the y-°?P of ATP was transferred mainly to phosphatidic acid (PA) and diphosphoinositide (DPI), while, in the mutants, we found that the phosphorylation of PA was drastically reduced, but that of DPI was not. The radioactivity incorporation into PA closely parallels with the degree of the mutant’ genes' expressivity among the three alleles of norpA tested. Enzymes related to the PA metabolism were studied. Diglyceride (DG) kinase activity was found to be almost absent in the photoreceptor cell of norpA. Other enzymes, such as monoacylglycero- phosphate acyltransferase and PA phosphatase, were normal in the mutants. The deficiency in PA content in the retinular cells of norpA, demonstrated by hed incorporation in vivo is therefore, due to abnormal reduction in DG kinase activity. PH 15 FINE STRUCTURE OF THE DISK MEMBRANES OF PHOTORECEPTORS IN THE VERTEBRATE RETINAS AS REVEALED BY THE RAPID FREEZING AND SUB- STITUTION FIXATION. Me pLOshiday insite. Blows, Ota, Univ. O1te. Isolated fresh retinas excised from the eye balls of carps, frogs, geckos and chikens were frozen rapidly by applying the metal contact freezing method at liq- uid nitrogen temperature, followed by the substitution fixation with acetone contain- ing two per cent osmium tetroxide. Thin sections of these materials embedded in Spurr's resin were examined with an elec- tron microscope. The rod disk of verte- brate photoreceptors so far examined ap- pears as a pentalaminar structure, which is composed of two trilaminar membranes and of a thin layer of intradiscal sub- stances fused at their opposing surfaces. Sometimes, the rod disk membrane showed a septalaminar structure, and also two tri- laminar membranes separated with a wide intradiscal space. The cone disk membrane was found to be much more delicate to the tissue preparation than rod disks, result- ing in the artifact of vesicle formation caused by swelling of intradiscal space, The present evidence supports the concept that there exists a distinction between rods and cones, in terms of the swelling tendency noted in the cone disk. PH 16 MORPHOLOGICAL DIFFERENCES IN ROD OUTER SEGMENTS AND MACROPHAGES BETWEEN THE DOR- SAL AND VENTRAL RETINA OF THE BULLFROG. Yo BUSUMANOMOS. WWE Dion a Oki) ANebis 4s Hy ORO OO. of Med., Nishinomiya , Hyogo. Photomicrographs of the vertical sec- tions of bullfrog eyes were obtained for measurements of some spatial parameters, including the curvatures of the cornea, the lens surfaces, and the retina, using whole eyes embedded in celloidin resin. The length, sectional area, and density of the red and green rod outer segments were measured along the meridional line using tissues embedded in Araldite resin. Both the rod outer segments are larger in length, smaller in sectional area, and higher in density at the dorso-central than at the ventro-central. It is Suggest- ed that visual acquity increases with al- most no decrease in sensitivity in the dorso-central "area centralis". The bullfrog retina had more abundant macrophages in the ventral than in the dorsal. Conversely the phagosomes of disc membranes in the pigment epithelium were more numerous in the dorsal than in the ventral. This implies that macrophages play supplementary roles in phagocytosis of detached disc membranes. These differences between the dorsal and ventral retinas are thought to adapt to the weaker light from the lower sur- rounding and the intenser light from the upper surrounding, respectively. 860 Physiology Pray PHOTORECEPTIVE MEMBRANE TURNOVER AND PHAGOCYTIC ACTIVITY OF PIGMENT CELLS IN THE EYE OF EUPLANARIA. N. Tamamaki Dept. Anatomy, Fukui Medical School, Fukui. Mechanism of photoreceptive membrane turnover, especially the mechanism of its removal, was investigated in the eye of euplanaria. At first, euplanaria col- lected from a mountain stream was dark adapted in order to accelerate the degra- dation of photoreceptor membrane. After the dark adaptation from 1 to 10 days, microvilli swelled and degradated into vesicles. These vesicles were accumulated among sensory processes of sensory cells and gradually taken into pigment cells by phagocytosis. At the same time increase of multivesicular body was observed in sensory cells. However the decrease in masses of microvilli is mainly attribut- able to scavenging capacity of pigment censls In daily cycle, small scale of micro- villi degradation and uptake by pigment cell was often observed at 4:00am. Pig- ment cells are working as a main scavenger also in daily cycle, just like pigment epithelium in vertebrate eyes. PH 18 OSMIC STAINING OF THREE DIFFERENT RHABDOMERIC PHOTORECEPTOR CELLS IN ONCHIDIUM. N. Katagiri, Wo Katagiri and K. Fujimoto” (Dept. of Anat’. and Physiol., Tokyo Women's Medical Coll., Tokyo, School of Law Senshu Univ., Tokyo) The cytoplasm of three different rhabdomeric photoreceptor cells (RPCs) in Onchidium verruculatum was studied by electron microscopy. The visual cell (VC) in the stalk eye is a typical RPC of the gastropod species. The lens cell (LC) in the dorsal eye is a unique RPC which is actually a constituent of a lens. The DPC is an extraocular RPC distributed separately in the connective tissue of the dorsal papillae. The cytoplasm of the VC and the DPC is characterized by photic vesicles of uniform size (80 nm in diameter). The LC resembles the DPC in fine structural features, but it con- tains irregularly arranged smooth endo- plasmic reticulum (SER) in the correspond- ing site to the area in which the photic vesicles are found in the DPC. The con- tents of photic vesicles and the cisternae of SER are similarly impregnated with the prolonged osmication ( 2% OsO4 aqueous solution, 40°C, 48 hr). The present study suggests that both photic vesicles and SER of three different RPC in Onchidium are the organelles involved in photoreception and contain components of photopigment. PH 19 LIGHT-INDUCED EXCITATORY RESPONSE AND ITS SPECTRAL SENSITIVITY OF AN IDENTIFIED ONCHIDIUM NEURON. T.Gotow. Dept. o& Phys voly \sich sso metedky, Kagoshima Univ., Kagoshima. The light-induced excitatory response of an extraretinal photoreceptor, the pho- tosensitive neuron (A-Pe-1), in the abdom- inal ganglion of Onchidium verruculatum, was studied using conventional microelec-— trode method and monochromatic light stim- ulation. Illumination of A-Pe-1 evoked a graded, depolarizing response (generator potential), which was accompanied by spikes at higher light intensities. The spectral sensitivity for the light re- sponse showed a single maximum at 490 nm. The threshold energy for minimally detec- table responses was about 1.2 X 10 ae watts per cm square at 490 nm, and this value is comparable to that reported for some mol- luscan retinal photoreceptors. The light— induced depolarization was associated with a decrease in membrane conductance. By changing the external K, the shift of the reversal potential for this depolarization was roughly similar to that predicted for the K-electrode. Decrease in external Na, Ca or Cl did not affect the response. The) results suggest chat they eenenatox potential ot VA>Pesl ass producedmpyea light-induced decrease in K permeability. PH 20 PHOTOEXCITATION RECEPTOR R1-6. K.Mimura. Nagasaki Univ., Fac. Liberal Arts, Nagasaki and Nat. Inst. Basic Biol., Okazaki. aE al See ITY EN ee IME AE CAMRY Ts A Experiments were carried out to de- termine the origin of dual peaks in UV and blue-green (BG) ranges, which appeared in the spectral sensitivity curves of the photoreceptors (R1-6) of the fly. ERG amplitude produced by local illumi- nation was maximum at a depth of about 200m to dim UV and at about 100um to dim BG. With increasing intensity of light, such wavelength-dependent, regional differences disappeared. Amplitude of the small M potential was maximum at a depth of about 200um in the UV-adapted state and at about 100yum in the BG-adapted state. However, when the large M potential was produced, the maximum potential appeared at the deeper site only. Results of microspectrophotometry of a sliced preparation from a white-eyed Musca compound eye indicate the existence of a UV-absorbing substance in the proximal region only of the photoreceptors R1-6. These results suggest that excitations by light of longer (400nm<) and shorter (400nm>) wavelengths are dependent on the region of the cell and that the exci- tations may be augmented in the proximal region by an intervening process. PROCESS IN FLY PHOTO- Physiology 861 PH 21 RHODOPSIN SYNTHESIS IN THE COMPOUND EYE BY SUPPLEMENT OF RETINOIDS AND CAROTENOIDS TO VITAMIN-A DEFICIENT FLY. K.Isono!, Y.Tsukahara’and T.Tanimura’. Research Center for Applied Information Sciences (Ojo Ken), Tohoku University, Sendai; *National Institute of Basic Biology (Kisei Ken), Okazaki. In situ spectrophotometry of the compound eye of Drosophila melanogaster showed that a rapid recovery of rhodopsin content was observed when vitamin A deficient flies were supplemented with a high dose of retinoid or carotenoid through ingestion. Svectral sensitivity of ERG and in vitro spectrophotometry of digitonin extracts from these supplemented flies showed no qualitative difference in the spectrum with those of control flies. HPLC analysis of oximes formed by treat- ing the head homogenates with hydro- xylamine has suggested that the chromo- phore of rhodopsin in the fly supplemented with any carotenoids or retinoids is 3-hydroxy-retinal which has been supposed to be the chromophore of rhodopsin in larger flies. Possible precursors of the chromophore were also identified by this method. Based on these observations, a possible metabolism of the fly rhodopsin chromophore is proposed. PH 22 PHOTOISOMERIZATION OF RETINAL CATALYSED BY RETINOCHROME. J.Uematsu. Dept. of Biol., Fac. of Sci., Osaka Univ. ,Toyonaka,Osaka. Irradiation of all-trans retinal with orange light(>560nm) in the presence of retinochrome at neutral pH results in al- most complete isomerization of retinal into the ll-cis form. This reaction con- sists of three processes, (A) Retino- chrome absorbs photon and turns to meta- retinochrome. (B) Meta-retinochrome dis- sociates to apo-retinochrome and ll-cis retinal. (C) Apo-retinochrome associates with all-trans retinal and retinochrome is regenerated. In the present work, the effects of detergents and salts on this reaction were investigated. Retinochrome was prepared from squid retina(Todarodes pacificus). Isomeric composition of retinal was estimated by HPLC, and reaction velocity was estimated by the change in absorbance at 390nm, Our results showed that process(B) was enhanced by 0.25 - 2 % laulyl ester of sugar (LM) or 10 % digitonin, Process (C) was suppressed by 1 -2 % LM or 10 % digi- tonin. Reaction velocity was changed by 0.5 -2 $ LM or 0.5 - 10 % digitonin. The higher concentration of them, the lower is the reaction velocity. At pH 6.5 1M NaCl or KCl, 0.25 - 1M MgCl.or cacl, low- erd reaction velocity by reduction of process (A). PH 23 FORMATION OF DIASTEREOMERIC PIGMENTS BY COMBINATION OF RACEMIC 5,6-EPOXY-3-DEHYDRO- RETINAL WITH APORETINOCHROME. T. Seki, Y. Kito and K. Shingu. Dept. of BiOlvy, LacweOr Sci. , Univ. Of Osaka. Osaka. Dept. of Chem., Nara Medical College. Nara. For an investigation of the specific isomerization of the prosthetic retinal in retinochrome by light, i.e., from all-trans to 11-cis, an analogue of retinal, 5,6- epoxy-3-dehydroretinal (EDR), was combined with aporetinochrome and the properties of products were examined. Methods for prep- aration of aporetinochrome and for analysis of EDR isomers have been described else- where (Eur. J. Biochem., submitted). Two diastereomeric pigments were produced by combination of racemic all-trans EDR with aporetinochrome. This was ascertained by measuring CD of two EDR isomers collected separately from a mixture of all-trans EDR and aporetinochrome after exposure to or- ange light; the all-trans EDR had a nega- tive CD band around 240 nm while the 11-cis EDR had a positive band in that region. The pigment containing all-trans EDR with negative CD band had a visible absorption maximum around 438 nm and showed some dif- ferent natures from the other pigment (A =464 nm) that exhibited properties simil3d?* to those of retinochrome. The difference in natures between the two diastereomeric pigments was supposed to be due to the dif- ference in conformation of the side chain between antipodes of EDR in aporetinochrome. PH 24 COMPETITION BETWEEN RETINAL AND 3-DEHYDRO- RETINAL FOR OPSIN IN THE REGENERATION OF VISUAL PIGMENT. T.Suzuki, M.Makino-Tasaka and S.Miyata Department of Pharmacology, Hyogo College of Medicine, Nishinomiya, Hyogo 663. Visual pigments with 1l-cis-3-dehydro- retinal as their chromophore are known in many freshwater vertebrates. Often, the vitamin A.,-based visual pigment coexists with a vitamin A,-based pigment to form a paired rhodopsin=porphyropsin pigment sys- tem. In this system, the composition of visual pigment in the retina does not al- ways match the composition of the A-vitam- in stores in the pigment epithelium. We found that rhodopsin regenerated faster than porphyropsin in the preparations of bullfrog opsin and rod outer segment mem- brane. When opsin was incubated with excess amount of an egqimolar mixture of ll-cis-retinal and 1l-cis-3-dehydroretinal, the composition of the regenerated pigment was simply dependent on the ratio of re- generation rates of rhodopsin and porphy- ropsin. This result can provide a mecha- nism to account for the discrepancy in vitamin A,/A., composition between the reti- na and thé p{gment epithelium, The pro- perty of opsin preferring retinal to 3- dehydroretinal may be one of the basic factors affecting vitamin A,/A, visual pigment system. ; 862 Physiology PH 25 EON ACCESS EBieleleiny = ONS ies Gili ©) MOH ORIE: BINDING SITE OF IODOPSIN. WaKeiee, ASSASavawa, WoSinieimicla aincl T.Yoshizawa. Dept. of Biophys., Fac. of SES 5 IKWOES Win Worn IXWVO"Oc lImmeKoies! Cog (ILE alopeale GwieSialefely Elialel digitonin concentration on the stability of iodopsin were investigated in order to clarify the ion accessiblity to the chro- mophore binding site of iodopsin. When a sample composed of 80% iod- opsin and 20% rhodopsin, was thoroughly deionised, the absorption maximum of iod- opsin shifted from 562 nm to 512 nm. It was recovered by addition of Cl but not by addition of NO3 . Thus iodopsin has two forms : Cl -free form and Cl -bound form, which were distinguishable on the absorption spectra. Concentration of Cl on the half saturation of this conversion was at 0.8 mM. The transfer of the chromophore of iodopsin to scotopsin was only observed at low concentration of Cl in the medium. The digitonin concentration and ionic strength were independent of the chromo- phore transfer. In the presence of Cl’ , iodopsin was stable against NH OH and scotopsin. Thus iodopsin in the medium at low Cl” concen- tration partly dissociates into 11-cis- retinal and photopsin so that the chromo- phore easily reacts to NH,OH and scotopsin to form retinal-oxime and rhodopsin, re- spectively. PH 26 THE PROPERTIES OF MONOCLONAL ANTIBODIES TO BOVINE RHODOPSIN T.Iwasa, M.Takao, F.Tokunaga and T.Take- Uchi + Dept. OL Physics, Lac MOS Cl mand *Dep. of Biology, College of Education, Tohoku University, Sendai The monoclonal antibodies were obtained from hybridoma cells cloned after fusion of mouse myeloma cells and spleen cells of a mouse immunized with purified rhodopsin by Con A sepharose column chromatography. Three monoclonal antibodies designated as Rh 29, Rh 112 and Rh 311 were specific for rhodopsin determined by enzyme-linked immuno absorbent assay and immuno blotting. Although Rh 29 and Rh 112 antibodies bound to solubilizedand purified opsin as well as rhodopsin, Rh 311 antibody recognized rhodopsin with higher affinity than opsin. Thermolysin digestion of bovine rod outer segment indicated that Rh 29 anti- body bound to larger fragment containing amino-terminal. The sealed disk membrane prepared according to Litman also had antigenic determinant for Rh 29. This indicates that Rh 29 recognized the cyto- plasmic side of rhodopsinmolecule; the possible antigenic sites for Rh 29 are in the fragments Tyr 60 - Lys 66, Cys 140- Gly 149 and Thr 228 - Ser 239 according to the structual model by Ovchinnikov. The other antibodies, Rh 112 and Rh 311 also cannot bind the carboxyl-terminal fragment from thermolysin digestion of Rhodopsin. rial 2 IMMUNOFLUORESCENT STAINING OF VISUAL CELLS USING ANTI-CATTLE RHODOPSIN MONOCLONAL ANTIBODIES. ) H. eel? Ks Kostner i, Togo and T. Tarenehin. S. Horiuchi 1) Dept. of Biol., Col. of Gen. Educ., Osaka Univ. Toyonaka, 2) Dept. of Phys., Fac. of Sci., Tohoku Univ. Sendai and 3) Dept. of Biol., Col. of Gen. Educ. Tohoku Univ. Sendai. Six monoclonal antibodies have been pro- duced against purified cattle rhodopsin by Takeuchi et al. 1983. By FITC-indirect im- munofluorescent method, the immunoreactivi- ty of these antibodies was examined on the polyethylene glycol embedded sections of the retinae of the cattle, cats, chickens, bullfrogs and Japanese newts. Cone cells were FITC-negative, while rod cells were FITC-positive in all species that we inves- tigated, although FITC fluorescent ataining intensity in rod cells differed among dif- ferent species with the antibodies employed. We haved analysed immunochemical specificity of these antibodies and discussed a possi- bility of their histochemical application to researches in development of rod cells. PH 28 TRIAL OF SEARCH FOR VISUAL PIGMENT GENES OF VARIOUS ANIMALS F. Tokunaga, S. Koike®, Y. Nabeshima**? and K. Ogata*: Dept. of Phys, Hace Otscise Tohoku Univ., Sendai and *Dept. of Biochen., Niigata Univ. School of Med., Niigata. Various animals which have color vision possess more than one kinds of visual pig- ments. They are different in apoprotein, opsin while they have the same chromophore, ll-cis retinal. It is difficult to isolate amounts of visual pigments enough to ana- lyze the difference. Therefore, we tried to isolate partial cDNA of bovine opsin and search for visual pigment genes of various animals. Bovine opsin cDNA's were cloned by using a mixture of 18-base-long synthetic DNA as a hybridization probe. The longest cDNA clone (pBO-1) containing an 811l-bp insert that included the 434 bp of the coding region corresponding to the C-terminal 144 amino acid residues of opsin peptide. By southern blot hybridization with the pBO-1 as a probe, digests of genomic DNA's by restriction enzymes were analyzed. Bo- vine liver DNA gave a rise toasingle band. In the case of chicken and human DNA's, strongly and weakly hybridizing fragments were observed. The strongly hybridizing fragments may contain rhodopsin genes and weakly one might contain iodopsin genes. Present address: °Dept. of Biochem., Fac., of Med., Univ. of Tokyo, Tokyo and tDept. of Bioche., Cancer Inst., Tokyo. Physiology 863 PH 29 SQUID METARETINOCHROME : METARETINOCHROME EXISTS IN TWO FORMS. R.Hara and T.Hara. Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka, Osaka. When retinochrome absorbs light, it bleaches to metaretinochrome, which may act as a direct supplier of 11-czis-retinal to protein opsin to form rhodopsin. The present experiments were aimed at eluci- dating the molecular state of metaretno- chrome. Retinochrome and metaretinochrome were mixéd each with sodium borohydride, sub- jected to sodium dodecyl sulfate-polyac- rylamide gel electrophoresis, and the re- sultant fluorescence bands were examined. The reduced retinochrome showed only one band due to N-retinyl protein, whereas the reduced metaretinochrome had two bands. When extracted with successive portions of n-hexane, metaretinochrome released the 11-cts-retinal in a time-course consisting of fast and slow phases. These findings indicated that metareti- nochrome exists in two forms, with loose and tight coupling of the chromophore to the protein moiety. Those two forms were usually balanced in a molar ratio of about 1 : 2, and the proportion of the tight form of metareti- nochrome was increased in the presence of excess 11-czs-retinal. Based upon these findings, the physiological role of meta- retinochrome in the visual cells was dis- cussed. PH 30 EFFERENT OPTIC NERVE IMPULSES IN RESPONSE TO ILLUMINATIONS OF THE EYES IN THE ORB WEAVING SPIDERS. S. Yamashita. Biol. Lab., Kyushu Inst. of Design, Fukuoka. The responses of the cerebral photo- sensitive neurons (the efferent neurons) in the orb weaving spiders, Argiope amoena and A. bruennichii to illuminations of the eyes and those to electrical stimulations of the optic nerves were studied by re- cording efferent optic nerve impulses. The frequency of efferent optic nerve impulses decreased rapidly, but then in- creased shortly after the onset of illumi- mation of the AL, PL or PM eye. This in- crease was observed for several seconds after illumination. A depolarizing cur- rent to the photoreceptor axons of the AL and PL eyes resulted in a sustained de- crease in the efferent activity, whereas the repolarization caused a transient in- crease in the efferent activity. In the AL, PL and PM eyes, I found that a certain kind of photoreceptor showed a gradual decrease of the amplitude of the depo- larizing receptor potential in the first 10-20 sec of illumination. These results Suggest that the decline of the depo- larization of the photoreceptors causes an increase in the efferent activity follow- ing the onset of illumination of the eyes. PH 31 IONIC MECHANISMS OF POTENTIAL OSCILLA- TIONS IN FROG RODS. K.-I. Takahashi, E.-I. Miyachi and M. Murakami. Dept. Physiol., Keio Univ. Sch. Med., Tokyo. It has been reported that, in rods of several animal species, the membrane po- tential oscillates at a frequency range of 1.5 - 3 Hz when the retinas were per- fused with solutions containing TEA, Ba, or low Cl. We found that this was also the case in rods of the bullfrog, Rana catesbeiana. In all the solutions tested, the oscillations were either damping or continuous, and were best observed at a certain membrane potential level. We also found that, in the bull- frog rods perfused with a normal Ringer solution, a similar oscillation was evoked when the rods were stimulated by a transretinal current pulse. An electri- cal stimulation greatly enhanced the os- cillations occurring in the solution of TEA, Ba, or low Cl, when the current pulse was given in phase of the oscilla- tions. No matter what the triggering mechanisms, all kinds of the oscillations were completely abolished by exposure of the retina to Co solution. These results suggest that the oscillatory potentials are fundamentally identical in respect that Ca conductance increase is involved, and that the membrane properties of rods may be common among animal species. PH 32 Spectral and Angular Sensitivity in Ligia exotica. T.Hariyama and Y.Tsukahara. Research Center for Applied Information Sciences, Tohoku Univ., Katahira 2 chome, Sendai 980 The ommatidial structure of Ligia exotica is an apposition type. In each ommatidiun, there are seven retinula cells with rhab- domeres. An eccentric cells occupies a central position and possesses a dendrite that extends into interrhabdomeric area where it is surrounded by the rhabdomeres of retinula cells R1-7. Retinula cells R4 and R5 are somewhat smaller than the others. Lucifer injection identified the cell whose spectral and angular sensiti- vity was measured. Amax of R4 was 340 nm, but that of R5 was still unknown. The larger cells’ Amax were 520 nm. Lucifer injection to the retinula cells has marked a single cell or intraommatidially coupled cells. In larger cells, it was observed that some retinula cells has only one peak, and others had two or more peaks of angular sensitivity. Some singly marked cells had two peaks. The angles between the two peaks were usually 1.5-2.0°. Morphological measurement of the retina gave the interommatidial angles of ca 1.5- 2202, 864 Physiology PH 33 DIURNAL CHANGES OF SENSITIVITY AND MORPHOLOGY OF HEMIGRAPSUS COMPOUND EYE. K.Arikawa, E.Eguchi & M.Nakamura. Dept Biol., Yokohama City Univ., Yokohama. Matic and Laughlin (1981) reported that the steepness of the linear part of the V-log I curve (n in the Naka & Rushton eq.) becomes much shallow- er in dark adapted locust and fly photoreceptors. Eguchi & Horikoshi (1984) found that the slope of V-log I curves are inversely proportional to the rhabdom occupation ratio (ROR) in the dark adapted eyes of 46 lepidopteran species. In the present study, we have chosen a crab, Hemigrapsus sanguineus as the experimental animal to reveal the relation between ROR and n in one particular species, because the ROR of this species shows a conspicuous diurnal change (ca 1% in the day, ca 9.5% in the night). We measured ROR and the rate of distribution of the screening pigment gra- nules(PM) by the light and electron micrographs and n and log K (light intensity which elicites the reponse of 5Vmax) values by electrophysiology(ERG). The relation between these morphological and physio- logical characters were examined. Strong correlations are observed in ROR-n, ROR- logK in the limited ROR range (less than 52%), and in PM-log K but nothing between PM and n. From these results, it is concluded that one of the determinants of the V-log I curve steepness (n) exists within the rhabdom but not in the effects of pigment migration, and log K is influenced both by the rhabdom size and the distribution of screening pigment granules in the retina. Matic & Laughlin 1981. J Comp Physiol,145,169-177. Eguchi & Horikoshi 1984. J Comp Physiol,154, 3-12. PH 34 LIGHT ADAPTATION MECHANISMS IN CATFISH RETINA. E. Hida and K.-I. Naka. National Institute for Basic Biology. Okazaki. Spatio-temporal dynamics of receptive field center of off-center bipolar cells and off-center ganglion cells were studied in catfish retina (Ictalurus punctatus) using micro-computer system. In both bipo- lar cells and ganglion cells, increase of the intensity of surround illumination resulted in the increase of the incremen- tal sensitivity of center response in addition to the improvement of the tempo- ral dynamics. In only ganglion cells, ill- umination of surround by spatio-temporal random noise of the same mean intensity as steady uniform light brought the marked decrement of the incremental sensitivity of center response without changing the temporal dynamics. The incremental sensi- tivity of center response of ganglion cells was monotonically decreased by enlarging the noise size. Spatial profiles of receptive field center of bipolar and ganglion cells did not depend on the mean intensity nor the contrast of surround illumination. These results suggested that regulation of the sensitivity and temporal dynamics dependent on the mean intensity of stimulus light was mediated by the surround of bipolar cells, and that regulation of the sensitivity by the contrast signal was carried out in the inner plexiform layer. Pil 3D SEROTONIN-LIKE IMMUNOREACTIVE NEURONS IN THE CATFISH RETINA. So RaGalkes, We Nasu ama Koel, Naka. National Inst. for Basic Biol., Okazaki. gxecOpe am EMG Cmiek retina, RO endogenous indoleamine cells were found by the Falck-Hillarp method although there were cells which accumualated exogenous indoleamines. We have found in channel GAELS, LGtaluenus puMmerarus, retina a class of cells with endogenous serotonin (serotonergic cells). Detached retinas were immunocytochemically processed in combination with the PAP method. The serotonergic cells had their somas in the amacrine cell layer but some cells had their somas in the ganglion cell layer. Judging from the shape of soma and disposition of dendrites some of the cells were the type N (or sustained) amacrine cells found in functional study. The cells had an extensive dendritic network which erisscrossed the entire retina and distribution of somas was not random but was not highly regular either. The size of somas in the central region was larger and variance of their size was larger than those in the near peripheral region. Electron microscopic observation showed that the sertonergic cells were postsynaptic to the ribbon synapse of bipolar cells which were probably off- center type. PH 36 CHAN NG WARIS IG PAW URIS OF CAM ILS In GANGLION CELL DISCHARGES Y. Hosokawa, K. Yokoyama and K.-I. Naka. National Institute for Basic Biology, Okazaki WSS S SD ewe ie TER We examined discharge patterns of a large number of channel catfish, Ictalurus punctatus, retinal ganglion cells in eye cup preparation. Post stimulus time (PST) histograms were produced for sikes discharges from about 1000 cells and a DATA base was produced by eight types of stimulus, seven photic generated by an INNISFREE pattern generator and one €Uurrpent injected anto) hordzontalaiceisss: Conclusions were: 1) most of the cells LaOmeawdecd hy Gxemracoliwilar spike discharges could be clasified into two types, on- and off-center ganglion cells, 2) the dichotomy was correlated with discharge patterns evoked by a spot of light, by an annulus of light or by current injected into the horizontal cell, 3) for the horizontal movement of light the magnitude of response could be different depending upon the movement's direction, and 4) many units responded more vigorously to the downward movement than the upward movement of light stripes. Physiology 865 PH 37 EFFECTS OF ORGANIC SOLVENTS ON THE AXONEMAL ATPase ACTIVITY OF CHLAMYDOMONAS FLAGELLA. M.Noguchi, N.Ishida, R.Adachi and K.Kubo. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. Effects of glycerol, ethylene glycol, DMSO and methanol on the axonemal ATPase of Chlamydomonas flagella were examined. Flagella were detached from cell body by exposing to the 30% glycerol solution, and demembranated by 0.05% Triton X-100. About 50% of the axonemes could be reactivated, but after several hours the stocked axonemes could no longer be reactivated. The difference of the ATPase activity between the moving and the stopped ayoneme was not detected. The Mg” -ATPase activity was stimulated by the addition of methanol or DMSO in the reaction mixture. Neither methanol nor DMSO activated the Ca” -ATPase activity of the axonemes. On,the other hand, glycerol inhibited the Mg” -ATPase activity, and ethylen glycol slightly inhibited the activity. These effects were reversible. In the presense of these solvents, the apparent Km was somewhat increased. On the other hand, the effects of these solvents on the Vmax were not uniform. Methanol and DMSO increased the Vmax, but glycerol lowered it. These results might suggest that methanol and DMSO increase the rate of the rate-limiting step in this enzyme system, whereas glycerol decreases it. PH 38 STUDIES ON ROLES OF CALMODULIN IN FLAGEL- LAR MOVEMENT OF SEA URCHIN SPERMATOZOA USING CALMODULIN ANTAGONIST (W-7). F.Iiwasa, Y.Hasegawa, S.Ishijima, N.Niitsu and H.Mohri Depre. of Biol., Univ. of Tokyo, Tokyo. We examined the features of sea urchin spermatozoa treated with a calmodulin an- tagonist, W-7, to elucidate roles of cal- modulin in flagellar movement. Spermatozoa of Hemicentrotus pulcherrimus and Antho- cidaris crassispina were used as materi- als. Flagellar movement of live spermato- zoa was arrested in a straight figure when exposed to W-7, whereas the drug had no effect on Triton-demembranated models. Measurement of stiffness of the arrested flagella revealed that they were ina re- laxed state. Concentrations of ATP and creatine phosphate were the same levels as those in the swimming spermatozoa, al- though oxygen consumption was markedly re- duced. Depolarization seemed to occur when spermatozg2 stopped beating. W-7 did not attect Ca, Furthermore W-7 did not ap- pear to inhibit initiation of motility. Spermatozoa restored flagellar beating completely by addition of calmodulin, and in part by theophilline. But NH, Cl which elevates pH, did not reinitiate motility. The results suggested that calmodulin is involved at least in determining concen- tration of cyclic nucleotide(s). Calmodul- in seems to have multiple targets concern- ing flagellar movement in spermatozoa. PH 39 THE CHANGES OF INTRACELLULAR CYCLIC NUCLEOTIDES DURING THE INITIATION OF STARFISH SPERM MOTILITY. N.Niitsul, K.Ishida? and H.Mohrit. lpept. Ob BLOW COlle On "ARCS and Sel... Univ. Tokyo, and 2Dept. Urol., sch, Med.; Teikyo Univ., Tokyo. We measured intracellular concentra- tions of» cyclic nucleotides and activities of related enzymes, using RIA method, to investigate the mechanism of the initi- ation of starfish sperm motility. When sperm were diluted into sea water, the intracellular concentration of cGMP decreased to about one fifth of the initial value in a few seconds, and then remained at the low level. In contrast, cAMP increased accompanied with the initi- ation of motility (about four times within one minute in Asterina pectinifera). When cAMP concentration was elevated, the activity of adenylate cyclase increased. On the other hand, the activity of gua- nylate cyclase remained constant. The activity of cAMP-phosphodiesterase was not detectable. We further examined the changes under low pH conditions and with sperm samples which were immotile in the absence of histidine. In both cases, cGMP decreased, whereas cAMP did not show any remarkable change. From these results, we conclude that; 1) cGMP decreases when sperm are diluted as a signal of dilution and 2) cAMP increases under the conditions causing sperm motility. PH 40 INHIBITION OF Ca-CHANNEL ACTIVITY BY NEO- MYCIN IN Paramecium caudatum. M.Takahashi. Inst.of Biol. Sci., Univ.of Tsukuba., Ibaraki When cells of Paramecium were stimy- lated by the solution containing high K , they respond with continuous backward swim- ming. Activation of voltage-dependent Ca- channels are involved in this reactiog, Neomycin has been known to inhibit Ca entrance at the neuromuscular junction. In this study, effect of Neomycin on the ciliary reversal induced by K stimulation. When Paramecium cells were transfer- red into the solution containing 20 mM KCl, 1 mM CaCl. and various concentration of Neomycin trom the solution containing 1 mM KCl and 1 mM Cacl., inhibition of backward swimming was observed. In more than 500 AM Neomycin, more than 90% inhibition occurred. Washing of Neomycin immediately restored.pormal duration of ciliary rever- sal. Ca showed an antagonistic action against Neomycin. This action of Neomycin was stronger in acidic solution (pH 5.6) and weaker in alkaline solution(pH 8.2). A possible mechanism of the action of Neomycin which is a polyvalent cation was discussed. 866 Physiology PH 41 REGULATION OF SWIMMING DIRECTION BY cCAMP- DEPENDENT PROTEIN PHOSPHORYLATION IN PARAMECIUM TRITON-EXTRACTED MODELS A.Izumi and Y.Nakaoka. Dept. of Biophys. Engineering, Fac. of Engineering Sci., Osaka Univ., Toyonaka It has been well known that the swimming direction of triton-extracted models of Paramecium is regulated by Ca concentrati- on. In this study, we found that not only Ca concentration but also cAMP regulated swimming direction. Calmodulin antagonists Trifluoperazine(TFP) and W-7, did not inh- ibit the Ca-induced backward swimming in the triton-extracted models prepared by the method of Naitoh and Kaneko(1972). Ho- wever, in the presence of cAMP, which is not enough to alter the swimming direction , TFP inhibited backward swimming. This TFP effect was inhibited by cAMP-dependent protein kinase inhibitor. These results suggest that the extent of cAMP-dependent protein phosphorylation increases by TFP, which causes forward swimming. Actually, addition of excess amount of cAMP inhibit- ed the Ca-induced backward swimming. Fur- thermore, addition of adenosine or adenine to reactivation medium, or treatment of triton-extracted models with sucrose-EDTA, inhibited forward swimming, even in the absence of Ca. cAMP released this inhibit- ion to cause forward swimming. These resu- lts suggest that cAMP plays an important role in forward swimming of triton-extrac- ted models. PH 42 PREPARATION AND THE MECHANICAL PROPERTIES OF THE IMMOTILE FLAGELLA FROM THE RAINBOW TROUT SPERMATOZOA DEMEMBRANATED WITH TRITON X-100. we OkKUM@., Weots OF BLOl., Coll, of Arcs elgel Seis Wiis Soir MOKVOR_ VOKWe Ss Two types of the immotile flagella were prepared from the demembranated sperm of the rainbow trout, Salmo gairdneri. One of them was obtained by removal of cAMP from the reactivating solution containing,MgATP. The other was made by addition of Ca When the Ca concentration was increased up to pCa=8 almost all of flagella stopped with fairly straight shape even in the presence of cAMP and MgATP. Some increase of beat asymmetry was observed a littls below the critical concentration of Ca“~ The change of cAMP concentration did not affect the flagellar responce against Cate The immotile spermatozoa were captured by a glass microneedle coated with poly- lysine at the proximal region of the fla- gellum for micromanipulation experiments. The stiffness measurements by hydrodynamic method revealed that both of the immotile flagella were flexible so that they were in relaxed state. When a bend was formed mechanically in the flagellum which were immotile in the absence of cAMP the bend propagated along the flagellum, although the amplitude was very small. On the other hand, ths immotile flagella in the presence of Ca** could not. These results might suggest that flagella in the absence of cAMP lost only the bend initiation. PH 43 HLECTROPHYSIOLOGICAL PROPERTIES OF ON-OFF AMACRINE CELLS IN THE CARP RETINA. T.Saito and T.kujiraoka, Dept. Physiol. St. Marianna Univ. Sch. of Med. Kanagawa. Retinal amacrine cells are postsynaptic Go) bipolar ceiiiss hey scesponGdmcomisneinits with either transient on-off depolarizations (ON-OFF type) or with sustained depolarizat- ions(ON-type) or hyperpolarizations (OFF- type). It has been suggested that ON-type and OFF-type amacrine cells receive inputs from depolarizing(ON) and hyperpolarizing (OFF) bipolars, respectively, and that ON- OFF amacrines receive inputs from both ON-and OFF-bipolars. Itmay be questioned how ON-OFF amacrine responses could be constructed by both ON and OFF bipolar inputs, since ON- OFF amacrine response are always depolariz-— ing and no hyperpolarizing components are seen. We tested effects of ions, drugs and currents on ON-OFF amacrine and effects of bipolar polarizations on them. As the cell was depolarized, both transient depolarizat- ions of ON-OFF amacrine reversed their po- larity in the range of +15-+50mV. Low Cr -medium,which suppressed on-responses selec- tively in the mudpuppy retina, didnot always suppress on-responses only in the carp retina. When an ON-bipolar was depolarized by a sustained current, the membrane potential of an ON-amacrine depolarized in sustained manner, while those of an ON-OFF amacrine in transient manner. These results suggest that ON-OFF amacrine receive inputs from ON-bipolars directly, but their synaptic mechanisms are different from those of ON- amacrines. PH 44 EFFECT OF SOME SYNAPTIC REAGENTS ON SPREADING DEPRESSION IN THE FROG RETINA. M.Fujimoto. DIOS OI Wiay~SilOll . , Sits Marianna Univ. Sch. of Med., Kawasaki. Recent studies have indicated that retinal spreading depression (SD) is triggered by abnormal synaptic activity in the inner plexiform layer which consists of many kinds of synapse and that Ach and glutamate have an important role in the eccurnence of SD. Using the spreading depression potential as an index, the effects of several transmitter candidates and their antagonists on SD, evoked in the dark with a low Cl” medium, was investi- gated. The £requency of SD | (ESD) iiwas increased in the media containing Ach, glutamate, noradrenaline, or dopamine, but overdoses of these substances frequently abolished SD. Unexpectedly, GABA and glycine also increased FSD in many cases. Curare, atropine and serotonine blocked SD in concentrations over 1 mM, but they enhanced SD in lower concentrations. Phentolamine and propranolol over 1 mM abolished SD. In lower concentrations, however, FSD was increased by phentolamine but decreased by propranolol in almost all the cases. Methysergide always decreased FSD. These results suggest that catechol— amine and indoleamine are also involved in the mechanism of SD generation. Physiology 867 PH 45 NONSPIKING VISUAL INTERNEURONS IN THE CRAYFISH BRAIN. Y.Okada and T.Yamaguchi. Dept. of Biol., Fac. of Sci., Univ. of Okayama. Okayama. It was found by means of intracellular staining technique of Lucifer yellow that there are six pairs of nonspiking visual interneurons which may connect post- synaptically to the sustaining fibers (third order visual interneurons). These interneurons have thick (25-50 um) and long (400-500 um) dendritic processes in the protocerebrum and their somata are located near the distal end of the optic tract. Electrophysiological study of these interneurons revealed that they are characterized by the spontaneous fluctu- ation of resting membrane potential (30-50 mV) without stimulus and by the initiation of depolarizing or hyperpolarizing poten- tial without spikes in response to the ipsilateral or contralateral illumination. Three pairs of them (G-type) have thick (50 um) dendritic processes and forma cluster in which they are interwound each other. The remaining three pairs (S-type) surround closely the cluster and they have thin (25 um) dendritic processes. The firing rate of the eye-up fiber (one of the compensatory oculomotor neurons) is increased or decreased by depolarizing or hyperpolarizing current injection into each of G-type nonspiking interneurons. This fact indicates that these nonspiking interneurons may function as premotor neurons. PH 46 CHROMATIC PROPERTIES AND INPUT STEMMATA OF THE MEDULLA NEURONS IN THE SWALLOWTAIL BUTTERFLY LARVA. i. senikawa. Depts of Biol.,. Pac. of Sci., Kyushu Univ., Fukuoka. The larvae have six stemmata on each side of the head. Spectral properties of the neurons in the second neuropile (medulla) of the optic lobe were intra- cellularly examined by stimulating the six stemmata individually by monochromatic flashes. Most medulla neurons received specific specral inputs from more than two stemmata. They can be classified into two groups. The first receive dominant inputs from one stemma and weak inputs from other two to five stemmata; the weak inputs are usually antagonistic to the dominant ones, when tested by white light stimuli. At least 14 types of neurons are identified, depending on the spectral properties of their dominant inputs. Ten of them show color-opponency in the dark and/or under light-adapted conditions. They usually include a few analogous neurons which receive dominant inputs of the same spectral type from different stemmata. Neurons in the second group receive inputs of similar magnitude from two to six stemmata. The stemmata feed some neurons with synergistic inputs and some neurons with antagonistic inputs. PH 47 ANATOMY AND RESPONSE OF THE HIGHER-ORDER NEURONS OF COCKROACH OCELLI. M.Mizunami, and H.Tateda. Dept. of Biol., Fac. of Sci., Kyushu Univ., Fukuoka. pn ed ES ae TE Ba ceed The neurons of the posterior slope neuropiles of the protocerebrum whose activities were affected by the ocellus were studied anatomically and physiologi- cally in the cockroach. These neurons, which are the 4th or 5th order ocellar neurons, are divided into three classes from their anatomy as follows. Class I. Neurons whose branches are restricted in the brain. They are further divided into four types. One type connects bilateral posterior slopes and exhibits inhibitory response during illumination, one has branches in the central body and in the alpha- and beta- lobes of the mushroom body and exhibits transient on-hyperpolarizing and off- depolarizing response, one has branches in the protocerebral bridge and exhibits transient off-hyperpolarizing response, and the other has branches in the trito- cerebrum and exhibits transient off- depolarizing response. Class II. A neuron with terminal branches in the lamina neuropile of the optic lobe. It exhibits tonic spike discharge during illumination. Class III. Descending neurons. They exhibit a few spikes or transient depolarizations when the light is off. PH 48 CYTOSKELTONS OF TWO TYPES OF INSECT MECHANORECEPTORS. Neo OMe DEDt. 1.0L LOL.» FaGs, OL SCivy Kyushu Univ., Fukuoka Cytoskeltons of two types of insect mechanoreceptors have been examined with reference to the receptor mechanism. The campaniform sensillum on the haltere of Drosophila possesses a single fan-shaped sensory cilium, which contains distally a tubular body. The tubular body consists of regularly arranged microtubules, which are interconnected to each other by dense substance, and connected to the ciliary membrane by fine filaments. The antennal chordotonal sensillum of Periplaneta con- tains two receptor cells: their sensory cilia are flagellar-shaped. The two cilia are free in the lumen formed by a scolopale cell. One cilium ends free in the cap, whereas the other ends with a swollen terminus trapped in the cap. Axonemal microtubules extend throughout the cilium. The microtubules and the Ciliary membrane are connected at some intervals. Membrane specializations are found around the distal margin of the sensory dendrite, where the cilium Originates. These results suggest that the sensory cilium itself may be a receptor site in the campaniform sensillum, whereas the cilium may be an accessory structure and the distal region of the dendrite may be a receptor site of the chrdotonal sensillum. 868 Physiology PH 49 MECHANICAL PROPERTIES AND ANGLE THRESH- OLDS OF CERCAL FILIFORM SENSILLA OF A CRICKET. T.Shimozawa and M. Kanou.,Div.Behav.Neuro- biol.,NIBB,Okazaki and Zool.Inst.Fac.Sci. Hokkaido Univ.,Sapporo. Sensory thresholds of cercal hairs of Gryllus bimaculatus to alternating air- current were measured at various frequen- cies. The hairs were range-fractionated depending on the length; long hairs were velocity sensitive and short ones were acceleration sensitive. The long-hair deprivation had no ef- fect on the thresholds of large interneu- rons LGI and MGI. Comparison of threshold between hairs and interneurons revealed that the long hairs converge upon inter- neurons‘ 10-2 and 10-3, and that the short ones do upon LGI and MGI. After the measurement of spring stiff- ness of hair base, the hair deflections were estimated by a numerical computation of the equation of motion. The boundary layer due to air viscosity was taken into account. Based on the estimated deflec- tions and the measured thresholds, the angle threshold of sensory neuron was de- ESOUINSG 45 Oo002> am long inate. Sensory neurons of short hair were sensitive to the rate of deflection but not to ae sta- tionary deflection. The combination of hair length, spring stiffness, and rate of relaxation of sen- SOry process, underlies the range frac- tionation of the filiform sensilla. PH 50 SENSORY HAIRS ON THE THORACIC TERGITE IN ISOPODA, LIGIA EXOTICA : ODOR AND WIND RE- CEPTION T.Hatanaka. Dept. Biol) Bacs of Hauck, UIEVe Of Chiba Chatbar ee eee Sensilla on the carapace of Ligia exo- tica were studied by scanning electron mi- croscopy and single-unit extracellular re- cording. The sensillum was composed of three structures which were arranged along the longitudinal axis of the animal body; anterior cuticullar creases, a blunty tip- ped sensory hair and a spoon-like foliate plate laying posteriorly. A single apical pore and an articulation of the hair sug- gested chemo- and mechano-sensitivity. Air current with various orientation and various odor puffs were applied asa mechanical and a chemical stimulations re- spectively. Wind blowing from behind of the animal was most effective, and this directional sensitivity probably reflected the morphological orientation of the sen- Sillum. Each unit responded to only one or two odors used. A relatively narrow spectral sensitivity and differences in threshold and slope of dose-response curve among units seemed to be available to odor discrimination. Chemical and mechanical reception sup- posed by morphological study was confirmed by electrophysiological study. il Bt THE EFFECT OF ABSTINENCE FROM WATER ON THE RESPONSE OF THE HYGRORECEPTORS OF THE COCKROACH. N.Doi, Y.Toh, and H.Tateda, Dept. of Biol., Fac. of Sci., Kyushu Univ., Fukuoka The sensillum capitulum of the cockroach Periplaneta contains moist and dry hygro- receptors, both responding to the relative humidity (Yokohari et al., 1976). In the present study effects of abstinence from water on the responses of hygroreceptors of Periplaneta were electrophysiologically investigated. After four days of abstinence from water, the responses to humidity stimuli increased in the moist receptor and decreased in the dry receptor as compared with those before the abstinence. After watered, responses recovered to the initial level. Therefore, the abstinence from water appears to increase probabilities of animals to detect water through change of their internal environment. After cockroaches were adapted to some humidity levels, responses of hygroreceptor were recorded. When the animals were adapted to lower humidity, sensitivities of moist receptors increased and those of dry receptors reduced. MThus, both abstinence from water and adaptation of animal to a dry environment produce a similar effect on the hygroreceptor responses. These results may show some steps of hygroreception may be simulta- neously controlled by both external and internal factors. PH 52 AN ANTENNAL COLD RECEPTOR OF THE COCKROACH, PERIPLANETA AMERICANA. M.Nishikawa, F.Yokohari and T.Ishibashi. Biol.Lab., Fac.Sci., Fukuoka Univ., Fukuoka The responses of a cold receptor were recorded from the thermo-olfactory sensil- lum on the cockroach antenna, in order to determine what the receptor respond to. Its candidates are as follows; temperature itself, temperature difference, temperature gradient, and change rate of temperature gradient. As for the temperature itself, the receptor activity at constant tempera- | ture had tendency to be somewhat high at 20 C to 30°C within 19-40°C examined, but it varied considerably from time to time. If the receptor were to respond exclu- Sively to the temperature gradient, the response to it should be constant and be independent of temperature itself. We meas- ured the responses to linearly falling tem- perature within the range of 23°C-27°C. Our results were F=29.1-0.37T (r=0.97) at dT/dt=-0.052 C/s and F=39.2-0.88T (r=0.97) at dT/dt=-0.014 C/s in a single receptor cell, where F is impulse activity (Hz), T is temperature and t is time. The relation between the response and temperature gradi- ent (dT/dt) was F=11-100dT/dt (r-0.93) at ABC. The response was highly dependent on temperature gradient but seemed to be somewhat dependent on temperature itself. The examination on the other candidates is in progress. Physiology 869 PH 53 CONTROL MECHANISMS INVOLVED IN THE REGULA- TION OF THE CERCAL NEUROMUSCULAR SYSTEM OF THE CRICKET (GRYLLUS BIMACULATUS). K.Mori and M.Sakai. Dept. of Biol., Fac. efssei., Univ. of Okayama. Ckayana -Ihe male cricket shows rhythmic move- ment of the cerci in response to tactile stimulation by a female during copulatory behavior. We analyzed it kinematically by a position sensing device in the decapi- tated preparation and examined neuro- muscular system involved in the pattern generation. The tip locus of the moving cercus is as follows; it starts with small upward twitch(Pl), followed by medio-ven- trally directed stroke(P2) in 50ms and then returns to the initial position(P3). The muscular system consists of six muscles(M1-M6) in radial arrangement, con- necting the cercal base with body cuticle. Electrical stimulation of those muscles elicited movements to five different di- rection. Intracellular recordings revealed that each muscle is innervated by one large, at least one small excitatory units and one common inhibitory unit. Extracellular re- cordings from the motor nerve indicated that the Pl is associated with an increase of M5 unit, and P2 with a decrease of M5 and an increase of three large units in- cluding M6. Those results suggest that both Pl and P2 phases are essential for generation of cercal rhythmic movement while P3 phase is a passive turning to the resting state. PH 54 THE STRUCTURE AND FUNCTION OF TEMPORAL ORGAN OF SCTIGEROMORPHA. K.Yamana, Y.Toh and H.Tateda. Dept. of Biol., Fac. of Sci., Kyushu univ. Fukuoka. A pair of temporal organs occur on the head of soil arthropod. There have been several structural accounts about the temporal organ, but few physiolosical reports. In the present study, the structure and function of the temporal organ have been examined in Thereuonema hilgendorfi, (Sctigeromorpha). The temporal organ contains about ten receptor cells and more than one hundred supporting cells. Each receptor cell possesses two sensory cilia. The cilia distally terminate in contact with a specialized surface cuticle, which is permeable to lutenium solution. The receptor cells show spontaneous discharge (15-30 impulses/s), which are depressed by CO, stimuli ina phasic-tonic manner. Impulse frequencies in both phasic and tonic phase decrease lineally with logarithmic increase in CO? concentration ranging from 0.001% to 0.1% (phasic phase) and from 0.05% to 5.0% (tonic phase). These ranges cover the CO? concentration where soil animals live. From these results, we conclude that the temporal organ of Sctigeromorpha is functional COz receptor. PH BS ODOR RESPONSES OF OLFACTORY RECEPTOR CELLS ON THE ANTENNAE IN DUNG BEETLES. J.Inouchi and T.Shibuya. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. This investigation was undertaken to study receptive mechanisms in single ol- factory receptor cells associated with s.basiconica in dung beetles (G.auratus and C.pecuarius) to the characteristic odors from cattle's dung (2-butanone, phenol, p-cresol, indole and skatole). Their receptor cells responded to the odors were divided two types (type I and type II). Type I cells responded strong- ly to 2-butanone only. Dose-response curves of type I cells were relatively steep. Threshold concentrations differed from cell to cell. 2-Butanone was an ef- fective stimulus for all cells of type II, while the responses to other effective odors differed from cell to cell. The dose-response curve of type II cells to 2-butanone resembled with that of type I cells, however, those to other odors didn't rise steeply. Individual type II cells had different threshold to each odor. These results suggest that the type I cells and type II cells may have dif- ferent coding mechanisms respectively. PH 56 PROCESS OF SLIDING DISINTEGRATION OF CILIALY AXONEMES. M.Suzuki and T.Miki-Noumura, Dept. of Biol. Ochanomizu Univ., Tokyo Demembranated ciliary axonemes of Tetrahymena pyriformis disintegrate spon- taneously without external proteolysis. We investigated the disintegration re- sponse of the axonemes, mesuring the tur- bidity change. At higher conc. of ATP than 40uM ATP, the time course of the decrease in 0.D. is divided into 3 steps. Duration of step II depends upon conc. of ATP. The rate and the magnitude of 0.D. decrease don't depend upon conc. of ATP through all steps. The duration of step II depends upon conc. of PEP in pyruvate kinase-PEP system. Based on the quantitative analysis of adenosine nucleotides in time course, ATP conc. at the transition point from step II toIII was determined to be about 40uM ATP. A dark-field microscopy revealed that one or two doublets are extrude from an axoneme at step II,and after step III, Sliding process is completely finished. Analysing the micrographs with a desitizer, we determined the mean number of extruded doublets to be 1.5 at step II,and 4.4 at step III. These results show at higher conc. of ATP, one or two doublets slide out from an axoneme at first, and the other doublets Slide out completely just when added ATP is hydlysed to 40uM ATP. 870 Physiology ime RESPONSES OF SWIMMING SEA-URCHIN SPERMATO- ZOA TO ELECTRIC STIMULATION. Go ShangyO7., ele aug camanda kes alwallialsinais AOYILA IMGs IMACS OH Seale, Wimawy5 WOlWOsp Tokyo. Echinoderm spermatozoa are not known to show a taxis, although recent studies have indicated that their beat form is con- trolled by calcium. In the present study, we have investigated the effects of elec- tric field stimulation on the behavior of free-swimming spermatozoa of Hemicentrotus pulcherrimus and Anthocidaris crassispina. A drop of sperm suspension in artificial sea water was placed between two Pt elec- trodes set 4 mm apart on a slide. A 30-ms clo6eGe joHlse or 5 coQ 100 W was aAjjpiliec between these electrodes. The response of the sperm swimming near the bottom surface was recorded by video. Stimuli of >75 V induced a response in >90 % of the swim- ming sperm within a fraction of a second. Typical responses were: quiescence fol- lowed by swimming with increased beat symmetry, an increase in beat asymmetry without quiescence, and an increase in Swimming velocity. The increased beat symmetry following quiescence made the Swimming path more straight than it was before the stimulus. During this period of increased symmetry, an increase of Swimming velocity was observed in many cases. These results might have implica- tions in the possible tactic behavior of the spermatozoa. PH 58 BENDING TRANSIENTS DURING RESPONSES TO ELECTRIC STIMULATION OF SEA-URCHIN EMBRYOS. So/\-Beloe eine VYoMOGemt. Deor, oF Biol. , Ochanomizu Univ., Tokyo. From high-speed cinefilm records we di- rectly plotted the course of responses of Sea-urchin embryo cilia stimulated in dif- ferent beat positions. The response of Cilia of the early developmental stages, blastulva and gastruila. (is Vsimalanmto ibuie different from ciliary reversal, which ap- pears only in the later stage, pluteus, and therefore designated ‘primitive re- sponse'. In these stages cilia stimulated at any beat position do NOE chanGger ner beating before stopping at the middle of Wile EiiECiEe Srwols, Winks wesuile Suge gests that the most sensitive period for stimulation is the period when cilia are at the middle of the effective stroke. In the ecxliary reversal cilia stimulated at any beat position change their bending forms with a definite short delay by pull- ing back into the start position of the SIEGE nVE Sele 5) Wine Clieiniete atin ielne course of the movement, when analyzed in EES OF ie Lehane enc Me wir elie Ci il teney rs, us Meweesic i@ir @ullig Sieimulleice) ain the early effective stroke; in these cilia change induced by stimulation is detected earlier in the basal-region of the cilia than in the distal. This suggestsa dis- tally-directed conduction of the induced (SEAN e rn SS) MOVEMENT OF THE SINGLE CILIUM ON THE ISO- LATED CELLS FROM SEA-URCHIN EMBRYOS. Y.Mogami, S.Sekiguchi and S.A.Baba. Dept. Or S1Ol>, Ochanonaizui Unive, Tokwo. Ciliated epithelial cells of "seasunelnaan embryos has a single cilium per a single cell. In order to obtain ene precise one tographic recording of the movement of the single cilium, Yeilvated! cells clnesca= urchin (Hemicentrotus pulcherrimus) em- bryos were isolated with Ca and Mg-free medium containing 0.9M glycine. The move- ment of a single cilium on the isolated cells, which were usually rotating, was recorded stroboscopically. After hatching through the pluteus stage both the beat frequency and the beat form did not appar- ently change. When the isolated cells were compaired with the cells in intact embryos in terms of ciliary movement, it was found that the isolation caused an increase in the period of the recovery ‘strokemohura decrease in thait olf “ehie epi ecbnsyemestaneicers As the result of the isolation the plateau phase in the curve of the tip angle dimin-= ished to be undetectable, which is seen between the effective and recovery phases reaching up to 30% of the beat cycle in UME SOLIS. i) Soc OF che changes in the beat pattern, isolated cells seemed Still to have the capability off ete ciliary responses, since they showed the Ciliary reversal spontaneously at the pluteus stage. PH 60 THE RELATION BETWEEN THE LENGTH AND THE SLIDING FORCE OF MICROTUBULES MEASURED UNDER 'ISOMETRIC' CONDITIONS. K. Oiwa and K. Takahashi. Zool. Inst., Fac. of Sci., Univ. of Tokyo., Tokyo. The force of microtubule sliding in demembranated sea urchin sperm flagella was measured by Kamimura and Takahashi (Nature, 293: 566, 11981). Using =basmcalaiky, the same method, we have studied the rela- tion between the length and the maximal sliding force of microtubules in Triton- extracted sperm flagella of the sea urchin Hemicentrotus pulcherrimus. We measured the maximal sliding force in a demembra- nated, elastase-treated axoneme, reacti- vated by a solution containing 200 uM ATP, by the bending displacement of a glass needle attached to the axoneme. The extent of the microtubule sliding was minimized by using a relatively stiff needle. Under such nearly isometric conditions the force was found to increase linearly with the length of the axonemal segment lying be- tween the needles. This indicates that the dynein arms are uniformly reactivated along the microtubules. The average iso- metric force generated by a dynein arm was estimated at about 1 pN. The histogram of the apparent force per dynein arm showed a skewed distribution, indicating that syn- chronized sliding may sometimes occur at two or more mechanically independent sites. Physiology 871 PH 61 CA-BINDING PROTEINS IN THE ANTERIOR BYSSAL RETRACTOR MUSCLE OF MYTILUS EDULIS. *T.Yamanobe, *T.Mimura and ~ HH. Sugi. *central Lab. Analyt. Biochem. and. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo. It has been shown that, in the anterior byssal retractor muscle (ABRM) of Mytilus edulis, Ca ions are accumulated along the inner surface of the plasma membrane, and are released into the myoplasm to cause con- traction[Atsumi and Sugi, J.Physiol,257,549- 560 (1976)]. To study the mechanism of the above Ca accumulation, which is entirely different from that by the sarcoplasmic re- ticulum, we started to detect Ca-binding proteins in the plasma membrene enriched fraction obtained by centrifugation of crude extracts of the ABRM with discontinuous su- crose gradient. SDS-polyacrylamide gel elec- trophresis combined with **Ca autoradiogra- phy showed a distinct peak of radioactivity at 450K and another less distinct peak at 17K. The 17K Ca-binding protein is regarded to be calmodulin [Yamanobe, Mimura and Sugi, Zool.Mag., 91, 504 (1982)]. The 450K Ca- binding protein has a marked Ca-binding ca- pacity, and we think that it may be mainly responsible for the Ca-accumula- tion along the inner surface of the plasma membrane in the ABRM. Experiments are in progress to clarify the properties of the 450K protein. PH 62 MODULATORY EFFECTS OF BIOGENIC AMINES ON MYTILUS SMOOTH MUSCLE. M.Matsuura, Y.Muneoka and M.Kobayashi. Fac. of Integrated Arts and Sciences, Hiroshima Univ., Hiroshima. Modulatory effects of serotonin, dop- amine and octopamine on the ABRM of Mytilus edulis were examined by using a sucrose-gap method. Serotonin potentiated muscle contraction by increasing membrane excit- ability at lower concentrations. At higher concentrations (10-5 M or higher) it first inhibited muscle contraction by hyper- polarizing the membrane, then potentiated the contraction by increasing the excit- ability. Dopamine also potentiated the contraction by increasing membrane excit- ability. In addition, it increased ampli- tude of excitatory junctional potentials. We could not observe dopamine-induced hyperpolarization of the membrane. However, at high concentrations (107-5 M or higher) dopamine depressed ACh contraction when it was applied simultaneously with ACh. Octopamine increased membrane excitability and amplitude of excitatory junctional potentials and, thus, potentiated muscle contraction. The amine did not seem to increase ACh potential. These results Support the idea that octopamine acts not only on muscle membrane but also on excitatory nerve terminals to increase release of excitatory transmitter, ACh. PH 63 CONTRACTION OF SINGLE SMOOTH MUSCLE CELLS ISOLATED FROM A MOLLUSCAN CATCH MUSCLE. Ns tei. ZOOL. Tnst., Fac. Of Seis Univ. of Tokyo. Tokyo. In order to obtain an insight into the regulation of catch contraction in the anterior byssus retractor muscle (ABRM) of Mytilus; mechanical and pharmacological properties of smooth muscle cells isolated from this muscle were studied by recording their isometric contractions following methods similar to those described pre- viously (Ishii & Takahashi, J. Muscle Res. Cera Motile, 3 25, 1962). Both singie (0.2 - 0.5 ms duration) and repetitive (5 - 10 Hz) electric stimulations elicited in the cells a tonic contraction with a slow relaxation that can be described by a single exponential (time constant T 2150 s ) or a sum of two exponential compo- iM siaueshe (et pas) y GRD oO) Ss We During the relaxation, no significant tension redevelopment was observed after a quick release by ca. 1 % of the cell length, indicating the absence of an active state. On the other hand, both single and repeti- tive stimulations elicited a phasic con- Podge on ("a= 20 es ) in the presence of 107 M 5-hydroxytryptamine (5-HT). The results suggest that, in the absence of 5- HT, the individual smooth muscle cells are capable of catch contraction in response to electric stimuli, irrespective of the mode of the stimulus. PH 64 PROPERTIES OF THE RAPID-COOLING CONTRAC- TURES IN GUINEA-PIG TAENIA COLI. A.Emura and H.Sugi. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo. The physiological properties of the rapid-cooling contractures in guinea-pig taenia coli were studied at various con- ditions. In normal Krebs solution, the Magnitude of the contracture tension, which amounted to about 60% of the maximum K-induced contracture tension, did not change appreciably when the temperature of the solution was suddenly lowered from 33 to 27-10°C. The mechanical response to rapid cooling was eliminated after the re- moval of external Ca ions (3mM EGTA pre- sent) or by the addition of Mn iong (5mM), but was not affected in the presence of procaine (2mM). These results indicate that the inward movement of external Ca ions may be responsible for the rapid- cooling contractures. The rapid-cooling contractures were also produced in pre- parations which had almost relaxed in a solution containing 120mM-K, and the mag- nitude of the mechanical response increas- ed as the magnitude of the temperature Step was increased. If the preparation was returned to the standard temperature (33°C) and again made to contract by rapid-cooling, the magnitude of the res-~- ponse decreased as the time in the stand- ard temperature was shortened. 872 Physiology PH 65 EFFECT OF TONICITY ON THE CONTRACTILE TENSION IN GUINEA-PIG TAENIA COLI. M.Toride and H.Sugi. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo The effect of tonicity of the bathing solution on K- and acetylcholine-induced contracture tension was studied on guinea- pig taenia coli. The contracture tension in response to 145mM-K or 10-3M acetyl- choline was reduced by about 50% in both a hypertonic solution (osmolarity 1.4 times the standard solution) and a hypotonic solution (osmolarity 0.6 times the stand- ard solution). This result contrasts with the effect of tonicity on vertebrate skel- etal muscle, in which the tension develop- ment is depressed inhypertonic and enhanced in hypotonic solutions. The depressing ef- fect of the hypotonic solution was not due to a reduction of Na ions, since the re- placement of Na by sucrose in solutions with normal tonicity did not affect the contracture tension. The force-velocity relation was also examined at various toni- cities by applying isotonic releases at the peak of contracture tension, and the maximum shortening velocity was found to be larger in a hypotonic solution than in a hypertonic solution having the same ten- sion depressing effect as the former. The rate of rise of contracture tension was markedly decreased in hypotonic solutions, but not in hypertonic solutions suggesting a slow and incomplete activation process. PH 66 STIFFNESS AND TENSION TRANSIENTS IN OSMOTICALLY COMPRESSED SKINNED MUSCLE FIBERS OF FROG. T. Tsuchiya and H. Sugi. Dep. of Physiol., sch. of Med., Teikyo Univ., Tokyo. The instantaneous stiffness and the tension transients as a function of rela- tive fiber width were measured in relaxed, activated and rigor skinned fibers from frog. Fiber width was reduced by adding PVP (polyvinylpyrrolidone) or Dextran to the bathing solution. The stiffness was detected in a relaxed reduced=-width fiber and it increased with degree of compres- Sion. Higher stiffness was observed in activating solution than in relaxing one at the same degree of compression. It is noticeable that the increased stiffness induced by activating solution in addition to that in relaxing one is nearly constant in a compressed fiber in the wide range of polymer concentration. This fact suggests that there are two kinds of stiffness, Ca sensitive and Ca insensitive, in a com- pressed fiber and they exist in parallelly arranged structure in a fiber. The obser-= vation of the tension transients of a relaxed reduced=width fiber and an acti-e- vated reduced=-width one supports the above view. In rigor reduced=width fiber also, two kinds of stiffness were suggested to exist. PH 67 MEASUREMENT OF LOCAL HEAT PRODUCTION IN FROG SKELETAL MUSCLE DURING ISOMETRIC CONTRACTION WITH AN INFRARED THERMOGRAPHY. T.Kobayashi and H.Sugi. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo Muscle heat measurements hitherto per- formed rest on the assumption that all the elementary segments of muscle respond in the same manner. To examine the validity of this assumption, We measured local heat production along the entire length of frog sartorius muscles by means of an infrared thermography with a temperature resolution of 20m°C. The muscle was divided into several segments by fine metal markers at-— tached to the muscle surface, and tetaniz-— ed isometrically for 5sec. The length and temperature changes in every segment were simultaneously recorded with the thermo- graphy. During isometric tetanus, there was always a non-uniformity in both length and temperature changes; in general, the segment near the pelvic end tended to shorten by stretching those near the tibi- al end, and the amount of heat produced was larger in the former than in the lat- ter. These results indicate that the amount of heat production is not uniform, but differs from segment to segment along the length of the muscle, and that the infrared thermography is useful for the experiments in the field of muscle energe- tics. PH 68 TENSION RESPONSE OF THE FROG SINGLE SKEL- ETAL MUSCLE FIBERS TO THE CONSTANT-VELOC-— ITY STRETCH. H. Iwamoto and H. Sugi*¥ Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo, *Dept. of Physiol., Sch. of Med., Teikyo Univ, Tokyo. Analysis of the tension responses of a muscle fiber to constant velocity stretches is a useful tool for studying the actin- myosin interactions during contraction, as well as the step and sinusoidal analyses. We characterized the dependence of the tension response on the stretching velocity which ranged over several orders. In order to check that the length change of the fiber was fully transmitted to the sarco- meres, their length was monitored using a photodiode array. In the tetanically contracting fibers, the magnitude of the tension response showed three phases as the stretching velocity was increased. In the first and the third phases, the magnitude of response increased with the stretching velocity, but in the second phase, the magnitude remained constant or reduced. On the contrary, in the fiber in a rigor state which was induced by monoiodoacetic acid, the tension response showed only a small velocity dependence and the magnitude of response remained relatively high over the range Of velocities employed. These results suggest that, in the tetanically con- tracting fiber, the state of myosin mole- cules is dynamic, whereas in the rigor, se al) GieeneslGig Physiology 873 PH 69 TIME-RESOLVED X-RAY DIFFRACTION STUDIES ON THE MOLECULAR MECHANISM OF FORCE ENHANCE- MENT BY STRETCH IN TETANIZED FROG SKELETAL MUSCLE. H.Sugi and T.Kobayashi. Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo Although it is well known that, when a tetanized skeletal muscle is stretched, the force attained after stretch is appre- Ciably higher than the normal isometric force at the same muscle length, the force enhancement by stretch is not readily ex- plained in terms of the sliding filament/ cross-bridge hypothesis. By the method of time-resolved X-ray diffraction combined with intense X-ray sources from synchro- tron radiation, we examined the change in the equatorial X-ray diffraction pattern from tetanized frog skeletal muscle during an applied slow stretch. It was found that the intensity of 1,1 equatorial reflection decreased during stretch, and showed a partial recovery after the completion of stretch; the above change in the intensity of 1,1 reflection was roughly an mirror image of the force change. On the other hand, the intensity of 1,0 reflection did not change significantly by stretch, due to a large scatter of the values measured. These results suggest that the force en- hancement by stretch is associated with a decrease in the regularity of the myofila- ment lattice, which results in an increase in the overall electrostatic repulsion forces in the whole filament lattice. PH 70 NEURALLY EVOKED ELECTROGENIC RESPONSE IN THE LARVAL BODY WALL MUSCLE OF DROSOPHILA. K.Yamaoka (Natur. Sci. Lab. , Toyo Univ.) and K.Ikeda (Div. of Neurosci., City of Hope Research Inst., U.S.A.). In Drosophila larval muscles supplied with air, an electrogenic response was evoked by transmembrane depolarizing current. This response had a double-peaked configulation; a fast graded spike followed by a plateau response (Yamaoka and Ikeda, 1982, 1983). The present report demonstrates that the neurally evoked response in larval muscles 6A and 7A is also electrogenic. The muscles were supplied with air through the tracheoles. The segmental nerve was stimulated by using a suction electrode. Responses were induced intracellularly. In standard saline, slow responses were elicited by weaker stimuli, while fast graded spikes were evoked by stronger stimuli. The spikes sometimes showed an inflection on their rising phase, and were infrequently followed by aplateau. Both the spike and the plateau exhibited refractori- ness. By extraperfusion with glutamate Saline, the spike gradually decreased in amplitude and a slow response remained. The remaining responses exhibited summation, so that in the process of desensitization the plateau was easily triggered by stimulation with a pulse train of an appropriate interval. Almost equivalent results were obtained by extraperfusion with high Mgt+ saline. The results suggest that the fast spike and the plateau are electrogenic, and that the slow responses are true EJP's. PH 71 INHIBITORY SUBSTANCE OF RHYTHMIC CONTRACTION IN SEA URCHIN GONAD. H. Nogi and M. Yoshida. Ushimado Mar. Lab., Fac. of Sci., Okayama Univ., Okayama, In response to a few drops of isotonic KCl applied to the test cavity, sea urchin gonads (Temnopleurus toreumaticus) show a large and long lasting contraction. Superposed on it, small and rhythmic contractions occur Yand the rhythm is synchronized in all the _ five gonads. According to Okada et al (Biol. Bull.,1984), the site of the rhythm center resides in the aboral nerve ring. We have observed bundles of nerve fibers running from the aboral nerve ring to the _ gonad via the wall of gonoduct. The rhythmic contraction is inhibited by glutamic acid (Glu) and aspartic acid at a concentration of 10°°M. The effect is not due to the acidity. GABA is facilitatory, inducing resumption of the rhythm in quiescent gonads. Homogenates of ovaries and testes have the same inhibitory effect as Glu and the effectiveness does not disappear upon boiling. When the homogenate is fractionated by Sephadex gel filtration (G-25), the inhibitory effect is most evident in the Glu rich fraction and is abolished by treatments with glutamic acid decarboxylase. These results suggest that the inhibitory substance in the homogenate is Glu. PH 72 THE EFFECT OF MYOSIN SULPHYDRIL MODIFICA- TION WITH p-PHENYLENEDIMALEIMIDE ON THE MECHANICAL RESPONSE OF GLYCERINATED RAB- BIT PSOAS MUSCLE FIBERS. *M.Shimada,**S.Chaen and**H.Sugi. *Central Lab. Analyt. Biochem. and**Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo. It has been reported that p-phenylene- dimaleimide (p-PDM) selectively reacts with the sulphydryls on the myosin head to result in the loss of its ability to combine with actin and to hydrolyse ATP. As an attempt to connect the knowledge of muscle biochemistry with that of muscle physiology, we examined the mechanical response of glycerinated rabbit psoas muscle fibers when they were treated’ with p-PDM at various concentrations. The mag- nitude of Ca-activated isometric tension (relative to the control value) in the p-PDM-treated fibers was found to be pro- portional to the square of the stiffness (also relative to the control value). If it is assumed that the probability to combine with p-PDM is the same between the two heads of each myosin molecule, the above relation between the isometric tension and the muscle fiber stiffness strongly suggests that (1) a cross-bridge can exert tension only when both heads are not modified by p-PDM, and (2) the stiffness represents the total number of unmodified myosin heads. 874 Physiology aly EFFECT OF STRETCH ON THE VENTRICULAR RHYTHM IN AN OYSTER Piece onal /J\JIeieg, linsieg Ox Wa@ie Seas q Wino Wie Wawel, Wieiieete at EE ————— EE In the heart of oyster, Classostrea eigas, interactions due to contraction are recognizable between beating rhythms of ventricle and atrium. In accordance with this fact, effects of stretch applied to the heart, especially to the ventricle were examined. Appiacation of trancileme arta tredaul stretches at early phases of the beat interval showed interval elongation, while stretches at later phases showed interval shortage. A similar tendency is seen when atrium acts as the source oie SerScen min Salew ging ViCe YWOLSA «Oi ENG WEmMtelcGle ALTOCENMSe ENS Berwin, Amount of elongation or shortage varied WHEN ElMNe® IMeCMOilcy OF SeeSecim, Om ene other hand, longer durations of stretch do not necessarily cause more interval shortage. There seems to be an adequate LGwigiein Off Giteaenom Wiere Line Meie@eesic amount is obtained. From these results, the beating rhythm of ventricle is apparently affected by the intensity of stretch, anid) sacconmdanics | tom etihves “pihialsie ss iwihiensie stretch is applied or released. And, it was suggested that beating rhythm of ventricle in situ may be under influence of atrial contraction in this same way. PH 74 NEURAL CONTROL OF CARDIAC OUTPUT TO THE ARTERIES IN BATHYNOMUS DOEDERLEINI. A.Kihara(l) and K.Kuwasawa(2). (1)Lab.Biol.,Hosei Univ. ,Tokyo. (2)Dept.Biol.,Tokyo Metropol.Univ.,Tokyo. Three anterior arteries and five pairs oie Ieleeieell enecereteg (AS) arise itireoem cae heart. The anterior median artery(AMA) gives off its branches into the celebral ganglion, the antennae, and the compound eyes, and the anterior lateral arteries (ALAs) ramify on the skeletal muscle in the cephalone. LAs run to the thorax and the abdomen, and extend to the thoracopods, the pleopods and the telson. There are the cardio-arterial valves at the junctions between the heart and arte- ries. All valves in three anterior arte- les se eLeCeCLVC Ed palin Or se xCiitalteOrne Walle nerves(VEs) from a pair of the anterior cardiac nerves(ACNs). The valves of both plus TECEIyS Zl jyelise Og z or MgCl> up to 10 mM to the purified hemoglobin at pH 8.0-8.1 induced increases in oxygen affinity and cooperativity and in the stability of the ~60S whole molecule. The effect on the oxygenation properties was greater with CaCl, than MgCl5 at the same molar concentration. The stabilizing effect on the ~60S molecule was almost the same with both CaCl, and MgCl>. (4) These results sug- gest that the dissociation property of the hemoglobin in whole blood is controlled by both Ca2+ and Mg2*, and that its oxygenation property is controlled by Ca?*. PH 87 A HIGH AMMONIA TOLERANCE IN THE MUDSKIPPER PERIOPHTHALMUS CANTONENSIS. K. Iwata. Biol. Lab., Fac. of Edu., Univ. OG Wakayama. Wakayama The mudskipper is able to survive for a long period out of water. During the pe- riod out of water, a great amount of ammo- nia is accumulated in the fish body. In keeping with this, this fish has a great tolerance to ammonia; it can survive for more than 7 days in 15 mM NH,Cl, while water-breathing gobid fishes died within 24 ht. iny10..mM Ne.GC1. In the fish exposed to 15 mM NH,Cl for 3-7 days, ammonia and FAA increased great- ly in the muscle and liver, while those levels remained low in the blood. In the brain, Glu and Gln levels increased great- ly, but ammonia remained a low level. No changes were found in urea levels in these tissues. Similar results were also ob- tained from the fish kept out of water. GluDH and Gln synthetase activities in the brain of the mudskipper were 10- and 1.6-fold higher than those of water-breath- ing gobid fishes respectively. GluDH and Gln synthetase seem to play a central role in removing ammonia from the tissue. In contrast to amphibia and ancestral fishes, it appears that the mudskipper does not modify a capacity toward ureotelism, but develop an ability to protect the central nervous system against ammonia toxicity in relation to the amphibious mode of life. PH 88 HYDROXYPROLINE CONCENTRATION IN SOLUBLE AND INSOLUBLE MATERIAL FROM SERUM IN POST- PARTUM MICE TREATED WITH TRICHLOROACETIC ACID. K.Shimizu,K.Honda,S.Takabe and M.Hokano. Dept. of Anatomy, Tokyo Med. Coll., Tokyo. It is generally thought that collagenase initially degrades collagen fibers to fragments and then other proteases degrade these fragments to amino acids. It is not clear to what extent proteases participate in uterine collagen degradation during postpartum involution. Since materials derived from the breakdown of uterine collagen are removed by the blood stream, a form of material containing hydroxy- proline ( Hyp ) in the serum should ,jhave an influence on the collagen degradating process in the postpartum uterus. Female mice of the IVCS strain were mated at 8 weeks of age. Blood was collected from day 0 ( parturition ) to day 3 postpartum from the femoral artery and vein. The Hyp concentration in both the soluble and in- soluble material from serum treated with trichloroacetic acid was determined ( Woessner's method ). The Hyp concen- tration of the insoluble material increased but that of the soluble material did not increase during the uterine involuting period. Our results indicated that the major form of the material containing Hyp in the serum is not an amino acid and that most protease,which can degrade a large peptide to amino acids, do not participate in postpartum collagen degradation. 878 Physiology PH 89 IDENTIFICATION AND PHEROMONE RESPONSES OF THE MUSHROOM BODY NEURONS IN THE MALE SILKWORM MOTH, Bombyx mori. R.Kanzaki and T.Shibuya. Inst. of Biol. Sci., Tsukuba Univ., Ibaraki The study was undertaken to know the neuronal processing mechanisms in the protocerebrum of the male silkworm moth's brain when the male moth showed the mating dance. Pheromone responses of the proto- cerebral neurons were mainly divided into three types, i.e. TA, TB, TC. TA and TB neurons showed long lasting high frequency spikes even if the stimulus ended. TC showed high frequency spikes especially during the stimulus. The pathway of these neurons became clear with lucifer intracellular staining. TA and TB branched near the a-lobe, then ran to the other side of the protocerebrum through the under side of the B-lobe and it was obserbed it went into the suboesophageal ganglion. MTC branched only in the ipsilateral proto- cerebrum. TA and TB responses were similar to those of the ventral nerve cord connects between the brain and the thoracic ganglion. The slope of the concentration response curves of the TA and TB became steep at threshold concentrations to the fluttering response. The curve was also Similar to that of the fluttering frequency. It is considered that the neural information patterns related to the fluttering behavior of the male may be already constructed in the mushroom body neurons responded with TA or TB type. PH 90 OLFACTORY NEURAL PATHWAY AND ODOR RESPONSES IN THE ANTENNAL LOBE OF THE HONEYBEE, Apis mellifera. A.Iwama and T.Shibuya. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki The study is an attempt to know the olfactory neural pathways and odor responses in the brain of the honeybee, Apis mellifera. For anatomical studies, the Bodian's method modified according to Ootsuka and the intraneuropilar cobalt injection method were used. In anatomical observations, indicate that the antennal lobes of the worker were much larger than those of the drones. The antennal lobe was compartmentalized glomelular neuropil. The number of glomeruli in the lobe was about 190 in the workers and about 120 in the drones. The drones had some macroglomerular complex. Output fibers from the antennal lobe went into the calyces of the mushroom body through the medial and the lateral antenno glomerular tracts. Branching regions of the output fibers was observed to locate at the lip zone of the calyces by cobalt staining. Neural activity was recorded extracellularly from the antennal lobe meurons in the worker brain. Olfactory stimuli used were geraniol (Nasonov pheromone and flower odor), phenethyl alcohol (rose odor) and isoamyl acetate (alarm pheromone). Some neurons responded excitedly to isoamyl acetate. Inhibitory responses were sometimes observed in other neurons to the same odor. Pll SU OLFACTORY NEURAL PATHWAY IN THE DEUTOCEREBRUM OF THE MOTH, Mamestra brassicae. M.Watanabe and T.Shibuya. Dept. of Biol., Fac. of Liberal Arts, Univ. of Yamaguchi, Yamaguchi, Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. For morphological investigation, the brains of the moth, Mamestra brassicae, were fixed, sectioned in three derections and then stained with the Bodian's method. Two main groups of neruon somata, the dorsomedial cell group and the ventrolateral cell group were observed in the deutocerebrum. The arrangement of glomeruli is conservative. In the male moths two macroglomeruli were observed, the other side, the female moths had homologous small size glomeruli. The axonal bundles from the deutocerebrum ran to the ipsilateral protocerebrum as the tractus olfactorio golbularis (TOG). To record electrical responses of the deutocerebrum neurons to odor compounds, microelectrode was inserted into the deutocerebrum of the male moth. As odor compounds, sex pheromones, citral (one of the plant's fragrances) and propionic acid (a component of artificial food at larva) were applied. Sex pheromones were extracted from the female moth with n-hexan, and caused electrical responses dose dependently. About half of deutocerebrum neurons responded to sex pheromones, responded to other odor compound (citral and/or propionic acid). Pil Sp SCANNING ELECTRON MICROSCOPY OF THE CERE- BRAL GANGLIONS OF INSECTS. T. Itoh and Y. Tominaga. Biol. Lab., Fac. of Sci., Fukuoka Univ., Fukuoka. After removal of connective tissue cov-= erings by trypsin digestion and HCl hy- drolysis, we observed the three-dimension- al architecture of the olfactory lobes and mushroom bodies of three different species of insects, Gryllus bimaculatus, Peri- planeta americana and Apis mellifera. The antennal glomeruli of the olfactory lobe are identifiable by the tangled shapes of many fine axonal branches, which come from olfactory cells in antennal sen- silla and the olfactory lobe neurons. Their spatial arrangement in the olfactory lobe varies with three insects. The spher- ical cell bodies of Kenyon cells are clearly visible on the dorsal surfaces of mushroom body calyces. Their size is only about one third as compared with that of the other surrounding neurons, and slightly differs according to calyx areas. In Gryllus, the calyx is subdi- vided into two parts,the anterior and posterior calyces, and many microglomeru- lar structures are found on the outer layer of the anterior calyx. They are probably the synaptic regions of Kenyon cell fibers and extrinsic nerve fibers. Physiology 879 PH 93 EFFECTS OF AMINO ACIDS ON PHOTOMACROGRAPHIC TRACKS OF AMOEBAE. H. Horikami and K. Ishii. Biol. Lab. Hosei Univ., Tokyo. Tracks of Amoeba proteus and Tricamgeba esseosaccus by dark field strobo-flash pho- tomacrography were analysed to investigate behavior of individuals. 1) In these hungry amoebae, a velocity of locomotion for 20 min shows no significant difference with the binomial test in 0-1 mM Na-glutamate or L-glu. However, the veloci- ty for 1 min is accelerated up to 25 % at 10 »pM of these amino acids. 2) The roughness of the velocity is ob- served significantly only at a short inter- val recording, but not at a longer. 3) When a body axis at the beginning of experiment is superimposed on the Y axis, the positions of individuals after 20 min tend to diverge at higher concentration in the range. 4) Profiles of most tracks for 20 min in these amoebae take wave shape of less than 1 cycle. 5) The nearing ratio to a stimulus source of 50 mM Na-glutamate after 80 min reaches a high value over 90 $. 6) From these results, ortho-, klinokinesis and taxis sensu strictg are at least relat- ed to the aggregation of amoebae to a stim- ulus. PH 94 MEMBRANE CURRENTS IN VOLTAGE CLAMPED NOCTILUCA K.Ooami and T.Sibaoka. Biol.Inst.,Fac.of Sci.,Tohoku Univ.,Sendai. Ionic currents in the membrane of a dinoflagellate Noctiluca were examined with a conventional two microelectrode voltage clamping technique under Ca- deficient condition. The voltage between the flotation vacuole and the external solution was clamped, so that the two cascaded membranes, the plasma membrane plus the vacuolar membrane, were involved in the clamped membrane system. When the vacuolar potential had pre- viously been kept at -80mV, a depolari- zation up to -50OmV or more produced a transient inward current. The inward current became maximum, at a depolarized level of -30mV. It became outward when the depolarization exceeded 25mV. When the vacuolar potential had previously been kept at -20mV, a membrane hyperpo- larization up to -30mV or more produced a transient outward current. The current became maximum at a hyperpolarized level of -40mV. It became inward when the hyperpolarization exceeded -90mV. The depolarization(hyperpolarization) activated inward(outward) current seems to be responsible for the depolarizing (hyperpolarizing) spike of the tentacle regulating potential(TRP). PH 95 LOCAL ION CURRENTS AND FEEDING INITIATION OF NOCTILUCA (MARINE DINOFLAGELLATE). T. NAWATA. “Biol. inst., Fac. Sei., Tohoku Univ., Sendai. An ultrasensitive vibrating probe was used to measure transcellular ion currents associated with the feeding initiation of Noctiluca. The feeding was experimentally induced by the removal of SO, ions from the bathing medium. Noctiluca cell has a specialized feeding apparatus (tentacle, cytostome,etc.) onthe longitudinal groove (sulcus). Just before and during the feeding, the large ion cur- rents were observed only around the sulcus, The ion currents entered the cell around cytostome (inward) and left from the distal ends of sulcus (outward). The inward currents (Max. 11 ypA/cm?=110 pmol/cm’sec of monovalent ions, recorded at 50 um far from the cell surface) decreased sharply with the decrease in extracellular concentration of Na* and Ca**, suggesting probably that the currents act like a Na*tinflux with some Ca involvement, whereas the decrease in Mg” elicited the oscillating current flow. In contrast, the outward (Max. 2 pA/cm’) did almost never decrease. It is still unknown that which ion(s) carried the outward ones. The present experiments show that there are some ion channels which open just before the feeding and that those are localized around the feeding apparatus. The possible ionic mechanism of feeding initiation would be that ions (eg,Ca*) carried into thecell by current cause the cytoplasmic movements, PH 96 ELECTROPHYSIOLOGICAL STUDY OF THE ALTER- ATION OF BEHAVIOR IN TERRESTRIAL PULMO- NATES. K.Hasegawa and N.Takeda. Dept. of Biol., Fac. of Sci., Toho Univ., Funabashi. Under moist conditions. terrestrial pulmonates tend to increase their general behavioral activity. Hydration of these animals resulted in decreased hemolymph osmolarity and increased activity. In the dehydrated condition, this phenomenon is reversible. Using the African giant snail, Achatina fulica, we examined the induction of behavior electrophysiologically. From their morphological appearance and the characteristics of intracellular record- ings, some giant neurons were identified on the surface of each ganglion. Using autoactive neurons, the effects of osmotic stress on electrical activity were examined by changing the bathing medium. Higher electrical activity was induced by hyper- tonic saline and lower activity was induced by hypotonic saline in isolated ganglia. On the contrary, in intact ganglion possessing the cerebral ganglion which contains the osmoreceptor, these cells became reversal. In some neurons such as the parietal ganglion and the pedal gangli- on, electrical activity was induced by hypotonic saline and suppressed by hyper- tonic saline. These results correspond well with our theory of the induction of behavior in terrestrial pulmonates by hemo- lymph osmotic pressure. 880 Physiology PH 97 POSSIBLE FUNCTIONAL FLEXIBILITY IN THE HIERARCHY OF CENTRAL RESPIRATORY ORGANIZATION IN THE LAMPREY. R. Kawasaki. Col. Biomed. Technol. Niigata Univ. Niigata. In vertebrates, local destruction of important central respiratory regions (DRG, VRG) does not abolish respiratory rhythm, indicating the 'strength' of the breathing rhythm generator function. The study tested in the lamprey wheth- er this strength is due to BRG existing exclusively in a very confined site in the medul- la, being fixed in the face of destructive invasions. Results 1) Mid-saggital slits through- out the brain stem revealed that each of the divided half showed respiratory discharges inde- pendently with almost the same frequency. 2) Long after immobilization, respiratory discharges be- came longer in duration and occurred irregularly at longer intervals. However, after the use of an artificial ventilator, this discharge pattern returned to the previous one, indicating the vul- nerability of respiratory neurons to their physiological conditions (pOo, &c). 3) Respira- tory neurons (showing respiratory spikes preceded by the slow depolarization) can be entrained (artificial pacemaking) by contralateral micro- Stimulation with frequencies higher than the spontaneous one. The results suggest that possi- ble pacemaker cells are functionally arranged in a hierarchical manner according to their degree of automaticity; in suppressive states of natural pacemaker cells, new ranking order can be estab-— lished. PH 98 ANALYSIS OF THE CENTRAL RESPIRATORY OSCILLATOR USING PHASE RESPONSE CURVE --- STIMULATION OF EXTERNAL INTERCOSTAL NERVE A.Komatsu & S.Kitano. Dept. Physiol. and Dept. Anesthesiol., Tokyo Women's Med. Colney, LOlsyOr In vagotomized rabbits, respiratory phrenic discharges were recorded. Short electrical stimuli were applied to the external intercostal nerve at various times in the respiratory cycle. Phase response curves (PRC's) were obtained from stimulus phase and phase shift in the respiratory activity. Stimuli during inspiration gave rise to all-or-none responses; no phase shift or a large phase advance occurred. In the latter case, the duration of expiration just after stimulation depended on the duration of preceeding inspiration. On tne contrary, stimuli during expiration resulted ina phase delay that occurred in graded manner. The duration of inspiration after stimulation did not depend on the duration of preceeding expiration. As a result, Two types of PRC's were obtained. When 10 stimulus pulses were applied, PRC of an average slope of 1 was obtained. When 2-8 pulses were applied, PRC's had an average slope between 0 and 0.3. In these PRC's, discontinuity appeared at the end of expiration. These results suggest that one respiratory cycle is a single course of events starting from inspiration and that the next cycle is brought about by a "restarting mechanism'. eal SIS FUNCTIONAL CONNECTIONS BETWEEN CERCAL INTERNEURONS AND LEG MOTONEURONS IN CRICKET M.Kanou and T.Shimozawa. Zool. Inst. Fac. Sci., Hokkaido Univ., Sapporo. Air-current stimulus affects the activi- ty of leg motoneurons(MNs), depressor and levator of mesothoracic trochanter, in two ways. One is excitatory and the other is inhibitory. Excitatory responses appeared in the slow and the fast depressor MNs when the acceleration of stimulus air-current was above 500 mm/s2. Inhibitions occured in the slow depressor and the levator MNs prior to the excitation when the velocity of the air-current was above 1 mm/s. Velocity thresholds of the MNS' response were measured at a various frequency of a Sinusoidal air-current stimulus. The frequency threshold curves of MNs were compared to those of cercal inter- neurons (Kanou and Shimozawa 1984, J CP ). The excitatory inputs of depressor MNS are identified as the large, acceleration sen- Sitive interneurons such as 9-1 (LGI) and 8-1 (MGI). The inhibitory inputs of the slow MNs are attributed to the small, ve- locity sensitive interneurons such as 10-2 and 10-3. The slow depressor MN sometimes showed a velocity dependent excitation at high threshold instead of the acceleration dependent one. A neural switching between the cercal interneurons and the leg MN is proposed. PH 100 THE ROLE OF LOCAL BILATERAL SPIKING INTER- NEURONS IN CRAYFISH AVOIDANCE REACTION. T.Nagayama, M.Takahata and M.Hisada. ZOOL. Inst., Fac. Of Sci.) HokkkadomUnbne Sapporo. Mechanical stimulation of either one side of uropods elicited the avoidance re- action in crayfish. Depending on the ani- mal's growth stage, either one of two al- ternative avoidance acts, "dart" and "turn" responses, was elicited. In both responses, the uropod on the contralateral side to the stimulus was closed. Both touching the exo- podite directly with a fine brush and stim- ulating electrically the 2nd root afferents of the terminal abdominal ganglion caused the reciprocal activation of the contra- lateral uropod motoneurons (MNs): spontane- ous discharge rate of the closer MNs in- creased and that of opener MNs decreased. Neither the 2nd root afferents nor the motoneuron dendrites did cross the midline of the ganglion. We found local bilateral spiking interneurons (LBSNs) which extended their branches bilaterally in the ganglion. They received monosynaptic excitatory input from the 2nd root afferents on the ipsi- lateral side to the somata. They could af- fect the motoneuron activity on the contra- lateral side when the current was injected intracellularly. We hence conclude that these LBSNs transmit the sensory informa- tion to the contralateral uropod MNs. Five different types of LBSNs were discriminated with different input and output pathways. Physiology 881 PH 101 VISUAL RESPONSES OF TOAD'S TECTAL NEURONS AND THEIR PROJECTION TO THE MEDULLA. M.Satou and J.-P.Ewert*. Meo inst., rac. Of°SCis, Univ. of ‘Tokyo, Tokyo. *Neuroethol]. Biocyber. Labs., Univ. of Kassel, Kassel, West Germany. Using paralyzed common. toads, Bufo bufo, we tested which of the known classes of tectal neurons can be activated anti- dromically in response to electrical stimuli applied to the tecto-bulbar/spinal tracts in the caudal medulla, hence specifying the tecto-motor output. Response properties of extracellularly recorded tectal neurons to moving visual stimuli were used for identifying the neuron classes. Criteria for antidromic activation were: constant latency response to electrical stimuli, following ability to two successive stimuli of short interval, and collision between visually and electrically evoked spikes. Various types of tectal neurons, e.g., ascelay (ce, TS, 64, °65.(2)-,6T5 (2), L5 (3), and 1T5(4) neurons, could be activated antidromically by stimuli applied to the contralateral caudal medulla. Class T7 neurons as well as class R2, R3, and R4 retinal neurons could not be activated antidromically. These results suggest a possibility that these tectal neurons which project to the medullary region carry motor-commands for visually guided behavior, such as prey-catching. PH 102 TOAD'S SNAPPING PATHWAY: EXCITATORY AND INHIBITORY PATHWAYS TO TONGUE-MUSCLE MOTONEURONS FROM THE OPTIC TECTUM. M.Satou, H.Takeuchi, T.Matsushima and K.Ueda. Zool e Soi. 7, haAG. Of SCs, Univ. Cf Tokyo, Tokyo. Using intracellular recording tech- niques in paralyzed Japanese toads, tongue-muscle motoneurons (protractor and retractor motoneurons: PMNs and RMNs respectively) were identified antidrom- ically and neural pathways to these motoneurons from the optic tectum were searched. Inhibitory postsynaptic potentials (IPSPs) as well as excitatory postsynaptic potentials (EPSPs) were evoked both in PMNS and RMNs in response to electrical stimuli applied to various parts in the optic tectum of either side. The size of these IPSPs as well as EPSPs was facili- tated when the electrical stimuli were repeated at short intervals and when the electrical stimuli were simultaneously applied to different parts of the optic tectum of either side. These results suggest that (1) inhibi- tory as well as excitatory interneurons intervene in descending pathways from the optic tectum to the tongue-muscle moto- neurons, and (2) the tectofugal descending volleys from different parts of the optic tectum of either side converge on these interneurons. PH 103 TOAD'S SNAPPING PATHWAY: RELATIONSHIP TO THE EXCITATORY PATHWAYS FROM GLOSSOPHARYN- GEAL NERVE TO TONGUE-MUSCLE MOTONEURONS. T.Matsushima, M.Satou and K.Ueda. Zool. Lusitws, hace Gf .«SCin;, sUniV..s0r Tokyo., Tokyo Neural pathways from glossopharyngeal nerve (IX) to tongue-muscle motoneurons (protractor and retractor motoneurons: PMNS and RMNS respectively) were intra- cellularly analyzed in paralyzed Japanese toads, with a special attention to their relationship to the excitatory pathways from the optic tectum (OT). Following ipsilateral IX stimuli, EPSPs of polysynaptic nature were evoked in PMNs. In RMNs, however, monosynaptic EPSP components of short latencies were evoked in addition to later polysynaptic compo- nents. Following contralateral IX stimuli, only polysynaptic EPSPs were evoked in PMNs as well as in RMNs. The size of these EPSPS was facilitated when LpSi=) 07 contralateral OT was Simultaneously stimulated. These results suggest that: (1) The excitatory neural pathways from IX to tongue-muscle motoneurons are composed of mono- and polysynaptic pathways, and (2) the latter polysynaptic pathways share common excitatory interneurons with the excitatory pathway from OT. These inter- neurons may, therefore, be responsible not only for snapping preys but also for rejecting unpalatable objects, such as bombardier beetles. PH 104 LOCALIZATION OF MOTONEURONS INVOLVED IN THE PREY-CATCHING BEHAVIOR IN THE JAPANESE TOAD. K.Takei, Y.Oka, M.Satou and K.Ueda. Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo. Some amphibian muscles in the head re- gion show the characteristic activity patterns during the prey-catching be- havior. In an attempt to elucidate the motor pattern generating mechanisms of that behavior in the Japanese toad, we studied the distribution of motoneurons innervating the tongue and jaw muscles, using the intramuscular injection tech- nique of horse radish peroxidase (HRP). We injected the HRP into the following’ eight relevant muscles: M. submentalis(Sment), M. masseter major (Mmaj), M. tempo- ralis(TP), M. depressor mandibulae(DM), M. genioglossus(GG), M. hyoglossus(HG), M. geniohyoideus(GH), M. sternohyoideus(SH). The HRP-labelled motoneurons were found in the motor trigeminal(Vm), facial(VIIm), and hypoglossal(XII) nuclei. The moto- neurons in each motor nucleus showed dis- tribution patterns characteristic to the target muscles. For example, the moto~ neurons innervating TP and DM were located more rostrally in Vm and VIIm_ respective- ly. The motoneurons innervating GG were located more caudally in the medial nu- cleus of XII, while the motoneurons inner- vating HG were located more rostrally in the same nucleus. 88? Physiology PH 105 DEVELOPMENT OF TONGUE MUSCLES AND HY POGLOSSAL MOTONEURONS DURING METAMORPHOSIS IN THE BULLFROG. Y.Oka, M.Umezawa, K.Takei, M.Satou and Kovedas ) 200!) InSite, haceOm SCs mlUlniauze of Tokyo, Tokyo. As one step to study the neural mechanisms underlying the metamorphic transformation of anuran feeding behavior (from herbivorous to carnivorous), we examined the morphological changes, during metamorphosis, in the tongue muscles and the hypoglossal motoneurons (XII-MNs) innervating these muscles. At stage xX undifferentiated muscle cells of the M. genioglossus (tongue protractor) and the M. hyoglossus (tongue retractor) already existed. These muscle cells showed a rapid differentiation around stage XX. By stage XXV (the end of metamorphosis) the tongue and its muscles attained full morphological differentiation, which enabled the froglet to perform prey- catching behavior. Associated with this tongue muscle development the XII-MNs showed concomitant morphological changes. Immature Or young XII-MNs could be distinguished, owing to their large size, from other neurons already at stage X. Maturation process of the XII-MNs remarkably proceeded from around stage XX Onward, including the increase in size of the cell body (especially the cytoplasm) as well as the development of Niss] bodies and dendritic arborizations. PH 106 STUDIES ON MICROTUBULE SLIDING USING THE STOPPED-FLOW-LIGHT-SCATTERING METHOD. S.Kamimura(1), M.Nakanishi(2), M.Yano(2) and H.Shimizu(2). (1)Research Development Corporation of Japan and (2)Fac. of Pharmaceutical Sci., Wiastyo ox Wolo, Tokyo. The dependency of the velocity of microtubule sliding on temperature (10- 21°C) and MgATP concentration (43-815 uM) was analyzed using the stopped-flow- light-scattering method. Flagella of sea- urchin (Hemicentrotus pulcherrimus) spermatozoa were demembranated and used after a brief treatment with trypsin. The change of turbidity (180° scattering of 350nm light) of the axonemal suspension (160ug protein /ml) during MgATP-induced disintegration (Summers and Gibbons,1971) was fitted to a single exponential function which yielded the rate of disintegration, R(1/s). R coincided well with the velocity of microtubule sliding, V(um/s) as determined by cinematographic analysis (Takahashi, Shingyoji and Kamimura, 1982), i.e. R = On22, al View ei — 0299725.) Lis) indicates that turbidimetry is a useful method to analyze the sliding velocity of microtubules. From the dependency on temperature, Q49 of the sliding velocity was found to be 2.0-2.3 at 43-815 uM of MgATP. PH 107 CYTOARCHITECTURE AND AFFERENT CONNECTIONS OF PREOPTIC AREA IN HIME SALMON. T.Shiga, Y.Oka, M. Satou, K. Ueda and N. Okumoto* Fac. of Sci., Univ. of Tokyo, Tokyo and *Nikko Branch, Nation. Res. Inst. of Aquacul., Nikko. The nucleus preopticus periventricularis (NPP) has been shown to play an important role in the sexual behavior of hime salmon (landlocked red salmon, Oncorhynchus nerka) (Satou et. al., 132 "84). The cytoarchitecture and afferent connections of NPP were studied by rapid Golgi and retrograde horseradish peroxidase (HRP) tracing methods, respectively. In Golgi preparations, we identified a tanycyte and 5 types of neurons. One of the neurons had a cerebrospinal fluid (CSF)-contacting dendrite, suggesting that it may receive humoral information from the GSE. The NPP and rostral part of nucleus preopticus received projections from area ventralis telencephali pars ventralis, area ventralis telencephali pars dorsalis, NPP, lateral part of preoptic area, nucleus anterioris periventricularis, nucleus ventromedialis thalami, nucleus lateral tuberis pars medialis, nucleus saccus vasculosus, nucleus recessus posterioris, nucleus posterior tuberis, and midbrain tegmentum. These findings seem to give an important basis for the understanding of neural mechanisms which are involved in the fish sexual behavior. Ph 108 NEURAL ACTIVITY IN THE OCULOMOTOR NUCLEUS OF THE JAPANESE DACE. N.Tsutsui and K.Aoki. Life Sci. Inst., Sophia Univ., Tokyo. We made simultaneous recordings of spontaneous eye movements in the horizon- tal plane and single-unit activity in the dark of the oculomotor nucleus which in- nevates the extraocular muscles, in Japanese dace (Tribolodon hakonensis). Fish, whose spinal cords are transected to suppress body movements, show spontane- ous eye movements which repeatedly consist of a quick eye movement (saccade) and the subsequent period of fixation. Several types of unit could be identi- fied; they showed different activity pat- terns during saccadic eye movements and periods of fixation. (1)Burst units. These units responded with a burst of spikes before and during saccades. (2) Pause units. These units showed a spontaneous discharge which was interrupted before and during saccades. (3)Phasic-Tonic units. These units responded with a burst of spikes before and during saccades and also showed a tonic response during periods of fixation. In some units, the tonic rate was proportional to the degree of poste- rior eye deviation, and bursts occured only in anterior saccades. The other units showed a tonic discharge not proportional to the position of the eye, and also res- ponded with a burst of spikes before and during both saccades. Physiology 883 PH 109 THE MECHANISM OF BLOCK OF GLUTAMATE SYNAPSES BY DIPICOLINIC ACID. D. Yamamoto and T. Miyamotol. Lab. of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, Tokyo., lDept. of Physiology, Nagasaki University School of Dentistry, Nagasaki. The effect of dipicolinic acid (2,6-pyridine dicarboxylic acid) on the mealworm neuromuscular junction was studied using conventional microelectrode recording techniques. Dipicolinic acid (10-5-10-3M) added to the bathing solution reversibly blocked neuromuscular transmission. The depolarization in response to iontophoretically applied L-glutamate (glutamate potential) was not affected by dipicolinic acid even when the neurally evoked excitatory postsynaptic potential (EPSP) was totally abolished. Focal extracellular recordings from single synaptic sites revealed that in the presence of 1 x 10-4M dipicolinic acid the presynaptic spike was unchanged, but the quantal content for evoked transmitter release was reduced. The calcium-dependent action potential elicited by direct stimulation of the muscle fiber was not impaired by dipicolinic acid. These results suggest that dipicolinic acid interferes with the transmitter-releasing mechanism from the presynaptic terminal. PH 110 ASCENDING INFORMATION EVOKED IN PROTHORACIC GANGLION FOR ORIENTATION TO AIR PUFF STIMU- LATION IN CRICKET,TELEOGRYLLUS EMMA. J.Tsukamoto and N.Ai. Dept. Biol., Tokyo Gakugei Univ., Tokyo. WL ae ed Air puff stimulation to long sensory hairs located in tibia of adult cricket makes wing beat and orientation behabior to air flow(N.Ai,M.Yoshino,'76). Induced- potential which recorded in primary neuron located in tibia was sent to the prothora- cic ganglion and condacted to mesothoracic and metathoracic ganglion to originate the muscle activities in flight(N.Ai,M.Ichimura '78). In this experiment, evoked impulses were observed in several sites of prothora- cic ganglion using metal electrode methods. These impulses were divided from three action patterns,that is "ON", "ON-OFF" and "Tonic" ones. Site of origination of impulses was determined by metal stainning method. But these impulse patterns were not smoothly and so it was tried to use calcu- lation machine. Burst activities were analysed by amplitude for simplification using comparater(hand made) and so it was easy to observe action patterns. These action potentials were corresponded to air puff stimulation to the long hairs. And also, they were corresponded to the activi- ty recorded from ascending path of subo- esophageal-prothoracic connective. In this experiment, several patterned activities makes little longer sustained spike trains in the ascending pathway. PH 111 MUSCLE DEGENERATION IN DROSOPHILA MUTANT, aperC, WITH ABNORMAL PROBOSCIS EXTENSION REFLEX K. Kimura, T. Shimozawa and T. Tanimura. Div. of Behaviour and Neurobiol., National Inst. for Basic Biol., Okazaki. The aperC mutation in D. melanogaster causes the defect in proboscis extension reflex (PER) in aged flies. In the age of 1-2 days after eclosion, all mutant flies showed normal PER. In the age of 5-6 days, over 90 % of mutant flies could not extend the proboscis. However, in the age of 10- ll days, over 50 % of the flies restored the ability of PER. The structure of a muscle, protractor of fulcrum, which is involved in the extension of the rostrum, was observed with the polarized light microscope. In the flies at the age of 3 days, the degeneration of the muscle was seen. The cross striation disappeared at the central portion of the muscle fibers, and then the birefringence of the fibers disappeared. The ability of PER was closely correlated with the degree of the muscle degeneration. The regeneration of the muscle was seen in the flies at the age of 10 days. In conclusion, the defect of PER in aperC mutants is caused by the degeneration of the muscle, protractor of fulcrum, and the recovery of PER is due to the regeneration of the muscle. The aperC gene seems to be required for maintenance of the muscle once formed. PH 412 MOVEMENT OF MALE ACCESSORY GLANDS AND ITS CONTROL BY DORSAL UNPAIRED MEDIAN NEURONS. T. Kimura, T. Yamaguchi and*K. Yasuyama. Dept. of Biol., Okayama Univ., Okayama and *Dept. of Biol., Kawasaki Med. Coll., Kurashiki. Three clusters of the dorsal unpaired median (DUM) neurons lie in the terminal abdominal ganglion of the male cricket (Gryllus bimaculatus), and their neurites emerging through the 7th roots innervate the accessory glands (AGs). In each AG, thin myofibrillae wind round the glandular epithelium and many terminals of neurons containing small and large vesicles are located on the outer surface of the myo- fibrillae. Usually AGs show rhythmic’ con- traction of small amplitude without stimu- lus and they respond with strong con- traction to the electrical stimulation of DUM neurons. The application of 10°*M Octopamine to AGs increases the frequency of rythmic contraction, while that of 10% M glutamate or 10M proctoline induces sustained contraction. The anlage of AGs from the 7th instar transplanted into the last instar develops the myofibrillae without the innervation of DUM neurons in synchrony with that of the host. After eclosion, the AGs transplanted show in- trinsic rhythm of slow contraction and they respond with sustained contraction to the application of 10M glutamate or 10°” M proctoline, but scarcely do to that of 10-*M octopamine. 884 Physiology PH 113 FUNCTIONAL PROPERTIES OF THE DUM NEURONES IN THE MESOTHORACIC GANGLION OF THE COCKROACH. Y. Tanaka and H. Washio. Lab. of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, Tokyo. The modulatory action of an octo- paminergic neurone (DUM) that projects to the coxal depressor muscles (135 d) of the cockroach middle leg has been studied. At low frequencies of the slow motor neurone stimulation (1 Hz or below), application of the octopamine (10-6m) causes the potentiation of twich tension in the depressor muscles and the increase in thet amplitude of the synaptic potential generated by slow motor neurone. At high frequencies (50-200 Hz), where tetanus is complete, the reduction of maintained tension becomes more prominant by the application of the octopamine (10-6). The application of higher concentrations of octopamine (107~*M) causes the increase in the amplitude of the maintained or basal tension. Two types of DUM neurones can be classfied functionally. One type of DUM neurone causes the significant decrease in the amplitude of the tetanic tension. The other type of DUM neurone causes the increase in the amplitude of the basal tension. Therefore, it is suggested that functional difference in the DUM neurones depends on the difference of the amount of octopamine released to the muscles. PH 114 NEUROMUSCULAR TRANSMISSION IN PECTORAL FIN RED MUSCLE OF SILVER CARP. T. Miyahara and T. Hidaka*. Dept. of Biol., Fac. OE (ScHe. sand “rac. of Gene sEadule- Kumamoto Univ., Kumamoto Some properties of the neuromuscular transmission were studied by the intrace- llular recording method to reveal how the junction potentials contribute to the mo- vement of a pectoral fin in silver carp, Carassius auratus. The excitatory junction potential (ejp), the inhibitory junction potential (ijp) and the complex of ejp and ijp were reco- rded from the fin red muscle in response to single nerve stimulation. A spike was rarely triggered. Repetitive stimulation (1-100 Hz) produced the summation of ejp but not the facilitation. Larger depola- rization attained by the summation of ejp did not result in spike generation. Hyper- polarization during the course of ijp was associated with an increase (maximum 1.5 times) of membrane conductance. The rever- sal potential of ijp was -107 mV at the resting potential of -74 mV. Both ijp and ejp were suppressed by dTC and were aug- mented by anti-ChE, indicating that both junction potentials were cholinergic. In addition to the previous report that the contraction of this muscle develops by ejp, the present result indicates that ijp also contributes to the fin movement of Silver carp. PH 115 PERIPHERAL NEURAL MECHANISMS OF THE GILL MOVEMENT IN APLYSIA M. Kurokawa and K. Kuwasawa. Dep. = BHolee Tokyo Metropolitan Univ., Tokyo. It has been reported that the gill move- ment in Aplysia californica was caused by motor neurons in the abdominal ganglion. We pointed out that there might be motor neurons inthe gill peripheral nervous system including the branchial ganglion. We studied farther the peripheral nervous system of the gill in Aplysia kurodai and A. juliana electro- physiologically. We found that the branchial ganglion itself contained motor neurons. Electrical stimulation of the branchial nerve running from the abdominal ganglion to the gill evoked the gill movement. The pinnule contraction in the movement was almost completely blocked by perfusing the branchial ganglion with the high Mg2+ saline. This result shows that the pinnule contrac- tion was mediated by the branchio-ganglionic neurons (BGNs). EJPs were recorded from the pinnule muscle one-for-one to impulses of a certain BGN with a constant latency, even when the branchial ganglion was perfused with the high Mg2+ saline. This represents that the branchial ganglion sent motor axons to the muscle. A neuron which produced common EPSPs in BGNs was identified in the abdominal ganglion. The pinnule contraction has been attributed only to the motor neurons in the abdominal ganglion. However, this shows that the contraction was shared with the motor neurons in the branchial ganglion. Ph 116 EFFECT OF D20 ON THE ACTION POTENTIAL AND CONTRACTION IN THE PAPILLARY MUSCLE OF GUINEA-PIG VENTRICLE. S.Gomi and H.Sugii. Dept. Physiol sen. Med., Teikyo Univ., Tokyo The effect Of DDO on the actionsporen. tial and isometric tension was studied on the papillary muscle isolated from the ventricle of guinea-pig. When the prepara- tion was transferred to the D20 solution, the magnitude of the mechanical response to a brief current pulse decreased marked- ly, while the action potential recorded with intracellular microelectrodes slightly prolonged. With prolonged immersion in the D20 solution, the mechanical response showed partial recovery to reach a steady Magnitude (less than 30% of the control response). On returning the preparation to the standard solution, the mechanical res- ponse exhibited a marked transient enhance- ment, in which the magnitude of the res- ponse increased to more than 200% of the control response; after the period of the transient enhancement, the mechanical res-— ponse returned to the control value. The reduction of the mechanical response in the D20 solution could be made less pro- nounced by increasing external Ca ion concentration, whereas the transient en- hancement could be made less marked by decreasing it. These results suggest that D20 primarily affects Ca-channels through which Ca enters into the cell. Physiology, Cell Biology 885 PH 117 INTERNAL PRESSURE CHANGES TO ELECTRICAL STIMULATION AND PROPAGATION OF CONTRACTION WAVE IN GUINEA-PIG URETER. N.Takei, T.Tsuchiya and H.Sugi. Dept. Physiol., Sch. Med., Teikyo Univ. Tokyo Physiological properties of guinea pig ureter were studied by recording the inter- nal pressure and the contraction wave pro- pagation with a pressure transducer and a video system respectively. When an isolated ureter was stimulated with a brief current pulse through massive electrodes, it showed an all-or none twitch-like pressure res- ponse, the magnitude of which increased with increasing internal pressure. If a part of the ureter was locally stimulated, a contraction wave was initiated at the point of stimulation and propagated along the length of the ureter with a velocity of 20-40mm/sec. The propagation velocity did not change appreciably irrespective of the direction of the contraction wave. With in- creasing internal pressure, the velocity of constriction at each elementary part of the ureter decreased while the extent of con- striction increased. Both the velocity and extent of constriction at each elementary part did not change markedly according to the direction of propagation. The duration of a constriction-reexpansion cycle was longer at the proximal region than at the distal region. The threshold for producing contraction was lowest at the most proximal region, and highest at the most distal region. CB 1 ATTACHMENT OF PARAMECIUM TO POLYSTYRENE SURFACES: IV. COMPARISON OF THE ADHESIVE- NESS IN SIX SPECIES OF THE GENUS PARAMECIUM AVEntocamnuna., Biol. inst... Fac. 0f-Scix., Tohoku Univ., Sendai. I have previously reported that cells of both P.caudatum and P.multimicronucleatum attach to polystyrene Petri dishes (Falcon 1007) at the tips of ventral cilia only when they are mating reactive. In the pres- ent study I examined the adhesiveness of cells to the dish surface in four other species of Paramecium. Cells of both P.tri- chium and P.tetraurelia also showed the mating-reactivity-dependent attachment to polystyrene. On the contrary, attachment was rarely seen in cells of P.bursaria. In all 20 stocks tested, both mating reactive and non-reactive cells failed to attach to the dish surface irrespective of extracel- lular ion concentration, temperature and swimming velocity. However, attachment was induced in mating reactive cells of P.bur- Saria by treatment with 0.5 mM benzylamine, 0.5 mM phenethylamine or 50 pg/ml trypsin. In P.duboscgui,strong adhesiveness of cells to the dish surface was seen even when cells are mating-non-reactive. They showed a maximum attachment (more than 90%) within 1 min after introduction to the dishes. Similar attachment took place when cells of P.duboscgui were applied into polystyrene dishes with reduced hydrophobicity. Differ- ences in cell surface hydrophobicity among six species of Paramecium were discussed. CB 2 A GENERAL METHOD FOR THE ISOLATION OF CROSS PAIRS IN PARAMECIUM CAUDATUM Y. Tsukii,; Biol. Labs, Hosei Univ., Chiyoda-ku, Tokyo 102 In a ciliated protozoan, P. caudatum, sexual cell agglutination occurs specifi- cally between cells of complementary mating types, but subsequent pair formation occurs nonspecifically. Mating mixtures, there- fore, produce not only cross pairs but also selfing pairs. For cross breeding analyses, only the cross pairs should be isolated. For that purpose, behavioral mutants, CNR, have been so far used for one of complemen- tary mating types. However, those mutants are available only in syngen 3 but not in other syngens of P. caudatum. I developed a general method (1) for discriminating cross pairs from selfing pairs, and (2)for inducing cross pairs be- tween any strains regardless of their mating type specificity. (1)Cells cultured with pea extract had transparent cytoplasm and looked white and those cultured with soybean flour (kinako) had opaque cytoplasm and looked black. Thus pairs consisting of white and black cells were easily discrimi- nated from both white-white and black-black pairs. By using gene markers, the former was identified as cross pairs andthe latter selfing pairs. (2)Cross pairs between cells with the same mating type and those between different syngens were induced by using cells paralyzed with nikel ions, of which Mating types were complementary to those of strains being crossed. CB 3 CHEMICALLY INDUCED CONJUGATION BY METHYL- ENE BLUE IN PARAMECIUM MULTIMICRONUCLEATUM. S.Mishima! and Y.Harada“. !-Biol. Lab., Coll. Gen. Educ. Lbaraki Univ., Mito. 2.Dept. Biophys. Engn., Fac. Engn. Sci., Osaka Univ., Toyonaka. It is known that chemically induced conjugation in P. multimicronucleatum cells occurs in KCl + acriflavine + Ca-poor conditions. We found that the induction occurred with methylene blue(MB) instead of acriflavine. The cells in the MB solu- tion formed pairs, micronuclei changed to cressent and macronuclear fragmentation occurred. The inducing effect by MB was as strong as by acriflavine; sometimes almost all cells formed pairs. It is known that conjugation is induced even though bacteria are added to the in- duction mixture. We found that MB-stained bacteria induced conjugation and the su- pernatant of MB-stained bacteria sometimes did not induce conjugation. However, when unstained bacteria were added to the MB solution, conjugation did not occur. It seems that MB's effect on conjug#tion 15s that of a solute. The cells in the presence of MB had very high photosensitivity i after expo- sure to strong light for se\ 11 minutes the cells died within 30 mi 886 Cell Biology CB 4 ANALYSIS OF EXPRESSION OF MATING REACTIVI- Ty IN PARAMECIUM BURSARIA. EL. Miwasebioleihabe cols. Gene ducky, Ibaraki Univ., Mito. Cell population of P. bursaria shows mating reactivity in the light period but not in the dark period in the condition of LD 12:12 cycle. When they were transferred to the constant light (LL) or constant da- rk (DD) condition they continued to show a circadian rhythm of mating reactivity in the biginning. When the cells of rhythmic population in the DD were transferred to the LL at non-reactive phase they began to appeare the mating reactivity 1.5 hrs aft- er onset of light and showed the resetted rhythm of mating reactivity in the LL of which the peak was shown 6 hrs after onset of light. When the rhythmic cells in the DD were exposed to light for 5 sec., 2 min. 1 hr at non-reactive phase they began to appeare the mating reactivity 1.5 hrs aft- er respectively but the phase shift of ea- ch rhythm did not occur after that. In these experiments it is suggested that the continuous exposure to light for more than 3 hrs is necessary to shift the phase of mating reactivity rhythm in rhy- thmic cell population in the DD and that the cells of non-reactive phase in the DD show the time lag of 1.5 hrs to appeare the mating reactivity after the exposure to light. CB 5 MATING-TYPE-SPECIFICITY OF CELL-CELL CONTACT DURING CONJUGATION IN TETRAHYMENA THERMOPHILA. Lisuga-aeeDepii. Baoller i baraleianUmenvesmMastor Conjugation in ciliate generally begins with mating-type-specific ciliary interac- tion or mating reaction and then proceeds to cell-cell contact which has low speci- ficity. T.thermophila seems to lack mating action; pairing begins with direct cell- iP contdct. (specificity, oO) paring was mined by determining the frequency of eropair using strains with small or ge micronuclei (A nullisomics, n and Re-pairing method was developed in ch the pair was detached by pipetting S were allowed to re-pair. If re some steps which make cells to ecifically and cell-cell contact is non-specific, re-pairing would e the specificity. When 2 mating af x @ ct OH 0 Of hon Oo @m Ww ls were mixed in various ratio, pairs reached the same level as theo- 1cal values. In this case and in re- ing system, pairing was specific. ific pairing in re-pairing system su- ts that mating substance is located on membrane of attachment site. Re-pair- at early stage of meiosis was also cific. This suggests that mating subs- nce exists long after pairing. Mating type self-incompatibility was observed in co-stimulation and in re-pair ing. Ke kaos 0 oe ct @ BO 1a{= a -O0 W'V (D rPnoas S 1 g 5 S oo td CB 6 HOMOTYPIC PAIR FORMATION IN TETRAHYMENA THERMOPHILA. A. Kitamura 1), T. Sugai 2) and Y. Kitamura 1) Biol. Inst., Fac. of Sci., Tohoku Univ., Sendai and 2)Dept. of Biol., Fac. of Sci., Ibaraki Univ., Mito. It has been generally beliebed that conjugation in Tetrahymena occurs only between cells of different mating types. However, little is known about occurrence of homotypic pairs during conjugation. To examine this we established cells of mating type III which have haploid micronuclei and cells of mating type II which have triploid micronuclei to distinguish them. During the first 10 min following the first pair for- mation, about 45 % of the pairs were homo- typic. Both haploid and triploid pairs appeared at the almost same ratio. The per- centage of homotypic pairs decreased to about 10 % in 30 min and to less than 5 % in 60 min. Micronuclear changes at stage 1 of meiotic prophase were observed in most homotypic pairs 60 min after the first pair formation, suggesting the retention of homotypic pairs for at least 30 min.Similar results were obtained when cells with weak mating reactivity were used. Homotypic pairs were also seen in the re-formed con- jugant pairs after mechanical separation of heterotypic pairs. Relationship between the mating type substances and molecules re- sponsible for mating type-non-specific adhesion of cells was discussed. A possible mechanism of self-incompatibility in conju- gation of Tetrahymena was proposed. C3 7/ CHANGE OF MICRONUCLEAR DNA CONTENT AFTER MEIOSIS IN PARAMECIUM CAUDATUM. A.Yanagi, Biol. Inst., Fac. of Sci., Tohoku Univ., Sendai. In Paramecium caudatum four haploid nu- clei(meiotic products) are produced after meiosis. Three of them degenerate and the remaining one survives in the paroral re- gion. To know the difference between de- generating and surviving nuclei, I exam- ined differences in their abilities of DNA synthesis and nuclear division. As to the ability of nuclear division, observation on living cells showed that some of the prospective degenerating nuclei had the ability of nuclear division as prospective surviving nuclei. DNA content of the mei- otic products at 0, 0.5, 1, 1.5 and about 2 hours(at 25°C) after the second meiotic telophase were measured by microspectro- photometry. All of meiotic products under- went DNA synthesis, the time course of which was also identical in all of them. DNA content increased linearly with time after the telophase. The DNA doubling time was about 1.5h. Moreover, the experiment using a inhibitor of DNA polymerase-a (aphidicolin) shawed that DNA synthesis was not necessary for the nucleus to sur- vive. These results indicate that abili- ties of DNA synthesis and nuclear division are not related to the determination of degeneration and survival of the meiotic products. —— o- » Cell CB 8 EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON TOTAL CONJUGATION IN PSEUDOUROSTYLA (CILIOPHORA). T. Takahashi. Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. Total conjugating pairs were continuously treated by 100 wg/ml actinomycin D (AmD) or 150 wg/ml puromycin (Pu). These treatment started at various times after the onset of conjugation. In this ciliate, total conju- gation processes are separable into two patterns. In a mating mixture, about a half of synconjugants underwent a physiological regeneration (P-II), while the remainders did not take part in the comparable process (P-I). If the AmD treatment on P-I cells began earlier at 7 hr, the cells did not enter a-RB stage in which RBs were formed in almost all of macronuclei. If the treat- ment began from 7 to 11 hr, the duration of developmental period was 30 hrs or more. If the Pu treatment on P-I cells began earlier at 7 hr and between 7 and 11 hr, the cells were arrested at a-RB stage and the early stage of the first cell division (CD), re- spectively. If the P-I synconjugants were treated later at 11 hr by AmD or Pu, the cells normally underwent CD at 21 hr. The P-II cells did not complete the developmen- tal process, even if the treatment of AmD or Pu began at 15 hr. These results suggest that RNA and proteins synthesized earlier at 7 hr are indispensable for the comple- tion of P-I, while P-II requires both RNA and proteins synthesized later at 15 hr. CB 9 RNA SYNTESIS DURING CONJUGATION OF STYLONYCHIA PUSTULATA (CILIOPHORA) fumeano.s ZOOL. inst., Fac. of. Sci, Hiroshima Univ. Hiroshima The role of RNA synthesis on nuclear and cortical changes during conjugation of S. pustulata was examined by the effects of actinomycin D (AmD) and the autoradiogra- phic technique. The conjugating pairs were continuously treated at various stages of conjugation with 50 weg/ml AmD which inhib- ited RNA synthesis. If AmD treatment began at meiosis I, developmental progresses were arrested at some defined stages through metaphase I and prezygotic divi- sion. If AmD treatment began at prezygotic division, nuclear changes were arrested at second postzygotic division, but the pairs could unaffectedly separate into exconju- gants, in which one of 1-4 nuclei often differentiated macronuclear anlage. If AmD treatment began between pronuclear differ- entiation and second postzygotic division, exconjugants were arrested at early stage of giant polytene chromosome. The conju- gating pairs were pulse labeled for 1 hr with *H-uridine. The macronuclei actively synthesized RNA during meiosis, and then the synthesis sharply dropped and remained at low level till later stages. But micro- nuclear RNA synthesis was not observed. These results suggest that RNA newly syn- thesized in macronuclei during meiosis is required for processes of conjugation. Biology 887 CB 10 FISSION LIFESPAN AND CALENDAR LIFESPAN IN PARAMECIUM TETRAURELIA: EFFECT OF NUTRI- TIONAL CONDITION. Y. Takagi and T. Nobuoka. Dept. of Biol., Nara Women's Univ., Nara 630. The question is if tthe length of lifespan measured in fissions and in days may change under different cultural condi- tions. Exconjugant clones of P. tetraure- lia, heterozygous for 2 marker genes, were used in order to check autogamy. Each clone was divided into 2 groups of differ- ent concentrations of Cerophyl (1X and 4X); each group was composed of 9 sub- clones each consisting of 2 daily reisola- tion lines, between which replacements were allowed. The lifespan of each sub- clone was represented by the longest-lived line, taking the last autogamy-free age into account as to the autogamy lines. In all of 4 clones’ studied, the mean fission-lifespan was significantly shorter in the subclones of 1X group than in those of 4X; while the mean calendar-lifespan remained unchanged between 2 groups. This contrasted strongly with the results of the other experiment in which fission rate was controlled with temperatures (25C and 20C): lifespan was shown to be coupled with fissions rather than days. We have shown that the answer to the opening question iS variable depending on what kind of methods to describe lifespan is used and which cell lines are selected, as well as how fission rate is controlled. GB Et MACRONUCLEAR DEVELOPMENT IN THE EXCONJU- GANTS OF EUPLOTES PATELLA (CILIOPHORA). Ke web. wOOL. Lnst., Fae. of Sciv, Hiroshima Univ., Hiroshima. The whole development process of macro- nuclear anlage (MA) was morphologically investigated in Euplotes patella syngen 2. Granular chromosomes(CH) appeared in a swelling young MA. The CH developed into thready CH with a weak banding pattern. In 3 h old MA, the thready CH began to coil, so that spiral CH were evidently observed in 6 h old MA. During these stages, the number of CH seemed to decrease gradually, although the CH became longer. Successive- ly, 24 h old MA was filled with giant poly tene CH. Following disintegration df the giant CH, MA became smaller, moving to an anterior part of cell. After this movement, the extrusion of a part of chromatin from the MA into cytoplasm appeared to occur. Because of the extrusion, 68 h old Mi Showed ring-like structure. However, the structure disappeared at the time when the MA became smaller. The chromatin MA at ring stage looked to be similar to that in a mature macronucleus. After elongation of MA, the replication band appeared at an anterior tip of elongating MA and migrated into a posterior end at least 4 times. Es- pecially with respect to the formation of polytene CH and the behavior of chromatin after the movement of MA, * e results provide new information in t MA develop- ment of Euplotes. B88 Cell Biology CB 12 MORPHOLOGICAL CHANGES ON ENCYSTMENT IN EUPLOTES (CILIOPHORA). MEE dolearaheso Oo, IbSibo5 HAC OF Silo p Hiroshima Univ., Hiroshima. Encystment of Euplotes can be easily induced by hanging drop method. The encyst- ment process was observed with light and electron microscope. Encystment was divided into the following 6 stages. (1) At 30-45 min after induction, frontal-ventral-trans- verse cirri broke down into component cilia except for their proximal parts. At this time cells lie quietly on the bottom of the drop. Irregular shaped vacuoles in the cytoplasm increased in number as com- pared with vegetative cells. (2) At 45-60 min cells became ellipsoidal shape. Dorsal ridges of the cell were not observed. AZM and frontal-ventral cirri were turned in the peristomal field of the cell. Cyto- plasmic vacuoles decreased in number as compared with stage 1 cells. The cyst wall began to be formed. (3) At 60-75 min cells became spherical shaped and had one layer eyst wall with 50-100 nm thickness. (4 At 75-180 min cells contained many electron dense bodies. A contractile vacuole was still functional. (5) At 60 hr the con- tractile vacuole was not functional. (6) At 72 hr the outer layer of the cyst wall formed ridges and the number of ridges was equal to that of dorsal ridges of vegetative cells. AZM and other ciliary organelles did not disappear in cysts of this species. CB 1S THE EXISTENCE OF TUBULIN IN THE MATURE CYST OF A HYPOTRICH CILIATE, HISTRICULUS MUSCORUM T. Nakamura and T. Matsusaka. Dept. of Biol., Fac. of Sci., Univ. of Kumamoto, Kumamoto. The tubulin was detected in the mature cyst of Histriculus muscorum by the use of (urea-) SDS polyacrylamide gel elec- trophoresis (PAGE) and immunoblotting tests. An antiserum was prepared against the tubulin bands separated by urea-SDS- PAGE of Tetrahymena ciliary axonemes. The specificity of this antiserum was contirmed stationary phase celis of H. muscorum by an indirect immunoflorescence method and immunoblotting tests. And we detected the tubulin in the mature cyst using immunoblotting tests. This tubulin band showed different mobility on SDS-PAGE with or without urea. On urea-SDS-PAGE, this tubulin band showed similar mobility to the rabbit brain # one and migrated slightly slower than the Tetrahymena ones. On SDS-PAGE, the tubulin band migrated slightly faster than the rabbit and Tetra- hymena ones. Cilia and microtubules of H. muscorum completely disappeared during the encystment, but these only disassem- bled to tubulin which existed in the mature cyst. This suggests that synthesis of tubulin protein is not required for ciliogenesis during the excystment. CB 14 THE POSITION OF THE CONTRACTILE VACUOLE PORE IN DOUBLETS OF THE CILIATE GLAUCOMA SCINTILLANS, MANIFESTING PATTERN REVERSAL. M.Suhama. Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. The position of the contractile vacuole pore(CVP) in various types of doublets de- rived from singlets(ROS-1) with a reversed oral apparatus(OA) of Glaucoma scintillans were examined. CVPs of normal and ROS-1 singlets were respectively located right and left to a kinety with an oral primor- dium. In most of the doublets derived two OAs were close to each other and the left OA was normal, but the right one was ab- normal and frequently manifested reversal. In these doublet lines the mean number of kineties was 42-53 and the mean distance between two OAs varied 9-20 in number of intermeridional spaces. When two OAS were sufficiently apart(20 spaces), two CVPs were located an equal distance from each OA between two OAs. When the distance be- came short(14-15), one CVP was situated near the middle point between two OAs. In cases of shorter distance, one CVP was lo- cated at the middle point and one more CVP occasionally appeared near the middle point on the opposite(dorsal) side of the cell. In cases of the shortest distance, the CVP on the dorsal side was dominant. These results suggest that in doublets manifesting pattern reversal the position of CVP orderly shifts depending on the distance between two OAs. CBS DISSOCIATION OF HOMOPOLAR TWIN CELLS OF PARAMECIUM THROUGH CELL DIVISION M.Sato. Biol. Inst., Fac. of Sci., Tohoku Univ., Sendai. Homopolar twin cells of Paramecium bur- Saria were induced by preventing conju- gating cells from pair separation with an osmotic shock. When the twin cells undergo repeated divisions, they produce daughter cells with different shapes and eventually separate single cells. In this process, first, the twin cell which has a notch at its anterior pole appears among daughter cells of ordinary twin cells. The notch is always formed in the right fields of the twin Cell. In the following cell divasrony the anterior notch is transformed only to the anterior daughter cell and gradually deepens. The deepening does not occur in the growth zone. The depth of the notch is a parameter of dissociation. When the notch deepens through cell divisions, two single cells are eventually derived from the anterior half. Thus, in Paramecium bursaria, single cells are always formed through the formation of notched cells. Formation and deepening of the notch may be explained as the change of relative positions of two long axes of a twin cell through cell divisions. Cell CB 16 ISOLATION OF ENDONUCLEAR SYMBIONT HOLOSPORA OBTUSA FROM MASS CULTURES OF PARAMECIUM CAUDATUM. M. Fujishima and K. Nagahara. Biol. Inst. Fac. of Sci., Yamaguchi Univ., Yamaguchi. The gram-negative bacterium Holospora obtusa is a macronucleus specific symbiont of ciliate Paramecium caudatum, which in- vades into the host cells via the food vac- oles, infects its macronucleus exclusively and grows in the nucleus. The bacterium never infect the micronucleus. To know what kinds of differences between the macro- and the micronucleus, of common genetic origin, can be recognized by the bacterium, we have aimed to establish in vitro infection system between the isolated bacteria and the isolated nuclei. In the present work, we succeeded to isolate the infectious bac- teria from Paramecium homogenates with Percoll density gradient centrifugation. Yields averaged 85 %, and the preparations were essentially free from contaminants such as mitochondria, cilia and food bac- teria. The isolated bacteria infected the macronucleus within 15 min after adding the bacteria into an external medium of Holospora-free paramecia. CB 17 EXTRACELLULAR PHOTOSYiITHETIC PRODUCTS OF THE APOSYHBIOTIC ALGAZ FROLi A CILIATE, STENTOR AMETHYSTINUS. H. Kawakami. Dept. of Nutrition, Suzugawine Women's College, Hirosnima. An endosymbiotic alga of Stentor amethy- stinus was isolated immediately after the release from the host ciliate which was col- lected in large number from the natural ha- bitat. The isolated endosymbiotic alga of S. amethystinus from another collection was cultivated on an agar slant and then grown in a liquid modified Bristol medium for 1 year in sunlight. The isolated endosymbiot- ic alga and the cultivated one were incubat- ed in a medium constituted of an inorganic salt solution, acetate buffer (pH 5.0) and NaHCO3 under 400 lux light at 25°C. At 0, 1, 2, 3, and 4 hr after incubation, the de- tection for sugar and amino acid in the ex- tracellular photosynthate was carried out. Maltose and alanine were produced in the incubation medium of the isolated endosyn- biotic alga and the quantity of the products increased with the prolonged incubation time. In the case of the cultivated endo- symbiotic alga, the photosynthetic products were small in quantity. 1 year culture med- um of the cultivated endosymbiotic alga contained a large amount of the extracellu- lar photosynthate. It was suggested that the endosymbiotic alga was competent to sup- ply the photosynthetic products to the host and that the quantity of the photosynthate released from the aposymbiotic alga decreas- ed with time after the isolation. Biology 889 CB 18 DISCRIMINATION OF FOOD IN PARAMECIUM MULTIMICRONUCLEATUM. PPT TEASE Y.Haradal and S.Mishima2. 1)pept. Biophys. Engn., Fac. Engn. Sci., Osaka Univ. ,Toyo- naka. Biol. Lab., Coll. Gen. Educ., Iba- rakz Univs, Mito. It is known that when starved Parameci- um is transferred to a medium rich in bac- teria, the first vacuole formed is always huge. On the other hand, Paramecium in a medium containing polystyrene latex parti- cles(PLP) forms only normal sized vacu- oles. This fact implies that Paramecium discriminates between food and indigesti- ble material. The volume of vacuoles formed during the first 1 min was measured after giving either bacteria or PLP of nearly the same size as bacteria. The ingestion rate of bacteria was several times large as that of PLP giving at the same concentration in the low concentration range. In the present study, both particles were given simultaniously in a medium. The volume of the vacuoles then formed was found to be the sum of those vacuoles formed when each kind of particle was given separately. This suggests that both particles are ingested indipendently with- out affecting each other and that Parame- cium may have a mechanism of selectively ingesting the digestible particle, bacte- rium, over the indigestible one, PLP. CB 19 CULTIVATION OF INTESTINAL PROTOZOA OF THE TERMITE ( RETICULITERMES SPERATUS ). l. I. Yamaoka, M. Moriyama and M. Endo. Biol. Inst., Fac. of Sci., Yamaguchi Univ., Yamaguch. Several conditions for the cultivation of intestinal protozoa of the termite were examined. Solution U ( Trager, 1934 ) was used as a basal saline. An optimum pH value for the cultivation was pH 7.0- 7.5. A suitabe O02 concentration was below 207% in the solution U. A survival ratio in this condition fell to bellow 5% at 12 hr. A high survival ratio was obtained by the cultivation in a conditioned medium which was prepared as follows: 10ml of the solution U contained the hindgut and its contents of 50 individuals of termite (worker-caste) was incubated at 25°C for 3 days. Twenty-four hours after the inocu- lation the survival ratios of Pyrsonympha OS Seen modesta, P.grandis and Trichonympha agilis were about fe 35 and 20%. By the addi- tion of a cellulose powder the high sur- vival ratio was able to hold for 465 hr or more time in three species of akg > oC scribed above and in Dinenym; eidy. When a_ filtrated conditioned medium ( membrane filter, 0.25 ~im) was used instead of the conditioned medium the survival ratio was reduced to that condi- tion. These results suggest hat a suit- able condition for the ration of the intestinal protozoa termite was prepared by bacteria. 890 Cell Biology CB 20 ELECTRON MICROSCOPIC VISUALIZATION OF CEL- LULAR ARCHITECTURE IN MOUSE MUCOGINGIVAL JUNCTION. T.Yamaai. Dept. .of OralvAnat., Sch. of Dent., Okayama Univ., Okayama. The cellular architecture of the muco- gingival junction was investigated with the scanning and transmission electron micro- copes. Adult male ddY mice were fixed with .5% glutaraldehyde and 1% paraformaldehyde in 0.05M Na-cacodylate buffer (pH7.4) by the perfusion through the left ventricle. After perfusion the oral epithelium was removed, trimmed and immersed in the same hixabave forean addutzonal Zhiegait a UCmpriesior GO Ghe) faxataenawathe125 72. OS O)eins 0). O.5hiaNa— cacodylate buffer (pH7.4) for 3hr at 4°C. Ultrastructural analysis of the muco- gingival junction revealed that two cell types, gingival epithelial cells and alveo- lar mucosal cells, were found in it and it represented mosaic of both gingival epithe- lial cells and alveolar mucosal cells. On the other hand, the type of surface of the cell in the mucogingival junction did not correspond with two cell types observed with the transmission electron microscope. For instance, some epithelial cell had two different types of cell surface, the gingival epithelial cell type and the alve- olar mucosal cell type. These suggest that the feature of the epithelial cell surface in the mucogingival junction depends upon the overlying cells which have been desquamated. mn CB 21 A COMPARATIVE INVESTIGATION ON THE STRUC- TURAL CHANGES IN THE SYMPATHETIC GANGLIA OF ADULT RATS INDUCED BY THE CHRONIC TREATMENT WITH HIGH DOSAGE OF GUANETHIDINE SULFATE. S. Kikuchi and K. Shiraishi. Dsjoien Re Into, Sells Cie ino, mes ancl Sesto, Iwate Med. Univ., Morioka. The structural changes in pelvic, celiac, Superior cervical, and stellate ganglia of adult rats were compared by light,glyoxylic acid-fluorescent and electron microscopical methods after chronic treatment for 7, 14, 30 and 75 days with guanethidine sulfate (Ismelin CIBA) (30 or 60 mg/Kg/day ip.). The fluorescent nerve fibers innervating blood vessels in every ganglion completely disap- peared already after 7 days treatment, while intraganglionic fluorescent neurons remained unchanged. After 30 days treatment the fluorescent nerve cell bodies and fi- bers almost disappeared in the pelvic and celiac ganglia, whereas a considerable number of them survived in both superior cervical and stellate ganglia even after 75 days treatment. Two weeks of cessation of the treatment resulted in marked prolifera- tion of fluorescent nerve fibers in every ganglion except the parasympathetic area of pelvic ganglion. No sign of degeneration was detected by electron microscopy in the neurons survived the treatment. The results Suggest that guanethidine-resistant neurons are more abundant in the paravertevral ganglia than the prevertevral ganglia in the rat sympathtic nervous system. GBaZ2 ULTRASTRUCTURAL ANALYSIS OF THE MORPHO- GENESIS OF HUMAN COLON CANCER CELLS IN ORGAN CULTURE. H. Fukamachi. Zool. Inst., Univ. of Tokyo, Tokyo. a ne Wenn AE ne Ine SE Fak Four lines of human colon cancer cells (LS174T, HT29, HCT48, and SKCO-1) were combined with forestomach mesenchyme of fetal rats, and organ-cultured on a mem- brane filter for 4 days. LS174T cells formed gland structure surrounded by mesenchymal cells, while HT29 cells formed only packed cell masses, and HCT48 and SKCO-1 cells were intermin- gled with mesenchymal cells. Microvilli, gap junctions, and desmosomes existed in all 4 cell lines. Basal laminae were observed in LS174T, HT29, and HCT48 cells. Tight junctions existed in LS174T and HT29 cells. As to the arrangement of these structures, gland-forming LS174T cells possessed similar arrangement to that of normal gland cells. However, in HT29 cells microvilli and tight junction were often observed at the epithelial-mesenchymal interface. These results indicated that the arrangement of these four structures plays essential roles in the establishment of cellular polarity and glandular morpho- genesis of human colon cancer cells, when they are cultured in association with mesenchymes. G3 2 DOMAIN ORGAMIZATION OF THE DESMOSOMAL GLYCOPROTEINS. H.Shida and *M.S.Steinberg. Department of Biology, University of Yamanashi Medical School, Tamaho, Yamanashi 409-38. *Department of Biology, Princeton University, Princeton, NJ 08544, U.S.A.. Desmosomal glycoproteins have been classified into three major antigenic families; desmoglein I(average MW=150KD), desmoglein II(a doublet, MW=118 and 97KD) and desmoglein III(a single band MW=22KD) (Cohen et al.,1983, J.Biol.Chem. ,258, 2621 ). We have studied the ultrastructural organization of desmoglein (DG) families by quantitative immunoelectron microscopy uSing a post-embedding labeling tech- nique. A thin slice of fixed bovine muzzle epidermis was embedded in a mixture containing twenty parts JB-4 A, one part divinylbenzen, one part methyl methacrylate and one part JB-4 B. The ultrathin sections were labeled specifi- caly with anti DG antibodies and protein A-gold particles. By the quantitative analysis of the specific labeling, three moleculer domains of DG I and DG II; cytoplasmic, intramembraneous and extra- cellular domain structure were confirmed. Although the extracellular domain of DG III were also confirmed, other domains of the DG III glycoprotein were not detected by the quantitative immunocyto- chemistry using a monoclonal antibody against DG III. Cell CB 24 CHROMATOPHORE DISTRIBUTION AND GLYCOSAMINO- GLYCANS IN THE INTEGUMENT OF THE LOWER VER- TEBRATES. Y.Kamishima. Dept.of Biol., Facult.of Sci. Okayama University, Okayama. The characteristic color pattern on the lower vertebrate skinis caused by the dis- tributional differences of three types of chromatophores, which are derived from the neural-crest and migrate to the skin. This study reports the relationship between the chromatophore distribution and the dermal component; glycosaminoglycans (GAGs). In frog and fish skin, melanophores were distributed where non-sulfated GAGs, such as hyaluronic acid, were histochemically detected, whereas iridophores were only observed where sulfated GAGs were abundant. In anon-pigmented area of the skin, strongly sulfated GAGs were demonstrated. On the chromatoblast migration in fish embryo, melanoblasts and iridoblasts did not migrate concomitantly inthe same path, while xanthoblasts were frequently found with other chromatoblasts inthe same path. In injured frog skin, where chromatophore unit had been distroyed and was re-forming again, all of chromatophores were randomly distributed at the first, when mesenchymal GAGs were not detectable histochemically, but later, they resumed the strict arrange- ment for the chromatophore unit. These observations will suggest a role of mesenchymal GAGs in the pattern form- ation of the dermal chromatophores. CB 25 ISOLATION AND CHARACTERIZATION OF A MONO- CLONAL ANTIBODY AGAINST MELANOSOME FRAC- TION FROM B16 MOUSE MELANQMA, CELL. N. Yanai and JT. Takeuchi’. Biological Institute and “Department of Biological Science, Tohoku University, Sendai. Melanosomes, the site of melanin forma- tion, are the unique and specialized organ- elles of melanocytes. These organelles con- tain tyrosinase, the key enzyme of melanin synthesis, and structural proteins that seem to be involved in melanosome architec- ture. Although the function of tyrosinase has been investigated intensively, the role of structural proteins are still unknown. For more detailed investigation of structural proteins, we have isolated a monoclonal antibody against one of melano- somal proteins. Melanosomal fraction from B16 mouse melanoma cells was used for im- munizing BALB/c mice. Hybridoma cells, secreting monoclonal antibodies, were formed by the fusion of mouse myeloma cells and spleen cells of immunized mice. Immuno- blot analysis indicates that the monoclonal antibody recognizes a low molecular weight protein (approx. 14K dalton). By protein A -gold technique, ultrastructural localiza- tion of antigenic sites were determined on the melanosome (between stage III and IV) and on microsome like structures. Biology 89] CB 26 DYNEIN LOCALIZATION AND EFFECT OF DYNEIN ATPASE INHIBITORS ON BRIJ PERMEABILIZED MELANOMA CELLS. S.Negishil, M.Obika!, Y.Wakamatsu2 and K.Ogawa’. IJpept. of Biol., Keio Univ., okohama. 2Biol. Lab., Kyoto Univ., Kyoto Dept. of Cell Biol., Natl. Inst. Basic Bio., Okazaki. In order to determine whether dynein ATPase participates in melanosome movement, we have investigated the effect of dynein ATPase inhibitors and the localization of dynein in cultured melanoma cells of pla- tyfish-swordtail hybrids. Permeabilized cell preparations obtained by the method of Clark and Rosenbaum (1983) were used. These preparations were capable of res- ponding to MCH or epinephrine with irre- versgible centripetal pigment translocation. Vanadate at 100 pM or 2 mM EHNA arrested melanosome aggregation in Brij-treated cells almost completely, implicating that dynein ATPase is required in this process. Indirect immunofluorescence microscopy using antidynein antibody revealed that melanoma cells with aggregated and dis- persed pigments were labeled in radial filaments running from cell center to the peripheral region. No appreciable differ- ence was observed in the distribution pattern of labeling between the cells with aggregated and dispersed pigments. Supported by a grant (No. 58540472) from the Ministry of Education. MCH was a generous gift from Dr. Mac E. Hadley. CB 27 ASSEMBLY AND DISASSEMBLY OF MICROTUBULES AND INTRACELLULAR TRANSPORT OF MELANOSOMES. M. Obika and S. Negishi. Dept. Biol., Keio Univ., Yokohama. Rapid pigment migration in melanophores of teleost is dependent on the presence of radially oriented microtubules(mt). In or- der to examine whether the disassembly and reassembly of mt are indispensable for pig- ment translocation, responses in isolated melanophores of Oryzias latipes were studi- ed in the presence of colchicine at 1 mM. At this concentration, the drug does not disrupt mt but inhibits mt reassembly in melanophores which have been exposed to low temperature. After 100-min incubation in the drug, melanophores responded to epine- phrine with complete aggregation and fol- lowing perfusion with theophylline produced a full pigment redispersion, indicating that the latter process does not require mt reassembly. If, however, melanophores were stimulated with epinephrine and theophylli- ne repetitively in the presence of colichi- cine, magnitude of pigment translocation to both directions decreased gradually. This Suggest that disassembly and re embly of mt are actually taking place d g chro- matophore responses probab! lependently of the mechanism by which the m fe force for pigment movement is gener ed. Colcemid has a similar effect as e. Supported by a grant or istry of Education no. 5854047 89? Cell CB 28 ULTRASTRUCTURE OF CIRCULATING HEMOLYMPH CELLS OF THE LAND SLUG, INCILARIA FRUKSTOR- FERI COLLINGE (GASTROPODA: PULMONATA) K. Yamaguchi E. Furuta*and A. Shimozawa ‘Lab. Med. Sci. and ?Dept. Anat., Dokkyo Univ. Sch. Med., Tochigi. Four morphologically distinct hemolymph cell types, Type I, I, III and WV, were found inthe hemolymph of the land slug, Incilaria frukstorferi Collinge. Type I cell, meaSuring 50um x 20um, possessed long filopodia with branched fibers and a polymorphic nucleus. Type I cell, approx- imately 35um in diameter, had extensive pseudopodia with supporting ribs and a kidney-shaped nucleus. Type I and I cells contained residual bodies, multivesicular bodies, Golgi bodies and free ribosomes. Type III cell, measuring approximately 5um in diameter, did not form large pseudopo- dia and had a round nucleus. It contained widespread free ribosomes around the large nucleus. Moreover, the nucleus to cyto- plasm ratio was higher. Type WV was a fi- broblast-like cell. It contained microfi- brils at the rim of perinuclear cytoplasm and possessed collagen-like fibers outside of the cytoplasmic membrane. Numerous solid elements, which had not been reported yet, were usually observed. They, measuring 1 to 5ym, were oval. They had no nucleus but only glycogen-like de- posits. GB e239 MAINTENANCE MECHANISM OF CONTRACTILE APPA- RATUS IN ASCIDIAN SMOOTH MUSCLE CELLS. K. Terakado. Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa. Organizations of contractile apparatus and cytoskeletal systems in the smooth mus- cle cells of an ascidian, Halocynthia rore- tzi were examined with an electron micro- scope. Small pieces of the body-wall mus- cle were fixed in 3% glutaraldehyde for 6- 12 hrs, and postfixed in 1% OsO, for 2 hrs. In sections of contracted muscle cells, many bundles of thick and thin filaments measuring about 0.2-0.5 pm in diameter were observed, though they were indistinct in relaxed cells. Numerous 10-nm filaments ran in groups between the bundles of filaments and terminated at the cell surface. Dense bodies were oval structures measuring about 0.05-0.1 wm and located mostly in the lat- eral surface of the bundles of filaments. They were attached by thin filaments at both sides and formed mini-sarcomeres with associated thick filaments. The dense bod- ies were further attached by an oblique network of 10-nm filaments running between the bundles of filaments. Thus, the ar- rangement of contractile apparatus of the smooth muscle cells may be mechanically maintained by the three-dimensinal associ- ations of 10-nm filaments to the dense bod- ies through contraction-relaxation cycles. The bundles of filaments seemed to be per- sistent structures having many contractile units and may be termed "myofibrils". Biology CB 30 ULTRASTRUCTURAL OBSERVATIONS OF THE CONT- RACTILE CELLS IN A FRESH-WATER SPONGE, EPHYDATIA FLUVIATILIS L. 1) A.Matsuno, H.Ishida and Y.Masuda Dep. Che Walolly 4 IMG, Oie SCis, Sinimeme Ura. Dep. of Biol., Kawasaki Med. Sch. ?? The osculum and the body wall of a Ephydatia are contractile. We observed these portions of the animal by the elect- ron microscope and recognized two types of cells regarded to be contractile, though these cells were not so many in number. The cells of the first type, which must play a main part of the movement, were ob- served in the mesoglea as free cells. They showed similar profiles to the other free cells, but were different in the intracel- lular structures; The cytoplasm of the cell was less electron dense and contained one or more bundles of filaments measuring about 0.2 pm in diameter. The filaments were measured 7-12 nm in diameter. The cells of second type were observed among internal epithelial cells and bore thin and long bundles of fine filaments meas- uring about 0.3 ym in diameter and 8-9 pm in length. The fine filaments were 5-7 nm in diameter. These filaments often con- nected to tono-filaments of the desmosome, but could be distinguished from the tono- filaments in their diameter, length and distributions. These cells also resembled in their profiles to other epitherial cells. Ce Sul EFFECTS OF Y{- IRRADIATION ON THE DEVELOP- MENT OF GEMMULES OF THE FRESH-WATER SPONGE, EPHYDATIA FLUVIATILIS. N. Egami, A. Shimada, E. Maeda, Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo and Y. Watanabe, Biol. Lab., Fac. of Sci., Ochanomizu Univ., Tokyo. We examined the effect of Y-ray irradi- ation on the asexual reproduction and the cell differentiation of the fresh-water sponge. Irradiation was carried out with a Y-ray source of about 4 kCi 137¢s at the dose rate of 0.7-2.4 kR/min. In order to observe differentiation of the cells the materials were fixed in toto with modyfied Carnoy's regent and stained with fluo- rescent dye or Giemsa solution. At less than the dose of 50 kR, the dose depend- ant delay of the hatch and the decrease in hatchability of the gemmules were observed. Especially the differentiation of cells into the choanocytes were inhibited. At more than the dose of 100 kR the cells came out from gemmules but did not spread and not attach to the Petri-dish. These results suggest that DNA replication and collagen synthesis were inhibited respec- tively. Cell Biology 893 CB 32 EFFECTS OF TOCOPHEROL ON LIPOFUSCIN ACCU- MULATION IN THE NEURONS OF RAT CEREBRAL CORTEX IN PRIMARY CULTURE. T.Fujii and S.Kawashima. Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. Accumulation of lipofuscin is the cyto- logical alteration with age in a variety of postmitotic cells. As for the origin of lipofuscin, it is claimed that lipofuscin is derived from peroxidated lipid compo- nents of the biological membranes, which are fused with lysosomes. Previous inves- tigators pointed out that lack of vitamin E in the diet caused an increase in lipo- fuscin storage in some postmitotic cells of mammals. In the present study we have cultured neurons dissociated from rat cerebral cortex in the medium enriched with vitamin E (dl-X-tocopherol or dl-«d- tocopherol acetate) or in the standard medium. The presence of lipofuscin pig- ments was verified by their autofluore- scence, periodic acid-Schiff stainability and acid phosphatase activity. The percen- tage of neurons containing lipofuscin was increased with age in culture in the stan- dard medium, but in the medium enriched with either kinds of vitamin E, the accu- mulation of lipofuscin was significantly reduced. Difference of effects between the two kinds of vitamin E was not detected. This effect was more marked in neurons than in non-neuronal cells. The present findings suggest that vitamin E was effec- tive as an antioxidant for neurons. CB 33 BARREL-SHAPED SPINDLE INDUCTION AND SUPPRESSION OF CYTOKINESIS IN SEA URCHIN EGGS BY T-1, THE MITOTIC ARRESTER. T.J.Itoh’, A.Kobayashi* and H.Sato! 1: Sugashima MBL, School of Sci., Nagoya Univ., Toba. 2: Dep. Agric. Chem., Fac. of Acric., Okayama Univ., Okayama. T-1, isolate of culture medium of soil bacteria, is known to possess arresting, and stabilizing effects on the assembly of mitotic spindle. We followed these drug effects using fertilized sea urchin eggs. T-1 induced barrel-shaped spindle with tiny asters in concentration dependent manner. Though anaphase chromosome movement could occur even in the barrel-shaped spindle, cleavage was usually suppressed in higher concentrations of T-l. To verify whether this suppression of cytokinesis was due to the direct T-l influence on egg surface or not, we examined the distribution pattern of egg microvilli in telophase by the SEM and the NBD-phallacidin staining. Results revealed that density of microvilli was higher in equatorial region than the other regions when T-1l was applied. Microvilli in equatorial region were longer than those in other regions. However, similar tendencies were observed in control experiments, and thus the suppression of cytokinesis was hardly due to the direct T-1 effects on egg cortex. Rather, we believe this effect might be introduced by the change of dis- tribution and orientation of microtubules in mitotic apparatus by T-1. CB 34 DIURNAL FLUCTUATIONS IN THE MITOTIC RATES OF STRATIFIED SQUAMOUS AND SIMPLE COLUMNAR EPITHELIA IN THE RAT DIGESTIVE TRACT. S.Toyoshima, K.Shirama, K.Shimizu and M.Ho- kano. Dept. of Anat., Tokyo Med. Col., Tokyo. Adult male rats were standardized to a 12L(0600-1800h)—12D cycle and fed ad lib. The environmental LD cycle was then re-_ versed (12D-—12L). When the rats were in the 12L—12D cycle, stratified squamous epi- thelia in the esophagus and forestomach had statistically significant rhythms. On the other hand, the rhythms of simple columnar epithelia in the duodenum and the glandular stomach were not significant. In the re- versed LD cycle, the phases of rhythms in the stratified epithelia were reversed. The amplitudes of rhythms in the simple co- lumnar epithelia increased and the ambigu- ous rhythms in the 12L—12D became statisti- cally significant when reversed. Since the change in LD caused the syn- chronized shifting of phases in the strati- fied epithelia, LD cycles seem to act as a dominant synchronizer for the rhythms of mitoses in these epithelia. But as it re- lates to the simple epithelia, the synchro- nous system seems more complicated. It is probable that some other factors can affect the mitotic rates of the simple epithelia. If these factors counterbalanced each other in these epithelia when standardized to the 12L—12D cycle, we can deduce that the re- verse in LD destroyed this balance and caused the increases of amplitudes. CB 35 ANALYSIS OF UNEQUAL CELL DIVISION IN BINUCLEATE GRASSHOPPER NEUROBLASTS INDUCED BY CYTOCHALASIN B TREATMENT. K.Kawamura. Biol. Lab., College of Dairyin Ebetu, Hokkaido. Grasshopper neuroblasts(NB) divide un- equally with a definite polarity. The present study deals with the induction of binucleate NBs by Cytochalasin B(CB) treatment and their behaviors in order to analyse cell division polarity. By CB action, two NB type nuclei were formed within a cell, their position and orienta- tion being at random. These binucleate NBs still maintained a unique hemispheri- cal shape. In the case of a low CB dose, there was a slight time delay in nuclear formation in the ganglion cell side. The nuclear type may be determined not only by cytoplasmic volume, as demonstrated in the previous study, but by a cortical struc- ture which is damaged by CB action. When the effect of CB was removed, the two nuclei entered mitosis synchronously. Two sets of mitotic apparatus(MA) were not always parallel at prometaphase. As the MAs grew, they became parallel and appear- ed to form a large metaphase spindle in the normal position and direction, al- though two centrosomes at diverged poles remained independent. Later the bi- nucleate cell with such an abnormal MA divided unequally. From these results, the direction of division axis and the inequality of daught cells seems to be under the control of the cortex. 804 Cell Biology CB 36 A NEW CELL LINE OF PORCINE DIPLOID AORTIC ENDOTHELIAL CELLS. K.Yamamoto, M.Yamamoto and *Y.Mitsui. Dept. Biol., Tokyo Metropol. Inst. Gerontol. and *Div. Cell Sci. Tech., Ferment. Res. Inst. A stable cell line of endothelial cells with normal chromosome number was’ estab- lished from porcine aorta for the study of vascular functions. Endothelial cells were isolated from fresh porcine aorta endothe- lium by scraping off method, while smooth muscle cells from the same aorta media by outgrowth method. The endothelial cells had much higher potential to produce pros- tacyclin, and much lower density of nega- tive charge on cell surface coat compared to smooth muscle cells.Among 26 cell lines from the individual pigs, 3 cell lines and 9 clonal lines were continueously subcul- tivated for more than one year. PAE-20 cells changed very slightly in the growth rate and DNA ploidy temporaly at about 80 PDL, but were subcultivated up to the pre- sent 231 PDL with normal range of cell e- lectrophoretic mobility, the relative dis- tribution of DNA contents, and normal mode of chromosome number. Karyotype analysis by G-banding of chromosomes was normal at 18 PDL. Abnormalities of chromosomes were observed in cells at 231 PDL. 3p+ was ob- served in all of 40 metaphase chromosomes at 231 PDL. Thus, PAE-20 cells with stable phenotypes are expected to be useful as a permanent cell line for the study of vas- cular diseases and functions. (om) Bown THE CELL LINES FROM FINS OF Sebastiscus marmoratus AND Sebastes inermis. DPKubo, Biol. Kabeyeuicch Unive Lokyver and N.Ebitani, Biol. Lab.,Shohoku Coll.,Atsugi. Two fibroblast-like cell lines were es- tablished from the fins of Sebastiscus ma- rmoratus and Sebastes inermis. The cells from S.marmoratus,named SMF have been sub- cultured over 120 times since March 1983. ] cells of SMF was shorter and thicker B) 1¢) YN as compared with that of the primary cul- ture.The chromosome number of SMF was 96 and that of the primary culture was 48. SMF was tetraploid. The cells from S.iner- mis, designated SIF have been subcultured Over 50 times since March 1984 in the me- dum onbdimine W5p POSvar 2500). Bie rd c= ference in the shape of cells between SIF and the primary culture was not found. The chromosome number of SIF and the pri- mary culture cells was both 48. The most effective percentage of FCS in the culture medium for the cell growth was 15-20%, and 5-10% was not so effective. The peak of the rate of cell growth in the temperature of incubation ranging 15°C to 30°C, showed at 25°C. SME and SIF showed the most growth rate when cells were inoculated 200 cells per 1 mm* in the flask. When SIF was cultured togeth- Caer alia SI atte fb aPileisikes S018 disanpeared after about 2 weeks and only SMF was found. CB 38 PROLIFERATION OF IRIDOPHORES FROM TOP-MIN- NOW, GAMBUSIA AFFINIS. M.Yasutomi. Biol. Lab., Aichi Medical Univ., Aichi. In order to get pigment cell lines from fish, pigment cells of some fishes were cultured. Among them iridophores of G. affinis proliferated successfully. This fish is ovoviviparous, so we can get fetuses sterilely from its uterus. The fetuses were minced by scissors and cul- tured in a Falcon dish at 25°C. As culture medium 199 medium containing 10% fetal calf serum, 100 units/ml penicillin and 100 ug/ml streptomycin was used. On the 2nd day of culture fibroblasts migrated actively from the explants and then irido- phores came to migrate on the fibroblasts. Many colonies of iridophores were formed in the dish one month after culture and each colony comprised 500-1000 cells. For subculture of iridophores, 199 medium was exchanged by Ca,Mg free Hanks solution containing 0.5% trypsin and the dish was incubated for 20 min at 25°C. Obtained iridophores and fibroblasts were cultured in a Falcon dish. The fibroblasts prolife- rated rapidly and cover the bottom of the dish within a week. Proliferation of iridophores occured on the fibroblasts 3-5 weeks after the inoculation. Doubling time of iridophores was about 5 days and their number in one colony attained to over 5000 cells. Addition of c-AMP (1074m) rather accelerated their proliferation rate. Cs 3S EFFECT OF FIBRONECTIN AND COLLAGEN ON THE SHAPE OF ISOLATED MELANOPHORE OF MEDAKA, ORYAEAS -EAER ES A. Iwata, M. Iwata and E. Nakano. Dept. of Biol., Nagoya Univ., Nagoya Melanophores of medaka, Oryzias lati- pes, were cultured on various substrata. On the substratum coated with collagen, melanophores became superdendritic in the presence of fibronectin(Fn), while they became less dendritic on the substra- tum coated with Fn or BSA. On the sub- stratum coated with both Fn and Collagen the cell shape was dependent of their con- centrations. The shape of melanophore was quantified by the concept of "complex— ity!) \(C=P 7A, Piperiphery,, Avanea)ide sumed by Kobatake (1983). By comparison of the complexity of melanophores on the scale with that of cultured melanophores on the coated substratum, the specific ratio of concentration of Fn and collagen was estimated. And cultured melanophores of Similar shapes as those in the scale were obtained. These results suggest that the shape of melanophores may be regulated by the coexistence of Fn and collagen in specif- Le Pe\wil© > Cell Biology 895 CB 40 ESTABLISHMENT OF CELL LINES FROM NORMAL MOUSE ~» Sipebaheone heat GoSatol. T. -TakguchiZand S. htos's I Biol... Inst Tohoku Univ., Dept. of Biol., Tohoku Univ. Sendai, 3Inst.Comp.Med.Sci., School of Med. Fujita-Gakuen Univ., Toyoake, Aichi. Mouse melanoma cells have been widely used for the study on melanotic expression of melanocytes. It seems probable, however, that their genetic constituents are distant from that of normal mouse. Then, it is required to establish cell lines which has normal properties of melanocyte for the study on the phenotypic expression. In order to establish cell lines of mouse melanocyte, epidermal cells from C57BL/6J mouse were cultured in the medium (MEM) containing phorbol 12-myristate 13-acetate (10 ng/ml) and chorela toxin (10-8M). In these conditions, melanocyte proliferated and became a homogeneous cell population. We cloned some cell lines from these cell populations. One of the Lines, TM clone 10, has normal chromosomes, ability to produce melanin and possesses tyrosinase activity. These cells seem to exhibit function of normal melanocytes and are useful for the study on the expression of melanocytes. Cells of TM clone 10 line were cultured in the medium containing dopa or cysteinyldopa. An increase in the content of pheomelanin in the cells cultured with dopa (2X10-4M) was observed, whereas no increase was seen in the cells cultured with cysteinyldopa (1.25X10-9-2X10-4M). CB 41 GROWTH OF ADULT FROG (Xenopus laevis) PARENCHYMAL HEPATOCYTES IN PRIMARY CULTURE. S.Ishikawa, A.Kawahara and M.Amano. Cell and Dev. Biol. Lab., Fac. of Integr. Arts and Sci., Univ. of Hiroshima. Hiroshima. Adult frog (Xenopus laevis) hepato- ¢ytes were found to proliferate in culture medium supplemented with adult homologous serum. Insulin and Dexa- methasone were required for a net prolif- eration of hepatocytes. Dose-response analysis showed that a low concentration of serum (more than 0.5%) was enough to induce DNA synthesis and mitosis. Fetal bovine serum had less proliferative activity and more cytotoxicity as com- pared to those of adult frog serum. Under an optimum condition examined, there was a two-three fold increase in nucleus number per culture. The autoradiographic analysis following 3y-thymidine-labeling indicated that the inducibility of DNA synthesis did not depend on cell density at inoculation untill at least 6.5x10* cells per cm Almost all of cells grown in the present culture system were characterized as parenchymal hepatocyte, histochemically and immunohistochemically. CB 42 IS ASERUM FROM THE OLD PERFORMING INHIBITION TO CELL PROLIFERATION? H. Kondo and T. A. Nomaguchi. Dept. Biol., Tokyo Metropol. Inst. Geront., Tokyo. It has generally been given an impres- Sion in which both serum and plasma from the old inhibit cell proliferation, after Carrel & Ebeling (1921) reported that those from old hens were more inhibitory to growth of chik fibroblasts inthe plasma clot cul- ture than those of the young. If old serum generally inhibits cell proliferation or not, we surveyed with rabbit and rat sera using an exact manner. Cultured cells of 8xl04 in 2 ml of 10% FBS= ot s BME were inoculated in a dish (8 cm2). After 14-16 hr, the medium was replaced by culture medium containing 10% serum from various ages, and then the cell number was counted on the 3- and 7-day. Serum from young rabbit (8 mo) stimulated proliferation of its skin fibroblasts as same as FBS, however, those of old rabbit (5-8 yr) demonstrated more stimulative ef- fect on the proliferation compared with those of the young. The same was also shown using human fetal lung fibroblasts (TIG-1l). Though the serum effects of rat (2-29 mo) on proliferation of its fibroblasts were measured, no activity changed during life- time. We confirmed that no inhibition to cell proliferation by sera from old rabbit and rat was performed. CB 43 HUMAN FIBROBLASTS CONDITIONED MEDIA CON- TAIN GROWTH STIMULATING ACTIVITIES FOR CELLS IN SERUM-FREE CONDITION, -AN AUTOCRINE GROWTH CONTROL- K. Kaji and M. Matsuo. Tokyo Metropolitan Institute of Gerontology, Tokyo. Previously, we developed a method for the growth of normal human fetal lung fibroblast, TIG=3 cells in serum-free media supplemented with peptide growth factors. This sensitive culture system have used to demonstrate that cells derived from human skin and lung release soluble factors which can stimulate fibro- blast growth. In MCDB-104 supplemented with insulin, transferrin, dexamethasone, epidermal growth factor and platelet- derived growth factor, TIG=-3 cells doubled 2 to 2.5 times in 72 hr. Quiescent human fibroblasts from embryos and donors ages 2i-yr-old to 77-yr-old were exposed tc MCDB-104 for 72 hr. Addition of 50% conditioned media collected from all of the fibroblasts including TIG-3 cells to the culture medium increased TIG-3 cells growth 25-40% above control. Human fibroblasts produced the cell growth stimulating activities throughout their lifespan. On the other hand, bovine smooth muscle cells did not produce the growth stimulating activities for human fibroblasts. The activities » > non- dialyzable and heat unstabl: C, Smin). These data showed existing « ience for autocrine growth control by normal human fibroblasts. 896 CB 44 MOLECULAR INTACTNESS OF TRANSFERRIN RECYCL- ED IN CHICKEN MYOGENIC CELL CULTURE. *IT.Kimura, **T.Hasegawa and *E.Ozawa. *Div. of Cell Biol., National Center for Nervous, Mental and Muscular Disorders, Tokyo.**Lab. of Mol. Biol., National Institute of Health Bethesda, U.S.A. Evidence has been accumulating that trans- ferrin(Tf£), an Fe-binding glycoprotein, is a unique ligand in that it recycled intact to the extracellular medium instead of be- ing directed to lysosome like other ligands However, the molecular intactness of re- cycled Tf has not been satisfactorily dem- onstrated. In this study we examined the properties of chicken Tf molecules recycled in chicken myogenic cell culture to confirm the intactness. Neither a difference in the SDS-PAGE mobil- ity and isoelectric focusing pattern before and after recycling, nor a significant change in total Tf concentration in the culture medium was observed during culture. Additionally, although recycled Tf exerted no myotrophic activity in vitro, it re- stored the former activity when reloaded with Fe. Combined, these results provide further evidence of the molecular intact- ness of recycled Tf. CB 45 THE EFFECT OF OXYGEN TENSION ON THE ATTACHMENT AND THE MAINTENANCE OF HEPATOCYTES IN VITRO. K.Sato, K.Nagayoshi*, S.Kusunoki*, H.Namiki and Yosh zZatoxcr-1 | Depite! Lot abi Ole ans hemor Educ., Waseda Univ., Tokyo; *ADVANCE R&P €o., LID., Lite Sei., Lab.; Tokyo; **Devel— Ops Biole Labia, Depe. of PlasmsungmalSchs of Med., Kitasato Univ., Kanagawa. Adult rat hepatocytes, isolated by the collagenase perfusion technique, were wilicwuaec! iEeie wh To) 2 Weeks tin a 52 CO atmosphere with various partial pressures of oxygen (PO,), in order to study the effect of oxygén tension on the attachment and the maintenance of the cells. PO examined was 0, 124, 285, 357, 428, and 496 mmHg . Viable cell number at a PO, of 214 mmHg waS approximately equal to that in air during aculture of 10 days. Attachment and maintenance of cells in the PO. of 285, 357 and 428 mmHg were better than in air with the best results at a PO Oe 357 mmHg, where 3 times more cells Survived at day 10 as compared to those in air. But cells ata PO. of 499 mmHg were worse than in air, which indicates that extremely high level of oxygen is rather toxic for hepato- cytes. These resultes Cemonstrated that the attachment and the maintenance of hepato- cytes in culture were dependent on oxygen tension of a gas phase, suggesting that oxygen tension is one of the key factors to be controlled for obtaining reliable re- Sults in the studies with cultured hepato- cytes. Cell Biology, Genetics CB 46 ISOLATION OF TADPOLE TAIL MESENCHYMAL CELLS AND THEIR CHARACTERIZATION IN VITRO. K. Yoshizato and A. Nishikawa*. Dept. of Plastic Surg. and *Dept of Biochem., Scholl] o£ Med: Kitasato Una) wanmagelwelr A method for isolating mesenchymal from the tail of bullfrog tadpoles developed and the character of the was studied, focussing on the hormonal effects on them. Epidermis-free tail fins were obtained as reported previously and digested with dispase and then collagenase - hyaluronidase. Cells liberated by the two enzymes were passed through a gradient medium of Percoll and called dispase-cells (D-cells) and collagenase cells (C-cells), respectively. D and C cells contained macrophage-like cells (M) and fibroblastic cells (F). M were rich in D cells and F in C cells. (1) M: The shape was similar to that of the peritoneal macrophages of guinea pig on plastic dishes and looked like a chestnut with burs on collagen fibers. The cells fused each other as a period of culture lenthened and became a giant cell. They survived in vitro at least for 20 days. Thyroid hormones did not affect their survival. (2) F: F could be maintained in vitro for 20 days in the thyroid hormone- deprived medium. The span of life shortend in the presence of the hormone in the dose- dependent mgnner, with a minimum effective dose at 10 “M. cells was cells GE 1 REDUCTION OF GERMINAL CHROMOSOMES IN TETRAHYMENA THERMOPHILA K.Ohmori and T.Sugai. Dept. Biol., Ibaraki UiNaLWo g IMELEO > Tetrahymena cell has a diploid germinal micronucleus (2n=10) but cell with haploid micronucleus has been recently isolated. Since haploid micronucleus as well as di- ploid micronucleus produces four meiotic products, we expect that those meiotic pro- ducts are highly aneuploid. The fertiliza- tion and later nuclear changes were cancel- ed by two methods; (1)crossing haploid to special strain (A*) which induces genomic exclusion, (2)detachment of conjugating pair. We have established clones with small micronuclei and examined the chromosome constitution of those clones at meiotic MmBcchoinelss IL Cv) (269 elomas)). Following results were obtained: (a)Those clones were confirmed to be aneuploid (n-1,n-2,n-3, 2n-2,2n-2-2 and 2n-2-2-2) except 11 clones. (b)Using telocentric-type no.5 chromosome as a marker, it was found that no.5 chrome— some never existed in n x A*® exconjugant. (c)There were clones with bivalent chromo- somes at MI(Cabout 50%). It is probable that univalent chromosomes endoreduplicated during clonal growth. By physically cutting the micronucleus at the stage of micronuclear extension (pachytene), we have isolated clones with reduced germinal chromosomes. Genetics 897 GE 2 PHENOTYPIC EXPRESSION AFTER CONJUGATION OF PARAMECIUM K. Mikami. Dept. of Biol., Miyagi Coll. of Educ., Sendai. Cells of Paramecium caudatum have germinal micronuclei (MIC) and somatic macronuclei (MAC). The MAC largely con- trols the phenotype of the cell and during sexual process it differentiates from the MIC. In order to to know when genes which derived from the MIC express, phenotypes of behavior CNR and trichocyst TND were investigated at 27+1°C after con- jJugation. To exclude affects of pre- zygotic MAC, heterokaryon clones were used; one of the heterokaryons, Het-I, had the MIC (cnrAt/cnrAt) of stock Yd and the MAC (cnrA/cnrA) of stock 16A380, and the other, Het-II, had the MIC (tnd-2+/tnd-2+) of stock Yd and the MAC (tnd-2/tnd-2)of stock Yg2VI. When Het-I conjugated with stock 16A1002 (cnrA/cnrA) and was fed soon after conjugation, wild type (avoiding reaction) began to appear mainly 40-45 hours after the critical stage of MAC differentiation. In the conjugation of Het-II with stock Yg2VI (tnd-2/tnd-2), the wild type (dis- charge of trichocysts) began to appear Mainly 38-45 hours after the critical stage. The expression was not closely associated with cell division after conju- gation. The results indicate that MAC anlagen produce genetic information by the age of 40 hours after the critical stage of MAC differentiation. GE 3 TRICHOCYST NON-DISCHARGE (TND) MUTANTS IN PARAMECIUM CAUDATUM.III. CHARACTORS OF THE DOUBLE TND MUTANTS. T.Watanabe, Biol. Inst., Tohoku Univ., Sendai. The trichocyst, a secretory organelle of Paramecium, provides a good model sys- tem for genetic analysis of exocytosis in eukaryotic cells. In P.caudatum, two loci of trichocyst non-discharge mutants (tndl and tnd2) have been known. In the present study, I obtained several double TND mu- tants (tndl/tndl-tnd2/tnd2) in the F2 pro- geny of the cross between TND1 and TND2, and characterized morphologically. Freeze fracture electron microscopy of the cell membrane revealed "parenthesis" shaped arrays of membrane intercalated particles at trichocyst attachment sites. The "pa- renthesis" shaped array indicate that the site is not occupied by any trichocyst ( Beisson et al.1976). Phase contrast mi- croscopy showed that the double TND mutant contain greatly reduced number of tricho- cysts. These trichocysts are mainly local- ized in the cytoplasm, and only a few exist in the cortex. TEM observation con- firmed mature trichocysts are arrested in the cytoplasm, but it remained uncertain whether the cortical trichocyst really at- tached to cell membrane or not. Upon squashing cells of double TNDs, no tricho- cyst is stretched. Thus, simultaneous mu- tation in the two loci resulted stronger inhibition of the trichocyst exocytosis. GE 4 MATING-TYPE DETERMINATION OF MALTIPLEX EXCONJUGANTS IN PARAMECIUM TETRAURELIA. 1§.Someya and ’S.KoOizumi. Inst. of Biol., Univ. of Tsukuba, Ibaraki and*Dept. of Biol., Miyagi Coll. of Educ., Sendai. Mating-type determination of P.tetraure- lia possessing two complementary types, _ O and E, is under cytoplasmic control when the new macronuclei are formed in each cell. In the present study, the qu- antitative effect of E cytoplasm in which the presence of E determining factor has been confirmed, was examined by using cy- toplasmically fused exconjugants. Animals used here were cells of stock d4-184, stock 51 killer and 51 sensitive (kappa free). Maltiplex exconjugants were obtai- ned by treatment with trypsin, 0.25% for lh, at a stage of 4h after mixing. The frequency of E produced by doublets from crosses between E and O singlets was 803%, and 47% by triplets from crosses between E singlets and O doublets. The Evidence for cytoplasmic exchange during the pro- cess of maltiplex formation was obtained by observing the transfer of kappa parti- cles in crosses between cells of 51 ki- ller and 51 sensitives: more than 50% of sensitive cells received particles by 8 h after the initiation of conjugation; par- ticles were uniformly distributed in both cells by 1l h. From these results, it seems that mating-type is determined to- wards E by the presence of 2/3 E cytopla- sm in maltiplex cells. GE 5 THE ODD FACTOR THAT FIXES MACRONUCLEAR MATING-TYPE DETERMINATION IN PARAMECIUM TETRAURELIA. S.Kobayashi and S.Koizumi. Dept. of Biol., Miyagi Coll. of Educ., Sendai. As a first step towards characteriza- tion of this factor produced by the young O macronucleus, whole or a part of macro- nuclear anlagen at the various developing stages were transplanted from donors of mating-type O into recipients of type E during the process of autogamy. Donors and recipients were cells of stock d4-186. Transplantation of O anlagen at a stage of 10 h after the first zygotic division into E autogamous cells produced O clones for the first time; the frequency of transfor- mation reached as high as 80%. The activi- ty of O factor was maintained by 21 h, be- gining of the third cell cycle. To examine the sensitive period of E anlagen operated by O factor, O karyoplasm from cells in the second cell cycle were transplanted into E autogamous cells. More than 90% of E clones shifted to O, when recipient cells were before the macronuclear differ- entiation. This high frequency of trans- formation was obtained by 7-8 h after the first zygotic division. These results indicate that (1) the O determining factor become functional by 10 h after the first zygotic division and maintained its activity just after the seco cell cycle and that (2) O factor acts developing anlagen of E cells during the first cell cycle. 898 Genetics GE 6 RELATIONSHIPS OF THE MEMBERS OF THE DROSOPHILA BIPECTINATA COMPLEX AS DETERMINED BY COMPARISONS OF POLYTENE CHROMOSOMES. Yo Tomimurasend You Na lobari ss Dept. Biol., Tokyo Metropolitan Univ., Tokyo. The 4 species, including 2 subspecies, of the Drosophila bipectinata complex (bipectinata, parabipectinata, malerkotliana and pseudoananassae) reveal a total of 59 arrangements of polytene chromosomes. Two of these arrangements are common to bipectinata and parabipectinata, and one is common to bipectinata and malerkotliana. MThis indicates close phylogenetic relation- ships among these three species. In contrast, D. pseudoananassae shares no arrangements with the other three species in addition to poor synapsis of polytene chromosomes in the interspecific hybrids. This suggests that pseudoananassae is most distant phylogenetically to the other members of the bipectinata complex. Many common gene arrangements are found at subspecies level. The two Subspecies of pseudoananassae share 10 of 15 arrangements and the 2 subspecies of malerkotliana share 8 of 14 arrangements. GE 7 GEOGRAPHICAL VARIATION IN THE REPRODUCTIVE DIAPAUSE OF DROSOPHILA BIFASCIATA. No lechiso, Zooiministh LacwiscimmHioecaado Univ., Sapporo. Geographical variation of photoperiodi- sm was studied in the reproductive dia- pause of Drosophila bifasciata. All strains from Hokkaidd(the northern island in Japan) and the northern part of Honshu (the main island in Japan) showed long- day photoperiodic response. The critical photoperiod(C.P.) significantly increased northward in the northern part of Honshi, but did not in HokkaidS. Furthermore, C. P, of the strain from northernmost part of Honshu was shorter than that of the strain from southernmost part Hokkaidd although latitude and climate do not differ between these localities. This difference of C.P. seems to be due to the geographical barri- er to gene flow because HokkaidSd and Hon- shi were separated from each other by Tsu- garu Strait which width seems very effec- tive geographical barrier to gene flow for drosophila. GE 8 EXTRACHROMOSOMAL FACTOR AFFECTING THE AC- TIVITY LEVEL OF GLUCOSE-6-P DEHYDROGENASE IN Drosophila melanogaster. S.H.Hori. Dept.of Zool, hac.of |SicieHokeae do University, Sapporo. We have previously reported that the major regulatory factor responsible for an increase of G6PD in D. melanogaster appears not to be linked to any of the chromosomes, but to reside in the nucleus as an extra- chromosomal replicon, and that the factor could occasionally be inserted and excised out of the X chromosome ( Jpn.J.Genet. 58: SME SOI » USS) o To corroborate these findings we re- peated similar experiments using flies re- cently collected from a natural population. The following reproducible results were obtained: (1) flies responded rapidly to artificial selection for high and low G6PD activity, but the high-activity trait can not be maintained without selection; (2) the trait was not X-linked in general, but it turned out to be X-linked during the course of continuous selection at an unex- pectedly high rate of incidence; (3) the X-linked factor appeared to be excised out of the chromosome upon outcrossing; (4)the factor had correlated effects on two other X-linked genes, Pgd and Had, but not on Gpt, r and Fum. It is thus highly probable that there may be a mobile extrachromosomal factor in the nucleus which is capable of stimulating the G6PD activity. Gig KARYOTYPE STUDIES OF SIX SPECIES OF ANGUI- HLLFORMES CPILSCIES )) A. Takai and Y. Ojima. Dept or Biol eaer of Sci., Kwansei Gakuin Univ., Nishinomiya. Karyotypes of six species of Anguilliformes were studied. The results are shown as follows; Conger japonicus (2n=38, 10M+10SM +18A, NF=58), Alloconger anagoides (2n=34, 10M+8SM+6ST+10A, NF=52), Gymnothorax kidako (2n=36, 16M+8SM+12A, NF=60), G. eurostus (2n =42, 12M,SM+30A, NF=54), Muraena pardalis (2n= 42, 10M,SM+32A, NF=52), and Muraenesox cinereus (2n=38, 8M+8SM+22A, NF=54). In C. japonicus and A. anagoides, the 2nd largest submetacentric pair was slightly heteromorphic. This condition, which would be induced by pericentric inversion, was very similar to that of the ZW sex chromo- some complex in C.myriaster shown by Ojima and Ueda. Conspicuous heteromorphic pair, suggesting the XY type sex chromosome con- stitution, was found in G. eurostus. Karyotypic comparisons above mentioned indicate that the chromosomal differentia-— tion would occur by not only pericentric inversion and Robertsonian translocation but also tandem translocation. Also, C-band and NOR distributions on the chromosomes of three muraenids were comparatively studied, and they seemed to have close relationships to the species differentiation. Furthermore the problem on phyletic evo- lution was discussed on the basis of cyto- genetic findings. Genetics 899 GE 10 CHROMOSOMAL DIFFERENTIATIONS IN APOGONIDAE (PERCIDA, PISCES). Y. Ojima, T. Kojima and T. Takai. Dept of Bigt._, Fac. Of Sci., Kwansei Gakuin Univ., Nishinomiya. Apogonidae fishes are widely distributed in coral reef and approximately 200 spe- cies have been known in the world. In Jap- an 29 species were noted up to the present. This kind of fish is well known as the mouth-breeder. Four species, Apogon orbi- cularis, A. semilineatus, A. doederleini and A. motatus were used for the karyolo- gical study. All these showed 46 chromo- somes in diploid. The chromosomal consti- tutions were as follows: A. orbicularis; 4SM + 42ST-A, NF=50, A. semilineatus; 2M + 6SM + 38ST-A, NF=54, A. doederleini; 2M + 6SM + 38ST-A, NF=54, A. notatus; 2M + 4SM + 40ST-A, NF=52. Noticeable is the appea- rance of the hetero- and homomorphic pair of chromosomes in both male and female. The heteromorphic chromosome would be in- duced by the deletion or translocation mechanism through the course of evolution. Sometimes these chromosomal features apt to make misunderstanding as the sex chro- mosomal constitutions such as XX-XY or ZW- ZZ. According to the Fish Chromosome Data Retreaval List by Ojima (1983), XX-XY, XX- XO, X)X)X2X9-X)X] Xo, X,X1X2XQ-X1X2¥Y, XX- XY)Y2 in male and ZW-ZZ, Z0-ZZ, ZW W2-22 in female have been reported in 72 species of fishes with sex chromosomes in the wor- ld. GE 11 ALBINOS IN THE MEDAKA(ORYZIAS LATIPES) H.Tomita. Lab. of Freshwater Fish Stock, Fac. of Sci., Nagoya Univ.. Two albino genes(i and i-3), recessive and autosomal, were found in the medaka. The albinos have pleiotropic effects on chromatophores, melanophores(no melanin), xanthophores (dilute color) and leucophores (increase in number). The two albinos are the same phenotype and can be distin- guished by test crosses. The i alleles are multiple (+>i>i). The color of the BibR type is variegated black at the embryonic and larval stages, and becomes to black at 10 mm body length. At the embryonic and larval stages, the i albino shows no tyrosinase reaction and the i-3 albino has weak tyrosinase in the histochemical tests. The offsprings of crosses between the i albino and the i-3 albino were all non- albino. The F., offsprings were segre- gated to non- -albino and albino in a ratio Or 9:7. The F., albino larvae were di- vided to tyrosifiase positive and negative in a ratio of 3:4. The results show that the i alleles are independent to the i-3 alleles and the i allele is epstatic to the i-3 allele. ~— The i albino was mated to the mutants (Da,df-1,df-2, £1,gu,1f,m1-3, pl and wl) and the i-3 albino to Da,dl, fm, gu,lf, ml- -3,pl and wl. No linkage g groups were found in the test crosses. GE 12 MORPHOLOGICAL VARIATION OF NO.10 CHROMO- SOME IN 9 POND TYPE HYNOBIUS FROM KOREA AND JAPAN. Cr Ikebe amide 8 Kuro-o “, R. Katakura Ss. Kusada- 7. and Ss Kohno’ . I: Dept. of Biol., Facul. of Gen. Educ.,.. 2: Dept. of Biol.,;, Bacul. Of.Ssci., TOho Univ., Chiba. 2 Chromosomes of east Asian salamanders: Hynobius leechii from Korea, and H. tsuensis, H. nebulosus, H. dunni, H. takedai, H. tokyoensis, H. lichenatus, H. nigrescens and H. retardatus from Japan were studied by the methods of conven- tional Giemsa staining and C-banding. The results of the study are as follows. 1). No. 10 chromosome in the 7 species from Japan, which is equivalent to No. 1l chromosome in H. retardatus, shows inter- and intra-species variation in its morpho- logy. In some instances, different species living in close localities possess the same type of No. 10 chromosome. The morphological changes of No. 10 chromosome would not change the taxonomic characteri- zation of these species, but they seem to be showing the process of the differenti- ation of these species, which should be valuable material for elucidating thea process. 2). A karyotype of H. leechii from Korea is similar to that of Japanese Hynobius from Kyushu (H. nebulosus and H. dunni ) except for No. 10 chromosome. The morphological type of No. 10 in H. leechii is similar to that of No. 11 chromosome in H. retardatus. GE 13 CHROMOSOMAL VARIATION OBSERVED IN A BREEDING COLONY OF TURKISH HAMSTERS. T.Shimada. Dept. of Hygiene and Preventive Medicine, Yamagata Univ. Sch. of Med., Yamagata. It has been known that the Turkish hamster (Mesocricetus brandti) from Malya has 42 chromosomes with two pairs of acrocentric chromosomes and the Turkish hamster from Ankara has 44 chromosomes with three pairs of acrocentric chromosomes. A total of 119 individuals of Turkish hamsters were analyzed for karyotypic variants by G-banding chromosomes. These chromosome analyses of our breeding colony which is supposed to be from the _ stock colony _of Malya hamsters revealed that they apparently possessed karyotypic features of the Ankara hamster with the chromosome number of 44, Besides, considerable heteromorphic variations in acrocentric chromosomes # 19 were observed. Further, some animals of the colony possessed only 43 chromosomes. Diplotene meiotic figures of these animals showed one chain and 18 bivalents including a pair of sex chromosomes. This suggests that one of chromosomes # 12 must have been translocated to yet undetermined chromosomes. Crossing between these animals produced offspring with the chromosome number of 44, 43 and 42. 900 Genetics, Immunology GE 14 ISOZYMES OF MOUSE TYROSINASE. H. Yamamoto and T. Takeuchi. Department of Biological Science, Tohoku University, Kawauchi, Sendai 980, Japan. Although three isozymes of mouse ty- rosinase (11 ,l2,13) have been well studied by using conventional PAGE (Davis, '64), little is known about their isoelectric points. ‘Recently anvour laboratory; epuni- fied tyrosinase from mouse melanoma was found to be separated into several iso- Zynes oon LEP Rgedis es lhuse resul:GaaledmuSmeo study mouse tyrosinase from several mutants by using not only conventional PAGE but also IEF to compare their isozymes. Dorsal skin of ee ses MmakeS (CS /Bib,// 6J-a/a black, C57BL/6A%/a lethal yellow, C57BL/6-e/e recessive yellow, BALB/c-c/¢ albino) was excised and solubilized with 2¢ Triton-X100. B16 mouse melanoma was also processed in the same way. The albino mice showed no tyrosinase activity. T2 isozymes was not detected in lethal yellow mice, whereas it was scarece- ly found in wWece sisal ve y.e low sm lCen assem nonagouti mice, broad tyrosinase bands were observed around at pH4.4, pH3.7-4.0 and pH3.4. Each of which consisted of several tyrosinase isozymes. It seems to be necessary to reestablish the nomenclature concerning tyrosinase isozymes. GE 15 INDUCTION OF EUMELANIN FORMATION IN THE MELANOCYTES OF GENETICALLY YELLOW MICE. T. Takeuchi and H. Yamamoto Vee BIOS SCtiscs VOhoku Uniwes Sencad Mouse melanocytes produce two kinds of melanins, eumelanin and pheomelanin. The shift in the pathways of the two melanins 1s controlled by the a (agouti) locus and the e (extension) locus. We have previous- ly shown by using organ culture that eu- melanin formation can be induced by a-MSH in the melanocytes of AY /a lethal yellow) mouse. We, however, failed to induce eu- melanin formation by a-MSH in the melano- cytes of e/e (recessive yellow) mouse. We then proposed that a-MSH competes with the product of the a locus at the a-MSH recep- tor site and that the e locus controls a mechanism that determines the functiona- bility of the a-MSH receptor. In order to verify our hypothesis, we examined the effects of forskolin, a potent activator of adenylate cyclase in membrane, on the melanocytes of the yellow mice. Skin explants from lethal yellow and recessive yellow mice were organ-cultured FOr 2 days_an thg medium containing for- Skolin (10 ~-10 "M). Significant eumelanin formation was observed in the melanocytes of A~/a hair follicles whereas melanocytes of e/e hair follicles contained exclusive- ly pheomelanin. It seems likely that e/e melanocytes possess a deficiency in a-MSH receptor-adenylate cyclase system. IM 1 ANALYSES OF TOLERANT STATE INDUCED BY INJECTION OF BLASTOMERES INTO GASTRULAE OF XENOPUS LAEVIS. IGMERLMOraC Oi. Kawagiwis ZOOL. las, Fac. of Science, Hokkaido Univ., Sapporo. Stage 10 Xenopus laevis (wild type) gastrulae were injected into the blasto-— coel with 100-250 dissociated blastomeres from the presumptive ectodermal area of stage-matched J strain gastrulae, to study their alloimmune reactivities at 2-12 month-old adult stages. Their responses to injection-donor in term of grafted skins (SKR) and one-way mixed leukocyte reaction (MLR) were either negative or positive in both SKR and MLR, or positive only in MLR, although both reactions ive the third party india vadualissiwemewapial: positive. IPEVOU IN Os ainjOCwocl COlIlsS vsinge diploid-triploid or borealis-laevis marker systems revealed that the donor cells were involved in all germ layers in 1-3 days post-injection, differentiated into hemo- poietic cells in variable degrees (0-312) in the frogs younger than 3 months old, but were scarcely found in hemopoietic GOlLILS av | year old rrogs. We suggest that the maintenance or breakdown of tolerant state is closely related to the degree of hemopoietic chimerisn. ili Z GRAFT-VERSUS-HOST DISEASE (GVHD) IN XENOPUS LAEVIS. HUST TINEMCEH DIT GOOG Mins555 WeElGo O# Sis; Hokkaido Univ., Sapporo. The (KxJ)F1 frogs were produced by mating of gynogenetically produced "K" strain with "J" strain. When the F1 hybrid frogs were injected with a high dose (3.2x10’ cells/body) of spleen cells from parental J, the hosts were dead due to the wasting and a local destruction of the skin. The symptoms always included a prominent splenomegaly, together with an involution of the thymus, color change of the liver, and vasodilatation, hemostasis and hemorrhage in various degrees throughout the body. Histologically, the Symptoms were associated with the systemic cellular infiltration and pigmental destruction, and a focal necrosis of lymphoid organs such as spleen, liver and kidney. The induction of these symptoms was specifically accelerated when the parental donors had been sensitized either by skin grafts (in vivo) or Y-irradiated leukocytes (in vitro) from F1 hybrids. These observations provide the first demonstration in poikilothermal vertebrates of the GVH reaction, as a function of T lymphocytes that is controlled by a major histocompatibility complex. Immunology 901 IM 3 ISOLATION AND CHARACTERIZATION OF THE FOURTH COMPONENT OF COMPLEMENT IN THE SERUM OF THE CLAWED FROG, XENOPUS LAEVIS. T. Fujii, A. Sekizawa and C. Katagiri. Zoot. iInst., Fac. Sci., Hokkaido Univ., Sapporo. The serum protein exhibiting a strong hemolytic activity against SRBC which had been sensitized with Xenopus antibodies was purified as a single entity from the Xenopus plasma following PEG precipita- tion, ion-exchange chromatographies and gel-filtrations. The protein required other components of the Xenopus serum to lyse sensitized SRBC, but failed to lyse unsensitized RRBC through alternate pathway. The protein, contained at 0.43 mg/ml in normal serum, showed an electro- phoretic mobility of B-globulin, with a mew. Of 202K comprising 3 distinct sub- units of 96K, 76K, and 26K which are linked by disulfide bonds. Digestion of 202K protein by trypsin resulted in a specific cleavage of the 96K-subunit and a conversion of its immunoelectrophoretic mobility to the anodal side, leaving the 76K- and 26K-subunits intact. The treat- ment with SDS/urea resulted in the split- ting of 96K-subunits into 48K and 45K, but this splitting was inhibited upon pre- treatment with methylamine, suggesting the presence of a thiol ester bond in the 96K- subunit. In all aspects, 202K protein is regarded as the C4 that play an essential role in the classical-hemolytic pathway. IM 4 CELL SURFACE MARKER FOR THYMUS-DERIVED CELLS IN XENOPUS LAEVIS THAT IS IDENTIFIED BY MONOCLONAL ANTIBODY. S. Nagata, Tokyo Metropol. Inst. Gerontol., Tokyo A monoclonal antibody, XT-1 (IgG,,X%), was prepared against J strain Xenopus laevis thymocytes and the expression of antigens recognized by this antibody (XT-1 antigens) was investigated by immuno- fluorescence method. XT-1 antibodies reacted with 93%-97% thymocytes and 28%- 42% spleen cells from intact J strain toads, as analysed by flow cytofluoro- metry. Early larval thymectomy depleted XT-1* cells in the splenic lympocyte popu- lation to less than 3%. Double immuno- fluorescence experiment demonstrated that there are 3 populations of lymphocy- tes in spleen, i.e., surface IgM (sIgM)*XT-1-, sIgM~XT-1* and sIgM"XT=1- - cells. The depletion of XT-] ells, however, did not abrogate proliferative responses of spleen cells to T cell mito- gens. Thus, both XT-1* and XT-1~ T cells would be present in the peripheral T cell population. The family study suggests the allelic polymorphism of XT-1 antigen mole- cules expressed on peripheral T cells. The surface antigen identified by the pre- sent XT-1 monoclonal antibody may provide an important marker for cells in T cell lineage in Xenopus laevis. IM 5 EFFECTS OF THYMECTOMY AND SPLENECTOMY ON THE ALLOGRAFT REJECTION IN ORYZIAS LATIPES. S. Kikuchi and S. Kambayashi. Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba. Allograft rejection was examined in the thymectomized and splenectomized fish, Oryzias latipes, at 25°C. The recipients of non-pigmented varieties were removed the thymus or the spleen. Scales from pigmented dorsal region of donor fish of wild-type were used as allografts both in the first- and in the second-set transplantation. Three scales were grafted into ventral side of each recipient. The first-set transplan- tation was performed just after the surgi- cal operation and the second-set was per- formed 15 days after the first-set. The de- struction of melanophores in the trans- planted scales was used as the indicator of rejection. Duration of survival of allo- grafts was about 5 days in the first-set and about 3 days in the second-set. No sig- nificant difference was observed between the experimental and the sham-operated con- trol. Thymic dependency of allograft rejec- tion was evidenced by allogeneic transplan- tation in developing fry of rainbow trout (Tatner and Manning, 1983) and by shielding experiment in x-irradiatedgoldfish (Desvaux and Charlemagne, 1983). The present re- sults, however, suggest that the thymus does not necessarily play an essential role in allograft rejection in Oryzias latipes. Splenectomy also did not affect survival period of the allografts. IM 6 BONE MARROW RECONSTITUTION OF ERYTHROPOIESIS AND IMMUNE RESPONSE FOLLOWING y-IRRADIATION IN THE MICE, T.A. Nomaguchi, K. Yamamoto, Y. Sakurai and M. Yamamoto. Dept. of Biol., Tokyo Metropol. Inst. of Gerontol., Tokyo. ~ The bone marrow(BM) is important source of cells capable of maintaining individual viavility, renewing erythrocytes and produc- ing plaque forming cells(PFC). We examined BM reconstitution on haemato-lymphopoiesis during lifetime in the mice. The SPF male mice in various month-olds, C57BL/6NCrj, were irradiated 1000R of y-rays and transplanted with BM cells of 5x10° in young or olddonors. Erythropoiesis was meas- ured with hematocrit and cell electrophbresis. PFC was determined at 2 months after treat- ment against sheep erythrocytes. The mice receivingirradiation all died before the 2nd week, but recipients which were transplanted with BM cells showed lifetime as likeas control. Radiation destroys ery- thropoiesis, but hematocrit of the recipients recovered at 2 weeks to control level. Though electrophoretic mobility of erythrocytes in- creased with growth and decreased with aging, those of the recipients in various ages kept the young level. Both PFC values of young re- cipients transplanted with BM cells from young (1 m.) and aged(22 m.) donors increased higher than control, but those of the adult recipi- ents(7m.) showed lower level. Recipients, except the aged(25m.), transplanted thymo- cytes of 5x10° before a week of immunization reached higher PFC levels than the others. 90? Immunology, Biochemistry IM 7 THE CHANGE OF GRANULES IN DENDRITIC RETI- CULAR CELLS THAT ARE CERVICAL LYMPH NODE IN MICE WITH DELAYED HYPERSENSITIVITY. S.Tanaka, H.Uda and T.Maruyama. Dept. of Path. Kagawa Med. School, Kagawa. BALB/c mice (8) aged 8 weeks were im- munized their ventral skin with picryl- chloride. A week after the sensitization they were painted with picrylchloride on their earlobes. The earthickness of the animals was measured after 24 hours, and their cervical lymph nodes were removed for examination. Ears and lymph nodes were fixed and embedded in the usual manner for electron microscopy. The dermis of the ear with allergy in mice was thickened and we could see lymphocytes and dendritic reticular cells (DRC) in it. The birbeck granule contain- ing cells decreased and the cored tubule containing cells increased in cervical lymph nodes. According to the results, it seems birbeck granules or cored tubules in the DRC have some role of recognition to an antigen or sending information. IM 8 Serological Survey of T-lymphocyte dif- ferentiation antigens in wild mice. Y.Kurihara and S.Kohno(Dep. of Pmysio— Chena Hace OL SCs Ulin Or ono) mang K.Moriwaki(Dept. of Cytogenetics, Natl. Inst. of Genetics, Mishima) We have surveyed geographical dis- tributions of Thy-1, Lyt-1 and Lyt-2 alleles in wild mice using serological methods. European subspecies, Mus muscu- lus domesticus and M.m.brevirostris, exhibited Thy-1.2 antigen, but Asian ones mostly Thy-1.1. Lyt-2.2 was unique to some European mice, and Lyt-2.1 was widely distributed both in Asian and European mice. Mus musculus subspecies from wild populations examined so far expressed only Lyt-1.2. We also surveyed two closely related species, Mus spretus and ee Though their lLympho- cytic phenotypes were similar to those of Asian mice, part of (SSE ets express-— ed Lyt-1.1 that colud not be found in the survey of M.musculus. Certain Asian mice related to M.m.- castaneus had weak reactivities to anti- Lyt-1.2 and anti-Lyt-2.1 monoclonal anti- bodies by complement-mediated cyto- toxicity test, which was confirmed by absorption test as well. At present, we can not decide whether the weak reacti- vities resulted from the less amount of expression or from the little change of the antigenic molecules on cell surface. Iie ANALYSIS OF THE LOCAL IMMUNE RESPONSES ELICITED IN THE UTERUS DURING EARLY STAGES OF IMPLANTATION IN MAMMALS. II. DISTRIBUTION OF IMMUNOCYTES IN THE MOUSE UTERUS. C. Tachi. Zoological Institute, Faculty of Science, Uni- verrsity of Tokyo, Bunkyo-ku, Tokyo. Crucial gap of knowledge exists concerning the chain of immunological reactions elicited locally in the endometrium during early period of implantation. We analyzed the dis- tribution of macrophages in the uterus during peri-nidatory period in the rat; the results strongly indicated that the decidua may serve as an immunological barrier preventing the access of Macrophages to the embryos during early period of implantation( C. Tachi et EWlgn Wo Iraq Hoolsan Ziv psis 198i ))- However, other immunocytes will be doubtlessly involved in the local endometrial responses to the implanting embryos. AS an approach to the problem, we analyzed the distribution of immunocytes as revealed by immuno- histochemistry of membrane-bound immuno- glubulins, such as IgG, IgE and IgA, in the mouse uterus during peri-nidatory period. The results demonstrated that the decidua may serve as an _ immunol- ogical barrier toward wide range of immunocytes, thus restricting the flow of immunological information from the embryo to the maternal immune system. gt It A NEW STRUCTURAL PROTEIN FROM ASCIDIAN SMOOTH MUSCLE M. Shirakata, T. Takagi & K. Konishi, BiO@l, ImSie., Bac. Sei., WOnoOkw Wmiw., Sendai We have purified 29K dalton protein from ascidian smooth muscle. Crude extracts from washed myofibrils with a solution containing ATP and EGTA at low lonic strength were purified by chroma- tography on DEAE- and CM-cellulose in the presence of urea. The purified protein showed a single band by SDS-PAGE Wakil Gl ilove C2I,OOO, Wae yaleilcl OIE jowiei= fied 29K protein was 40 mg from 160 g of muscle. After dialysis of 29K protein aeinsie O53 WM Wacl, 10 mul Wels, joa 7.5 at room temperature, the filaments with a diameter of 10-15 nm were obser- ved by electron microscopy. Occasionally this filament assembled laterally and became large bundles. The filaments of 29K protein were resemble to those of desmin which is known as the interme- diate filament protein of vertebrate smooth muscle cells. 29k protein was also extracted with urea from high con- centration of KCI-KI insoluble mate- EES COE MHVOIE/oien Sq This method is generally used for the extraction of desmin. From these results 29K protein seems to be one of the components of intermediate filaments of ascidian smooth muscle cells. Biochemistry 903 BI 2 AMINO ACID SEQUENCE OF 29K _ PROTEIN OBTAINED FROM ASCIDIAN BODY WALL MUSCLE H. Yasunaga, T. Takagi & K. Konishi, Peat. inSt.,. Sac. -Sci., Tohoku. Univ.., Sendai Desmin or vimentin is known as a component of intermediate filament of vertebrate smooth muscle cells and its molecular weight is more than 50,000. The 29K protein obtained from ascidian (Halocynthia roretzi) body wall muscle is smaller than desmin or vimentin, however, it is thought to be one of the components of intermediate filament as shown by Shirakata et al. in the prece- ding presentation (lI 0900). The partial amino acid sequence of 29K protein has been determined by using its CNBr pep- tides and tryptic peptides of citra- conylated protein. The N-terminus of 29K protein was blocked and the N-termi- mal sequence was S LMPFODODF Y/Y P Y QL. The C-terminal sequence, ca. 145 residues, was determined as following: Beeson ©: DEL VR.K VD RR FEF T GM See DOPE PFA R-ON TS V-E. EB Seok Th ODF NM K VD VQ Paes Vv KV OIG GOVE V HA KR Meme Mm rOAY SC oS &F KR A-F IT G:P Eee SAC: Robt S| Sl. Sv. RCLO -res..)T fT V V V (15 res.)-OH The sequence homology with desmin or vimentin is not so high. BI 3 THE AMINO ACID SEQUENCE OF ASCIDIAN MYOSIN LIGHT CHAINS T. Takagi, S. Kudoh & K. Konishi, Biol. isc. , rac, Sci.,;, Tohoku Univ: , Sendai The amino acid sequences of two myosin light chains of ascidian (Halocy- nthia roretzi) body wall muscle were determined as following: Regulatory light chain ATS NV OS Mer Dp Ors Ort OE Fr K- EA Pema eee UR DG F x°D'O EB DL KD T Pore ae na Gt KD EB RL KR DP Mb ASB S ae wow fr eO rr b ChE GD-K’ Lb S GT wees Soe | bE AY KR i) b DA DW R GV mao wos b> A oS te Mh EY OA DR F S O'S Hewee oom F D Li-S° Pe iD VA GOON L D-Y¥ K momee © tt THGOQOBEE Essential light chain Seer Ss » DR" TE COS A ETE EB LF. D'R San GKVF:BGQOVGDILRAL GQ BE TNGDVTKVLGWNPPREBEELA mney o Yr S*E Fb PM GL AQ iE ROT mimes k & DEV EG L RV F DR E ND GRASBGAELRAVL STL GERMS Pops Vb eo be Oo GO PTD PN GC i 8 YEESBFSKYLLBEG Regulatory light chain is composed of 153 amino acid residues, has one EF-hand type calcium-binding site and its N- terinus is not blocked. On the other hand essential light chain is composed of 151 amino acid residues and its N- terminus is blocked. BI 4 COFILIN, A PROTEIN IN PORCINE BRAIN THAT BINDS TO ACTIN FILAMENTS AND INHIBITS THEIR INTERACTIONS WITH MYOSIN AND TROPO- MYOSIN. E. Nishida, S. Maekawa, and H. Sakai. Dept. Biophys. & Biochem., Fac. Sci., Univ. of Tokyo, Tokyo Cofilin, a monomeric protein with a molecular weight of 21,000 has been purified from porcine brain by DNase I- agarose affinity chromatography, hydroxy- apatite and phosphocellulose chromato- graphies, and gel filtration on Sephadex G-75. Studies with N-(l-pyrenyl) iodoacet- amide-labeled actin showed that cofilin reacts stoichiometrically with actin ina 1:1 molar ratio. Cofilin increases the steady state monomer concentration in equilibrium with F-actin to a limited extent. The pelleting assay demonstrated that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament. Cofilin is also able to bind to monomeric actin, as demon- strated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. Cofilin blocks the binding of tropomyosin to actin filaments. Cofilin was found to inhibit the actin- activated myosin ATPase as well as the superprecipitation of the actin-myosin mixture. All these results suggest that cofilin is a new type of actin-associated protein. BI 5 EFFECTS OF CYTOCHALASIN D ON THE POLYMERIZATION OF ACTIN. A. Tashiro, Y. Inoue and K. Maruyama. Dept. Biol.,Fac. Sci.,Chiba Univ., Chiba Cytochalasins are well known to inhibit the elongation of an actin filament at the barbed end, and also to enhance the rate of polymerization of actin under certain conditions. The present work confirms and extends the previous studies with the action of cytochalasin D (cytD) on actin. The acceleration of actin polymeriza- tion was remarkable at 1-8 pM cytD in the presence of 75 mM KCl.When 4 pM actin was incubated with 6 pM cytD for 4.5 min, the number of formed actin filaments greatly increased, and the number average length was as short as 0.6 pm as compared with more than 1 of control. It appears that actin nucleates more effectively in the presence of cytD than in its absence. When actin polymerization was completed and reached the steady state, however, the the extent of polymerization was much lower than that of control, as judged by fluorescence measurements. The final state was the same when fully polymerized actin was further incubated with cytD for a sufficiently long time. The mechanism of the action of cytD on actin polymerization and G-F equilibrium is discussed on the basis of the present results and information available. 904 Biochemistry BI 6 C-PROTEIN-ACTIN INTERACTION IN THE PRESENCE OR THE ABSENCE OF TROPOMYOSIN. M. Higuchi and T. Obinata, Dept. of Biol., Insvon Ce Seals 5 Cowl Winshyo p Elaullog, It has been demonstrated that C-protein, one of the myosin-linked regulatory proteins in striated muscles, can bind to both purified myosin and actin and that C-protein may play some regulatory role in actin-myosin interaction (Moos et alley LITS, CLOTS eS) In this study, binding of C-protein to actin filaments with or without tropomyosin (TM) was examined by both centrifugation method and electron microscopy. Actin, C-protein and TM were obtained from rabbit skeletal muscle. MTM-containing actin filaments were prepared either by polymerizing actin in the presence of TM or by incubating F-actin with TM. Free TM was removed by centrifugation. Native thin filaments were directly separated from myofibrils. Tropomyosin-free F-actin could bind to C-protein to form filament bundles aS previously reported (Moos et al., 1978). However, binding of C-protein to TM-containing F-actin was not remarkable: the aggregation of neither reconstituted filaments nor native thin filaments by the addition of C-protein was not observed under an electron microscope and C-protein mostly stayed in the supernatant when the mixture of C-protein and the TM-containing actin filaments was centrifuged. BI 7 ASSEMBLY OF FITC-LABELLED ACTIN INTRODUCED INTO CULTURED SKELETAL MUSCLE MYOTUBES. ©. Saitoh and T. Obinata. Dept. cf BHol., lneiels @ie Seis, Goulloe Wino, Ciniloe In order to investigate how newly synthesized actin is assembled into myofibrillar structure in developing muscle cells, FITC-labelled actin was introduced to skeletal muscle myotubes which were cultured in vitro by a microinjection method and actin assembly was examined under a fluorescence microscope. Our observations are summarized as follows. 1) When FITC-actin was introduced into immature myotubes: 2 hrs after injection, FITC-actin was mostly detected as filamentous structures, but discontinuous distribution of actin with a constant periodicity along filamentous structures was also observed. One day after injection, FITC-actin was diffused into whole area of myotubes and incorporated into I-band region of myofibrils. 2) When FITC-actin was microinjected into myotubes having well-organized myofibrils, FITC-actin appeared as narrow bands near Z-lines shortly after injection, but in most cases, the bands with fluorescence became wider one day after injection. From these observations, it is suggested that FITC-actin could be transiently assembled to cytoplasmic filamentous structures in the beginning, but later localized in I-band region of myofibrils. BI 8 ON THE HETEROGENEITY AND TISSUE SPECIFICITY OF SCALLOP TROPOMYOSIN T.Ishimoda-Takagi, M.Kobayashi and M.Yaguchi. Dept. of Biol., Tokyo Gakugei Univ. Heterogeneity and tissue specificity of tropomyosin (TM) in the scallop, Patinopec- ten yessoensts, were investigated. TM was isolated from adductor striated and smooth muscles, heart, foot, mantle, intestine and gonad, after they were treated with trichlo- roacetic acid to inactivate the proteolytic enzymes involved in these muscle sources. Adductor striated muscle contained a single TM component in SDS-polyacrylamide gel elec-— trophoresis, whereas, adductor smooth muscle contained two TM components, one of which had the same mobility as that of striated muscle TM and the other had slightly lower mobility. As pI value of the former in iso- electric focusing was slightly higher than that of striated muscle TM, this TM mole- cule seemed to be different from that ob- served in striated muscle. Cardiac muscle contained a single TM component which was identical with a TM subunit with slightly lower mobility of smooth muscle TM subunits. Foot and gonad involved two TM components which were indistinguishable from smooth muscle TM subunits, and mantle contained all of three TM components. Further, we in- vestigated the antigenicity of these three TM components. As each TM component formed two precipitin lines with the antiserum, as many as six kinds of TM isoforms may exist in scallop muscles. Bile SARCOPLASMIC CALCIUM-BINDING FROM SCALLOP ADDUCTOR MUSCLE Y. Takasaki, K. Konishi, H. Yamamoto* & Ww, Wakeueink, I3ito@il, -linsit., Bac. SC. & Dept. Biol. Seis LohokumUninvermoomCl cel PROTEIN Sarcoplasmic Ca**-binding protein, SCP, was obtained from the adductor striated muscle of scallops. It was not found in the smooth muscle. Indirect immunofluorescent microscopy showed that SCP located on I band and H zone. We prepared two monoclonal antibodies, D-4 and D-5, against scallop BOR tee cross- reactivities with other -binding proteins were tested by ere: Both D-4 and D-5 strongly crossreacted with asci- GllElio ieielojXoinalin C, eiacl D=5 CieosSiceaccec! also with SDS-denatured scallop SCP and shrimp SCP beta chain, whereas D-4 did weakely with these proteins. These re- sults suggest that these antibodies recognize different epitopes. To deter- mine the epitopes of these antibodies, we tested reactivities of CNBr peptides (CN) of scallop SCP with these antibodi-— es by dot-immunobinding assay. Both antibodies reacted with all of CNBr pep- tides which we tested; the reaction with CNl was remarkg le. These CN peptides contain the Ca -binding sites. There- fore, these results suggest bere these antibodies recognize the Ca binding sites or the vicinities of them. Biochemistry 905 BI 10 ADENOSINETRIPHOSPHATASE ACTIVITY OF FRUITFLY AND CRICKET MYOSINS. M. Tanikawa, A. Ueyama and K. Maruyama. Dept. Biol.,Fac. Sci.,Chiba Univ.,Chiba. Myosins were purified from whole homo- genate of Drosophila melanogaster and thoracic muscle of Gryllus bimaculatus. SDS gel electrophoresis revealed that only a trace amount of actin was present in the myosin preparations. The calcium-activated ATPase activities of both myosins greatly decreased on stor- age an 0.6 M RCI at O0°C. Thus the activi- ty dropped to one fifth within 2 days after preparation. Rabbit skeletal muscle myosin ATPase activity did not appreciably change for 3 days' storage. The denatu- ration of insect myosins was fully protec- ted by the addition of rabbit actin. The specific ATPase activity of Drosophila myosin was as high as 1.5 oles P/mg/min immediately after preparation. The ATPase activity of Gryllus myosin was approximat-— ely 0.5 pmoles P/mg/min similar to that of rabbit myosin. The KCl-activated ATPase activity specific to myosin increased by about 30% during a few hours after emergence of adult fruitfly and remained constant thereafter. The enzyme activity was meas- ured in the presence of 10 mM EDTA and 1 M KCl, using a crude KCl extract of total homogenate of flies. BI 11 TROPONIN ISOFORMS IN CHICKEN STRIATED MUSCLES: ANALYSES BY TWO DIMENSIONAL GEL ELECTROPHORESIS. H. Abe and T. Obinata. Dept. of Biol., Fac. Ge oc1., Chiba Univ., Chiba. We established an electrophoretic condition to characterize multiple isoforms of troponin components, TN-T, TN-I and TN-C, in chicken striated muscles. Troponin-complex was separated from the other proteins by lst dimensional isoelectric focusing electrophoresis in the presence of 3 M urea at pH range 5-9, and then troponin cmponents were separated by 2nd dimensional SDS-PAGE. Troponin fraction, even crude preparation, was preferable for this electrophoresis to whole protein extracts. By this method, we were able to distinguish not only troponin components of fast-skeletal, Slow-skeletal and cardiac type, but also TN-T isoforms present in chicken fast skeletal muscles, namely breast TN-T, leg TN-T, and leg TN-T., (according to the terminology by Wilkinson Ot .alvs.», d984;).2 breast muscle contains only breast TN-T, leg muscle contains two classes of leg-type TN-T, and PLD muscle contains breast TN-T and leg TN-T.. Leg TN-T, may exist in only small amount in PLD. The expression of leg TN-T in neonatal, denervated and dystrophic breast muscles was observed by this method, but the types of leg TN-T expressed in these muscle differed from each other. BI 12 EXPRESSION OF EMBRYO-SPECIFIC MYOSIN LIGHT CHAIN (L 3) IN VARIOUS TISSUES. H. Takan62Ohmuro(1), T. Obinatal(i2)),; OIF WoL ,, BAS, Gr Sel, , Chloe Wai, Chiba. C-protein isoforms expressed in embryonic, neonatal and adult chicken breast muscles differ from each other, and when adult muscle is denervated, C-protein isoform expression goes back to neonatal state. In this study, the breast muscle of adult chicken was focally cold injured to induce muscle regeneration, and the C-protein isoforms expressed were examined with the antibodies specific to fast-, Slow-, or cardiac-type C protein by immunohistochemistry. Cardiac-type C-protein became detectable in the myofibers of small diameter which appeared in the peripheral area of the injured muscle at about 3 days after injury. About 1 day later, two skeletal C-protein isoforms, fast- and slow-type C-proteins, also appeared in most of myofibers in the regenerating area. Cardiac-type C-protein disappeared from majority of myofibers by about 14 days after injury. Slow-type C-protein continued to exist even 2 weeks after injury, but disappeared at about 3 weeks after injury. These results indicate that C-protein isoform transitions as observed during muscle development from embryo to adult also occur in the regenerating muscle. BI 16 EFFECT OF BESTATIN ON CHICKEN MUSCULAR DYSTROPHY. K. Shinbo, T. Obinata and K. Maruyama. DANES Oie WiOls, HAG, GO Sei, Cailoa Univ. , Chiba. We previously reported that slow-type C-protein appears in dystrophic chicken breast muscle, while the normal adult muscle does not contain this protein, and therefore, slow C-protein can be usable for a marker to detect the early phase of chicken muscular dystrophy. In this study, we examined the effect of bestatin on chicken muscular dystrophy using the slow C-protein expression as a marker of muscular dystrophy. Bestatin (10 mg/Kg body weight) was administered subctaneously every day. Serial cryosections of the breast muscle were treated with anti-slow or anti-fast C-protein antibody and examined under an immunofluorescence microscope. At the time point of one month after hatching, the expression of slow C-protein in dystrophic chicken breast muscle was significantly repressed by the adimistration of bestatin. However, slow C-protein appeared in the breast muscle of the dystrophic chicken of 3-month old irrespetvie of bestatin administration, although the percentage of myofibers positive to anti-slow C-protein antibody was lower than in the dystrophic chicken muscle without bestatin. It is suggested that bestatin may suppress the development of muscular dystrophy to some extent. Bl CHANGES IN CONNECTIN ISOFORMS IN CHICKEN BREAST MUSCLE DURING EMBRYONIC AND POST- NATAL DEVELOPMENT. H. Yoshidomi, K. Ohashi and K. Maruyama. Dept. Bilol., Fac. Sci.) Chiba Until Chulloce It has been well established that there are several isoforms in some myofibrillar structural proteins and the gene express- ion undergoes characteristic changes during differentiation of muscle cells. In the present work, the changes of connectin isoforms in chicken breast muscle were investigated by SDS gel electrophore- sis during embryonic and postnatal develop- ment. There are three isoforms in X- connectin: embryonic, neonatal, and adult types. The MWs are larger in this order. The embryonic type is present from 7 day embryo to 7 day postnatal chick. The neonatal type is found from after hatch to 10 day chick. The adult type appears on the fifth day after hatch. On the other hand, there are embryonic and adult types in @-connectin. The MW of the former is larger than the latter. The adult type appears on the 5th day after hatch. All the isoforms described above react- ed with anti-native @-connectin of adult type, as revealed by an immunoblot techni- que. There was not any low molecular wei- ght protein detectable with anti-connect-— in in embryonic and postnatal muscles. Biochemistry 907 BI 18 PROTEOLYSIS OF CONNECTIN, AN ELASTIC PROTE- IN OF VERTEBRATE SKELETAL MUSCLE. D. H. Hu, S. Kimura and K. Maruyama. Dept. Biol.,Fac. Sci.,Chiba Univ.,Chiba. In vertebrate skeletal muscle, there are two forms of connectin, giant elastic protein: &-connectin (MW,2800 kD) and@- connectin (MW,2100 kD). In the present study, it has been shown that @-connectin is a proteolytic product of Q&-connectin, using chicken breast muscle. When freshly excised muscle was kept at 0°C,Q was converted into@within a day, whereas it took 4 days, when well washed myofibrils were stored in 50 mM KCl and 1 mM NaHCO3 at 0°C. Addition of EGTA delayed the breakdown, but PMSF or PCMB did not affect at all, suggesting that calcium- activated protease is involved in this process in situ. When myofibrils were stored in 15 mM sodium phosphate buffer (pH 6-8) at 0°C, the conversion of X to was delayed up to two weeks. On the other hand, treatment of myofibrils with trypsin 1/500 by weight, resulted in a complete breakdown of €to@within a minute at 25°C. The@ form of connectin was fairly stable: no further proteolysis occurred for a month in muscle stored at 0°C. However, in myofibrils, a slow hydrolysis off toy form (MW,1700 kD) took place, and after two weeks at 0°C the ratio of B tog was approximately 3:1. BI 19 Z-PROTEIN OF CHICKEN GIZZARD SMOOTH MUSCLB K. Ohashi and K. Maruyama. Dept. of Biol., Pac. of Sci., Chiba Univ., Chiba. - Z-protein, whose molecular weight is 55,000, 1s a component of the myofibrillar Z-band. Immunoblotting test revealed that antibodies against myofibrillar Z-protein reacted with a 35 k dalton peptide’ in the SDS extract of chicken gizzard. This” pep- tide was, to some extent, extracted with the Hasselbach-Schneider's solution. The extract was dialyzed against 7 vol of dis- tilled water. The white precipitate was collected by centrifugation. This pellet was dissolved in a 6 M urea solution con- taining 20 mM phosphate buffer, pH 7.2. After ultracentrifugation at 50,000 rpm for 1 hr, the solution was applied:onto a DEAZ- Sephacel column. The 35 k dalton peptide reacted with the antiserum against myo- fibrillar Z-protein. Chicken gizzard smooth muscle cells were isolated into a solution containing 0.2 # sucrose, 10 mM EGTA, and 30 mM phosphate buffer, pH 7.0. The cells were collected by centrifugation at 1,500 rpm for 5 min and Suspended into a 50 % glycerin solution containing 0.1 M NaCl, 10 mM EGTA, and 10 mM phosphate buffer, pH 7.2. FITC-labelled anti-myofibrillar Z-protein antibodies Stained the cells at the dense bodies in the same manner as anti-@-actinin anti- bodies that was described by Fay et al. (J Cell Biol., 96, 1983) BI 20 EFFECTS OF THE ACTIN DEGRADING ENZYME ON MYOFIBRILS. ve Motobayashi, Ke WOshizato*,...and- +L. Horiguchi**. Dept. | of Biochem. and *Develop.Biol. Lab., Dep. of Plastic Surg., Sch. of Med., Kitasato Univ., Kanagawa. * (PL-A2)- mediated hydrolysis. The experimental re- sults demonstrated that the treated lipo- protein retains their physiological func- tion of taking up diacylglycerol (DG) from fat body, and that PL-A2-hydrolyzed lipo- phorin receives DG from fat body 20 - 30% more than the intact one does. Since PL-A2 removes the fatty acid from the 2 position of PL, the monolayer surface of the hydro- lyzed lipophorin particle would be becom- ing more loosely organized. PL located in the surface of lipophorin seems to con- tribute to the integrity of the particle structure but not to its physiological function. Bl 31 THE POSSIBLE BINDING-SITE OF LIPOPHORIN AT THE CELL SURFACE OF FAT BODY AND MUSCLE OF THE LOCUST. Y.Hayakawa and H.Chino. Biochem. Lab., Inst. of Low Temp. Sci., Hokkaido Univ., Sapporo. Insect lipophorin is a major serum lipoprotein which functions as a reusable shuttle in transporting a variety of lip- ids, diacylglycerol, cholesterol and hy- drocarbon. However, the mechanism by which lipophorin loads and unloads the lipids is unknown. In order to investigate the mech- anism, we tried to find out the specific Site with which the lipophorin associates on the surface of target organs. We first homogenized the locust fat body (loading site) and muscle (unloading site) with a buffer containing 0.2 % Tween 20 and cen- trifuged at 8,000 g for 5 min. The super- natant obtained was used for measuring the affinity to lipophorin. Several lines of evidence obtained suggest the existence in the fat body and muscle of a specific binding site for lipophorin. The injection of adipokinetic hormone (AKH) caused the appreciable increase of the affinity of lipophorin to the fat body and muscle. The effect of AKH observed for the muscle was greater than that observed for the fat body. St By TRIACYLGLYCEROL LIPASE OF THE FAT BODY OF THE LOCUST, LOCUSTA MIGRATORIA. Y. Hirayama and H. Chino. Biochem. Lab., Inst. of Low Temp. Sci., Hokkaido Univ., Sapporo. An enzyme assay is reported in which triacylglycerol (TG) was micelled by Tri- ton X-100 to measure the lipase activity in the fat body of the locusts. In insects, diacylglycerol (DG) is predominantly re- leased from the fat body and associates with a specific serum lipoprotein, lipo- phorin. Since neutral fat is stored in the fat body as TG, a specific lipase that hydrolyzes TG to DG and free fatty acid should be present in the fat body. However, the appreciable activity has not been dem- onstrated in the locust fat body as far as the usual techniques are used. We develop- ed a new method in which TG was treated with ethanol and Triton X-100 (mol ratio of TG to Triton X-100 = 1:150) and sub- sequently the ethanol was removed by N,- stream. This process provided a clear micelles of TG and we succeeded in demon- strating the lipase activity of the fat body by the use of this micelle as the substrate. The activity obtained under the optimal conditions (pH 6.5-7.0, ionic strength:0.5) was almost 100 times greater than that obtained using other substrates, e.g., TG emulsified with Arabic gum or polyvinylalcohol. Biles PEPTIDOGLYCAN-INDUCED ACTIVATION OF THE SERINE ENZYMES AND PRO-PHENOLOXIDASE IN PLASMA OF SILKWORM, BOMBYX MORI. H.Yoshida and M.Ashida. Biochem. Lab., The Institute of Low Temperature Science, Hokkaido Univ.,Sapporo. Bacterial cell walls and zymosan induce the activation of two serine enzymes in plasma of silkworm,Bombyx mori. One of them is pro-phenoloxidase(pro-PO) activa- ting enzyme (PPAE) which activates pro-PO in fashon of limited proteolysis. The other is an esterase (referred as BAEEase) which hydrolyzes BAEE,a synthetic substrate of bovine trypsin. We obtained the following results in the present study. 1)Lipopolysaccharides did not cause activation of these enzymes. 2) Lysozyme from chicken egg white almost completely lost the abilities as an elic- itor of cell walls and heat-killed cells of Gram-positive and -negative bacteria. 3)Purified peptidoglycan from a Gram-posi- tive bacteria caused the activation of the enzymes. 4)Intact cells of Escherichia coli induced no activation of pro-PO in contrast to Gram-positive bacteria and heatrikiTledsr..cola The above results lead to a conclusion that peptidoglycan is active principle of bacterial cell walls to cause activa- tion of BAEEase and PPAE in plasma of silkworm. Biochemistry 911 BI 34 PURIFICATION OF ARGINASE FROM THE SILKMOTH, BOMBYX MORI. T. Aigaki and M. Osanai, Dept. Biol., Tokyo Metropol. Inst. of Geront., Tokyo. Adult fatbody contains the highest arginase activity in saturniid silkworm, Hyalophora gloveri (Reddy and Campbell, 1969). However, in Bombyx mori, the highest arginase activity was recognized in vesicula seminalis of male moths. After Bombyx-arginase was purified, some properties of the enzyme were examined and compared with those of adult fatbody arginase of H. gloveri. Purifica- tion of the enzyme was performed as follows: 1) Extraction by homogenization of male moth abdomens with 0.1 % Cetyl- trimethylammonium bromide. 2)Ammonium sulfate precipitation (20-60 % sat.). 3) Gel filtration by Sephacryl S-300. 4) Chromatofocusing with PBE94 and Polybuffer 74. 5) Preparative electrophoresis. Specific activity of purified enzyme was 270-fold higher than that of the original extracts. Molecular weight estimated by gel filtration was 230,000. pI of the enzyme was estimated as between 5.3 and 5.5. The optimum pH for the enzyme reac- tign was e2>° The gnzyme was activated by Mn“*, Ni“* and Co“*, but inhibited by L- ornithine. The apparent K, for L-arginine was 26 mM and K; for L-ornithine was 3.4 mM. Most of the properties of Bombyx- arginase were very similar to those of adult fatbody arginase of H. gloveri. BI 35 ACTIVATION OF LACCASE-TYPE PRO-PHENOL- OXIDASE IN THE CUTICLE OF INSECTS. I. ACTIVATION BY PROTEASES. H.1I.Yamazaki and S.Kambara. Biol. Lab. Atomi Gakuen Women's Univ., Saitama. ae ee ee eee Cuticle-bound phenoloxidase, having properties of laccase plays an important role in the sclerotizing process of insect cuticle. It was found that the enzyme occurs as an inactive pro-enzyme bound to cuticle. Therefore, the mechanism of the appearance of the enzyme activity at critical stage of pupation is considered to be activation of pro-enzyme to active form. In order to confirm the mechanism of the activation, pro-enzyme was solubilized and purified from the cuticle of silkworm pupae. The purified preparation was homogenous on electro- phoresis. The pro-enzyme was activated by trypsin with linear fashion and the activation depended on the amount of trypsin. The activation by trypsin was suppressed by lima-bean inhibitor, DFP, leupeptin, and also by NaCl of higher concentration. The pro-enzyme was also activated by thermolysin, pepsin, pronase E and proteinase K. The patterns of active enzyme produced by proteolytic enzymes were examined by activity staining after electrophoresis. The results suggest that the pro-enzyme is activated by a proteolytic enzyme(s) which might be present in cuticle. Bl 36 BETA-ALANINE AND KETOCATECHOLS IN THE HYDROLYSATE OF THE PUPARIAL CASE OF DROSOPHILA MELANOGASTER, Y, Umebachi and S, Kiyokawa, Dept, of Biol,, Fac, of Sci., Kanazawa Univ,, Kanazawa. Be Ae Naan ae SEs SE SORE eR nae Ss UE ae eee Umebachi and Aburano (1978, 1979) pre- viously reported that there was an inverse relation between the #-alanine and ketoca- techols produced by hydrolysis of wing- scales of butterflies, The present expe- riments have been made to see if the in- verse relation is true of cuticle, For this purpose, puparial cases of three ki- nds of mutant strains (y, b, and e) of D, melanogaster were used, They were hydro- lyzed in 1 N HCl at 100°C for 5 hr, and the hydrolysates were examined for @-ala- nine and ketocatechols, The amount of @- alanine was largest in y, small in b, and zero ine, On the contrary, the amount of ketocatechols was largest in e, medium in b, and smallest in y, Furthermore, when b was fed on a #-alanine-containing medi- um, 6-alanine of puparial case increased, while ketocatechols produced on hydrolysis decreased, These results indicate that the inverse relation between the #-alanine and ketocatechols released by hydrolysis is valid also for cuticle and suggest that there may be an inverse relation between the amount of f-alanine in cutic- le and the extent of #-sclerotization, Bl 37 THE AMINO ACID SEQUENCE OF CALCIUM- BINDING PROTEIN (TCBP-10) OBTAINED FROM Tetrahymena T. Kobayashi, T. Takagi, K. Konishi, K. Ohnishi* & Y. Watanabe*. Biol. Inst., Fac. Sci., Tohoku Univ., Sendai, *Inst. Biol. Sci., Univ. Tsukuba, Ibaraki A a et taniel A calcium-binding protein, diffe- rent from calmodulin, has been obtained from the cilium and cell body of Tetra- hymena (1). This protein was designated as TCBP-10 (Tetrahymena Calcium-Binding Protein with m.w. 10,000). TCBP-10 was first reported that it had no methionine (1), but recent amino acid analysis showed TCBP-10 had three methionine residues. Therefore we have determined its sequence by using CNBr peptides. The overlapping of CNBr pepties were obtained by its tryptic peptides. The amino acid sequence of TCBP-10 is follo- wing: S..S> Kee Re Von? EB V5 K LDVAR EAR. RY DK DG S:G'O LD QDLA EB i i Belk San SMG MASON T PY Res D VKIWLQMADTNSAGSV LEE R Bape es oO R AG IRV K QS VFL It is composed of 103 aminc sid resi- dues and has one EF-hand type calcium binding site at residues 69-80. (1) Ohnishi, K. & Watar e, Y. (1983) J. Biol. Chem. 258, 13978- : 912 Biochemistry BI 38 CALMODULIN-BINDING PROTEINS IN TETRAHYMENA CILIA. J.Hirano and Y. Watanabe. Inst. of Biol. Sei., Uuiv.a of Tsukuba, tbharaka: Previously, we have detected at least 36 kinds of calmodulin-binding proteins (CaMBPs) in Tetrahymena cilia by using a radioiodinated CaM overlay method. As a first step to know the role of CaMBPs in Ca-dependent ciliary reversal, we have tried to prepare such CaMBPs of native form. For this purpose, we used a CaM- agarose affinity column. As an absorbant, Tetrahymena CaM purified by phenyl - sepharose and hydroxylapatite columns was coupled to Affi-Gel 15. A protein sample from Tetrahymena cilia or from ciliary subfraction was loaded first into a phenothiazine column to remove endogenous CaM and the void fraction was loaded secondly into a CaM column in the presence of 1mM Ca and eluted with 5mM EGTA. When the whole ciliary fraction and outer- doublet microtubule fractions were sub- jected to these column chromatographies, nearly the same results were obtained: several proteins having the molecular weights of 92k, 66k, 63k, 45k and 27k were clearly detected in their EGTA eluants. Almost all of the eluted proteins could bind radioiodinated CaM by the overlay method, and most of them corresponded to some of the CaMBPs previously detected. On the other hand, few CaMBPs were found in the membrane and matrix fraction and the crude dynein fraction. BI 39 CALMODULINE-BINDING PROTEINS (CaM-BPs) IN CILIARY MEMBRANE. 1. STUDY ON CALCIUM CHANNEL DEFECTIVE MUTANTS IN CILIATES. T.Takemasa, J.Hirano, M.Takahashi and YeWatanabery sinisie., On) BaroleaeSicuy..- University of Tsukuba,Ibaraki. A set of mutants affecting calcium channel function has been isolated in ciliates as behavioral mutants (ciliary non-reversal mutants) and designated as tnr in Tetrahymena thermophila, cnr in Paramecium caudatum, and pawn in Pe retrawnedsiar On the other hand, we previously detected about 20 kinds of CaM-BPs in ciliary membrane & matrix fraction of wild-type T.thermophila by using a highly sensitive, radiolabeled CaM overlay method. In this paper, we have investigated both protein constituents and CaM-BPs of the wild-type and mutant ciliary membranes using above-mentioned mutants, considering the possibility to obtain a clue for elucidating a molecular mechanism of calcium channel. This would be possible, if we succeed in finding a certain difference between wild-type and mutant cells. However, as far as our experimental results are concerned, there exists no Significant difference between tnr, cnr, pown mutants and the respective wild-type cells. BI 40 Ca°t-DEPENDENT PHOSPHOPROTEIN PHOSPHATASES FROM EGGS AND SPERMATOZOA OF SEA URCHIN. F.Iwasa and K.Ishiguro* Dept. of Biol., *Dept. of Biophys. and Biochem., Univ. of Tokyo, Tokyo. In order to elucidate role(s) of cal- modulin in eggs, we surveyed calmodulin- binding proteins in sea urchin eggs and found a calmodulin-binding protein which consists of two subunits (55K and 17K dal- tons). In this paper we describe about en- Zymatic activity of this protein. Klee et al reported that calcineurin (a calmodulin-binding protein from mamma- lian brain) is a phosphatase. One dimen- sional peptide mapping revealed similarity of the 17K subunit to 19K subunit of cal- cineurin. However, the 55K subunit and 61K subunit of calcineurin were different from each other. Nevertheless the 55K+1/K pro- tein showed phosphatase activity against inhibitor-l as calcineurin. Km and Vmax values of both phosphatases were similar. The 55K+17K phosphatase had a strict sub- strate specificity. We found a few sub- strates in sea urchin egg homogenate but none in isolated cortices and mitotic ap- paratuses. a4 In spermatozoa there was Ca -depend- ent phosphatase activity in Tris-EDTA ex- tract of demembranated flagella, which did not dephosphorylate inhibitor-l. The ac- tivity was retained on calmodulin-affinity column. RI 41 FURTHER PURIFICATION AND CHARACTERIZATION OF THE MOTILITY ACTIVATOR OF SEA URCHIN SPERM. K.Ishiguro, H.Murofushi and H.Sakai. Dept. Qs lV oIMyS>o SdocMems 5 PACs Oi Sis ; Univ. of Tokyo. Bunkyo-ku, Tokyo. Phosphorylation of a protein factor by cAMP-dependent protein kinase is required for motility reactivation of sea urchin sperm models. We call the factor motility activator. This activator was further purified by the use of hydroxy- apatite column chromatography, DEAE- Sephacel column chromatography and iso- electric precipitation. SDS-polyacryl- amide electrophoresis and autoradiography showed that 46K dalton protein was phosphorylated by purified cAMP-dependent protein kinase. The phosphorylated motility activator reactivated the flagellar movement of sperm models. The activator also reactivated the sliding of the outer doublet microtubules, but had little effect on dynein ATPase activity. It was suggested that the motility activator interacts with a machinary coupling dynein ATPase activity with the Saeaalnicne Biochemistry 913 BI 42 PROTEASE INHIBITORS,LEUPEPTIN AND ANTIPAIN STIMULATE THE RESPIRATION OF SEA URCHIN SPERM 1) 2) K. Nomura ‘and S. Isaka’’, 1)Dept. Biochem., Tokyo Metro. Inst. of Gerontol. (Tokyo) and 2)Noto Marine Lab., Kanazawa Univ. (Uchiura) Sperm Activating Peptides(SAPs) from the jelly coat of sea urchin eggs can stimulate the sperm respiration at pH 6.8. Various analogue peptides have been synthesized to elucidate the structure-activity relation- ships. In the course of studies on sub- stances which modulate the SAP activity, we have found that leupeptin and antipain, mi- crobial inhibitors of Ser- and SH-proteases, can stimulate the respiration of sperm of Anthoctdaris crassispina and Hemicentrotus pulcherrimus in a Nat- and Mg2+-dependent manner without inducing acrosome reaction. The ACsg was 60 uM for leupeptin and 200 uM for antipain. Leupeptin acid, benzamidine and TLCK were moderate stimulators, whereas soybean trypsin inhibitor, p-aminobenzami- dine, TAME and L-arginine were almost with- out effect. Elastatinal, chymostatin and E-64 were weak stimulators, while TPCK was rather inhibitory. These results suggest that leupeptin and antipain stimulate the sperm respiration by a mechanism not asso- ciated with any protease activity. Interestingly, the inhibitors are more active on A. crassispina sperm than on Jd. puleherrimus sperm, in contrast to SAPs which are more potent in #. pulcherrimus sperm than in A. crassispina sperm. BI 43 LOCALIZATION OF CYTOPLASMIC DYNEIN OF SEA URCHIN EGGS DURING MITOSIS. S.Hisanagal, T.Tanaka2, T.Masaki3 and H. Sakai4. Ipiv. of Cell Prolif. ,Natl.Inst.for Basic Biol., Okazaki, 2Japan Red Cross Sai- tama Branch, Yono, 3Inst. of Basic Med. Sci -,Univ. of Tsukuba, Niihari-gun and 4Dept. of Biophys. and Biochem., Fuc. of Sci., Univ. of Tokyo, Tokyo. We prepared several monoclonal anti- bodies (mcAbs) against sea urchin sperm flagellar 21S dynein. Three of them were used to identify and localize cytoplasmic dynein in sea urchin eggs by immunoblotting and immunofluorescent microscopy. One mcAb (D57) that was directed against the Ag chain of flagellar 21S dynein crossreacted with the heavy chain of cytoplasmic dynein. D57 stained both spindle and spindle poles of isolated mitotic apparatus, although the fluorescent intensity was not so strong as when stained with the other two mcAbs. Ano- ther mcAb (D6) that reacted with A and B chains of flagellar dynein also stained both spindle poles and spindle, mainly in the region near chromosomes. The third mcAb (D5), that showed weak reactivity to flagellar dynein A and B chains, crossre- acted with a 110K polypeptide in addition to the heavy chain of cytoplasmic dynein. D5 also stained both spindle and spindle poles, staining of the spindle by D5 being the brightest of the three mcAbs. These results suggest the presence of cytoplasmic dynein in the spindle and the spindle pole. BI 44 ALPHA-ACTININ FROM SEA URCHIN EGGS: PROPER- TIES AND INTRACELLULAR LOCALIZATION DURING FERTILIZATION I.Mabuchil and Y.Hamaguchi2, IDept. Bia O dine Worl. Arts and “Sci., Unive Tokyo, Tokyo; and Dept. Cell Biol., NIBB, Okazaki, and 2Dept. Biol., Tokyo Inst. Technol., Tokyo. ETN al he ES RS I ee a GQ-actinin was isolated from unfertilized eggs of Hemicentrotus pulcherrimus. The interaction of egg a-actinin with rabbi¢c skeletal muscle actin was investigated by means of low-shear viscometry using a fall- ing ball apparatus. An F-actin solution was gelledby egg a-actinin at a molar ratio more than 1:20. The gelation took place at pHs between 6.0 and 8.0 and Ca ion concen- tration less than 1 pM. Rabbit skeletal muscle tropomyosin inhibited the gelation. To study the intracellular localization of Q-actinin, we labeled this protein with rhodamine. When microinjected into unferti- lized eggs, rhodamine-a-actinin distributed almost evenly. Upon fertilization, however, it was first accumulated in a limited cor- tical region where sperm penetrated in. Then the fertilization cone grew up, which also contained this protein. And then the entire cortical layer became fluorescent. Microinjection of rhodamine-a-actinin into fertilized eggs also resulted in its con- centration in the cortical layer. At cyto- kinesis, the cortical layer at the furrow region seemed slightly thicker than the rest of the cortical layer as viewed by the fluorescence. BI 45 45,000 MW PROTEIN FROM UNFERTILIZED SEA URCHIN EGGS AND CHANGES IN THE PROPERTY OF 45,000 MW PROTEIN-ACTIN COMPLEX AFTER FERTILIZATION H.Hosoya, I.Mabuchi'’and H.Sakai. Dept. of Biophys.& Biochem., Fac. of Sci., Univ. of Tokyo., Tokyo. ‘Dept... OF .Bi01., .Coll«esof Arts & Sci., Univ. of Tokyo, Tokyo, and Dept. of Biol., NIBB, Okazaki. 45,000 MW protein-actin complex (45K-A) is an actin filament capping-protein puri- fied from unfertilized sea urchin eggs. The complex is detected in the soluble fraction of the egg but not inthe insoluble fraction. We have used affinity purified antibody against the 45K protein to investigate its distribution in several echinoderms and mammalian brain. Immunoblotting demonst - rated that eggs of five species of sea urchins contained the antigen showing an MW of 45K that reacted with the antibody. In contrast, eggs of starfish as well as por- cine brain extracts did not react with the antibody. We compared the properties of the 45K-A from fertilized eggs (F-45K-A) with those from unfertilized eggs (U- 45K-A). Both the 45K-A did not depolymerize F-actin and inhibited the annealing of fragmented F-actin. The U-45K-A accelerated polyme- rization of actin but the F-45K-A did not. Two dimensional gel electrophoresis showed the presence of some microheterogeneity in both the 45K proteins from unfertilized and fertilized sea urchin eggs. 914 Biochemistry BI 46 PURIFICATION AND CARACTERIZATION OF DNASE I-BINDING PROTEIN FROM TETRAHYMENA M.Hirono, O.Numata and Y.Watanabe. Inst. of Biol. Sci., Univ. of Tsukuba., Ibaraki. To detect and isolate actin from Tetra- hymena, we have applied the DNase I- affinity column method to the extract of ethanol-ether powder of Tetrahymena cell. The DNase I-binding fraction included a 35k protein predominantly. Since the 35k protein is a major constituent of NP-40- extracted cell model, we have tried to isolate this protein, and scceeded in it. The procedure is as follows. The model was washed with 0.6 M KI, and solubilized with a buffer containing 7 M urea, and then urea concentration of resulting super- natant was reduced to 3 M. As a result, 35k protein was exclusively recovered as a precipitate. This protein was separated into three major bands (pl; 5.93-6.05) in an isoelectric focusing gel. Using the purified protein, we analyzed its amino acid composition. The composition was not resembled to those of actins from several species. We prepared antibody specific for 35k protein. The fluorescein- labeled antibody stained the cortical region and nuclear envelope of Tetrahymena cell. These data suggest that 35k protein is a major cytoskeletal protein of Tetrahymena, although it is not always actin-like. BI 47 A POSSIBLE FUNCTION OF TETRAHYMENA 14-NM FILAMENT PROTEIN DURING CONJUGATION oO. Numata , T. Sugai~ and Y. Watanabe’. EDSe. Oi Biol. Sci., Univ. of Tsukuba., tbanalos) Dept. Of —Biole, etbarakaniUnaive, Ibaraki. We have previously described the prop- erties of Tetrahymena 14-nm filament pro- tein (49K protein). The 49K protein resembled intermediate filament protein from mammalian cells, in its molecular weight, amino acid compositions, filament Size (14-nm) and conditions for assembly and disassembly of the filament. We here report possible biological functions of 49K protein relevant to the nuclear events of fertilization during conjugation, based on the immunofluores- cence localization of 49K protein. From the results obtained in this study, 49K protein is considered to exert some significant influences upon (i) the deter- mination of one functional nucleus among 4 haploid nuclei generated by meiosis, (ii) the division of the functional nucleus to generate migratory and stationary pronu- clei, (iii) the determination of migratory pronucleus, (iv) the migratory pronucleus exchange, (v) the selection of two gametic pronuclei among 5 nuclei including 3 relic ones to form fertilization nucleus, and (vi) the fusion process of gametic pronu- clei. BI 48 PURIFICATION OF A MUTANT GENE PRODUCT FROM A TETRAHYMENA THERMOPHILA MUTANT AFFECTING CELL DIVISION, cdaA1 , H.Ohba, I.Ohmori and Y.Watanabe. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. A cdaAil(cell division arrest) mutant is known to have a defect on a mechanism of determination of fission zone formation in temperature-sensitive manner. We have investigated the differences in protein constituents between cdaAl and wild type by 2 dimensional gel electrophoresis. As a result, we detected a difference ona single spot (Mw=85k,pI=4.7).The result of cross between cdaAil and wild type showed that protein amount of the spot from cdaAl correlated well with the mutant phenotypic expression. We then tried to purify this protein(p85) from wild-type cells. First the cells were solubilized with 6M Guanidine HCl, fractionated with 0-30% ammonium sulfate, and followed by solubilizing in 1M KI, precipitating in 20mM K-PO4 buffer, and re-solubilizing in 1M urea. The protein sample were then subjected to DEAE-sepharose column chro- matography, and finally p85 was purified by 2 dimensional gei electrophoresis. We prepared anti-p85 antiserum in guinea pigs, and revealed that p85 was localized in the oral apparatus and its anterior vortical structure. Further examination on dividing cdaAl mutants at permissive and nonpermissive temperature is in progress. BI 49 PURIFICATION AND CHARACTERIZATION OF THE THREE COMPONENTS OF HIGH-MOLECULAR WEIGHT MICROTUBULE-ASSOCIATED PROTEINS FROM RAT BRAIN. 1 2 2 T. Kobayashi? S.Tsukita;, S.Tsukita and G. Matsumoto. Dept. Biochem. ,Jikei Univ., School of Med.,“Dept. Anat.,Tokyo Univ., Faculty of Med. ,2Optoelectronics, Electro- technical Lab. Microtubules purified from rat brain contained three components of high molecular weight microtubule-associated proteins—— MAP-1(350k,doublet and con- tained equal amountof each of the two polypeptides), MAP-X(320k, singlet) and MAP-2(300k, doublet). MAP-1 and MAP-X were heat-sensitive, while MAP-2 was heat-resistant. On the other hand, micro- tubules from squid axoplasm contained two components of MAPs——320k and 260k. Neither of MAP-1 and MAP-2 was found in squid axoplasm. The three components of rat brain MAPs were purified by DEAE-cellulose column chromatography and high performance gel filtration through TSK-Gel G4000. All the MAPs from rat brain could co- sediment with tubulin and actin in vitro, while 260k from squid axoplasm could co- sediment with tubulin but could not with actin. MAP-X was found in brain, liver, spleen and kidney in rats, MAP-1 and MAP-2, however,in only brain. Biochemistry 915 BI 50 DYSPROPTERIN SYNTHETASE FROM FAT BODIES OF SILKWORM LARVAE. M.Masada, S.Yoshioka and M.Akino. Dept. of Biol. Tokyo Met. Univ., Tokyo. Dyspropterin synthetase that catalyzes the conversion of dihydroneopterin tri- phosphate to dyspropterin in the presence of Mg2+, was purified from fat bodies of silkworm larvae by procedures including heat treatment, ammonium sulfate fractio- mation and column chromatography on hydro- xylapatite and DEAE-Toyopearl. Finally, the preparation was purified to homogenei- ty by means of disc electrophoresis. The specific activity was raised 1100-fold over the crude extract. The K, value of dihydro- neopterin triphosphate for the enzyme was calculated to be 5.1 x 10-6 M. Ultraviolet absorption spectrum of the enzyme showed a Single peak around 280 nm. The molecular weight was estimated as 87,000 by gel fil- tration and 72,000 by the method of Hedrick and Smith. SDS-electrophoresis revealed that the enzyme is composed of two subunits having molecular weights of 35,000 and 18,000, respectively. The enzyme activity was inhibited by sulfhydryl reagents such as PCMB, monoiodoacetate and N-ethylmale- imide. BI 51 BIOSYNTHESIS OF TETRAHYDROBIOPTERIN. M.Masada, M.Akino, T.Sueoka* and S.Katoh*. Dept. of Biol. Tokyo Met. Univ., Tokyo and *Dept. of Biochem. Josai Dent. Univ., Saitama. The structure of an intermediate named dyspropterin, which is formed from dihydro- neopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin (BH,), has been studied. Dyspropterin was found to be reduced to BH4g in the presence of NADPH by sepiapterin reductase which has an ability to reduce dioxo compounds to dihydroxy de- rivatives. Stoichiometric analysis reveal- ed that there is 1:2 relation between the production of biopterin and the oxidation of NADPH during the reductase-catalyzed reduction of dyspropterin. The BH, pro- duction from dyspropterin was stimulated by dihydropteridine reductase. Dyspropte- rin could also serve as a cofactor for phenylalanine hydroxylase. These results are consistent with the view that dyspro- pterin is 6-(1,2-dioxopropyl)-5,6,7,8- tetrahydropterin. Based on the findings a revised biosynthetic pathway of BH, from dihydroneopterin triphosphate has been proposed. BI 52 GLUCAGON STIMULATION OF HEPATIC SEPIAPTERIN REDUCTASE in Vivo. T. Sueoka and S. Katoh., Dept. of Biochem., Josai Dental Univ., Sakado. Tetrahydrobiopterin is required as a co- factor of hepatic phenylalanine hydroxylase which is activated by glucagon through phos phorylation in vivo. In this presentation, transient stimulation of sepiapterin re- ductase, an enzyme involved in the biosyn- thesis of tetrahydrobiopterin, and level alteration of total biopterin (reduced and oxidized forms) in rat liver by glucagon treatment were studied. Activity of sepiapterin reductase inthe extract of liver was assayed in the pres- ence of 50M sepiapterin and 100M NADPH at PH 6.4. Total and tetrahydro- biopterins were determined by HPLC following a differ- ential oxidation procedure with iodine by the method of Fukushima & Nixon(1980). When 400ng glucagon was injected per 100 g body weight of male Sprague-Dawley rat, about 150% activity of sepiapterin reduct- ase was showed in the liver after 30 min and this activation decayed after lh of the injection. Level of total biopterin in the liver was found to alter during lh after the injection of glucagon within pM concentration. A mechanism to control the activity of hepatic phenylalanine hydroxyl- ase by alteration of pteridine level in the liver,besides the phosphorylation of the enzyme,by glucagon was suggested in vivo. BI 53 A NEW PTERIN DERIVATIVE FROM LARVAL SKIN OF RANA CATESBIANA. T. Yoshida and Y. Iwanami. Dept. Chem. Keio Univ. Hiyoshi 4 Yokohama. Recently molybdopterin and methanopter- in have been found anew from sources of molybdenum enzymes and Methanobacterium, respectively, to constitute a new class of pterins. We now report the occurrence of a new pterin derivative in the larval skin of Rana catesbiana. Extraction was performed with 50 mM AcOH followed by successive co- lumn chromatographies starting from ECTEO- LA cellulose. The UV absorption spectra of the isolated compound were closely resemb- led those of isoxanthopterin(7-hydroxypte- rin). We encountered with difficulties to obtain good MS and NMR spectra probably because of poor vapor pressure and extre- mely low solubility to proper solvents. Permanganate oxidation of the compound ga- ve isoxanthopterin-6-carboxylic acid. The compound showed less acidic nature than isoxanthopterin and ichthyopterin(7-hydro- xybiopterin) being eluted faster on ECTEO- LA Sephadex column, These resuits suggest that the compound is a 7-hydroxylated pte- rin with a side chain at 6-position. The side chain appears to be labile. This fea- ture is presumably associated with intra- molecular hydrogen bonding formed therein. 916 Biochemistry BI 54 COMPARATIVE BIOCHEMICAL STUDIES ON LIPIDS FROM THE SKIN OF THREE FROG SPECIES. M. Ryuzaki. Dept. of Biol., Kitasato Univ. School of Medicine. Sagamihara. Phosphatidylcholine (PC) and Phosphati- dylethanolamine (PE) molecules were isola- ted from the skin of Xenopus laevis, Bufo japonicus and Rana catesbeliana, and the positional distribution of the constituent fatty acids in their molecules was examin- ed in order to clarify the relationship between the differences in phospholipids and the species specificity. Phospholipids were fractionated by one-demensional thin- layer chromatography (Skipski et al.,1964) Phospholipase Aj was used to remove the fatty acid residues from the 2-position of PC and PE molecules (Harverkate et al.,195 7). The isolated fatty acids and lysophos- phoglycerides were methanolyzed, and the fatty acid methyl esters were analyzed at 185°C by Shimadzu GC-5A unit equipped with a 3.0 m X 3 mm glass column packed with 25 % EGS on Celite 545 HMDS. Except for some minor variations, the gross pattern of the positional distribution of the constituent fatty acids in PC molecules from the skin showed no remarkable changes among the three frog species. However there were marked changes in the positional distribu- tion of the constituent fatty acids in the PE molecules. The significance of these changes in the skin of the three frog species was discussed. BI 55 DEVELOPMENTAL CHANGES OF GABA AND PUTATIVE AMINO ACID NEUROTRANSMITTERS IN THE ORGAN- OTYPIC CULTURE OF NEWBORN MOUSE CEREBELLUM D-aSéeroni, Dept, Ot BiGl,, Coll, of Ares and Sci., Univ. of Tokyo, Tokyo. The quantitative changes and metabolism GABA and putative amino acid neurotran- mitters during early developmental stages the organotypic culture of newborn mou- cerebellum were examined using a HPLC chnique. D-[U-'“C]glucose was used as a recursor of amino acids. Explants were incubated for 4 weeks under standard cond- itions. The amount of GABA increased line- arly from 8.7+1.3 nmol/mg protein(2 day in vitro,2DIV) to 26.5+6.1 nmol/mg protein (15 DIV) and saturated thereafter. The pe riod of GABA increase was overlapped with the onset of synapse formation in vitro. During the same period, the ability of GABA synthesis from ['"C]glucose increased rapidly. These results were in good agree- ment with those obtained in vivo. In case of glutamic acid, the level in explants was nearly constant during incubation. While, the ability of synthesis increased during 8-22 DIV. This fact indicates that the turnover rate of glutamic acid in exp- lants increased accompanying with develop- ment. The changing patterns of aspartic acid and glycine in vitro were also simil- ar to those in vivo. It was concluded that this organotypic culture developed normal- ly in respect to neurotransmitter synthes-— dL Se Lr (p) yah Sy te SO oN Ho © 14) BI 56 EFFECTS OF METHYLMERCURY ON ENZYME ACTIVITIES IN RAT TISSUES. S.Omata, Y.Hamada, M.Atsumi, K.Hasegawa, H.Hasegawa and H.Sugano. Dept. of Biochem., Fac. of Sci., Niigata Univ., Niigata. In order to examine the biochemical mechanism of methylmercury intoxication, we investigated the changes in enzyme activities in rat tissues during the period from an early phase of intoxciation to the appearance of neurological symptoms after ad- ministration of methylmercury chloride. Activities of neurotransmitter-metabolizing enzymes examined, glutamate decarboxylase, GABA-aminotransferase, catechol-o-methyltransferase and monoamine oxidase (type A and B) showed no significant changes when homogenates or subcellular fractions from whole brain were used. On the other hand, monoamine oxi- dase activities in brain regions such as, cerebral cortex, cerebellum and hippocampus, decreased sig- nificantly at the symptomatic period. 2',3'-cyclic nucleotide-3'-phosphohydrolase activity in the brain was not altered by methylmercury. Lipid per- oxidation in the liver was largely increased while that in sciatic nerve was decreased. Glutathione peroxidase activities in the brain, liver, kidney and blood were significantly decreased at the sym- ptomatic period. Compared with the enzyme activi- ties in the tissues of the pair-fed rats, it is concluded that the increase in lipid peroxidation in the liver of the poisoned rats was largely due to nutritional deficiency, but other changes in enzyme activities described above resulted from the direct action of methylmercury on these tis- sues. Effects of methylmercury on enzyme activit- ies in vivo were discussed in relation to those in vitro. Bley BIOCHEMICAL CHARACTERISTICS OF RAT LUNGS EXPOSED TO OZONE Z.Nambu, I.Ichikawa and E.Yokoyama. Department of Industrial Health, The Institute of Public Health., Tokyo. Sensitive biochemical indices of the ef- fects of exposure to ozone on rat lung, lung lavage and blood were searched. Male rats were exposed to 0.3, 0.5 and 0.6 ppm ozone for 24 hrs and killed immediately. Hematocrit of the exposed rats increased by 10 %, and no effects were shown on serum trypsin inhibitor capacity, concen- trations of nonprotein SH, protein and sugar in blood of the exposed. DNA and RNA of free cells in the lavage were increased by 0.3 ppm ozone and DNA was decreased by 0.6 ppm ozone, while protein of the cells was not affected. Ascorbic acid in the la- vage fluid of the exposed rats decreased. Protein and sugar in the lavage fluid in- creased very much in proportion to ozone- concentration and the age of rats, and Sugar was more sensitive than protein. There was a good correlation between the exposure-period and the contents of the Sugar and the protein. Total lung choles- terol decreased by ozone-exposure. No ef- fects of the exposure were found on total lipids, phospholipids and cholesterol in the white layer, and protein, collagen, elastin, sugar, total lipids, phospho- lipids, ascorbic acid, nonprotein SH, TBA- value, SOD, CAT and GSH-Px in rat lung. Biochemistry, Developmental Biology 917 BI 58 CHANGES IN DNA POLYMERASE 8 ACTIVITY IN NASCENT NUCLEOSOME OF RAT LIVER AFTER PARTIAL HEPATECTOMY. T.Yoshikawa, H.Shimada and T.Shiroya. Zool. fasee, fac. Of Sci., University of Tokyo, Tokyo. Using DNA polymerase8g (pol 8) as a marker for non-histone protein, we studied the mode of incorporation of non-histone protein into nescent nucleosomal structures in the liver of partially hepatectomized rat. The chromatin fraction obtained from regenerating liver was digested by micro- coccal nuclease and the nuclease-sensitive fraction was separated by centrifugation. Centrifugal analysis of the nuclease- sensitive fraction suggests that polf is firmly bound to the nucleosome core. The pol 8 content of mononucleosome started to drop 24hr after partial hepatectomy, reached about 40% of the original content at 28hr and recovered to normal level at 69hr. The distribution of radioactivity in the liver chromatin of partially hepatecto- mized rat after pulse-labeling with *H-TdR or *H-leucine suggests that the nuclease- sensitive chromatin consists of nascent nucleosomes. It was tentatively concluded that the incorporation of non-histone protein such as pols into the newly-formed nucleosome occurs later than the formation of the DNA-histone octamer complex. BI 59 HIGH TEMPERATURE INDUCES APOSYMBIOTIC APHIDS H. Ishikawa and M. Yamaji Dept. Biol., Coll. Arts §& Sci., Univ. Tokyo, Tokyo. An intracellular symbiont of the pea aph- id, A. pisum, when harbored by the myceto- cyte, synthesizes only one protein speci- es, symbionin. In an effort to know biol- ogical functions of symbionin, conditions under which the aposymbiotic insect is pr- oduced were investigated. Injection of rifampicin, a specific inhibitor against the prokaryotic RNA polymerase, into the insect induced completely aposymbiotic progeny (Zool. Sci. 1, 509-522 (1984)). When the aphid nymphs born at 15°C were kept at 37°C for the first 15 hr, they gr- ew into extremely undersized and sterile adults. Just like the rifampicin-induced aposymbiotic host, these insects neither contained nor synthesized symbionin. High sensitivity to elevated temperature of the aphid endosymbiont is reminiscent of Rhiz- obium and those in amebae in which large plasmids play crucial roles in their endo- symbiosis with the host cell. It is likely that symbionin is synthesiz- ed by the intracellular symbiont in the embryo developing in the ovariole and pla- ys an important role in the embryonic and post-embryonic development of the host insect. BI 60 AN IMMUNOCHEMICAL STUDY ON HUMAN LENS LOW MOLECULAR WEIGHT CRYSTALLIN. M.Fujishita, Dept. o£ Appl. Biol. Sci., Science University of Tokyo, Noda. Se aheawetnedtan-teneetractecsi are teins, ¥-crystallin has been classified as a group of proteins which have the lowest molecular weight and the lowest electropho- retic mobility at alkaline pH. In mouse and calf lenses, the final peak from gel- filtration fractionation contains only basic proteins. Hense, these low-molecu- lar-weight fraction(LMW)s have been called yYecrystallin and used for further investi- gations. LMW from human lenses has been found highly heterogenous. It contains proteins with isoelectric points ranging from pHS.5 Or Oe Ul To study the relation of human LMW, especially its non-baSic components to Y-crystallin, immunoblot experiments were performed with antiserum which bound to mouse ¥= but neither to mouse &= nor A= crystallin. This antisérum recognized human LMW. Further fractionation of human LMW revealed that components with higher molecular weight(YH, M.W.=ca.24,000) bound to the antiserum very weakly, while lower molecular weight species(Y¥L, M.W.=ca.19,000 ) did apparently. Almost all bands found after electrophoresis of ML were recognized by the antiserum after immunoblotting to nitrocellulose membrane. The result suggest the possibility that human lens contains non=-baSic low molecular weight proteins which are at least immuno- chemically classified to Y=crystallin. DB 1 ACROSOME REACTION-INDUCING SUBSTANCE OF SEA URCHIN EGG JELLY. K.Mikami, K.Ishihara and K.Schmid. Dept.of Reg.Biol.,Saitama Univ.,Urawa and Dept.of Biochem. ,Boston Univ., Boston. It has been not fully elucidated which component of egg jelly that contains main- ly fucose-sulfate,sialic acid and protein is active for inducing acrosome reaction of spermatozoa. We tried to separate the fucose-rich,sialic acid-rich and protein- rich fractions, and estimate their indivi- dual ability to induce acrosome reaction. Using gel filtration of crude jelly subs- tance on Sepharose CL-4B,three fractions which are fucose-rich,sialic acid-rich , and protein-ricn,respectively,were obtain- ed. Only the fucose-rich fraction which contained also protein was active for acr- osome reaction, while both sialic acid- rich and low molecular weight protein fra- ctions were inactive. All of tnree sialic acid fractions separated by DEAE-Sephadex A-25 were also inactive. Both crude jelly substance and fucose fraction which conta- ined protein lost the activity by the tre- atment with pronase at 37°C for 448 hrs, suggesting that the activity resides in protein part of polyfucose-protein complex. To eliminate the sugar moiety of the com- plex, P-elimination of crude jelly subst- ance and fucose fraction were carried out and the resulted materials were separated by gel filtration on Sepharose CL-4B. Bio- logical activity remained in fucose frac- tion with protein and in protein fraction. 918 Developmental Biology DB 2 ACROSOME REACTION-INDUCING SUBSTANCE (ARIS) IN THE JELLY COAT OF ASTERIAS AMURENSIS. T. Matsui, I. Nishiyama and M. Hoshi. Dept. of Biol., Nagoya Univ. Nagoya Acrosome reaction inducing capacity of the egg jelly has been attributed to two components; a sulfated glycoprotein (ARIS) and its cofactor (Co-ARIS). In normal sea water (10 mM Ca2+), ARIS requires Co-ARIS for its full activity, while in sea water enriched with Ca2+(50 mM) it induces acro- some reaction even inthe absence of Co-ARIS, Once spermatozoa were treated with jelly, or either or both of ARIS and Co-ARIS under improper conditions for inducing acrosome reaction, they never underwent the acrosome reaction even after the conditions were im- proved. Pronase digested ARIS (P-ARIS) re- tains the capacity of inducing acrosome re- action in the presence of Co-ARIS or 50 mM Ca2+,. Spermatozoa which have been treated with P-ARIS in normal sea water never under- go the acrosome reaction even after the addition of whole jelly or Co-ARIS, or Ca2+, Treatment of spermatozoa with either ARIS or Co-ARIS seems to cause an irreversible change of the cells. Spermatozoa pretreated with heterologous P-ARIS or various sac- charides still respond to the jelly, ARIS- Co-ARIS, or ARIS-50 mM Ca2+, Periodate ox- idation of P-ARIS destroyed its capacities of inducing acrosome reaction under proper conditions and of causing an irreversible change under improper conditions, which suggests the importance of saccharide chain(s) for the biological activities. DB 3 CO-ARIS, A COFACTOR FOR ACROSOME REACTION- INDUCING SUBSTANCE, IN THE JELLY COAT OF ASTERIAS AMURENSIS. I.Nishiyama, T.Matsui and M.Hoshi. Dept. of Biol., Fac. of Sci., Nagoya Univ., Nagoya Two components in the egg jelly are re- guired for full induction of the acrosome reaction in the starfish, Asterias amuren- sis ;a sulfated glycoprotein(ARIS) and a dialysable factor(Co-ARIS). ARIS obtained by gel filtration has only a low activity to induce the acrosome reaction. This ac- tivity was greatly enhanced by the addition of Co-ARIS which has no capacity to induce the acrosome reaction by itself. Similar factors were also found in the egg jelly of another starfish and sea ur- chins. The action of Asterias Co-ARIS was species-specific, whereas Asterias sperm respond to Co-ARIS from other animals. Co-ARIS was partially purified from the Asterias egg jelly by using Sep-Pak(C,,) and DEAE-Sephadex A-25. Co-ARIS was heat- stable but was susceptible to the treatment with 1N HCl or NaOH. Neither Pronase E nor any of glycosidases so far tested diminish the activity. The spermatozoa pretreated with Co-ARIS did not undergo the acrosome reaction even after the addition of ARIS or egg jelly. If Co-ARIS was added to the spermatozoa that had been treated with ARIS, no additional acrosome reaction was induced. Therefore Co-ARIS should simulta- neously be added to spermatozoa for induc- tion of the acrosome reaction. DB 4 RESACT: A SPERM ACTIVATING PEPTIDE OBTAINED FROM ARBACIA PUNCTULATA EGG JELLY. N. Suzuki#, H. Shimomura* & D.L. Garbers* #) RI Lab., Teikyo Univ. School of Medicine *) Dept. of Pharmacology, Vanderbilt Univ. School of Medicine. A peptide(resact) associated with the eggs of the sea urchin, Arbacta punctulata, which stimulates sperm respiration rates by 5-10 fold, was purified and its amino acid sequence was determined. The sequence was found to be Cys-Val-Thr-Gly-Ala-Pro-Gly- Cys-Val-Gly-Gly-Gly-Arg-Leu-NH?. Resact did not cross-react with sperm cells obtained from Lytechtnus pictus or Strongylocentro- tus purpuratus ; speract which was contain- ed in egg jelly of Hemicentritus pulcherr- tmus and S. purpuratus did not stimulate A. punetulata spermatozoa. Resact caused an apparent shift in the molecular weight (160,000 to 150,000) of a major sperm plasma membrane protein; as with cyclic GMP elevations, this response was evident at extracellular pH values of both 6.6 and 8.0. The protein exists in the cell as a phosphoprotein and 32P is released coincident with the molecular weight change. Resact caused the apparent molecular weight conversion within 5 s and appeared to do so in an irreversible mann- er. The membrane protein appears to be the enzyme guanylate cyclase. DB 5 ADENOSINE RELIEVES SEA URCHIN EGGS AND EMBRYOS FROM DEATH, K,Aketa. Akkeshi Marine Biol,Stat., Hokkaido Univ, ,Akkeshi,Hokkaido When spawned eggs of the sea urchin, Strongylocentrotus intermedius,are kept in sea water containing adenosine, they remain fertilizable even after complete loss of fertilizability of those kept in ordinary sea water, Recovery from decay of fertilizability by aging also is ob- served by administration of adenosine in many cases,but revival is not always suc- cessful probably by over-aging or by un- known factor(s). Abnormal cleavage and development caus- ed by over-maturation,inherent factor of fertilized spermatozoon* or by artificial factors can well be overcome by adminis- tration of adenosine. Adenosine can not be replaced by guano- sine, Adenine is toxic,causing morbid de- velopment such as exogastrulation, Even if increase in cAMP level of eggs and embryos may occur by adenosine,it seems not relate directly to the improve- ment of development, Unlike cAMP,adenosine does not accelerate development of St. intermedius, *High percentage of abnormal cleavage was observed in eggs from several females in- seminated by sperm from one and the same male, Developmental Biology 919 DB 6 INTRACELLULAR CA** RELEASE AND MORPHOLOGI- CAL CHANGES UPON FERTILIZATION IN EGGS OF THE TELEOST, ORYZIAS LATIPES. Y. Hiramoto!’?, Y. Yoshimoto*, and T. Iwa- matsu*. ‘Biol. Lab., Tokyo Inst. of Tech- nol., Tokyo, *Dept. Cell Biol., National Inst. for Basic Biol., Okazaki and *Dept. Biol., Aichi Univ. Educ., Kariya. Intracellular Ca” release upon fertili- zation in the egg of the teleost, Oryzias latipes was determined by the luminescence of aequorin which had been microinjected into the egg. The luminescence was rec- orded with a video-intensified microscope consisting of a microscope, an ultra-high sensitivity TV-camera with an image proc- esser (C-1966-20, Hamamatsu Photonics K.K), an ordinary TV-camera, and a video-tape recorder. The luminescence increase indicating Ca** release in the egg cytoplasm was ini- tiated at the site of sperm entrance 20-30 sec after the entrance of a spermatozoon into the micropile, and the luminescence wave propagated along the egg cortex to- ward the vegetal pole of the egg with a velocity of about 10 um/sec. By comparing microscopic images by luminescence and by transmitted light recorded in the same egg, it was concluded that the breakdown of cortical alveoli started at the site of sperm entrance about 20 sec after the start of Ca” release and propagated along the cortex with a velocity similar to the propagation velocity of the Ca** wave. DB 7 MICROINJECTION OF DIVALENT CATIONS AND CORTICAL REACTION IN ORYZIAS EGGS. T. Iwamatsu, K. Onitake, Y. Yoshimoto and Y. Hiramoto. Dept. Bicl., Aichi Univ. Edua, Kariya-city, Biol. Lab., Nagoya Univ.Coll. Med. Technol., Nagoya, Basic Biol. Instit., Okazaki-city, and Biol. Lab., Tokyo Inst. of Technol., Tokyo. Cortical reaction of Oryzias eggs ina CaeMg-free saline was examined by microin- jection of several divalent cations and aequorin. Cac¢*, Bact, Sr@+(0.5 mM) micro- injected into the cytoplasm elicited a transient increase in intracellular Ca°* which was propagated throughout the whole cortical cytoplasm. This increase began less than & sec after injection of each di- valent cation, followed by cortical vesicle | isp aemge Microinjection of Mg into each blastomere at 8-cell stage. The embryos were allowed to develop to stage 24 tail bud, then fixed, and frozen serial sections were made. We have succeeded in defining all 4 kinds of clones in tail bud. Our results are for the most part consistent with earlier works with vital dye mapping or time lapse video observation. One of new findings is that whole notochord consists of the progeny of animal-dorsal micromeres which contribute to the deep marginal zone in stage 10 early gastrula. Clonal organization in somites was also defined for the first time. Innermost region at the level of notochord is occupied by the progeny of animal-dorsal micromeres. Outside this area, progenies of vegetal-dorsal and animal-ventral blastomeres are located. Vegetal-ventral progeny occupies the outermost region of the somites and continues to the lateral plate. This organization is easily understood if the process of somitogenesis is taken into account. Further observa- tions will show how presumptive mesoderm is organized into notochord or somites. DB 12 DELAYED FERTILIZATION OF XENOPUS LAEVIS AND DEVELOPMENT OF PRIMORDIAL GERM CELLS. M. Wakahara#tT A. W. Neff? and G. M. Mala- cinski€ Zool. Inst., Hokkaido Univ., Sap- poro? and Dept. Biol., Indiana Univ., Bloomington, IN. USA? Unfertilized Xenopus eggs were maintain- ed in a physiological saline at 10 C, and then inseminated in a delayed fertiliza- . tion (DelF) condition. Several features of the development of the DelF eggs were described. The frequency of normal deve- lopment varied according to the length of time eggs were maintained in the DelF con- dition. Various developmental abnormali- ties were observed during gastrulation, neurulation, and organogenesis. Most ab- normalities appeared, however, to be rela- ted to morphogenesis of the endoderm. Primordial germ cell (PGC) development was examined in DelF eggs which displayed normal external morphological features at swimming tadpole stage. PGC counts varied considerably in short-duration DelF from tadpole to tadpole, but substantially re- duced or completely diminished in longer- duration DelF tadpoles. The structural integrity of the germ plasm was disorder- ed. During cleavage, the germ plasm was not segregated into the endoderm as dis- crete islands. Rather, it streaked into the internal cytoplasm. That observation May account for low PGC counts in DelF tadpoles. DB 13 A POSSIBILITY IN TRANSFORMATION OF SOMATIC CELL INTO PGCS IN XENOPUS, WITH A MICRO- INJECTION OF 20,000g PELLET OF VEGETAL POLE CYTOPLASM. K.Ikenishi, T.Okuda and S.Nakazato. Dept. Ose IstOILG p WES Ole Sel, 5 OSalkel Gira Vinay. Osaka. A lot of works suggesting that "germ plasm" plays an important role in the dif- ferentiation of PGCs in anura have been done so far. However, there is no work to show that germ cell determinant resides in the "germ plasm". To clarify whether germ cell determinant exists in the "germ plasm" 20,000g pellet of vegetal pole cytoplasm, probably including the "germ plasm", from 2-cell stage embryos was microinjected in- to somatic blastomeres isolated from Xeno- pus 32-cell stage embryos. The blasto- meres were cultured with tritiated thymi- dine until the normal control embryos deve-+ loped to neurula stage. The labeled ex- plants were implanted into unlabeled host neurula embryos, which were allowed to de- velop to tadpole stage. The labeled PGCs were found in the genital ridges of the ex= perimental tadpoles, while any labeled PGC was not seen in the control tadpoles, into which the labeled explants from non-injec- ted blastomeres had been implanted. fThese facts lead us to conclude that 20,000g pe- llet of vegetal pole cytoplasm can make transform somatic cells into PGCs or it contains germ cell determinant. Developmental Biology 921 DB 14 MONOCLONAL ANTIBODY AGAINST VEGETAL POLE CYTOPLASM OF XENOPUS 2-CELL STAGE EMBRYOS. S.Nakazato, T.Okuda and K.Ikenishi. Dept. Senos, Frac.) of Scis, Osaka City Univ. Osaka. It is well known in anuran amphibians that "germ plasm" plays an important role in the differentiation of primordial germ cells. ricted in polar plasm are confirmed to be composed of unique, basic protein of 95K dalton. contains germinal granules which are cor- responding to the polar granules, we tried to prepare monoclonal antibody using par- tially purified vegetal pole cytoplasm, probably including the "germ plasm", as an antigen, i.e., to remove common proteins in both animal and vegetal hemispheres, 20,000g pellet of vegetal pole cytoplasm of 2-cell stage embryos prepared after Wa- kahara (1978) was immunoabsorbed with the rabbit antibody against the 20,000g pellet of cytoplasm in animal hemisphere of Xeno- pus. More than 300 hybridoma culture me- dia (antibodies) were checked by indirect immunofluorescent staining for sgqash prepa- rations both of the blastomeres with and without the "germ plasm". both blastomeres and some didn't react at all. Only one antibody was confirmed to react to the region, which seemed to be a region of the "germ plasm", in the blasto- meres with the plasm, and not to react to the blastomeres without the plasm. DB 15 ELECTRON-MICROSCOPICAL OBSERVATION OF CELL ORGANELLE DURING THE DEVELOPMENT OF Xenopus laevis. M.Asashima and Ze eouseaki o be ee BON. Yokohama City Univ. Yokohama., Dept.Anat. Saitama Med.School.Saitama. Full grown oocyte yolk platelets were found to contain heterogeous components such as a main body and certain dense layers. During the morula and blastula stages, yolk degradation was observed to occur in the superficial layer, but its extend and characteristics were not as easily discernible as in the case of other amphibian embryos. The number of r-ER increased gradually following cleavage to the early gastrula stage near the animal pole. The r-ER of Xenopus laevis embryos were free from yolk platelets and of course did not complex with yoik platelets and mitochondria, as in the case of Cynops pyrrhogaster. At the early gastrula stage, r-ER in the outer layer were twice as many as in the inner layer, but the number of mitochon- dria in these layers and on the ventral and dorsal sides of the embryo was essentially the same. The endoderm and endo-mesoderm areas contained only a few mitochondria, r-ER and Golgi bodies, but were rich in lipid drops and yolk granules. The number and characteristics of Xenopus cell organelles were compared with those of other amphibian embryos. In Drosophila, polar granules rest- Since the "germ plasm" in Xenopus Some reacted to DB 16 PIGMENT GRANULE ACCUMULATION AROUND NUCLEI IN EARLY EMBRYO OF AMPHIBIA (2) Y.Kobayakawa. Biol. Lab., Col. of genl. ed., Kyushu Univ., Fukuoka It was reported previously (1983) that pigment granules accumulate around nuclei. with the advance of development during . cleavage period in some species of amphibian embryos (Hyla arborea, Rana rugosa, Rana japonica and Bufo bufo japonics). And it was discussed _ that this phenomenon suggests the general existence of intracellular motility toward nuclei in blastomeres of early amphibian embryos. In this time, this phenomenon was observed in detail with a dark field microscope. And to investigate whether the accumulation of pigment granules around nuclei depends on microtubules or microfilaments, effects of microtubule inhibitors (colchicine and podophyllin) and of microfilament inhibitor (cytochalasin B) were studied. The results of this study (colchicine and podophyllin suppressed this phenomenon, Duce eyeochalasiny dud "inot) suggest that similar to intracellular motility in melanophore, erythrophore and other cultured cells (Beckerle & Porter,1983; etc.) the accumulation of pigment granules around nuclei (migration of Pigment granules toward nuclei) does not depend on microfilaments but on microtubules mainly. DB 17 SCANNING ELECTRON MICROSCOPIC OBSERVATIONS ON SPERM PENETRATION IN THE NEWT. K. Onitake! and T. Iwamatsu*. 'Biol. Lab. Nagoya Univ. College of Med. Technol., Nagoya. “Dept. of Biol., Aichi Univ. of Hdtw, ADehies The process by which a spermatozoon penetrates into the egg cytoplasm in the newt, Cynops pyrrhogaster, was examined with scanning electron microscopy. The vitelline envelope was removed from egg surface in order to observe the early 4 events of sperm-egg plasma membrane inter + action. An acrosomal cap of the spermato- zoon passed through the vitelline enve-» lope disappeared. A spermatozoon effectively to the egg plasma membrar about 1 minute after insemination. I initial step of penetration, the zoon attached obliquely at its +p +4 a4 a (perforatorium region) membrane. A blister-like cone with a microvillus-f1 peared on the egg surface penetrating sperma was engulfed slowly face without microvi disappeared before +he Parts —p ae Bi sil : 4+asnar 9?? Developmental Biology DB 18 PROPERTIES OF FERTILIZATION POTENTIAL OF AMPHIBIAN EGGS IN RESPONSE TO NEWT SPERM. Y. Iwao! and K. Onitake*. ‘Dept. Biol., Fac. Sci., Yamaguchi Univ., Yamaguchi. 2Biol. Lab., Nagoya Univ. College of Med. Technol., Nagoya. To understand mechanisms of electri- cally mediated block to polyspermy in amphibian eggs, we recorded fertilization potentials in mono- and polyspermic ferti- lization. Fully jellied eggs of Cynops pyrrhogaster generated several recurring hyperpolarizations (2-10 mV) upon insemi- nation with homologous sperm in 1/10 Steinberg's solution (SB). When dejellied eggs were inseminated in 1/10 SB, a large amplitude of hyperpolarization (40-50 mV) was observed, regardless of egg-activation or fertilization. This potential was mainly dependent upon concentration of sperm- suspension and external concentration of monovalent cations. The supernatant of sperm-suspension at 3,000 rpm for 15 min had no effect on egg-membrane potential. The eggs of Xenopus laevis inseminated with Cynops sperm underwent a gradual hyperpolarization from -10 mV to -35 mV in 1/10 SB, followed by a rapid depolariza- tion to +30 mV. The former was,mediated by increased permeability to Na , whereas the latter was mediated by Cl-permeability, These results suggest that the hyper- polarizations observed in eggs of both species were mediated by a specific factor of sperm plasma membrane, rather than by egg-activation. DB 19 A MORPHOLOGICAL ANALYSIS OF SURFACE CON- TRACTION WAVES IN COLCHICINE INJECTED EGGS OF JAPANESE NEWT. T. Esaka. Biol. Lab., Science Education Inst. of Osaka Pref., Osaka. Surface contraction waves (SCWs) in eggs of Cynops pyrrhogaster is more distinctly observed than that in normal eggs. Eggs were placed on an elliptic shallow dimple on agar plate, and were injected with 60 nl of colchicine solution (16 mg/ml modi- fied Steinberg's solution). They were fix- ed in Bouin's fixative during passage of SCWs along the egg surface of animal hemi- sphere which was monitored by time-lapse VTR. Serial sections were made and stained with azan method. An area of yolk-free cytoplasm stained in light blue was observed in the corti- cal layer of the egg fixed when the SCW-1 (bright wave) was passing along the egg surface. The area corresponded to the propagation of SCW-1. Occasionally the SCW-1 started from two points of the egg surface at the same time, and the bright area of dumbbell shape was thus formed. In such cases, the distribution of the yolk-free cytoplasm was conformed to the dumbbell shaped area. It is indicated that the appearance of SCW-1 is due to the accumulation of yolk-free cytoplasm in the egg cortex. DB 20 CLEAVAGE CYCLE IN NEWT EMBRYOS IS COMPLETED BY MODURATING THEIR CYTOPLASMIC CYCLE. Shinagawa,A., Nakatsuka,M., and Nonaka,M. Dept. Biol., Fac. Sci., Yamagata Univ. Uncleaved eggs of Cynopus pyrrhogaster were bisected by placing a fine glass rod on the boundary line between the animal and vegetal hemispheres immediately after the eggs had been rotated 90° off the vertical axis. The resulting vegetal halves, injected with colchicine, show much slower cyclic rounding-up and flattening than that seen in the animal partners, prevented from cleavage by colchicine. The ratio of the former periodicity to the latter one is about 1.5 on average. Furthermore, the cleaving vegetal halves without cochicine-injection, occasionally induced by polyspermy, also show slower periodicity than that shown in the cleaving animal partners. The ratio of the former to the latter is about 1.5 on average. The similarity between the both above ratios strongly suggests that the cleavage cycle of newt eggs is completed by modulating ( shortening ) the original cycle in cytoplasm but not newly produced independently of the cytoplasmic cycle. This result is consistant with the previous studies ( Sluder, 1979,1983; Sakai & Shinagawa, 1983; Shinagawa, 1983 Ne DB 21 POSSIBLE ROLE OF CALCIUM ON THE CYCLIC SURFACE CHANGES OF AMPHIBIAN EGGS. M. Sakai. Dept.of Biol., Fac. of Sci., Kagoshima Univ. Kagoshima. Colchicine injected eggs or non- nucleate egg fragments of Xenopus laevis were incubated in the following solutions; 1)50mM NaCl, 2mM CaCl.2, and 2)50mM NaCl, 2mM EGTA. In both solutions, the egg and the egg fragment showed cyclic rounding-up and relaxing movements and surface contraction waves as in Steinberg's solution. On the other hand, microinjection of calcium or EGTA caused drastic changes on the fragment and the cyclic surface changes. When 15-50nl of 100mM CaCl. was injected into the egg or the egg fragment, the cell membrane protruded at that point within 30 sec suggesting that the membrane softened remarkably. Injection of 100mM EGTA(15-50nl1) caused no such rapid changes on the egg or the egg fragment however, it usually caused prolongation of the cytoplasmic cycle. In some cases, the duration of the cytoplasmic cycle became more than twice as long as the original cycle. These results suggest that 1) cyclic surface changes of Xenopus need no influx of external calcium and 2) changes of intracellular calcium may possibly be involved in the cyclic surface changes of Xenopus egg. Developmental Biology 923 DB 22 DB 24 INHIBITORY EFFECTS OF CALCIUM ANTAGONISTS SURFACE MOVEMENT IN THE REGION ALONG THE AND CALMODULIN ANTAGONISTS ON THE CLEAVAGE CLEAVAGE FURROW IN NEWT'S EGGS DURING IN SEA URCHIN EGGS. 2 THE FIRST CLEAVAGE. E. Tatara’,,A.,Fujiwara’, Y. Fujino” and T.Sawai. Dept. of Biol., Fac, of General I. Yasumasu’. Dept..,of Biol., Sch.of Educ. Education, Yamagata Univ., Yamagata. Waseda Univ. Tokyo. “Dept of Pharma., Teikyo Univ., Sch. of Med., Tokyo. In the first cleavage stage in eggs of the newt, Cynops pyrrhogaster, surface movement was ob- The lst cleavage in sea urchin eggs, served to examine the mechanism of formation of Hemicentrotus pulcherrimus, was inhibited the unpigmented pale surface which expanded along by calcium antagonists, verapamil and dil- both sides of a deepening furrow, by a method tiazem, which were added within 30 min marking the egg surface with small carbon parti- after insemination, and marked delay of cles. From the behavior of the particles, the the lst cleavage (at least 4 hr delay) process of the pale surface formation could be occurred when they were added between 30 divided into three phases. The first phase was min and 80 min after insemination. By add- a period of a sudden appearance of the pale sur- ing these compounds during the period of face in both sides of the furrow at the early first cleavage, further cleavage did not occur at cleavage stage, which was brought about by an least 2 hr from the time of the additions. extreme expansion of a bit of pigmented surface DNA synthesis before the first cleavage was between double strands of the initial furrow. not inhibited by verapamil or diltiazem, The second phase was characterized by a gradual when they were added at times between fer- expansion of the pale area from its appearance to tilization and just after the first cleav- a maximum growth. The surface growth in this age. By adding these compounds about 10 stage was largerin the region close to the furrow, min after the end of the first cleavage, but less in the region distant from it. In the DNA synthesis occurred but the second last phase, the pale surface further grew at the cleavage was inhibited. W-7 caused a delay site adjacent to the furrow but diminished at the of egg cleavage and reduced the rate of D A opposite site adjacent to the pigmented surface. synthesis, but W-5 did not. Change in Ca Therefore, this period was apparently a steady concentration and calmodulin dependent re- state with little change in the area of the pale actions probably support, even though in- surface. These observations might conclude that directly, egg-cleavage and DNA synthesis. the pale surface was initially formed by the expan- sion of the pigmentec surface of the furrow region, but its succeeding growth was brought about by a membrane addition at the bottom of the furrow. DB 23 DB 25 PROCESS OF CLEAVAGE IN THE EGG OF MEDAKA, ELECTRON MICROSCOPIC ANALYSIS OF THE MALE ORYZIAS LATIPES PRONUCLEUS FORMATION IN THE BRITTLE-STAR Seiko Nakashima. Lab. of Freshwater Fish (AMPHIPHOLIS KOCHII) EGGS. Stock, Nagoya Univ., Nagoya. MO Yanashita. Zool. Inst., Fac. of Sci., oe Se ee ee ee eee Hokkaido Univ., Sapporo. The process of cleavage of the normal egg in medaka, Oryzias latipes was examin- ed and compared with that of neutral red (NR) stained eggs. Unfertilized eggs were stained with 0.001% NR solution and then fertilized. During the early cleavage stage, both sides of the cleavage plate were homogene- ously stained and NR-stained granules were localized in the cytoplasm adjacent to the yolk region. In normal eggs, the protrusion of yolk is formed adjacent to the central region of the blastodisc in correspondence with the cell cycle. In NR-stained eggs, how- ever, the protrusion of the yolk was en- larged and the blastomeres became flatten- ed and separated each other. Sometimes cytokinesis was obstructed. In the both normal and NR-stained eggs, the cortex of The male pronucleus formation of the brittle-star was analyzed with transmis- sion electron microscope both under normal and experimental conditions. A rapid sperm chromatin dispersion before the nuclear envelope formation (the first dispersion) and a slow chromatin dispersion on and after the formation (the second disper- sion) were observed in the normal eggs. When meiosis was inhibited with colchi- cine, the first dispersion occurred on schedule but the nuclear envelope was not formed and the second dispersion was also inhibited. When spermatozoa were intro- duced into the germinal vesicle-intact oo- cyte, the sperm chromatin did not disperse at all. Treatment of the spawned eggs at the first meiotic metaphase with cyclo- heximide had no effect on the pronucleus s +hoa + ’ the furrow region was thicker than other formation. These results re egy tH: region and a meshwork of microfilaments male pronucleus formation of tI eh pete was observed there. star consists of at ROBAt sale gis se These results suggest that the pro- processes, the aoe oese_suLepend’s ed eta trusion of yolk region may play some im- (the first See hia sbcend diececatael, portant roles in the early cleavage and dependent process A ng pcg 1 fest neutral red may disturb the formation of a een Poel le te “eid protrusion of yolk and it results in ir- sctite y 2p orerlagelrts Pp aS eaakhte regular cleavage or obstruction of cyto- cytoplasm after the g: re a ae Maes breakdown. kinesis. 924 Developmental Biology DB 26 APPEARANCE AND BEHAVIOR OF THE SUBCORTICAL FIBER STRUCTURE FOLLOWING CORTICAL CHANGE OF THE SEA URCHIN EGG. M.Ishikawa and K.Kanatani. Dept.of Biol.,Fac.of Sei.,Ehime Univ., Matsuyama. When sea urchin eggs were immeresed in 4% hexyleneglycol-sea water soon after ertilization, the fibrous structure mainly composed of microtubules appeares beneath the egg cortex. However, this subcortical fiber(SCF) does not appear after onset of nuclear division. To sutdy on the relation between the formation of SCF and cortical change, and onset of nuclear division, the following experments were performed. (1) When eggs were pretreated with procain and then inseminated, or artificially activated by procain and ammonia, cortical change was Suppressed. SCF appeared as similar to that of fertilized eggs. (2) When eggs weretreated with cycloheximide and emetine after fertilization, nuclear division was ceased at streak stage. SCF was also formed. (3) When eggs just after insemi- nation were put into Nat-free sea water, SCF did not appear even though the eggs have not cortical granules. Afterward, many cytospots with radiated microtubules were formed. The results show that both breakdown of cortical granules and onset of nuclear division do not correlate to SCF formation DB 27 CYCLIC AMP ACCELERATES ELONGATION OF ASTRAL RAY OF MITOTIC APPARATUS OF SEA URCHIN EGG. “A. puzuki, **i.Yasumasu and ***kK i sinmday. *Cnt. eS. Lab., Meiji Seika, Yokohama, wIDisjore, WBaloll,- {SClaic Ichwic,, Waseca Walw.> 5 INOI 0) eravol IDS OiG5 Wille, SCla_o MOC, , Wowkyo 1onophosphate (cAMP) shortened clea- ILS Gi SSE Wlieelailin Sgeso Siimee wails ide hadynowacceleiracicme mic CicOm ocity of DNA synthesis, the cleavage I(ojal \iels) CUS 1) ZL MOCWISi~ICEMm) Oi iM AS was reported previously, adenosine Cc n order to elucidate how the M phase was reduced by cAMP, we succesSively observ— intracellular morphological change in urchin eggs (Strongylocentrotus nudus) g the first cleavage cycle using a differential interference microscope equi- pped with Nomarsky prism. Disappearance of nucleus membrane occurred with no signifi- Cant difference at 57 min after fertiliza— tion both in the control eggs and in the experimental eggs treated with 1 mM cAMP. However, interval between the disappearance of nucleus membrane and the occurrence of cleavage furrow was 24.6min in the control while that of the experimental group was 16.8min. Microscopical observation revealed that astral rays of mitotic apparatus of the experimental eggs elongated at higher velocity than those of the control. These results suggest strongly that cAMP ICC Coed the elongation of astral rays of nitotic apparatus of the sea urchin eggs. an oo i) c ty pb. =] sad) (@) DB 28 TWO SURFACE REGIONS OF SEA URCHIN BLASTO- MERES APPEARED AFTER CLEAVAGE. T.Yazaki., Dept. Of Biol yeehace mOtmes Cheer Tokyo Metropolitan Univ., Tokyo. Sea urchin blastomeres have two surface regions, one of which consists of the membrane to which an antibody against the egg surface(anti-ES) binds, the other con- sists of the membrane to which anti-ES does not bind. Uncleaved fertilized eggs of H. pulche- rrimus was labelled with FITC-anti-ES which had been previously digested into monovalent, and let to be cleaved in nor- mal sea water. Fluorescence covered all over the sur- face of just divided daughter blastomeres. At that time, an increase of the surface area of each blastomere had completed. 8 or 4 min after the lst or 2nd cleavage respectively, a dark area(new surface area) appeared in the furrowed region. The new surface area continued to enlarge for a several minutes accompanying by a decrease of the anti-ES-binding surface region. The movement of the anti-ES-binding sur- face was independent of the hyaline layer resided on the surface of eggs. The new surface area WaSostulated to be a membrane remained after a retreat of the anti-ES-binding surface rather than an internalization of the anti-ES-binding Sites in that region. DB 29 DEVELOPMENTAL FATE OF THE VEGETAL POLE CELLS IN SEA URCHIN EMBRYOS. S.Amemiya. Misaki Marine Biol. Station Univ. of Tokyo. Kanagawa. The developmental fate of the vegetal pole cells in the sea urchin embryos cul- tured in normal sea water (NSW) or in Ca-low sea water (CLSW) was examined by scanning electron microscopy (SEM). Four species, Hemicentrotus pulcherrimus, Arbacia lixula, Anthocidaris crassispina and Paracentrotus lividus were used as materials. Ca-low treatment was performed as follows. The embryos of H. pulcherrimus cultured at 20°C were transferre rom NSW to CLSW (1 vol of NSW and 19 vol of Ca''-free sea water) at hatching blastula stage. This treatment brought about exo- gastrulation at later stage. SEM observa- tions on the embryos cultured in CLSW as well as in NSW revealed that a few cells remain in the vegetal pole region after the releasing of primary mesenchyme cells into blastocoel. The numbers of the cells were estimated to be four in some species (H. pulcherrimus and A. lixula) and to be eight in others (A. crassispina and P. lividus). It was suggested from SEM observations on the early developmental course of normal embryos and on the process of exo-gastrulation that the cells remaining in the vegetal pole without releasing into blastocoel are the descend- ant of smaller micromeres, and that the cells differentiate into pharynx. Developmental Biology 925 DB 30 VISUALIZATION OF ACTIN FILAMENTS IN SEA URCHIN EGGS WITH NBD-PHALLACIDIN Y. Tanaka and H. Sato, Sugashima Marine Biol. Lab., Nagoya Univ., Toba, Mie 517 NBD-phallacidin (NBD-ph.) is a fluores- cent probe specifically bound to F-actin, so is suited for the staining of actin fil- aments only. Formalin-fixed or saponin-per- meabilized eggs of the sea urchin, Antho- cidaris crassispina were stained with NBD- ph. Unfertilized eggs had a number of micro- villi 1-2 am long which were stained with NBD-ph. Microvilli of fertilized eggs were preserved and well stained in permeabiliz- ed eggs, however, were shortened and less stained in formalin-fixed eggs. Contractile ring (CR) of sea urchin eggs which con- stricts the cytoplasm in cytokinesis and May be consisted of actin filaments was visualized in formalin-fixed eggs, but not in permeabilized eggs. Actin filaments were circumferentially arrayed in about 8 ym width within the cortex of furrow region. CR was already formed when eggs slightly elongated towards the poles (the initia- tion of furrowing). When blastomeres ad- hered tightly each other in the inter- kinetic phase, the fringe of the adhering area was well stained with NBD-ph. However the adhering area itself was less stained than the other area. This observation assumes that the concentration of micro- villi from the adhering area to the fringe occurs. DB 31 THE ROLE OF MICROTUBULES IN PRONUCLEAR MIGRATION IN SAND DOLLAR EGGS ANALYZED BY UV-COLCEMID METHOD. M. S. Hamaguchi'and Y. Hiramoto??. Biol. Lab., Tokyo Inst. of Technol., Tokyo, and *Dept. Cell Biol., National Inst. for Basic Biol., Okazaki. The formation and migration of the sperm aster and the migration of male and female pronuclei during fertilization were investigated in eggs of the sand dollar, Clypeaster japonicus using Colcemid-UV method. When the egg in sea water con- taining Colcemid was irradiated with UV light about 365 nm wavelength at a limited region containing sperm centriolar region, a sperm aster formed and migrated to the center of the UV region, until finally it occupied the entire UV region. When the UV region was displaced or the shape of re- gion was changed after the formation of the sperm aster, its center migrated to the new UV region. When a region containing both the centriolar region and the female pronucleus was irradiated with UV-light, the female pronucleus migrated to the as- tral center after some astral rays reached it. In any cases, the male pronucleus mi- grated accompanying the center of the sperm aster. It was concluded that the female pronucleus migrates to the astral center along the astral rays and that the sperm aster and the male pronucleus move in the egg cytoplasm by forces generated by astral rays radiating into the cytoplasm. DB 32 THE MITOTIC APPARATUS WITH UNUSUALLY MANY MICROTUBULES FROM SEA URCHIN EGGS TREATED BY HEXYLENEGLYCOL S.Endo, M.Toriyama and H.Sakai. Dept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo The effects of hexyleneglycol on the structure of the mitotic apparatus of the sea urchin eggs were studied by electron microscopy. The mitotic apparatus was found to become remarkably decorated with unusually many astral microtubules, which were uniform in length conspicuously at prometaphase and metaphase. These micro- tubules appeared to be associated with the granular materials which are most likely microtubule organizing centers. When treated at metaphase, many miniasters were appeared throughout the cytoplasm and small dots of the granular materials were in the center of each miniaster. When treated at anaphase, numerous spindle microtubules were newly formed, which were uniform in length but astral microtubules were not. When treated at telophase, a large monaster was found to grow from the clusters of granular materials which re- mained in the vicinity of each daughter nucleus. Chromosomes were seen to have no connection with the spindle microtubules. In conclusion it is suggested that hexy- leneglycol disperses and reactivates the clusters of granular materials to enhance the microtubule organizing activity. DB 33 DISTRIBUTION OF FLUORESCENTLY LABELED TUBULIN INJECTED INTO SAND DOLLAR EGGS DURING CLEAVAGES. Yukihisa Hamaguchi!,Masaru Toriyama’, Hikoichi Sakai? and Yukio Hiramoto!}’, iBiol. Lab., Tokyo Inst. of Technol., Tokyo, *Dept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo, and spapt., of ‘Cell. Biol.,; Natl. Inst. for Basic Biol., Okazaki. When porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was injected into fertilized eggs of the sand dollar, Clypeaster japonicus, the tubulin was guickly incorporated into astral cent- ers. Upon reaching metaphase, the labeled tubulin accumulated into the mitotic appa- ratus and later dispersed during inter- phase, which repeated at successive cleav- ages. Fluorescent fibrous structures of the mitotic apparatus were coincided with the structures observed by polarization and differential interference microscopy. The tubulin was incorporated into the mito- tic apparatus within 30 sec when injected at metaphase or anaphase, suggest y that the equilibrium between microtubules and tubulin is attained very rapid n the mitotic eggs. FITC-labeled tubulin of starfish sperm flagella was 9 incorpo- rated into microtubular st res, These results suggest that FITC- heteroge- neous tubulins undergo spat and stage- specific regulation of ass y-disassembly in the same manner a tubulin. 926 Developmental Biology DB 34 FURTHER DEVELOPMENT OF THE EQUAL-DIVIDED BLASTOMERES OF THE SEA URCHIN EMBRYOS. M. Miura and J. Tsukahara. Dept. of Biol., Fac. of Sci., Kagoshima Univ. Kagoshima Micromere formation is inhibited by the equal cleavage if the embryos are pre- treated with SDS (TANAKA,1976). To study the later development of these equal divided cells, they were dissociated into single cells at 16 cell stage and cultured Hm \Walercto}— ~ Gametes of H. pulcherrimus and A. crassispina were used as materials. Denuded eggs were treated with SDS at 4 cell stage and dissociated into single cells. When 1/4 embryos developed into 16 cell stage, they were re-dissociated into 4 single cells by glass needles. 1/16 embryos were cultured in natural sea water containing horse serum. 1/4 embryos performed horizontal equal Givision at 4th cleavege. 2 animal blastomeres of 1/4 embryos developed equally into animalized blastulae with many long cilia. In many cases, 2 vegetal blastomeres of 1/4 embryos developed into the different embryos each other, e.g. blastula and migrating cells, or gastrula and migrating cells, which formed spicules or not. These results suggested that in most experiments vegetal pole cytoplasm of SDS treated blastomeres may be unequal distribution at 4th cleavage in spite of horizontal equal division. DB 35 DEVELOPMENT OF BASAL LAMINA IN SEA URCHIN EMBRYO. Y. Nakajimal and Y. D. Noda2. 1Dept. Biol., Keio Univ., Yokohama and 2Dept. Biol., Ehime Univ., Matsuyama. Development of basal lamina(BL) in sea urchin embryo was studied by electron mi- croscopy. Embryos of Mespilia globulus were fixed in osmic acid followed by tannic acid treatment. Precursor of BL becomes discern- ible at 4th cleavage stage by TEM. At 8-9th cleavage stage, the cell surface facing to blastocoel is lined with BL constructed of discontinuous, branched filamentous struc-— ture decorated with amorphous materials. Fragments of filamentous materials similar to BL are also seen in the blastocoel. Three dimentional structure of BL was ob- served by SEM using the embryos dissected by tungsten needle. At 4th cleavage stage, three dimensional, beaded meshwork is ob- served on cell surface and in blastocoel. The amount of the materials increases with the age, and the diameter becomes thinner finally filling up blastocoel with filamen- tous network. A part of this network adhere closely on the surface of the cells at 8- 9th cleavage stage. Thus, BL of sea urchin is not a contin-= uous sheet of membrane but a network over- laying on the inner surface of the blasto- meres whose structure and the developmental Origin are indistinguishable from the ex- tracellular matrix present in blastocoel. DB 36 INTERVAL OF THE CYTOPLASMIC CYCLE IN NON-NUCLEATE FRAGMENTS OF ECHINODERM EGGS. M. Yoneda and K. Yamamoto. Dept. Zool., Kyoto Univ., Kyoto. Cyclic changes in the cortical tension of activated non-nucleate fragments derived from unfertilized eggs of the sea-urchin, Hemicentrotus pulcherrimus, were compared with the cleavage interval of its nucleate counterpart. The interval of the tension cycle was longer than the cleavage interval of the nucleate partner by about 30 $%. Non-nucleate fragments derived from fertilized eggs still exhibited longer duration of the tension cycle than the cleavage cycle of the nucleate partner. This was also the case with non-nucleate egg fragments of the starfish, Asterina pectinifera. Such a prolongation in the cytoplasmic cycle, originally reported in egg fragments of Xenopus laevis by Sakai & Kubota ('81) and Shinagawa ('83), thus appears to be common among non-nucleate cytoplasm of animal eggs, and points to participation of nuclear events in modulating the autonomous cyclic activity of cytoplasm in normally developing eggs. DB 3/ Occurrence of vimentin associated with primary mesenchyme cell formation in the sea urchin, Anthocidaris crassispina. H. Katow. Biol. Lab., Rikkyo University. Tokyo. Occurrence of vimentin type inter- mediate filament was examined in the embryos at hatched and mesenchyme blastula and early and mid-gastrula stages using monoclonal anti-human fibroblast vimentin mouse IgM and FITC-conjugated anti-mouse IgM rabbit antibodies following fixation in -20°C methanol. Vimentin was detected only in the primary mesenchyme cells obtained from mesenchyme blastulae and early gastrulae, but not these from mid-gastrulae in which these cells forming spicules. Epithelial cells, including blastocoel wall cells at hatched blastula stage possessed no vimentin throughout early developmental stages, at least, up to mid-gastrula stage. Vimentin in primary mesenchyme cells appears to be associated with a spherical cell configuration after ingression, hence after reduction of cell-cell contact, but not directly with cell migration. This work was supported in part by a Grant-in-Aid from The Ministry of Education Science and Culture of Japan #59340047. Developmental Biology 927 DB 38 CYTOSKELETAL FRAMEWORK OF SEA URCHIN EMBRYO. I. MICROTUBULES. I. Uemura. Dept. of Biol., Tokyo Metro- politan Univ., Tokyo. Stage-specific change in distribution pattern of microtubules (mts) is studied by immunocytochemistry. Embryos of the sea urchin, Hemicentrotus pulcherrimus, are treated with isolation medium containing 0.5% Triton X-100 at several stages from hatched blastula to pluteus. Embryos retain their three- dimentional topology following this treatment. After fixation, embryos are incubated in mono-clonal anti-tubulin antibody, then in protein A-colloidal gold. Ectoderm cells of hatched blastula show numerous red lines (color of colloidal gold) running along outer- inner axis of cells with no apparent localization. Electron microscopic ex- amination confirms that these red lines are mts decorated with gold particles. From prism stage on, and most clearly at pluteus stage, however, mts localize in the cells at cilial bands on oral side including four arms and mouth. Few mts are found in other region, ventral and dorsal sides, where ectoderm cells are flattened in contrast to the columnnar cells at cilial bands. This correlation between cell shape and the presence of mts leads to a hypothesis that mts play a role as mechanical support in morpho- genesis. DB 39 EFFECTS OF A SURFACTANT, SODIUM DODECYL SULFATE (SDS) ON SPICULE FORMATION IN SEA URCHIN EMBRYOS. M.Miyagishi and M.K.Kojima. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. Sea urchin embryos were not only con- tinuously exposed to 0.0001-0.001% SDS sea water solutions from various developmental stages but also temporarily treated with 0.001-0.005% SDS solutions. It was found that there are three SDS-sensitive periods as to the spicule formation; that is, 1) the period from the 4-cell stage to the 8- cell stage, 2) the period from the hatching blastula stage to the early gastrula stage and 3) the late gastrula period (the early stage in elongation of triradiate spic- ules). When embryos were exposed to SDS so- lutions at these three periods, both forma- tion and elongation of their spicules were markedly inhibited. In other series of ex- periments, it became clear that SDS can disturb the behaviors and distribution pat- terns of the primary mesenchymal cells as well as the formation of micromeres. From these results, therefore, it may be con- cluded that, at least, three different events at three different developmental periods, (such as occurrence of the micro- mere formation, migration and adhesion of the primary mesenchymal cells, and chages in nature of triradiate spicules at the early stage of their elongation), play impor- tant roles in normal spicule formation of sea urchin embryos. DB 40 ULTRASTRUCTURAL OBSERVATION CONCERNING EFFECTS OF PENICILLAMINE ON SPICULE FORMATION IN SEA URCHIN EMBRYOS. N.Uto, M.Fujie and Y.Muranaka. Dept. of Biology and Central laboratory of Electron Microscopy, Hamamatsu Univ. Sch. of Med., Hamamatsu. We have reported that collagen cross- linking inhibitors such as 8-aminopropio- nitrile and penicillamine(Pen) inhibit embryogenesis especially spicule formation in sea urchin embryos. In this experiment, we investigated migration, arrangement and function for spicule formation in mesenchyme of the embryos treated with Pen after invagina- tian. Failure of primordia formation, retarded growth or deformation of spicule were observed depending on concentration of Pen. In this case, mesenchyme cells responsible for spicule formation do not gather and arrange successfully, as ob- served by scanning electron microscope. Also, fibrous components connecting these cells are poor and fused each other or snapped, whereas hydroxyproline synthesis was not inhibited. However, calcium was detected in mesenchyme cells of embryos treated with Pen, as well as in untreated embryos by X-ray Micro Analysis. In conclusion, mesenchyme cells accu- mulate calcium but can not form spicule successfully due to irregular migration and arrangement. DB 41 ENHANCEMENT OF SPICULE FORMATION AND CAL- CIUM UPTAKE BY MONOCLONAL ANTIBODIES TO FIBRONECTIN-BINDING ACID POLYSACCHARIDE IN MICROMERE-DERIVED CELLS OF THE SEA URCHIN IN VITRO. M. Iwata and E. Nakano. Dept. of Biol., Nagoya Univ., Nagoya. We found a novel fibronectin-binding acid polysaccharide (named FAPS) in the extracellular matrix of sea urchin em- bryos. When monoclonal antibodies to FAPS (anti-FAPS) were microinjected into sea urchin embryos, malformation of spicule occurred. In the present study, we examined the effect of anti-FAPS on differentiation of micromere-derived célis.in vitro: Spicule formation of micromere-derived cells was enhanced by anti-FAPS at a con- centration-dependent manner.The increase of spicule formation was correlated t< the increase of calcium uptake int spi- cules. Furthermore, calcium uptake was inhibited by calcium channel er: Radioautographic studies revealed tiat calcium deposited in spicule: i el] surface regions of micror -derived cells. A core-like struct spicule was observed and cal if accumulated along the core. These results APS, component of the bla xtracellu- lar matrix, may eC} level of calcium uptake and ferentiation of micromere-der 928 Developmental Biology DB 42 EFFECT OF ZINC AND LITHIUM IONS ON CELL PROLIFERATION OF SEA URCHIN EMBRYOS DURING SWIMMING BLASTULA AND _ EARLY GASTRULA STAGE. H. Bugisawa. Bilol, Depe., wasieama, Unaiver, Urawa, Saitama Morphological aspects of sea urchin em- bryos effected by zinc and lithium ions have been well-known, however,quantitative analyses such as strength of cell adhesion or cell number of the embryos treated with these ions have scarcely been undertaken. In this work I have examined the effect of these ions on cell proliferation of embryos of Anthocidaris crassispina and Hemicentrotus pulcherrimus during the Stages from swimming blastula and early gastrula. In both embryos zinc ions did not affect the increase in cell number, while lithium ions suppressed the increase remarkably. I also examined the effect of a protein extracted from blastcoelic fluid Of GAC crassispina embryos ( gift of K. Ishihara, Saitama Univ. ) whick affects embryos at blastula stage to form exogas- trula when embryos are cultured in the presence of this protein. This substance did not affect the cell proliferation. From these results it may be suggested that the cell proliferation in blastula stage does not correlate with formation of archenteron, and the exogastrula effected by a protein extracted from the blast- coelic fluid is not vegitalized ones such as one caused by lithium ions. DB 43 Changes of intracellular localization of the silver stained proteins during early development in Hemicentrotus eulcherrimus. R. M. Amikura. Dept. of Anatomy, Tokyo Women’s Medical College. Tokyo. The previous report on the changes of the argyrophile proteins, visualized as Silver stained granules (SSGs) by NOR- Silver staining, showed that the SSGs were observed in the germinal vesicle of the oocyte but distributed in both the pro- nucleus and cytoplasm in the egg (Amikura and Ihnuma, in press). We further studied the fate of the SSGs in the egg cytoplasm during early development. From the first to seventh division, SSGs are observed in both cytoplasm and nucleus at interphase, in the chromosomal NOR-regions at meta- phase, and in both cytoplasm and chromo- some vesicles at telophase. In the 128- cell stage embryos, SSGs were observed within the nucleus but not observed in the cytoplasm, suggesting that the silver stained proteins in SSGs of the blastomeres are moved to the nucleus before the eighth division. In the sperm cell, the acrosomal region and the basal part of the sperm head were densely stained but not the sperm nucleus by NOR- Silver staining, implying the movement of SSGs of egg cytoplasm to the sperm nucleus during the process of male pronucleus formation. DB 44 PARTIAL PURIFICATION AND CHARACTERIZATION OF THE ACID SEA WATER EXTRACT FROM UNFERTI- LIZED SEA URCHIN EGGS. M.Fujitake, S.Nakamura and M.K.Kojima. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. Previous studies have demonstrated that the acid sea water extract from the unferti- lized sea urchin eggs (egg extract: EE) has the following effects: activation of unferti- lized eggs, acceleration of cleavage, and induction of animalization (Kojima, '69,'71, '73,'74,'77). In this study we have attempted to purify and cahracterize accelerating factors of the cleavage in EE. EE from the unfertilized sea urchin eggs (Anthocidaris crassispina) was concentrated to about 100-fold and applied to ion exchange chromatography on DEAE Sephadex A-50. Samples were eluted stepwise with 0.2M, 0.5M,0.8M, 1.0Mand2.0M NaCl inthe Tris buffer (pH 7.0) and 3.5-ml fractions were collected. Four peaks were detected by absorbance at 280 nm and these fractions were tested for the cleavage accelerating activity.Theiractivi- ties differed to some extent from experiment to experiment, the fraction of the fourth peak (P4) always had the highest activity. The spectrum of ultraviolet absorption showed that P4 fraction had one peak at 260 nm. SDS-polyacrylamide gel electrophoresis of P4 fraction indicated the presence of nucle- otides. These results suggest that the nu- cleotides contained P4 fraction are acceler- ating factors of the cleavage. DB 45 PHOSPHORYLATION OF 15K PROTEIN IS THE FINAL TRIGGER TO INDUCE THE INITIATION OF SPERM MOTILITY IN RAINBOW TROUT. M.Morisawatand H.Hayashit Ocean Res. Inst. Univ. Tokyo, Tokyoland Fac. Sci. Nagoya Univ. Nagoya‘ We have demonstrated that cAMP is the internal factor to induce the initiation of sperm motility. We show here that cAMP de- pendent phosphorylation of a protein with molecular weight of 15 kilodaltons(15K)is the final trigger in the initiation of sperm motility in rainbow trout. The semen was suspended in the extraction medium contain- ing 0.04% Triton X-100 to remove the plasma membrane. The demembranated sperm were re- suspended and incubated in reactivation medium containing [y-32P]ATP and cAMP for one second. Under these conditions ,axonemal movement occurred, and Pi incorporation was observed mainly in 15K,42K and 45K proteins as determined by SDS-polyacrylamidegel electrophoresis and autoradiography. Neither axonemal movement nor Pi incorpo- ration occurred in the absence of cAMP. When radioactivity was measured, Pi incorpo -ration ratio(+cAMP/-cAMP) at one sec. was 5.0 in 15K protein, 1.4 in 42K protein and 0.8 in 45K protein. This suggests that short- term cAMP-dependent phosphorylation occurr- ed in 15K protein when spermatozoa are re- leased into freshwater at spawning and the phosphoprotein or phosphorylation itself triggers the initiation of sperm motility in rainbow trout. Developmental Biology 929 DB 46 THE INTERACTION OF STARFISH OOCYTES WITH ACROSOMAL PROCESS-CUT SPERM. K.Kyozuka and K.Osanai. Marine Biological Stn Tohoku Univ., Aomori. In the fertilization process, sperm interact with the egg surface successively by two sites, an acorosmal process and a head. The role of them during fertili- zation was examined using acrosomal process-cut sperm (AP-cut sperm). The material used was Asterina pectinifera. After the addition of egg water to induce acrosome,reaction, sperm Suspension (2 ml, 3.6x10 /ml) was treated with a disperser (Yamato, LK-21) for 10 sec. The acrosomal process and tail were cut from the sperm head. These AP-cut sperm could not fertilize mature oocytes. They did not penetrate the jelly coat and the vitelline coat. When intact sperm were treated with a disperser, they lost their tails. However, they could undergo the acrosome reaction and fertilize the oocytes. AP-cut sperm were added to vitelline coat-free oocytes. They were engulfed by microvilli from the denuded egg surface, but the sperm head did not fuse with egg plasma membrane. The conclusions are as follows: (1) the head of acrosome-reacted sperm induces the engulfing response of egg surface and (2) the acrosomal process is necessary for the fusion with egg plasma membrane and for leading the sperm head to egg plasma membrane. DB 47 CHANGES IN DISTRIBUTION OF MEMBRANE PARTICLES DURING THE ACROSOME REACTION IN JAPANESE ABALONE SPERMATOZOA Y.T.Sakai, F.Suzuki’ and Y.Shiroya, Biol. Lab., Wayo Women's Univ., Chiba. 1Dept. of Anat., Sch. of Med., Chiba Univ., Chiba. Distribution of the intramembranous particles of the plasma and acrosomal mem- branes and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome reveals sparse distribution of small particles except the apical area that includes the trigger and truncated cone regions. Inthis area larger particles about 10 nmin diameter are located. The E face of the plasma membrane has few particles. Of the outer acrosomal membrane, numerous particles are randomly distributed throughout the P face. On the corresponding E face, a few particles are scattered. The P face of the inner acrosomal membrane ex- hibits a densely packed lattice of particles. The initial change of the acrosome reaction accompanies the clearance of the membrane particles from the P faces of the plasma and outer acrosomal membranes around the apical region, where the fusionof both the membranes occurs. As the acrosomal process elongates, the dense lattice pattern on the P face of the inner acrosomal membrane changes through a coarse lattice into a patched one with the appearance of particle-free areas. The arrangement of particles is further disorganized into a scattered random distribution. DB 48 ACROSOMAL CONTENTS OF ABALONE SPERMATOZOA 1.PURIFICATION AND LOCALIZATION OF VITEL- LINE COAT-LYSIN FROM HALIOTIS DISCUS. K.Haino-Fukushima & N.Usui¥ Dept.of Biol., Fac.of Sci.,Tokyo Metropolitan Univ.& Dept. of Anat.,Teikyo Univ.Sch.of Medt,Tokyo. Soluble components of abalone acrosomes are known to contain two major proteins and one of them is the vitelline coat- lysin. Electron microscopy on acrosomal vesicle showed the presence of two distinct parts different in electron density. Following experiment was carried out to determine the localization of vitelline coat-lysin in the acrosome. Haliotis discus sperm spawned by expos- ing matured shell to UV-irradiated sea water were induced acrosome reaction in high-calcium sea water. The supernatant of acrosome-reacted sperm was contained two major proteins having molecular weights of 15.5K and 20K. They were separated by the difference of solubility in 50mM Tris,10mM NaCl,pH 7.4. Only 15.5K protein had a lytic activity. The 15.5K lysin was further puri- fied by preparative SDS-PAGE and HPLC. Antibodies against the purified lysin were prepared in a rabbit. This study is the first to demonstrate intra-acrosomal localization of the 15.5K lysin using immunocytochemical protein A- gold technique. The anti-15.5K lysin anti- bodies were well located at the posterior half of the acrosomal vesicle. DB 49 ACROSOMAL CONTENTS OF ABALONE SPERMATOZOA 2. EFFECTS OF THE CONTENTS ON THE FINE STRUCTURE OF THE EGG VITELLINE COAT. N. Usui and K. Haino-Fukushima. Dept. of Anatomy, Teikyo Univ. Sch. of Med. and Dept. of Biology, Fac. of Sci., Tokyo Metropolitan Univ. Tokyo. Effects of the sperm acrosomal contents on the egg vitelline coat (VC) of the Japanease abalone Haliotis discus were studied by electron microscopy. The intact VC was 1 in thickness and composed of a feltwork of fine filaments, among which 3-4 layers of large ovoid structures with electron-dense margin were contained. Penetraing through the VC, two types of pores were observed; a large’pore 1-2 pm in diameter and a number of thin pores with a diameter of 0.2-0.3 pm. A treatment with crude acrosomal contents released from the spermatozoa attending to the acrosome reaction led to an extreme loosening of the feltwork and disappearance of the ovoid structures and the thin pores, resulting in a fivefold increase of the VC in thickness. On the other hand, when being treated with 15.5K protein; one of the two major proteins from the acrosome and known as the "VC-lysin", the feltwork of the VC was also loosened and the ovoid structures were swollen, but not so much as those of crude acrosomal substance-treated oocytes. It is indicated that not only 15.5K but also 20K protein and/or some minor com- ponents of the acrosome are concerned with the lysis of the egg VC. 930 Developmental Biology DB 50 A ROLE FOR SPERM GLYCOSIDASES IN THE ASCID- IAN FERTILIZATION. M.Hoshi. Dept. Biol., Nagoya Univ., Nagoya. Specific binding of spermatozoa to the vitelline coat (VC) is an obligatory step for fertilization in various animals. Bind- ing depends upon the association between a glycoprotein(s) in the VC, sperm receptor, and a complementary carbohydrate-binding protein in the spermatozoa, VC-binding pro- tein(VCBP). In the ascidians, sperm glyco- Sidases have been implied as the VCBP(Hoshi et al., 1983) basing partly upon the fact that the strongest activity is the one di- rected toward the sugar residue that is suggested to be a ligand for sperm binding. Our hypothesis that a-L-fucosidase is a VC- BP in the ascidian, Ciona intestinalis, was further supported by the following evidence: 1.The enzyme was mostly located at the sur- face of sperm tip; 2.The binding was inhib- ited by monoclonal antibodies against the enzyme and by synthetic substrates as well as competitive inhibitor of it; 3.All the spermatozoa bound to the VC detached within a few minutes of decreasing the pH at 20°C but not at 0°C to a value close to the op- timum for the hydrolysis by the enzyme. Thus two categories of saccharide recog- nition protein have been proposed as VCBP; a lectin for acrosome reacted sperm in the sea urchin(Vacquier & Moy,1977) and two en- zymes for unreacted sperm, glycosyltrans- ferase in the mouse(Shur & Hall,1982) and glycosidase in the ascidians(this paper). DB 51 PARTIAL PURIFICATION AND ENZYMOLOGICAL STU- DY OF PROTEASE IN SEA URCHIN SPERM Y.Taniguchi,kK.Akasaka,H.Shimada,T.Shiroya Inst.of Zool.,Fac.of Sci.,Univ.of Tokyo. It is suggested that the proteolytic de- gradation of sperm histones may be one of the important steps in the decondensation process of sperm chromatin on fertilization, In the present study,protease was extract-— ed with 1% TritonX-100 solution from sea urchin sperm,and partially purified by DEAE -cellulose and SephadexG-100.The protease activity was assayed by SDS-PAGE using H1 histone of sea urchin sperm as a substrate. The enzyme rapidly degraded Hl histone,whi- le it degraded other histones only slightly even after prolonged incubation.Casein was not digested by this enzyme.Using various peptidyl-MCA substrates,it was found that this protease solely hydrolyzed Boc-Val-Leu -Lys-MCA.The enzyme showed maximal activity at the wide pH range between 7 and 11, and its molecular weight was about 41,000.The protease activity was not inhibited by DFP, STI,Leupeptin,Antipain,TLCK,Bestatin,PCMB, NEM,HgC1l3,CuClz and EDTA,but inhibited by o-Phenanthroline.Moreover,the activity of this enzyme was stimulated by ZnClz2 at 0.1 mM and inhibited by ZnCl2z2 at 1lmM.Therefore, this protease seems to be a metalloprotease It is assumed that this protease enters into egg cytoplasm after fertlization and plays some role in the decondensation proc-— ess of sperm chromatin through the hydroly- Ss) Oe Hela hetsivomner. DB 52 TRYPSIN INHIBITOR FROM SPERM OF THE SEA URCHIN, STRONGYLOCENTROTUS INTERMEDIUS K.Matsumura, Y,Yamada and K.Aketa. Akkeshi Mar. Biol, Stat,, Hokkaido Univ., Akkeshi Hokkaido, Trypsin-like enzyme exists in sea urchin sperm(Levine et al,,1978; Yamada and Aketa, 1982), The role of this enzyme, however, is unknown, In the present study, sperm tryp- sin inhibitor(STI), which appears to con- trol the activity of trypsin-like enzyme, was purified partially from sperm of the sea urchin, Strongylocentrotus intermedius and partially characterized, Partial puri- fication was performed by affinity chroma- tography on trypsin-Sepharose 4B. The in- hibitory activity of STI was assayed by measurement of inhibition of BAEE esterase activity of bovine pancreatic trypsin. The optimal pH of the inhibitory activity was 7-7.5, and the activity was suppressed at above pH Ps It was enhanced in the pesos of 1 mM Ca abe but suppressed by 10 mM Ca”~ . STI was stable at 80°C for 5 min, Release of STI from intact sperm_was observed by the addition of 30 mM Ca¢?. Suppression of the inhibitory activity of STI under the conditions inducing acro- some reaction (high pH, high ca2+ concen- tration) suggests that STI is released from the sperm surface before or at the time of the acrosome reaction and inactivated after dispersion into the surrounding sea water, DB 53 OOGENESIS IN THE DEMOSPONGE, HALICHONDRIA OKADAT. K.Tanakal and Y.Watanabe2. l!shimoda Kita High Sch., Shimoda and 2Dept. of Biol., Fac. of Sci., Ochanomizu Univ., Tokyo. The origin of nurse cells and their contribution to oogenesis were studied in the viviparous sponge, H. okadai. During the reproductive period, remarkable changes were observed in the sponge. In the meso- hyl, the population of amoeboid cells in- creased proportionately with the decrease of choanocyte chambers. Some choanocytes lost their collars and flagella, migrated into the mesohyl and grew larger engulfing other cells, while the cytoplasm became electron dense. These cells transformed into nurse cells and accumulated around young oocytes. In early stage,oocytes were associated with a small number of nurse cells, but in later stages, they were sur- rounded by several layers of them. Young oocytes ingested some nurse cells and formed dense yolk granules. These granules first appeared in the peripheral cytoplasm and increased in number as the oocytes grew larger. Vitellogeneic oocytes were surrounded by single layers of follicular epithelium; numerous nurse cells remained outside. Some nurse cells broke into frag- ments which were taken up by the oocytes through the gaps of the epithelium and became light yolk granules. We consider that choanocytes play an important role as nutrients in the oogen- esis of H. okadai. Developmental Biology 931 DB 54 YOLK FORMATION ON THE OOCYTE OF N. POMPILIUS. Jd. Tsukahara. Dept. of Biol., Fac. of Sci., Kagoshima Univ. Kagoshima. Nautilus oocytes possess great quantities of amber-colored fluid yolks during vitellogenesis. In order to study the formation and chemical nature of yolks, cytological and cytochemical investigation have been undertaken. When the oocyte increase in size to 500um in diameter, the germinal vesicle is carried to periphery, where the nucleus and cytoplasm may become blastodisc in future. On the opposite side follicle cells begin to fold and invade the oocyte surface. Many microvilli grow in clusters on the surface of oocyte and follicle cell. When the oocyte diameter is increase to 800nm, follicle cells become columnar and have many ER and electron dense granules in the cytoplasm. Many electron ill-dense vesicles appear in the cortical layer. They may supply the yolk substances. The yolk in the oocyte is well-stained with PAS and show strong metachromagy with toluidine blue (pH 4.0). There are many PAS positive granules also in the cytoplasm of the follicle cell. When the oocyte stained with Sudan black B, the well-stained granules were found at the periphery of the yolk fluid. The full-grown oocytes in the ovary increase in size to 22-25mm in diameter and 2.3-2.7g in weight. DB 55 EARLY OOGENESIS AND THE STRUCTURE OF THE OVARY OF AN ASCIDIAN, CIONA SAVIGNYI. Y.M.Sugino and Y.Takashima. Dept. Anat., Ehime Univ. Sch. Med., Ehime. The purpose of this study is to under- stand cytodifferentiation and cell inter- actions during early oogenesis and to describe the histology and ultrastructure of ovarian epithelia (OE) of ascidians. The OE are composed of ciliated epithelium (CE) and germinal epithelium (GE). The GE consists of three regions: the stratified, egg-follicle and terminal regions. There are young oocytes at two stages in the stratified region. Synaptonemal complexes (SCs) are observed in the nuclei of the oocytes 5-7 pm in diameter. The SCs disappear in the larger oocytes. The lat- ter oocytes have mitochondria arranged very near the nuclear envelope and three kinds of electron dense materials. Two kinds of small cells are observed adjacent to the oocytes. The nucleoplasm and cyto- plasm of one of these cell types is dense, and of the other is clear. There are cell junctions between these cells. During cy- tolysis cells with phagosomes also appear. In the boundary areas, there are some cells not in any of these foregoing cate- gories, which may be undifferentiated. The apical surface of the cell of the CE is rather smooth, and the basal is convoluted. The cells have striated rootlets, glycogen particles and lipid droplets. There are also junctions between these cells. DB 56 FINE STRUCTURE OF THE MICROPYLAR CELL AND GRANULOSA CELL IN THE OVARIAN FOLLICLE OF CHUM SALMON, ONCORHYNCHUS KETA. W.Kobayashi and T.S.Yamamoto. Zool. Inst., Fac. of Sci., Hokkaido Univ. Sapporo. The fine structure of micropylar cell (MPC) was compared with that of granulosa cell (GC) in the ovarian follicle with the oocyte at the stage of germinal vesicle migration. Both types of the cells were divided into two portions: the main cell body and the long cytoplasmic process. Although a remarkable dilation of rER was observed, the fine structure of the main cell body in the MPC was nearly the same as that in the GC. The cytoplasmic processes expanded when they passed through the length of the radial canals or of the micropylar canal in the egg casing. The thickness of process was considerably large in the case of the MPC. The distal end of the process of MPC was inserted into an indentation of the ooplasmic sur- face. Although a similar insertion was also observed in some cases, the expanded end of the process of GC usually termi- nated either in the lumen of radial canals or was placed on a slight depression of the ooplasmic surface. Various types of junctional structures were involved in the oocyte-GC as well as -MPC associations. These observations suggest that the MPC participating in the formation of micro- pylar apparatus has a similar origin as the GC in the chum salmon ovary. DB 5/ OVERRIPENING PHENOMENA IN THE MEDAKA, ORYZIAS LATIPES, R. Hori and T.J. Lam, Dept. of Zool., Natl. Univ. of Singapore, Singapore. It is well known that ovulated eggs of oviparous teleosts become 'overripe' if retained within the ovarian or coelomic cavity for a varying period of time. In this study the overripening process of the medaka, Oryzias latipes, was described and also the fertilizability of overripe eggs was observed by artificial insemination. The morphology and number of eggs released and retained within the fish were observed in previously spawned females now isolated from the males. The overripening process of eggs may be divided into 4 stages: (1) eggs that closely resemble normal ova immediately after ovulation; (2)eggs with partial breakdown of cortical alveoli; (3) eggs with only cortical alveoli in animal pole and oil globules in vegetal pole; (4) eggs with concentration of protoplasm in the central region with few or no cortical alveoli. Artificial insemination was applied to the overripening eggs. For eggs of stage (1) and (2), the percentage of complete breakdown of cortical alveoli and further development was relatively high, and the processes were similar to those of normally fertilized egg. However, no success was obtained with eggs of stage (3) and (4). In conclusion, the overripe- ning egg may be fertilized provided the cortical alveoli are still present through- out the cortical region of the egg. 932 Developmental Biology DB 58 CHORION GLYCOPROTEIN-LIKE IMMUNOREACTIVE SUBSTANCE IN THE ESTROGENIZED ADULT MALE MEDAKA. T. Hamazaki, I. Iuchi and K. Yamagami. Life Sci. Inst., Sophia Univ., Tokyo. The antigenic substance reactive to anti- chorion (egg envelope) glycoprotein antibody exists not only in the ovary but also in the liver and blood plasma of a spawning female medaka, Oryzias latipes (Hamazaki et al., 1983, 1984). The substance is considered to relate closely to oogenesis on account of its absence in tissues of the non-spawning female as well as of the male fish. This substance (mw ca 60,000) is evidently distinct from probable vitellogenin (yolk lipo- phosphoprotein, mw 420,000) which is also found in blood plasma of the female fish. Immunohistoche- mical observation revealed that after the cessation of oviposition, the diminution of the substance was first observed in hepatic cells while other tissues are still immunoreactive. These results strongly suggest that the substance is synthesized in the liver of the female fish. The present experiment showed that the antige- nic substance was also synthesized in the liver of the estrogenized adult male like vitellogenin. Both of those products were accumulated in the ascites under such an unnatural condition. In the hepatic cells of the estrogenized male, a number of vacuolar deposits were observed, which seem to consists mostly of vitellogenin and partially of this substance. A large amount of the antigenic substance as well as vitellogenin is now able to obtained from the ascites of the estrogenized males. The antigenic substance is to be isolated to clarify its role in oogenesis of medaka. DB 59 ESTABLISHMENT Cir POLARITY IN DEVELOPING GERM CELLS DURi™G XENOPUS EARLY OOGENESIS. K.Takamoto. Biot. Inst., Kyoto Pref. Univ. of Med. Kyoto. Previous electron microscopic study nas revealed that developing germ cells(in oo- gonia,preleptotene, leptotene, zygotene, pachytene stages) in the newly-metamorpho- sed Xenopus ovaries are orten connected one another by intercellular bridges and exhibit a highly developed Golgi area which is routinely located near the cytoplasmic continuities. The purpose of the present study is to obtain more detailed informati- on about the relationship between the pola- rized distribution of organelle and the location of intercellular bridges within developing germ cells. Although this study has not determined the total number of bri- dges connected to a single germ COI, it has been frequently observed ona single section that two or three bridges open in- to one germ cell. in such a case, these connections are always found to be located adjacent to each other facing the conspi- cuous Golgi area. The Golgi areas, includ- ing the mitochondrial cloud and the centri- ole, are interconnected by a bundle of mic- rotubules passing through the bridges. Dur- ing preleptotene interphase the centriole become attached to the cell membrane adja- cent to the intercellular bridges. These findings suggest that the cytoplasmic bri- dges play an important role in the establi- shment of the polarity of developing germ cells. DB 60 ULTRASTRUCTURES OF XENOPUS EGG ENVELOPES AND THE OVIDUCT PRODUCING THEM. N. Yoshizakie ~Depis Oe TOlN ee bacrmons Ceinerrealt MCE. 5 Gaieu Waiwe, Gatu. a ga Light and scanning or transmission electron microscopic observations were made on the total length of the Xenopus laevis oviduct in order to correlate each of the egg envelopes with the producing sites. During the passage through the oviduct, a uterine egg acquires the following substances or envelopes; electron dense particles in the vitelline coat, the pre-fertilization layer around the vitvelline coat, and four layers won jelly. The former two were produced at the pars recva i and 2 portalonis on ovel@ulen and jelly Mayers 1% 25 3) andl waaulemecrms econvoluta 15 2, 3 and: 4 portions eecesuce= tively. The relative length of these portions were 0-0.044, 0.044-0.055, 0.055- O25 Wes52505 7/05 OoVO-—Os8reiac O. 871 from anverior Go) POS lelrnOire Ee TLonmemnrinc whole length of oviduct, the epithelium was comprised of cilia cells and various types Of secretory cells. Except for the pars recta 1 portion, there were two or three types of secretory cells at each portion of oviduct, indicating that the stratum structure of each egg envelope is formed by an interaction of the products from both types of secretory cells specif- 1G WO SECA OOMTILOIM OF Cle OwLCuUert - DB 61 BIOCHEMICAL DIFFERENCE BETWEEN ZONAE PELLU- CIDAE OF OVARIAN AND OVIDUCAL EGGS OF THE GOLDEN HAMSTER, MESOCRICETUS AURATUS. T. OITKAWA AND Y. SENDAI, DEPT. OF BIOL., FAC. OF SCI., YAMAGATA UNIV., YAMAGATA 990, JAPAN. Living eggs collected from either ovi- ducts or ovaries were stained with fluo- rescein-conjugated Bandeiraea simplici- folia lectin (FITC-BSA-1) that specifical- ly binds to alpha-galactose-like terminal saccharide residues. The biochemical dif- ference between the zonae of ovarian eggs (ZP-OVA) and those of oviduct eggs (ZP-OVI) was obvious when they stained with FITC- BSA-1. ZP-OVI bound uniformly and intense- ly with FITC-STA-1, whereas ZP-OVA does not at all. ZP-OVA did not bind to FITC- BSA-1 regardless of its stage of matura- tion even immediately before ovulation. When ZP-OVA was exposed to oviduct extract, it bound with FITC-BSA-1 just like ZP-OVI. Apparently, biochemical properties of the zona, at least the configulation of the terminal saccharide residues of zona-glyco- proteins, alter drastically after entry of egg into the oviduct. It is tempting to speculate that this alteration is due, at least in part, to addition of some materi- als from the oviduct or an alteration of zona material by some substance from the oviduct. This study was supported by the grant to T.O. from Yagai Chikusan Kogyo Co., Ltd., Yamagata 990, Japan. Developmental Biology 933 DB 62 INVOLVEMENT OF AN ASPARAGINE-LINKED OLIGO- SACCHARIDE LOCATED IN ZONA PELLUCIDA IN MOUSE FERTILIZATION IN VITRO. T.Yamagata, M.Ito and N.Takahashi*, Lab. Advanced Res., Mitsubishi-Kasei Inst. Life Sci., Tokyo and *Dept. Biochem., Nagoya City Univ., Nagoya. When zonae pellucidae of mouse eggs were treated with glycopeptidase, which cleaves specifically N-acetylglucosaminyl asparagine linkage of N-linked glyco- proteins, mouse sperm failed to become attached to zonae pellucidae. Oligosac- charides thus obtained from unfertilized eggs were compared with those from 2-cell stage embryos, using a reverse phase column by high performance liquid chro- matography, after oligosaccharides were labeled by a fluorescent dye. Patterns were quite similar except for a marked decrease in one of the oligosaccharides from 2-cell stage embryos. It is, there- fore, most likely that this asparagine- linked oligosaccharide of glycoproteins constituting zona pellucida is involved in recognition mechanism between mouse gametes. DB 63 STARFISH RADIAL NERVE SUBSTANCE INDUCING OOCYTE MATURATION OF SEA CUCUMBERS. Y.K.Maruyama. Dept. of Zoology, Fac. of Science, Kyoto Univ., Kyoto and Friday Harbor Laboratories, WA. Cross-action of starfish radial nerve extracts to sea cucumber oocyte maturation was re-examined. Radial nerve extracts from starfishes, Pycnopodia helianthoides and Pisaster ochraceus, were found to act directly on follicle cell-free oocytes of the sea cucumber Stichopus californicus to mature. The mafuration-inducing activity occurred in Ca -free seawater as well as in normal seawater. Heating (90°C for 15 min) of the radial nerve extracts did not inactivate the maturation-inducing activity, but treatments of the extracts with pronase or trypsin inactivated the activity. Fractionation of Pycnopodia radial nerve extracts through a Sephadex G 50 column showed that the active fractions to Stichopus oocytes are such fractions that have high GSS(gonad-stimulating substance) activity to starfish_ovarian oocytes. 1-MeAde (5X10 ~-1x10 ~M) did not induce the maturation of Stichopus oocytes. These results show that starfish radial nerve extracts contain a factor inducing the maturation of follicle cell-free oocytes of sea cucumbers, and indicate that the factor is either a peptide with a molecular weight similar to that of GSS or GSS itself. DB 64 EFFECT OF CYSTEINE AND ITS DERIVATIVES ON 1-METHYLADENINE PRODUCTION IN FOLLICLE CELLS OF THE STARFISH, Asterina pectinifera. M. Mita and H. Kanatani. Lab. of Reprod. Biol., Nat. Inst. Basic Biol., Okazaki. Follicle cells in starfish produce the oocyte maturation inducing substance, l- methyladenine (1l-MeAde) in response to gonad-stimulating substnace (GSS). Cysteine inhibited GSS-induced 1-MeAde production in follicle cells. The half maximum inhibition was obtained at the concentration of 2.6 mML-cysteine. Similar effect was observed with D-cysteine. Though cystamine, cysteamine, homocysteine and cysteine- methylester also inhibited 1-MeAde product- ion, cysteic acid, methionine and serine had no effect. This suggests that SH group and S-S bond of these compounds are related to inhibition 1-MeAde production, since 2- mercaptoethanol, dithiothreitol and glutathione had inhibitory effect. Cysteine inhibited GSS-induced Spawning and oocyte maturation. Another experiment indicated that the site of cysteine block is not at the cell surface. It is known that cysteine inhibits conversion from methionine to S-adenosyl- methionine (SAM). SAM is a candidate methyldonor for 1-MeAde production. These data suggest that synthesis of 1-MeAde in follicle cells is inhibited by cysteine and its derivatives, resulting from the block of SAM production. DB 65 PARTIAL PURIFICATION AND CHARACTERIZATION OF MATURATION-PROMOTING FACTOR FROM STARFISH OOCYTES. T.Kishimoto, H.Kondo and H.Kanatani. Dept. of Develop. Biol., Natl. Inst. for Basic Bide, Okazaki. Maturation-promoting factor(MPF), which is produced in the cytoplasm of oocytes to trigger meiotic maturation, was extracted from oocytes of the starfish, Asterina pectinifera, in order to characterize it. Isolated immature oocytes were treated with l-methyladenine and washed with Ca- free seawater containing EGTA and Na-glyc- erophosphate. After the removal of jelly and excess medium, maturing oocytes were crushed by centrifugation. The supernatant was homogenized with a buffer containing NaF, Na-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF in the supernatant was purified by ammo- nium sulfate precipitation(50% saturation), hydrophobic chromatography on pentyl- agarose and gel-filtration on Sephacryl S-300. The final material induced matura- tion in the recipient starfish oocytes when 1 ng of protein was injected ina volume of 500 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronu- cleus. Further, MPF was found to be a heat -labile protein and its molecular weight was estimated as 300K daltons by gel- filtration on Sephacryl S-300. 934 Developmental Biology DB 66 CYCLIC GMP IS AN INHIBITORY FACTOR OF MEIOSIS REINITIATION OF STARFISH OOCYTES. S. Nemoto, T. Kominami* and K. Ishida**., Tateyama Marine Lab., Ochanomizu Univ., Tateyama, *Dept. of Biol. Ehime Univ., Matsuyama and **Dep. of Urol., Sch. Med. Teikyo Univ., Tokyo. As we previously reported (Exp. Cell Res.,'83), an intracellular level of cGMP decreased on meiosis reinitiation of star- fish oocytes, and exogenously applied cGMP and methylxanthines inhibited oocyte matu- ration induced by l-methyladenine (1-MA). These results suggest that a decrease in cGMP level is a prerequisite for meiosis reinitiation of starfish oocytes. To confirm this further, we determined cGMP levels of Astertas amurensts oocytes whose maturation was inhibited by treating with phosphodiesterase inhibitor (caffeine) , cyclic nucleotide-synthetase activator (forskolin) and exogenously applied cGMP. Five mM caffeine, 20 pM forskolin and 10 mM dibutyryl cGMP inhibited oocyte maturation under the presence of 1 pM 1-MA, and increased intracellular cGMP levels to about 200%, 150% and 300%, respectively. Microinjection of cGMP into oocytes (final conc., 0.5 mM) inhibited 1—-MA-induced maturation. These results indicate that increase in Or suppression of decrease in cGMP levels inhibits meiosis reinitiation of starfish oocytes. DB 67 # ReoOPLRATORY BURST OF CN CHLL-FREA SYSTEM. akezawe. Devot. of 55 Wolo Wasi. U2. consumption in sea urchin (Hemicen— trotus pnulcherrimus) egg homogenates was Stimuleted by the addition of calcium (Hultin, 1950). This activity was local— ized in vost-mitochondrial supernatant ( PMS fraction). The rete of Ca-induced (2 consumption wes further increased in the presence of C.2 mM srachidonic acid (Ca- AA-purst), but not of the same concentra-— tion of linoleic or linolenic acid. Ca— 44-burst was inhibited by 2 mM SKF-525A. However, neither NADE nor NADPH is effec-— tive on Ca-AA-burst and elso the exis- tence of cytochrome P-450 or b was not recognized in PMS fraction. On the other hend, O.3 mii indomethacin or 10 mM aspi- rin (both of them is the inhibitor of cyclooxygenase) inhibited Ca—-AA-burst. Therefore, it is suggested that the Ca- AA-purst reflects the activation of arachidonate cascade in microsome. Res-- piretory burst of the intact egg just efter fertilization was inhibited by pro- cein dose dependently, in contrast with no inhibition in Ca—AA-burst. The inhi- bition by procain may be dependent on the intactness of egg membrane. DB 68 EFFECTS OF KCN ON OXYGEN CONSUMPTION IN PROCAINE-TREATED SEA URCHIN EGGS. M.K.Kojima” and M.Kaji® . Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. 2) Ishi- kawa Blind School, Kanazawa. It has been reported that when unferti- lized sea urchin eggs are pre-treated with procaine sea water for a short period and then fertilized, they can develop much ear- lier than the controls. Further, it was found that Oz consumption is enhanced in such procaine-treated eggs. Therefore, it was determined whether KCN has inhibitory effects on the increase of 02 consumption induced by procaine treatments. When KCN is added to unfertilized eggs whose respira- tion has been previously enhanced with pro- Caine, the increased respiration is strong- ly reduced, as compared with the case of Oz uptake in the control eggs which are treated with KCN only- On the (contrary meee procaine is added to eggs whose respiration has been previously reduced with KCN, addi- tion of procaine results in a considerable increase of Oz consumption. Those results suggest that 1) 0g consumption enhanced by procaine treatments is mostly due to the mitochondrial and KCN-sensitive oxidation which is activated after fertilization; 2) On the other hand, procaine can activate a non-mitochondrial and KCN-insensitive res- piratory system which may involve a oxida- tion of unsaturated fatty acids, such as arachidonic acid, and may be stimulated by calcium. DB 69 A Ca-BINDING PROTEIN ISOLATED FROM THE AGGREGATION FACTOR COMPLEX OF SEA URCHIN EMBRYO II. H.Kurihara, S.Naitou and Y.Tonegawa. Dept.Regulation Biol. ,Saitama Univ. ,Urawa. A Ca-binding protein was isolated from the aggregation factor complex(AFX) of sea urchin embryo(H.pulcherrimus). Aggregation factor complex, extracted from blastula embryos by CMF-SW, was separated into pro- tein and proteoglycan(PG) species by DEAE-— cellulose and Sepharose 2B chromatography. The protein species embodied the cell agg- regating activity of the AFX and also the Ca-binding activity. Whereas, the PG spe- cies showed inhibitory activity to the cell aggregation induced by AFX but syner- getic activity to that by isolated protein. The cell aggregating activity of this pro- tein was found to be inactivated by EDTA- treatment and reactivated by following treatment with Ca. In the process of in- activation and reactivation, two different types of Ca-binding was demonstrated. One occured coincident with cell aggregating activity, apparent in active and reacti- vated protein, suggesting its involvement in cell adhesion. Another became prominent after EDTA treatment with the loss of cell aggregating activity, and disappeared with the recovery of the activity. This second type of Ca-binding is assumed to be relat- ed to the maintenance of protein structure needed for cell aggregating activity. Developmental Biology 935 DB 70 TRYPSIN INHIBITOR FROM SEA URCHIN EGG II, Y, Yamada, K, Matsumura, and K, Aketa, Akkeshi Mar, Biol, Stat,., Hokkaido Univ,, Akkeshi, Hokkaido, A trypsin inhibitor (ETI) has been puri- fied from unfertilized and fertilized eggs of the sea urchin, Strongylocentrotus intermedius, and partially characterized ( Yamada et al., Zool, Mag., 92, 469, 1983). In the present study, ETI activity was measured in eggs and embryos at various stages of development ranging from unfer- tilized to the pluteus stage, ETI activity decreased after fertilization, which was about 55 % that of the unfertilized eggs, and it remained unchanged throughout the developmental stages studied. Soybean trypsin inhibitor (SBTI) is known to induce polyspermy of sea urchin eggs (Hagstrém, B.E., Ark. Zool., 10, 307, 1956). Unlike SBTI, ETI neither affected formation of the fertilization membrane nor induced polyspermy, The development normal- ly occurred in the presence of ETI, Indirect immunofluorescence technique revealed that ETI exists in the cytoplasm of unfertilized eggs and embryos. Fibrous structure in coelom of prism embryo was fluoresced, Fluorescence was observed also on the fertilization membrane, Isolated cortical granules (CGs) had sites reactive with the antibody against ETI, and these sites disappeared after the addition of Ca2+, These results suggest that ETI is present in CGs and released by the exocy- tosis of CGs upon fertilization, DB 71 B-N-ACETYLGLUCOSAMINIDASE IN THE SEA URCHIN ANTHOCIDARIS CRASSISPINA 3 Y. YOKOTA , M. G. Cacace and E. NAKANO IBiol. Lab., Aichi Pref. Univ., Nagoya, 2Inst. Mol. Embryol., Natl. Council of Res. Italy, Arco Felice., 3Dept. of Biol., Nago- ya Univ., Nagoya. 8-N-Acetylglucosaminidase from the ga- metes of the sea urchin, Anthocidaris crassispina, was partially purified and characterized. The purification proce- dures involved ammonium sulfate fractiona- tion, gel filtration, chromatography on SP-Sephadex C-25 and on hydroxylapatite followed by affinity chromatography on p-aminopheny1-N-acetylglucosaminide-Sepha- rose 4B. The enzyme from eggs was 410- fold purified with 13% recovery. When the enzyme was subjected to isoelectric focusing, a pl peak between 4.8 and 5.0 was observed for both egg and sperm en- zymes. Kinetic properties and molecular weight of the enzymes from both sources were similar, the Km being 1 mM and 0.8 mM for egg and sperm enzymes, respective- ly. Tne apparent molecular weight was 205,000 + 10,000 for both enzymes. The egg enzyme was associated with yolk gran- ules and released by treatment with acid citrate-phosphate buffer, while the sperm enzyme was loosely bound to sperm and re- leased by mild osmotic shock or sheering stress. DB 72 THE EFFECTS OF CALCIUM ANTAGONISTS AND CALMODULIN ANTAGONISTS ON ACID PRODUCTION FOLLOWING. FERTILIZATION. M.Komukai;,,A.Fujiwara; Y.Fujino*and I.Yasumasus 1. Dept. Biol., Sch. Edu., Waseda Univ., Tokyo. 2. Dept. Pharmacol., Teikyo Univ. Sch. Med., Tokyo. Fertilization-induced acid production in the eggs of sea urchin, Hemicentrotus pulcherrius and Anthocidaris crassispina was inhibited by calcium antagonists, verapamil and diltiazem, by calmodulin antagonists, W-7 and chlorpromazine and by an anion transport inhibitor, DIDS when these compounds were added to the egg suspension 30 sec after insemination. W-5, a calmodulin antagonist, amiloride, an inhibitor of Na‘/H* exchange and furosemide, a Cl transport inhibitor failed to inhibit acid production. The inhibition of verapamil and diltiazem on acid production were effective when these compounds were added to the egg suspension by 2 and 3_min after insemina- tion, respectively. Ca uptake, which was enhanced in its rate following ferti- lization, was inhibited by calcium antago- nists and DIDS, not by calmodulin antago- nists. These results suggest that ferti- lization-induced acid production results from calmodulin-dependent reactions which are stimulated by Ca*+ influx and Ca*t influx may be coupled with anion rihpbellepre- DB 73 INHIBITION OF CYANIDE INSENSITIVE RESPIRA- TION IN JUST FERTILIZED EGGS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS, BY CALMODULIN ANTAGONISTS AND CALCIUM ANTAGO- NISTS. 1 2 E. Tuchida, A., Fujiwara a ed ee oC I. Yasumasu’. Dept.,of Biol. Sch.of Educ. Waseda Univ., Tokyo. “Dept of Pharma., Teikyo Univ., Sch. of Med., Tokyo. Sea urchin eggs exhibited a marked in- creased in the respiratory rate followed by gradual decrease during about 10 min after insemination. During increasing phase of fertilization-induced respiration, the respiration was insensitive to cyanide. Its sensitivity to cyanide increased there- after and finally became completely sensi- tive to CN about 10 min after insemination where the rate of respiration was higher than in unfertilized eggs. This cyanide- insensitive respiration was inhibited by adding calcium antagonists, verapamil and diltiazem, and calmodulin antagonist, W-7, whereas cyanide sensitive respiration was not. Calcium uptake following fertiliza- tion was inhibited by calcium antagonists but was not by calmodulin antagonists. Cyanide-insensitive respiration seems to be initiated by calmodulin dependent reactions which is stimulated by Ca influx. 936 Developmental Biology DB 74 EFFECTS OF CALCIUM ANTAGONISTS AND CALMO- DULIN ANTAGONISTS ON THE FERTILIZATION MEMBRANE ELEVATION, ACID PRODUCTION, GLY- COLYSIS SYSTM AND RESPIRATION IN JUST FER- TILYZED EGGS OF THE ECHIUROID, URECHIS UNICINCTUS. E. Tazawa, M. Komukai, A. Fujiwara, Y. Fujino and I. Yasumasu. BOIL, IOASIES - Yokohama City Univ., Yokohama, Dept. of Biol., Waseda Univ., Tokyo and “Fac. of Med., Teikyo Univ., Tokyo. Fertilization in the echiuroid eggs was inhibited in the presence of calcium antagonist, verapamil and diltiazem, and by a calmodulin antagonist, W-7. By adding these componds 30 sec after insemination, fertilization membrane elevation occurred with a marked delay. Acid production, which occurred normally prior to fertili- zation membrane elevation, was markedly reduced in its rate by adding these com- pounds 30 sec after insemination. W-5, failed to inhibit fertilization, fertili- zation membrane elevation and acid produc-— tion. An increase in glucose-1-phosphate following fertilization, indicating an activation of phosphorylase reaction, was also inhibited by these, ¢ompounds, but the respiration was not. Ca uptake, which was enhanced in its rate following ferti- lization, was inhibited by calcium antago- nists but not by calmodulin antagonists. Fertilization membrane formation, acid production, stimulation of ph. reaction Sem EQ weEsuultc sereom Cell. dependgnt react-— i1ens which are stimulated by Ca influx. Ds) 75 Canis TRANSPORT IN SPICULE FORMATION OF SEA URCHIN EMBRYOS. 1 K.Mitsunaga~-, Y.Fujino , A.Fujiwara’ and i.Yasumasuy. IDept. Of Biloll., Sch. of Educ., Waseda Univ., Tokyo. 2Dept. of Pharmacol., Teikyo Univ. Sch. Med., Tokyo. Effects of ion transport inhibitors on the spicule calcification, morphogenesis and Ca uptake were studied in the sea urchin embryos, Hemicentrotus pulcherrimus, to analyze the mechanisms of Ca2+ trans- port in the cells. Embryos, treated with calcium antago- nists, diltiazem and verapamil, or an an- ion transport inhibitor, 4,4'-diisothio- cyano-2,2'-disulfonic acid stilbene (DIDS) between the mesenchyme blastula and the pluteus corresponding stage, became spheri- cal plutei with quite small spicules. The treatment with Cl transport inhibitors, ethacrynic acid and furosemide, during the same period yielded quasi-normal plutei with poor spicules and somewhat developed arms. In the gastrulae, Dena uptake was inhibited by all these compounds. In the swimming blastulae, 45ca2+ uptake was blocked only by calcium antagonists and DIDS. Cl transport inhibitors blocked 45ca2+ uptake only in the bag fraction but not in the embryo-wall cell fraction ob- tained from gastrulae, though calcium antagonists and DIDS inhibited in the both fractions. Ca“ _ uptake probably becomes coupled with Cl influx only in the embryos in which spicule calcification occurs. DB 76 EFBECTS OF ANION TRANSPORT INHIBITORS ON Ca UPTAKE IN SEA URCHIN EMBRYOS. Y.Fujinol, K.Mitsunaga“, A.Fujiwara and I.Yasumasu 1Dept. Parmacol., Teikyo Univ. Sich. 9Medy, Wokyo.. 2Depiicas saoler, Sch. Educ., Waseda Univ., Tokyo. In the sea urghin, Anthocidaris Crassispina, Ca uptake just after fertilization was inhibited by calcium antagonists, diltiazem and verapamil, and by an anion transport inhibitor, 4,4'- diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), but Cl transport inhibi- tors, ethacrynic acid and furosemide, failed to inhibit 4°ca¢? uptake in the eggs. On the other hand, in the embryos at the stages where spicule formation occurred, ianhibitors of CIN transporte reduced the rate of 4°ca uptake. In the embryos between the two cell and they. 24 swimming blastula stage, the rate of “Ca uptake was very low and these inhibitors were not effective on Ca transport across the membrane. These suggest that Ca++ concentration in the cytosol is main- ly regulated by calcium stores in the cells between the two cell and the swim- ming blastula stage. These also suggest that electrosilent ca2t transport across the membrane just after fertilization is established mainly by concomitant trans- MOIS Of EMLOGMS, Ca uptake coupled with Cl transport also occurs in the embryos, in which spicule formation occurs. DB 77 EFFECT OF PALMITOYL CoA ON cAMP DEPENDENT PROTEIN KINASE IN SEA URCHIN EGGS. 1 h. Fujiwara, Y. Fujino™ and I. Yasumasu’. Dept. @f Biol.,Sch. of Educ., Waseda Univ. Tokyo. “Dept. of Pharma., Teikyo Univ., Sch. of Med. Tokyo. ~ Partially purified cAMP dependent pro- tein kinase in sea urchin eggs was activat- ed by palmitoyl CoA at concentrations be- tween 1 and 20 uM, and was inhibited at higher concentrations than 30 uM, in the presence og lower concentrations of cAMP than 5xlQ M. At concentrations @£ CAMP than 10 “M, palmitoyl CoA exerted only slight effects on cAMP dependent protein kinase. Palmitoyl CoA enhanced cAMP-bind- ing capacity at concentrations between 1 and 20 uM and reduced the capacity at high- er concentrations than 25 uM. Palmitoyl CoA did not exerted any effect on catalytio subunit of the enzyme. The effects of palmitoyl CoA on cAMP dependent protein kinase probably result from the interaction between regulatory subunit and palmitoyl CoA, which alters cAMP binding capacity of regulatory subunit. Myristoyl CoA and stearoyl CoA exerted similar effects as palmitoyl CoA. Decanoyl CoA, butyryl CoA, propionyl CoA and acetyl CoA, as well as CoA, palmitate-Na, and stearate-Na, did not alter the activity and cAMP binding capacity of partially purified enzyme. Developmental Biology 937 DB 78 PHOSPHORYLASE A ACTIVITY IN SEA URCHIN EGGS KEPT IN THE ANOXIA AND IN THE COLD, WITH SPECIAL REFERENCE TO THE CONCENTRATION OF LONG CHAIN ACYL CoA'S. I. Yasumasu and A. Fujiwara. Dept. of Biol. Sch. of Educ., Waseda Univ., Tokyo In sea urchin eggs at the 16-32 cell stage, kept in anaerobic condition or in the cold, long chain acyl CoA's. increased in their levels, and the development was arrested. These embryos thus treated deve- loped further and long chain acyl CoA level decreased to a normal level, when they were kept in aerobic condition and at 20°C. ATP level is maintaned during rearing in the anoxia or in the cold and glycogen decrease qgcompanied by lactate increase occurred. P incorporation into protein was enhanced 42Phosphorylase a activity increased and P incorporation into RNA reduced in these conditions. Carnitine, which reduced in- tracellulgr long chain acyl CoA level, reduced P incorporation into protein, phosphorylase activity and ATP level in the embryos kept . the anoxia and in the cold but enhanced P incorporation into RNA. Long chain acyl CoA's probably inhibit energy-utilizing reactions, such as RNA synthesis, and stimulate glycolysis due to an increase in phosphorylase a activity to maintain ATP level. The eggs, kept in carnitine, developed normally, but those, kept in the anoxia and in the cold in the presence of carnitine, failed to develop further, even when they were kept in aero- bic condition and at 20°C. DB 79 CHANGE IN THE ACTIVITY OF CAMP-DEPENDENT PROTEIN KINASE IN NUCLEI OF SEA URCHIN EMBRYOS DURING EARLY DEVELOPMENT M. Motozima and I. Yasumasu. Dept. of Biol.,Sch. of Educ., Waseda Univ., Tokyo In the eggs and the embryos of the sea urchin, Anthocidaris crassispina, CAMP- dependent protein kinase in soluble frac- tion, obtained by centrifugation at 10,000g for 1 hr was high in unfertilized eggs and gradually increased up to the swimming blastula stage, then, suddenly decreased to a quite low level. In the precipitate fraction, the enzyme activity was quite low and undetectable up to the morula stage and became detectable at the blstula stage. Then, it became maximum at the mesenchyme blastula stage and then decreased. In nuclei, the activity became detectable at the blastula stage, and was undetectable at the time just after hatching and then became detectable at the mesenchyme blas- tula stage. In the eggs at the 32-64 cell stage, where cAMP-dependent protein kinase activity is undetectable in nuclei, poly(ADP-ribose) polymerase activity in nuclei has been reported to be high, and decreased at the blastula stage, where the protein kinase activity become detect- able. Provided that these embryos cause modifications of nuclear protein to con- tribute to cell differentiation, the roles of these enzymes in the cell differentia- tion seem to be stage specific. DB 80 POLY (ADP-RIBOSYL) ATION OF HISTONES IN NUCLEI ISOLATED FROM SEA URCHIN EMBRYOS. A.Kanai, A.Isoai and I.Yasumasu. Dept.Biol Sch.Edu. Waseda Univ. Tokyo. ADP-ribosyltransferase activity in the nuclei isolated from embryos of the sea urchin, Hemicentrotus pulcherrimus, has been reported to be high at the 16-32 cell stage. The activity becomes quite low at the mesenchyme blastula stage and slightly increased at the onset of gastrulation (Isoai and Yasumasu '84). It is also well known that poly (ADP-ribosyl) ation of histones,as well as of non-histone proteins in nuclei occurs as the result of the reaction catalyzed by this enzyme, using NAD as the substrate. In the present study, poly (ADP-ribosyl) ation of proteins in chromation as well as in isolated nuclei from the 16-32 cell stage embryos were found in a reaction system using [-+%C- adenine]NAD as the substrate. Poly (ADP- ribosyl) ation of histones among the pro- teins in the chromatin was examined in the present study. Poly(ADP-ribosyl) ation was predominant in Hl among histones. DB 81 IN VITRO DNA SYNTHESIZING SYSTEM USING SEA URCHIN EMBRYOS. (1)NUCLEAR MEMBRANE-DNA POLYMERASE a COMPLEX. Masaki Shioda. Dept.of Physiological Chemistry & Nutrition, Fac.of Medicine, Univ.of Tokyo, Tokyo. Crude nuclear membrane-DNA polymerase @ complex fraction was prepared from 15h-old embryos (at 20°C) of sea urchin (Hemicentrotus pulcherrimus) by isopycnic centrifugation and DNA synthesizing activity was examined in vitro. The complex synth- esized DNA using fd phage single stranded circular DNA as template depending on NTPs. Agarose gel analysis of the product showed that about 100-500 bases of DNA was synthesized on the template DNA for 30 min at 26°C. The activity was sensitive to aphidicolin (20 ug/ml) and N-ethylmaleimide (20 mM) but it was resistant to ddTTP (ddTTP/dTTP=2) and G-amanitin (100 ug/ml), suggesting participation of DNA polymerase @ in the activity. DNA replicase (DNA polymerase @ - DNA primase) was released from the complex by NaCl treatment and separated by pucrose gradient centrifugation. However, de novo DNA synthesizing activity of the nuclear membrane- DNA polymerase & complex was much higher than that of membrane free DNA replicase when single stranded DNA was used as template. These results suggested existence of a possible DNA replication machinery which contained DNA replicase on the nuclear mem- brane. Further analysis of DNA product and a possible DNA replication machinery is under investi- gation. 938 Developmental Biology DB 82 STUDY ON DNA METHYLATION IN CHROMATIN OF SEA URCHIN EMBRYOS. T.Sakurai, K.Akasaka, H.Shimada, T.Shiroya Inst.of Zool., HRac.of Sci-., Univ-of Tokyo =, Tokyo. The extent of methylation of chromatin DNA was compared between blustula and pluteus of sea urchin. Purified chromatins were sheared for 2 minutes with Polytron and separated by glycerol gradient (7.6-76%) centrifugation into rapidly sedimenting(RF) and slowly sedimenting fraction (SF). Since only the latter fraction showed high template activity for RNA synthesis in the presence of E.coli RNA polymerase, it seems that SF is an active chromatin and RF is an inactive chromatin. DNA extracted from these chromatins was digested by two restriction endonucleases (HpaJl and Mspl), and separated by electrophoresis througn 1.2% agarose gel. The size of DNA fragments resulted from MspI digestion was smaller than those of Hpaf digestion with respect to both SF and RF. The computer analysis of these data sugests that no difference exists in the degree of methylation of chromatin DNA between either SF and RF or blustula and pluteus. The extent of DNA methylation at CCGG site was estimated about 50% or more. DB 83 ENDOGENEOUS ENDONUCLEASE IN ISOLATED NUCLEI FROM SEA URCHIN EMBRYOS. A. Isoai and I. Yasumasu. Dept. Biol. Sch. Edu. Waseda Univ. Tokyo. Previously, we have reported that acid-soluble DNA fragments are released from nuclei isolated from sea urchin embryos during the incubation in the pres- ence of Ca2t or Mg*t. In the present study, nuclease activity in the presence of Ca and/or Mg 2+ was observed in the extract with 0.4M NaCl from nuclei, isolated from the embryos of Hemicentrotus pulcherrimus. Nuclease was extracted from isolated nuclei of the sea urchin embryos by 0.4M NaCl treatment. The activity was assayed using \} phage DNA or plasmid pBR322. One unit of activity was defined as that amount of enzyme required to convert 0.5ug of superherical DNA to other forms in 1 min at 37°C. This nuclease was found to be endo- nuclease which cleaves closed-circular superherical pBR322. This enzyme was maxi- mally stimulated in the presence of Ca2t and Mg2+ ions (0.5-1.0 and 10mM, respec- tively). Nat and DTT were not required for its activity. At the 16-32 cell stage, the IMaximum activity of endonuclease was obtained. The activity became low at the blastula stage before hatching out and then decresed slightly at the swimming blastula and middle gastrula stage. DB 84 ISOLATION OF CALMODULIN IN THE NUCLEI ISOLATED FROM SEA URCHIN EGGS. T. Seki, A. Isoai and I. Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. In chromatin isolated from pea bud, cal- modulin has been found(Matsumoto et al,'83) - In the present study, we obtained calmo- dulin from chromatin isolated from the sea urchin eggs at the 16-32 cell stage. Assay of calmodulin activity was performed by the calmodulin caused stimulation of cAMP-phos- phodiesterase (Boehringer Manheim Co. W- Germany), according to the method of Murray and Togers,('78). Chromatin isolated from the sea urchin embryos at the 32-64 cell stage was incubated for 15 min in a solu- tion containing 5 mM EGTA and 50 mM Tris- HCl, pH 8.0 in the cold and then heated for 5 min in boiling water. Supernatant, ob- tained by centrifugation at 15,000g for 15 min, was dialyzed against 0.3M NaCl solu- tion containing 1 mM CaCl.,, 1 mM mercapta- ethanol 50 mM Tris-HCl (pf ToDo awinevel affinity gel chromatography (Affigel pheno- thiazine) was performed, and yielded sub- stances with phosphodiesterase stimulating activity. This fraction contains proteins other than calmodulin-like protein. This sample was applied on DEAE cellulase column and elution was performed by linear NaCl gradient. Two fractions, which contained calmodulin-like proteins, were obtained. By electrophoresis, these also contained small amounts of the other proteins. DB 85 INHIBITION OF EGG CLEAVAGE BY 5-BROMO-2'- DEOXYURIDINE IN THE EGGS OF THE SEA URCHIN ANTHOCIDARIS CRASSISPINA. K. Niikura, A. Fujiwara and I. Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ. Tokyo. In the eggs of the sea urchin, Hemicent- rotus pulcherrimus, egg cleavage occurred normally in the presence of 5-bromo-2'- deoxyuridine (BUdR), but was arrested in the eggs of Anthocidaris crassispina. 5- fluoro-2'-deoxyuridine (FUdR) inhibited egg cleavage in both species. Aminopterin, known to reduce the rate of thymidylate synthetase reaction, exerted stronger in Anthocidaris eggs than in Hemicentrotus eggs, which was reversed by exogenous dihy- drofolate or thymidine. BUdR-caused inhi- bition of egg cleavage in A. crassispina was canceled, to some extent, by exogenous thymidine. In the eggs of A. crassispina, dTTP level was quite lower than in those of H. pulcherrimus. If BUdR exerted lower inhibitory effect on thymidylate synthesis, than FUdR, quite low level of dTTP and low rate of production of thymidylate probably fail to support DNA synthesis, resulting in an arrest of egg cleavage. One of main reasons why BUdR inhibit the egg cleavage in Anthocidaris eggs but not in Hemicentro- tus eggs is probably the difference in dTTP level in the eggs between these species. Developmental Biology 939 DB 86 CLEAVAGE AND DNA SYNTHESIS OF STARFISH EMB- RYOS IN THE PRESENCE OF HYDROXYUREA. H. Yamada, R. Kuraishi, S. Hirai (Tohoku Univ), Y. Katoh, A. Shinagawa, N. Fusetani (Tokyo Univ), K. Okano (Teikyo Univ) and H. Nagano (Yamanouchi, Co Ltd). Hydroxyurea (HU) is an inhibitor of rib- onucleotide reductase which converts rNDP to dNDP. When starfish embryos were cultur- ed in the presence of HU and/or injected with HU at the l1-cell stage, cleavage was achromosomal and DNA synthesis was inhibit- ed after 16-32-cell stage. These results indicate that DNA synthesis of the first 4 or 5 S-phases is resistant to HU and taht of the stages later than 16-32-cell stage is sensitive. It has been reported that increase in dNTP pools is triggered by the 1-methyladenine treatment and dNTP pools expand during oocyte maturation and early embryogenesis. In the presence of HU, however, the expansion of dNTP pools was not obvious compared with the control, sug- gesting that HU has an inhibitory effect on the another unknown step of dNTP biosynthe- sis. The achromosomal cleavage and inhibit- ion of DNA synthesis caused by HU were released by adding AdR and CdR to the cult- ure medium, indicating that deamination of these bases occurs in oocytes. DB 87 RNA SYNTHESIS DURING EARLY DEVELOPMENT OF THE STARFISH, ASTERINA PECTINIFERA Sa eeecami, J. imayoshi, TL. Higaki ands. Sasaki. Dept. Applied Biochem., Hiroshima Univ., Fukuyama-shi, Hiroshima. We have examined the nature of RNA synthesized in blastulae of the starfish, Asterina pectinifera. The rate of RNA Synthesis begins to increase at the 128- to 256-cell stage which corresponds to the onset of blastulation. Actinomycin D at 25mg/1l inhibited incorporation of tritiated uridine into RNA of early blas- tulae by 882. In the presence of actino- mycin D (25mg/1), fertilized eggs clove eight or nine times but were unable to become blastulae. In the presence of aphidicolin (10mg/1), a selective inhibitor of DNA polymerase alpha, fertilized eggs clove achromosomally and the rate of synthesis of RNA was quite low as compared with that of the normal embryos of the corre- sponding stage. Sucrose gradient profile of tritiated RNA of early blastulae shows that synthe- sis of ribosomal RNA is quite low. These results indicate that most of newly syn- thesized RNA of early blastulae of the starfish, Asterina pectinifera, is derived from nuclear genomes and that these genomic informations are required for blastulation. DB 88 EXPRESSION OF SPICULE FORMING STAGE SPECIFIC PROTEINS DURING DIFFERENTIATION OF SEA URCHIN MICROMERES. R. Matsuda, T. Kitajima, C. Obata, H. Oohinata and T. Higashinakagawa. Dept. of Biol. Tokyo Metropolitan Univ., Tokyo. Cultured micromeres of sea urchin embryos, Hemicentrotous pulcherrimus, synthesize five specific proteins during differentiation to the spicule forming cells (Kitajima & Matsuda. Zool. Mag. 91: 200-205, 1982). The synthetic rates of these proteins increased at the onset of spicule formation. To know whether the expression of these proteins requires a new transcription of mRNAs or a selective activation of stored mRNAs, RNA was isolated from unfertilized eggs and embryos at various developmental stages and translated in vitro. The translation product was analyzed by 2D-PAGE. It was found that unfertilized eggs contained mRNAs coding for at least two of five spicule forming stage specific proteins. This result suggests that there is a selective activation of stored mRNAs during differentiation of sea urchin micromeres, DB 89 INTRACELLULAR AP4A CONTENT DRASTICALLY CHANGES PRIOR TO THE ONSET OF S-PHASE. M.Morioka and H.Shimada. Zool. Inst., Fac. Sci., University of Tokyo, Tokyo. We have reported that there is an active synthesis of AP4A in sea urchin emb- ryos during early development. To know if there is a specific period for the synthe- sis of AP4gA during a cell cycle, we attem- pted to deterimine the intracellular level of AP4gA in sea urchin embryos. AP4A was separated by DEAE-cellulose column chromatography from the soluble nucleotide fraction predigested by alkaline phosphatase and was determined by biolumi- nescence assay method using lusiferin-luci- ferase in the presence of phosphodiéste- rase. Unfertilized eggs contained significa- nt amount of AP4A, but it disappeared almost completely within 20min after ferti- lization. Prior to the onset of every S- phase, AP4A content increased markedly. Thereafter, prior to the onset of every S- phase, a marked and abrupt increase in AP4A content followed by its rapid disappearance was observed. When aphidicolin and emetin were added to the embryos just after ferti- lization, although the normal increase of AP4A was observed just before S-phase, the rapid disappearance of AP4A was not obser- ved, and the significant amount of AP4A were remained even 100-130min after ferti- lization. These results seem to suggest that AP,A is somehow involved in the initi- ation of S-phase in the sea urchin embryos, 940 Developmental Biology DB 90 BLASTOGENESIS AND GAMETOGENESIS IN INLAND- CULTURED COLONIAL ASCIDIANS. K. Kawamura and M. Nakauchi. Dept. of Biol., Fac. of Souls np Iola Winalwys , IKoGinal The method of laboratory culture and the process of sexual and asexual reproduction of the colonial ascidian, Botrylloides si- modensis, are described. The colonies were fed on Nitzschia. Natural sea water in which they were kept at 20°C was aerated and changed everyday. The used sea water was filtered to recycle. Under these condi- tions, colonies could be raised very satis- factorily. Single zooids produced two or three palleal buds. When a colony was vas- cularized, vascular budding took place. Histological studies showed that one day after vascularization two to five hemobla- sts of about 5 um in diameter aggregated in the test vessel. The aggregates were always enclosed by a single accessory cell which was autolyzed later. By three days they expanded to form a closed vesicle that con- stituted the inner epithelial layer of a vascualr bud. Germ cells at the youngest stage, about 7 um in diameter, appeared just behind the budding zone of a prefunc- tional zooid. They were also associated with single accessory cells. Oocytes of about 50 um in diameter were transfered from prefunctional zooids to their palleal buds. When a colony became mature, vitello- genesis was achieved in about a week. Self- fertilization took place. Embryos developed into tadpole larvae in about five days. DB 91 EFFECT OF CAUTERIZING THE NEURAL GANGLION UPON THE GAMETE RELEASE OF THE ASCIDIAN, HALOCYNTHIA RORETZI1. T.Numakunai, S.Kajiwara and Z.Hoshino. Wns Isao, Swe, Wolnolkan Weblo, Jour. Mar. Biol. Stat., Okayama Univ., Ushimado, Biol. Lab., Fac. of Educat., Univ. of Iwa- te, Morioka Neural ganglia of adult animals of three intra-specific groups of Halocynthia rore- tzi were cauterized, which have spawning seasons and spawning times different from one another. After operation they were kept under natural light condition (LD) and continuous light condition (LL) for a week or more. The animals of each group under LD discharged gametes not only at the original spawning time, but also at extraordinary time. In extreme cases they kept to spawn all day for several days. The animals of each group under LL also discharged gametes continuously, although the intact animals did not spawn under LL, except for one of three groups. The quan- tity of gametes discharged at extraordina- ry time are various. In some cases they released gametes as much as the intact an- mals did. All of them were mature and fer- tilizable. The neural ganglion seems to have some inhibitory effect upon the ga- mete release. To remove some inhibitory effect by the neural ganglion is one of factors that give rise to gamete release. SELECTIVE ISOLATION OF MATURE HAMSTER SPERM USING PERCOLL DENSITY GRADIENT CENTRIFUGA- TION. S. Oshio and H. Mohri. Dept. of Biol., Univ. Tokyo, Tokyo Density distribution of golden hamster sperm was examined by Percoll density gra- dient centrifugation. Spermatozoa collect- ed from distal cauda epididymides were washed with modified Tyrode's solution. Then lml of the sperm suspension was lay- ered onto 9ml of 60% Percoll and centrifug- ed at 30,000xg for 30 min at 25°C with an angle rotor. After centrifugation, the content of the tube was collected through a pinhole at the bottom into 20 fractions. The density of sperm was estimated from the elution profile of color density marker beads. The elution pattern showed separat-— ed two main peaks. The first peak of 1.12 g/ml contained both morphologically mature and motile sperm, whereas the second peak of 1.07g/ml was mainly composed of poorly motile sperm with cytoplasmic droplet. Thus the density of mature sperm was larger than that of immature sperm. To obtain mature and motile sperm more simply, monolayer of 1, 09¢/ml, Pereoll! was wsied.) Onlem millmonsmstaite sperm suspension was layered onto 4ml of 1.09g/ml Percoll and centrifuged at 250xg for 15 min at 25°C with a swing out rotor. After centrifugation, immature sperm with poor motility formed single peak in the upper layer, whereas loose pellet of mature and motile sperm was obtained at the bottom. DB 93 SPERM STORAGE AND ITS MODE IN HIBERNATING BATS. T. Mori and T. A. Uchida., Zool. Lab., Fac. of Agr., Kyushu Univ. Fukuoka Since the first discovery of the dis- tended uterus with live spermatozoa during winter in Pipistrellus pipistrellus (Pagen= stecher, 1859), the reproductive biology of bats has aroused interest in the variety of sites and modes of sperm storage. The pres- ent study investigated electron microscopi- cally the principal site of sperm storage and its mode in hibernating bats, which have a reproductive pattern involving pro- longed sperm storage. In vespertilionine bats (Myotis lucifu- gus and Pipistrellus abramus) the principal site of sperm storage is the intramural part of the oviduct. In a rhinolophid bat (Rhinolophus ferrumequinum), and in Myotis formosus and Pipistrellus endoi, the prin- cipal site of sperm storage is the caudal isthmus of the oviduct. The spermatozoa, wherever they are stored in the female re- productive tract, have close relationships with the microvilli or indentations of the epithelial cells. Such a mutual associa- tion suggests that the epithelial cells ac- tively provide the spermatozoa with nutri- ents for their prolonged survival. Further- more, infiltration of polymorphonuclear leucocytes does not occur in the principal site of sperm storage, although engulfment of spermatozoa by the epithelial cells is recognized. Developmental Biology 941 DB 94 ON THE FORMATION OF THE CYCLE OF THE SEMI- NIFEROUS EPITHELIUM IN THE MOUSE. M. Chiba. Dept. of Biol., Fukushima Med. Coll. Fukushima. A survey was made in order to ascer- tain when and how the first normal cycle of the seminiferous epithelium becomes es- tablished in the mouse. Testes at 10, 20 and 30 days were fixed in Bouin's, embed- ded in paraffine and cut serially at 10pm. Mallory's stained the basement membranes of the tubules distinctly. Two-dimentional reconstruction maps of tubules were made through serial photomicrographs. The site in meiotic metaphases was used to discri- minate the limits of the spermatogenic waves in the tubule. A reconstruction of a tubule from a 30-day mouse, in which the descent of the testis had already taken place, showed the normal stages in sperma- togenesis. The site of reversal of the waves was found in the vicinity of a large blood vessel situated opposite to the rete and beneath the tunica albuginea. The mean length of the waves on both sides of the site of reversal (11220pm) was much longer as compared with the average length of the waves measured elsewhere (5760pm). This Suggests that the spermatogenic waves progress in the distal direction in the tubule, e.g. toward the site of reversal and the first normal cycle of the semini- ferous epithelium becomes gradually estab- lished after the descent of the testis. DB 95 ULTRASTRUCTURAL OBSERVATIONS OF THE EMBRYONIC TESTIS IN THE LIZARD, TAKYDRO- MUS TACHYDROMOIDES. T. Oka. Dept. of Biol., Tokyo Gakugei Univ., Tokyo The development and differentiation of the testes were observed by light- and electron- microscopy in Takydromus embry- os incubated at 28 C and 30 C. Dark and light Sertoli cells were recognized. The organelles of both cells were similar, but those of the light cells were more dispersed than those of the dark cells. The endoplasmic reticulum of these cells was in the tubular form. From stage 36 to stage 38 the amount of endoplasmic retic- ulum increased. The membranes of the end- oplasmic reticulum were partly smooth but they were occupied widely by clusters of ribosomes, Ultrastructural alterations of the endoplasmic reticulum in this study were coincided with the increase of 38- hydroxysteroid dehydrogenase activity in Lacerta(Morat, 1971). However, it is cur- rently believed that much of the enzyme consists in the membranes of the smooth reticulum, The typical smooth reticulum could not observe during testicular diff- erentiation in Takydromus,. The interstit- ial cells did not show major cytological alterations as differentiation proceeded, except for a little increased amount of rough reticulum. DB 96 AUTORADIOGRAPHIC STUDY OF THE ORIGIN OF THE MEDULLARY CELLS IN THE PRIMORDIAL TESTIS OF RHACOPHORUS ARBOREUS. Toshikatsu Takasu and Hisaaki Iwasawa. Biol. Inst., Niigata Univ., Niigata. The medullary cords of the primordial testis: are formed by the cells which line the ovarian cavity of the ovary-like primor- dial gonad in Rhacophorus arboreus (Takasu and Iwasawa 1983). Mitotic activity in the somatic cells of the primordial gonads was examined throughout the process of testi- cular differentiation in the present study. Tritium thymidine (Spec. Act. 58 Ci / mMol) of lpCi / g body weight was injected intra- peritoneally into larvae in stages 40-46 (Iwasawa and Kawasaki 1979). After 2hrs the gonadal regions were fixed in Karnovsky's solution and osmic acid, and embedded in Epok 812. In stages 05-04 and ee) (Iwasawa's stages of gonadal differentiation in this species, 1969), in which testicular differ- entiation was occurring, each 4 blocks at one stage were sectioned serially at l pm and examined autoradiographically. An in- crease in the labeling index was observed in the ovarian cavity-lining cells at stage T,, and the laveling index in the mesenchy- mal cells increased at stage Tj. These re- sults support those of our previous study. DB 97 AN ELECTRON MICROSCOPIC STUDY ON THE BLOOD-TESTIS BARRIER DNS HE, SEEDS i; ORYZIAS LATIPES Naoki Shibata* and Satoshi Hamaguchi** VEVtae DIO. .fhiGe Sluis 2 sDEpUe DTU liau Golle——Gens—Edues 5 “Niigata Unive, Niigata. In mammals, the presence of the blood-testis barrier (BTB) between leptotene and zygotene spermatocytes has been demonstrated by electron microscopy. Recent studies revealed that the BIB in lower vertebrates exists at later stages of spermatogenesis. The present study was carried out cin!) order? to »cillarihy the existence of BIB in the teleost Oryzias latipes. We used horseradish peroxidase (HRP) and lanthanum chloride (La) as tracers and introduced them into the testis by the following two methods. IN VIVO STUDY: HRP was injected into the heart of the fish. IN VITRO STUDY: the testes were dissected out and immersed in saline containing HRP or La. The testis is composed of cysts of germ cells, and the differentiation of germ cells proceeds Synchronously in each cyst. The tracers penetrated all cysts and were observed between spermatogonia, spermatocytes, and spermatids, but were not noticed in the lumen of the efferent duct where the spermatozoa are located. These results indicate that the BIB exists in the wall of the efferent duct. The role of the BTB in QO. latipes seems to be different from that in mammals. 94? Developmental Biology DB 98 FINE STRUCTURE OF TWO TYPES OF SPERMATOZOA IN DROSOPHILA SUBOBSCURA. H. Takamori and H. Kurokawa. LMS. Olt Rayell, Geal5, Wins, Ox Gein, , Woeuceilsal . The polymorphic condition in spermato- zoa charactaristically exists in the Dro- sophila obscura species subgroup. D. sub- obscura produces long and short sized spermatozoa. None of short spermatozoon was found in seminal reseptacle of ferti- lized female fly. Fine structure of both long and short spermatozoa was investi- gated with an electron microscope. In each of the cases, the nucleus runs paral- lel with the axoneme and the major mito- chondrial derivative, the basal body is consequently adjacent to the acrosome. Though, the space construction of the short spermatozoon is similar to that of the long one, the lengths of several com- ponents in the former are much shorter than in the latter. The structures of the both long and short spermatozoa are Sie Wehie WE) TaINOSS ie@PisCl im ID. leases ( Takamori and Kurokawa, 1982; 1983 ), excepting cross profile of the nucleus. DB 99 MIGRATION MODE OF TWO TYPES OF SPERMATOZOA OF BOMBYX MORI FROM TESTIS TO RECEPTACULUM SEMINIS. H. Kasuga and M. OSanai, Dept. Biol., Tokyo Metropol. Inst. Geront., Tokyo. Migration of two types of spermatozoa of Bombyx mori, eupyrenes and apyrenes, was studied by observing ultrastructures of testis, seminal duct and s. vesicle in male pupae and adults, and receptaculum seminis and bursa copulatrix in mated female moths. Already in follicles of testis inner structures of eupyrenes and apyrenes were differently characterized. Eupyrene having a nucleus, where a pair of mitochondrial helices fused to form a semicircular nebenkern, while apyrene having no nucleus, where they existed separately as two oval nebenkerns. Such structural differences between two types of spermatozoa remained unchanged in the entire migration process. Even in recepta- culum in mated females apyrene spermatozoa were still existed with eupyrenes. In testis 256 spermatozoa derived from single spermatogonium were wrapped by a cyst envelope to form a bandle. The apyrene bandle was broken down, when it passed through the basilar membrane of testis, whereas eupyrenes migrated in a form of bandle until bursa copulatrix. The envelope of eupyrene bandle disappeared in seminal duct, reappeared in seminal vesicle and disappeared again in bursa copulatrix, then the bandle was broken. DB 100 LATE DEVELOPMENT OF ASTERINA PECTINIFERA: SCANNING ELECTRON MICROSCOPIC OBSERVATIONS OF THE SKELETON AND LARVA, Mi Komatsu) Dept. of Boll) Shae moss clam Toyama Univ., Toyama. Development of the skeletal system and the external fine structure of the early brachiolaria of the sea-star, Asterina ectinifere was observed with a light microscope and a SEM, About 15 days after fertilization, rudiment) of thes temnuanal plates appeared as a spicule on the poste- rior part of a bipinnaria. Five days there- after, the rudiment of the cemtra ioiame was formed at the middle of 5 terminal plates on the future aboral side. Five in- terradial plates, and 5 pairs of oral ~ plates became obvious on the future aboral, and oral sides, respectively, in 20 day early brachiolaria. Juveniles immediately after metamorphosis had 5 pairs of ambula- cral plates between the Ist and 2nd tube- feet, in addition to plates already formed, which were porous and furnished with Spines. No trace of the madreporic plate was seen even in the juvenile bearing 5 pairs of tube-feet on each arm, which was covered with many dorsal plates. Therefore, formation of madreporic plate of this spe- Giles, of which development as €heindiwnece type, Seems to be difterene fromeciaemson the sea-stars undergoing the nonbrachio- larian type of development, in which the madreporic plate develops at the early stage of primary skeletal system formation. DB 101 SCANNING ELECTRON MICROSCOPY OF MALFORMED EMBRYOS OF SEA URCHIN INDUCED BY PARTIAL UV-IRRADIATION. Y. Akimoto, T. Shiroya and S. Amemiya*. Zool: Inst.) hace omeseis Univ. of Tokyo. Tokyo and *Misaki Marine BHolls Stata, Hac. Of Sel.) Uni Or alokvor Morphological abnormalities induced by partial UV-inradiation to 16) cell vembsyos were Studied in sea urchin by using scann- ing electron microscope. UV-irradiation on the animal hemisphere of 16 cell embryos inhibited the invagination of archenteron and caused the formation of exogastrula. The blastocoel of the exogastrula was smaller than that of normal gastrula and was filled with a lot of cells. UV-irradi- ation on the vegetal hemisphere inhibited the gastrulation and the skeleton formation and gave rise to permanent blastula. A lot of cells which had few pseudopodia located at the vegetal hemisphere. UV-induced mor- phological abnormalities were due to the impairment of intercellular organization of mesenchyme (primary and secondary) cells and blastular wall. These UV-induced mal- formations were compared with chemicals ( ZnCl9, LiCl)-induced malformations. Intra- coelomic organization was different between them. By exposing the embryos to visible light immediately after UV-irradiation, morphological abnormalities were reversed almost completely, showing primary lesions were most probably pyrimidine dimers indu- ced in 16 cell embryos by partial UV-irrad- lation. Developmental Biology 943 DB 102 COLLAGEN SYNTHESIS IN SEA URCHIN EMBRYONIC ORGANS . H.Mizoguchi! andI.Yasumasu?.1.Div. of Biol., Jun. Col. of Rissh6 Univ., Saitama. 2.Dept. of Biol.,Sch. of Educ.,Waseda Univ., Tokyo. From sea urchin embryos, dissociated em- bryo-wall cells, gut and skeleton were sep- arated by washing them in Ca2+ and Mg2+ free sea water, 1M glycine containing 2mM ethylene-diamine-tetraacetic acid, and Ca?t+ and Mg2+ free sea water containing 1M su- crose, followed by hand-driven centrifuge. The spicules were isolated by another wash- ing with 0.2% sodium dodecyl sulfate and 0.01% CaCl2. In these isolated embryonic organs from the embryo exposed to 14C-pro- line for 3 hr, 14C-radioactivity was found in non-dialyzable matter extracted with hot 5% trichloroacetic acid, in which more than 43 of 14cC-proline residue was convert- ed to 14¢-hydroxyproline residue. Among these organs, the rate of collagen synthe- Sis was predominant in the archenteron at the gastrula stage, in the embryo-wall at the prism stage and in the spicule at the pluteus stage. The radioactivity in col- lagen fraction became low in the isolated gut at post-gastrula stage and gradually increased thereafter. In the embryo-wall, the rate became maxium at the prism stage. It is probable that the differentiation in sea urchin embryos in the endodermal cells occurs at first, then in ectodermal cells and fially in the mesodermal cells during early development. DB 103 CELL SURFACE OLIGOSACCHARIDES AS THE RE- CEPTOR RESPONSIVE TO THE NEURAL-INDUCING EFFECT OF CON A IN CYNOPS GASTRULA ECTO- DERM. K. Takata and K. Yamazaki-Yamamoto, Radio- isotope Center, Nagoya Univ., Nagoya. For the purpose to obtain further evi- dence on asparagine-linked oligosaccharid- es which possibly participate in Con A- mediated neural induction in Cynops pre- sumptive ectoderm, we used ovo-albumin, fibrinogen and IgG in combination with Con A-Sepharose which binds to oligosaccharid- es having the particular structures and causes neural induction. Before sandwiched by ectoderm pieces, Con A-Sepharose beads were immersed in solutions of the respec- tive glycoprotein, kept for 30 min at 20° C, and rinsed in Steinberg's solution. Sandwich explants thus prepared were cul- tured for 2 weeks. Histological examina- tion of explants showed that among glyco- proteins tested only ovo-albumin had the intensive inhibitory activity to the neu- ral-inducing effect of Con A-Sepharose. Results obtained suggest that cell surface Oligosaccharides involved in the first step of neural-inducing machinery are of the high-mannose type and/or the hybrid type, since each ovo-albumin molecule has one or the other of two types of sugar chain. HPLC analysis of cell surface sugar chains released by almond glycopeptidase revealed occurrence of several kinds of high-mannose type oligosaccharides. DB 104 DORSALIZING EFFECT OF CON A ON THE VENTRAL MARGINAL ZONE OF CYNOPS EARLY GASTRULA. K. Yamazaki-Yamamoto and K. Takata, Radio- isotope Center, Nagoya Univ., Nagoya The ventral marginal zone of early gas- trula (St 11) differentiated into the dor- sal tissues such as notochord and somites when treated by Con A (300 ug/ml), whereas it developed into blood island or nephric tubules in the isolation culture without Con A treatment. To analyze the mode of action of Con A, mannose (2 mM) was added to the Con A solution before use. Differ- ent from the results on the ectoderm, the dorsalizing effect of Con A on the ventral marginal zone could not be inhibited by mannose. *H-Con A bound to the explants at 20°C and 6°C was measured and visual- ized by autoradiography. Some difference in radioactivity and distribution of sil- ver grains were found between the differ- ent temperature experiments. Addition of Mannose reduced the bound Con A amount to the 1/7 level at 20°C and the 1/10 at 6° C, and resulted in disappearence of silver grains from the surface of explants but not from the cytoplasm where a small num- ber of grains were found. Inhibition of Con A effect could be achieved only in the condition of presence of mannose at 6°C. Present experiments suggest that Con A acts on the ventral marginal zone to cause the dorsalization of differentiative poten- cy through the cellular mechanism differ- ent from that in the presumptive ectoderm. DB 105 EFFECT OF GS(1)N441 GENE ON NUCLEAR MIGRATION IN DROSOPHILA EMBRYOS. K.Hatanaka and M.Okada. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. Early development of embryos of a ma- ternal effect mutant, s(1)N441 was ana- lyzed to elucidate the function of its wildtype gene. Microscopy of sectioned, and whole mount preparations, and time lapse videos were used for this work. In wildtype embryos, nuclei penetrated the periplasm in the posterior pole region immediately after the 8th nuclear div- ision. In mutant embryos nuclear arrival in the periplasm was retarded in the pos- terior region. To learn the cause’of retarded nuclear arrival in the posterior region, embryos at earlier stages were observed. Both in the wildtype and mutant, the number of nuclei increased in a 2" fashion and no asynchrony in mitotic cycles was noticed. However, posteriorward nuclear migration was clearly retarded in the mutant as early as_ the first mitosis. These results suggest that a function of maternal gs(1)N441° gene product is neces- sary for normal nuclear migration, from very early cleavage stages. Treatment of wildtype embryos with 2 pg/ml cytochalasin B for 5 min immediately after egg laying had no effect on mitosis, but prevented the nuclei from migrating normally, resul- ting in the treated embryos showing Similar nuclear distribution to that in the mutant embryos. 944 Developmental Biology DB 106 A PROBABLE CUE TO INITIATE CELLULARIZATION IN DROSOPHILA SYNCYTIAL BLASTODERMS. M.Okada and K.Hatanaka. Inst. Biol. Sci., Univ. of Tsukuba., Ibaraki. In wildtype Drosophila embryos, cleav- age furrow formation or cellularilzation starts after the 4th syncytial blasto- dermal mitosis to form cellular blasto- derms. In hope of answering the question, on what signal the cellularization in- itiates, a mutant with abnormal blastoderm formation (gs(1)N26) was observed for the early development. Microscopy of whole mount preparations and time lapse videos were used. Total mitotic cycles that syn- cytial blastodermal nuclei follow before cellularization starts varied from 0 to 6 nid GSU)NZG, Baws, cla® GOUIMES O% Maicoesle cycles are unlikely to be a cue to cellu- larization, although in wildtype embryos cellularization always follows the 4th syncytial blastodermal mitosis. Density of nuclei in the periplasm at and before cellularization was measured by_counting MUIGIEL im 2 Squace ©£ £00 pam Lin ielNe@ periplasm. The nuclear density in cellu- law lXlasicocdermms was 1360 2 A263 am gs(1)N26, and 13.8 + 1.0 in wildtype. In the mutant, cellularization was never seen in regions where nuclear density was less than 8, and it was always seen in the area where’ the density was more than 10. The results suggest that density of nuclei in the periplasm may give a cue to the ini- tiation of cellularization. DB 107 INDUCTION AND SUPPRESSION OF HEAT SHOCK PUFF FORMATION IN DROSOPHILA SALIVARY GLANDS TREATED WITH DETERGENTS. M.Myohara and M.Okada. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. To establish an assay system of cyto- plasmic activity on gene regulation, we treated salivary glands with detergents to make them permeable to exterior cytoplas- mic factors. Treatment with 0.005% saponin (or with 0.01% digitonin) for 10 min ren- dered a salivary glands permeable to DNase-I (MW=31K), and treatment with 0.005% saponin (or with 0.1% Nonidet-P 40, @ie \yalicll Oo 1% GWremicom—< OO) ster QO malin rendered it permeable to B-galctosidase (MW=540K) as well. Heat shock puffs were formed in non-heat shocked salivary glands when the glands had been permeabilized and incubated with a homogenate from heat shocked cells. Heat shock puffs were also observed in the permeabilized salivary glands incubated with a homogenate from non-heat shocked cells, suggesting that the heat shock response was initiated by the permeabilization itself. However, the LIMNEGWOCIEiLen Qi O01 GlacgiieComim—cieeacec salivary glands from larvae that had been kept ati3s7°@ for 20 mimvthen at 2326 fom | h did not induce heat shock puff forma- tion. The results suggest that a salivary gland preheat shocked then permeabilized with detergent for high molecules is a promising assay system for a gene regula- ting activity of extranuclear molecules. DB 108 STRUCTURAL CHANGES IN MALPIGHIAN TUBULES OF EYE COLOR MUTANT OF DROSOPHILA MELANO- GASTER LINKED WITH TRYPTOPHAN METABOLISM. SiYagal. Dept. Biol, Coll. Gs Educ, sumanz Tokushima. Tokushima. A wild type, Oregon-R and an eye color mutant, Cinnabar brown (cn bw) were used to investigate the fine structure of the cytoplasmic granules in Malpighian tubules of Drosophila melanogaster. Malpighian tubules were fixed 2.5% glutaraldehyde with 0.1% phosphate buffer for lh and postfixed in 1% OsO, with the same buffer for lh. Tissues were stained by uranyl acetate and lead citrate. The differences in the fine structure between the two strains as well as the size and fluoreées- cence in the larva were recognized. The large granules in Oregon-R which reach the diameter of 3-4 am reveal the vesicular forms with almost completely electron- lucent interior. The small. granules of about 1 ~m in diameter of cn bw made fair- ly homogenous structure. Larvae of cn bw were given tryptophan metabolite, 3-hydro- xykynurenine orally. About 2h after giv- ing the compound, the electron dense granules appeared amorphous and then, numerous myeline-like figure occured. 5h later, the granules of 2 pm in diameter with network appeared and myeline-like figure further developed. 24-30h later, the granules of 3 ~m in diameter showed a clear vacuole form. DB 109 ELECTRON MICROSCOPIC STUDY ON DIFFERENTIA- TED CELLS FROM DROSOPHILA EMBRYOS IN CUL- TURE. l Dh Y; Kurodas and Y. Shimaday,) sLabeeotpebheno- genet., Dept. of Ontogenet., Nat. Inst. Genet., Mishima, and 4Dept. of Anat., Sch. Of eMedia ChatbamUnanven a Ghealinas Each cell of blastoderm embryos of Drosophila is predetermined for its pros- pective fate in normal development. When cells from post-gastrula embryos were cultured in vitro in the absence of ecdy- sterone, they differentiated into charac- teristic cells of embryonic or larval tissues or organs, such as muscle cells, epithelial cells and nerve cells. On the other hand, cells from post-gastrula were cultured in the presence of ecdysterone, they differentiated and formed some struc- tures of adult tissues or organs, such as compound eyes, wings, and legs. The ultra- fine structures of cells which showed characteristic differentiated features were examined under an electron microscope. In muscle cells, the formation of typical myofibrils was observed. In syncytium of fused muscle cells, the M and Z lines in the A and I bands in sarcomeres were clear- ly found. In nerve cells, many neurofila- ments were parallel running in nerve fi- bers. In epithelial cells, the formation of interdigitations, gap junctions and desmosomes was observed. Developmental Biology 945 DB 110 REGULATION OF ERYTHROID DIFFERENTIATION OF GOLDEN HAMSTER BONE MARROW CELLS STUDIED IN VITRO. K.Morioka and T.Ono. The Tokyo Metropolitan Inst. Med. Sci., Tokyo. Subcultured supernatant cells of long term hamster (Mesocricetus auratus) bone Marrow culture could be induced to differe- ntiate into hemoglobin-producing cells by the addition of erythropoietin. Addition of each of "Friend inducers" alone such as dimethyl sulfoxide, nicotinamide, or pro- staglandin E, had no remarkable effect, though they could enhance the effect of erythropoietin. Exceptionally 5-azacytidine induced erythropoiesis without exogenous addition of erythropoietin. We have recently shown that m-aminobenzamide is a moderate inducer of Friend cell differenti- ation, but it strongly repressed the eryth- roid differentiation of the present system. In contrast, 12-o-tetradecanoylphorbol-13- acetate and glucocorticoid, which are known to be inducers of Friend cells, also strong- ly repressed the in vitro erythropoiesis of hamster bone marrow cells suggestive of somewhat common characteristics between differentiation mechanisms of normal and neoplastic cells. Thus the present system is quite useful for the study concerning regulatory mechanisms of erythropoiesis. DB 111 FURTHER STUDIES OF CHICK AND QUAIL LIMB BUDS IN ORGAN CULTURE. H.R.Suzuki and H.Ide. Biol. Inst., Tohoku Univ., Sendai We have previoursly reported that leg buds from stages 20-22 chick embryo develop into leg structure with adult limb pattern. In the present study, we have tried organ culture of quail wing buds and investigated the effects of cAMP On chick leg buds in organ culture. The wing buds of stage 23 quail embryo were Cultured on a membrane filter with culture medium consisting of Ham’s F-12, chick serum and chick embryo extract. After 6 days in culture, well-developed cartilage pattern of wing including Shoulder girdle, humerus, radius, ulna and digits was observed. Previous attempt to culture chick wing buds was unsuccessful. Thus, quail wing buds seem to be hopeful material to analyze fore- limb development. To investigate the effects of cAMP, leg buds from stages 20-23 chick embryo were cultured for 6 days in the medium with or without 1lmM cAMP and 1mM theophylline. In the presence of cAMP and theophylline, the development of distal elements were suppressed and the extent of suppression was more intense in younger limb buds. These results indicate the presence of developmentaily labile distal region which is sensitive to cAMP. DB 112 LOCAL APPLICATION OF FGF TO THE LIMB BUD MIMICS THE ACTION OF ZPA. TlHattcor1, and H-lde. Biol. Inst:, Tohoku Univ., Sendai. It 1s assumed that ZPA of chick limb buds is the area synthesizing "morphogen" associated with the limb pattern formation. But the entity is still obscure. Cooke et al. (1980) thought that ZPA provided two signals influencing cell proliferation and assigning position value. To elucidate the relationship between cell proliferation and Limb pattern formation, we examined the effect of locally applied FGF on the pat- tern formation. The limb bud tissues were dissected out in a wedge shape, treated in 70% ethanol at 60°C for 5 min and dehydrated by freeze drying. The blocks soaked in FGF (10pg/m1) were used for donors. Anterior border of a host bud was slit and the donor was insert- ed into the slit. After 6 days, extra digit-2 was induced with a frequency of 10% and the digit pat- tern became 2234. Branching structures of humerus and radius were also observed. Furthermore in cell culture, it was found that FGF inhibited the chondrogenesis of limb bud cells at low density. The possible functions of FGF in extra digit formation would seem to be Dimas— signing of positional signal which controls the fate of cells in the pattern specifi- cation, 2) increasing of anteroposterior width, 3) inhibition of chondrogenic dif- ferentiation. DB 113 SPATIAL INDIVIDUALITY IN CHICK LIMB BUD DETECTED BY IMMUNOHISTOCHEMISTRY WITH MONOCLONAL ANTIBODY. K.Ohsugi and H.Ide. Biol. Inst., Tohoku Univ. Sandai. To analyze the molecular mechanism of pattern formation, monoclonal antibodies have been made against antigens from chick limb buds. The immunogen used was the homogenate of stage 23 chick limb buds. Antibodies were examined by indirect immunofluorescence for reactivities with frozen sections of stage 23 chick limb buds. In a total of more than 2500 clones, one antibody (AV-1d) reacted with the specific region of chick limb buds. AV-1 recognized a distal portion of anterio-ventral mesoderm. This antigen was detectable by immuno- fluorescence in stage 20-32 in wing bud and in stage 20-30 in leg bud. No fluo- rescence was detected in any other tis- sues of chick embryo. These immunohisto- chemical patterns indicate that this antigen plays an important role in limb pattern formation. This is the first evidence that spatial individuality at molecular level really exists in limb bud. 946 Developmental Biology DB 114 A LIGHT- AND ELECTRON-MICROSCOPIC OBSERVA- TION ON THE SEX-REVERSAL PROCESS IN THE GENETIC FEMALE GONAD OF THE MEDAKA (ORYZIAS LATIPES) BY ANDROGEN. Hemkobayasha and) i. Hashuida. Weptsot. Baolus, Grew CONGO Meme. ,Gisevi. Newly hatched fry were dialy given meth- yltestosterone-diet(50p/g) during the juve- MILI jOSTOIUOG 4 IU SIE AISA VEIMES Ose iclnS wile gonocytes in the female gonad was at 10-11 mm of body length. Many gonocytes had al- ready differentiated into enlarged oocytes which usually had large germinal vesicles With peripheral nucileolis One ehewocheneha> nd,some of the remaining had begun to pro- liferate into small-sized spermatogonia connected each other with intercellular br- idgesyresuLeimeg in ches fonnaelonmomespe ama togonial cysts. Thereafter,the gonad became tO enlarge, increasing the eyst in) sazesand number,together with the growth of oocyte, and consequently it became to show a herma- phroditic state. At 15-18 mm, oocytes stop- ped the more auxocytic growth and then they turned@tordegenerate followanig \therneicno tae changes in ooplasm and nucleoplasm. It is most likely that oocytes degenerate into a number of large debris of myelin-like figu- res. These debris and the residual sperma- tid cytoplasm were captured by the columnar Cyst epitherial cells which show signs of active phagocytosis by the 20-25 mm stage when the gonad was occupied by the great numbers of cysts filled with all kinds of male gonocytes in spermatogenesis. DB 115 ISOLATION AND CHARACTERIZATION OF GALAC- TOSE-BINDING PROTEINS FROM NEW BORN MICE. T.Ohara and T.Yamagata. Lab. Advanced INS 5, MONE MoMISinatSierjeal Insc Cie IbseS SE@abio , Tokyo. Cell-cell interactions via glycoconju- gates on cell surfaces are reported to be important in early differentiation of Mammalian embryos. Since the occurrence of lactosaminoglycans was shown on the surface of early mouse embryos, we have been interested in searching for their counterparts, i.e., lectins specific for lactosaminoglycans in mouse embryos. We will report here the occurrence of two galactose-binding proteins (M.W. of which are 15K and 16K daltons) purified from soluble extracts of new born mice by affinity chromatography using a column of lactamyl-Sepharose. They agglutinated formaldehyde fixed trypsinized rabbit erythrocytes. Their hemagglutinating ac- tivity was inhibited by galactose-contain- ing saccharides and glycopeptides. N-Ace- tyllactosamine and asialo-glycopeptides having N-acetyllactosamine at their non- reducing termini were found to be most inhibitory. Judged from the specificity of these galactose-binding proteins, it is likely that they play an important role on differentiation of mouse embryos. DB 116 APPEARANCE OF TESTOSTERONE BINDING CAPACITY IN THE GENITAL TUBERCLE OF FETAL RATS. R. Murakami and T. Mizuno. Zool. sinsita, Unave Of eLokyojm lokyor Two steps are discernible in the development of os penis in rats: the first is the formation of the rudiment in male and female fetuses, and the second is the androgen-dependent chondrogenesis and osteogenesis after birth. We studied the localization of testos- terone binding site in the genital tuber- cle of fetal rats by the thaw-mount steroid autoradiography (Stumpf and Sar, 1975). The binding specificity of the labelling was confirmed by the competition experi- ments with cold testosterone and dihydro- testosterone. Nuclear labelling appeared at 16.5 days in the condensed mesenchymal cells forming the rudiments of corpus cavernosum penis and os penis. The nuclear labelling in these regions increased after 17.5 days. Mesenchyme of the future pre- puce, urethral epithelium, and the urethral mesenchyme were also labelled intensely. The surface epithelium of genital tubercle and the epithelium of preputial glands were scarecely labelled. In female fetuses, the labelling pattern was similar to that in males though the labelling intensity was weaker than that in males. These results indicate that the acquisi- tion of the testosterone binding capacity is involved in the first step of the devel- opmental process of os penis in rats. DB 117 MODE OF ACTION OF ANDROGEN DURING PROSTATIC BUD FORMATION IN THE RAT UROGENITAL SINUS. H. Takeda and T. Mizuno. Zool. Inst., Univ. of Tokyo, Tokyo. Prostatic glands develop as epithelial buds projecting from the urogenital sinus epithelium. This process is androgen- dependent and previous autoradiographic studies have revealed that the mesenchyme is the actual target for androgen. In the present study, we examined the relationship between the bud formation in the sinus after a brief exposure to androgen and the uptake of androgen by the mesenchyme by Stumpf's steroid autoradiography. Uro- genital sinuses of 16.5-day rat fetuses were cultured in vitro with a physiological dose of testosterone for appropriate time, then transferred to control medium. The minimum exposure time required for the bud formation was 12 hours in male and 24 hours in female sinuses. The lower sensitivity of female sinuses was accounted by auto- radiographic observations, that the female mesenchyme incorporated androgen more slowly for the first 12 hours. The number of induced buds was increased as the duration of testosterone-treatment was pro- longed. These observations indicate that the sinus mesenchyme required at least 12 to 24 hours' exposure to androgen to induce prostatic buds from the sinus epithelium and the number of induced buds is dependent on the duration of androgen treatment. Developmental Biology 947 DB 118 ANDROGEN INCORPORATION INTO THE EPITHELIUM AND INITIATION OF CELL DIFFERENTIATION IN THE RAT PROSTATE. N. Hironaka, H. Takeda and T. Mizuno. Zool. Inst., Univ. of Tokyo, Tokyo. Prostate glands of the rat develop as epithelial buds projecting from the uro- genital sinus epithelium into the surround- ing mesenchyme during fetal stages. The formation of the lumen begins among these prostatic buds at about 10-postnatal days. Steroid autoradiographical studies revealed that nuclear labelling with 3H-testosterone is restricted to the mesenchyme during fetal stages, but the epithelium begins to show nuclear labelling before the lumen formation. Electron microscopical obser- vation demonstrated that the size of the Golgi apparatus and amount of granular endoplasmic reticulum increased as the process of lumen formation proceeded. Thiamine-pyrophosphatase activity appeared in the centre of the prostatic buds just before the lumen formation, and then localized in the Golgi region of prostatic epithelium. These results indicate that the initiation of androgen incorporation into the epithelium is involved in the initiation of the functional differentia - tion of the prostate gland. DB 119 BLADDER EPITHELIUM OF ADULT RATS HAS POTENCY TO FORM PROSTATE-LIKE GLANDS. N. Suematsu and T. Mizuno. Zool. Inst., Univ. of Tokyo, Tokyo. Fully differentiated, stratified transi- tional, bladder epithelium of adult rats was examined whether the expression of the epithelium can be modified when recombined with ventral urogenital sinus mesenchyme of fetal rats. Adult bladder epithelium and fetal uro- genital sinus mesenchyme were isolated by collagenase treatment and recombined. The recombinants were transplanted under the kidney capsule of adult male rats. At 1 week after the transplantation, the epithe lium invaginated into the mesenchyme. This process is similar to the bud for- mation observed in the normal development of prostatic glands. After 3-4 weeks, complex glandular structures were formed in the recombinants. The histological features of these glands were similar to those of dorsal prostate glands, and rather different from those of ventral prostate glands in several points: rich in mesenchyme and less active in secretion. Nevertheless, these results indicated that adult rat bladder epithelium still has competence to form prostate-like glandular epithelium under the influence of fetal urogenital sinus mesenchyme and androgens. DB 120 MORPHOGENESIS OF CHICK PROVENTRICULAR GLAND, K. Hayashi and T. Mizuno, Zool. Inst., Univ. of Tokyo, Tokyo. The earliest phase of the proven- tricular gland formation in chick embryos is small invaginations of luminal surface of the proventricular endoderm of 6 day- embryos. At about 6.5 days, the glands develop as spherical buds, and then they begin to elongate into the surrounding mesenchyme. This gland formation has been known to be induced by proventricular mesen- chyme, though its mechanism remains uncertain. First, we studied the basement membrane by immunofluorescence with anti-laminin antibody and electron microscopy. It was demonstrated that the intensity of fluorescence and the thickness of basal lamina was uniformly strong and thick during luminal invaginations at 6 days. However, they became very weak and thin at the tip of the elongating glands after 6.5 days. Second, mitotic activity was measured by counting the metaphase nuclei accumu- lated by treatment with colcemid for 4 hr. High mitotic activity was observed in the region where the basal lamina was thane The relationship between high mitotic activity and thin basal lamina is under investigation. DB 121 INDUCTION OF EMBRYO-SPECIFIC PEPSINOGENS IN THE GIZZARD EPITHELIUM OF CHICK EMBRYOS. K. Takiguchi, S. Yasugi and T. Mizuno. Zool. Inst., Univ. of Tokyo, Tokyo. In normal development, gizzard epithe- lium never forms proventricular glands and never synthesizes pepsinogens. In the present study, we investigated whether the proventricular mesenchyme can modify pre- sumptive fate of the gizzard epithelium. The 4.5-or 6-day gizzard epithelium was combined with the 6-day proventricular mesenchyme, and grafted on the chorio- allantoic membrane. Grafts were cultured for 9 days. The gizzard epithelium com- bined with the proventricular mesenchyme formed glands similar to normal proven- tricular glands. Using the immunofluore- scent technique, we found that those glandular epithelial cells synthesized a chick embryo-specific pepsinogen. Bio- chemical studies demonstrated that the crude extract of the grafts possessed acid protease activity at pH 2.2. Electro- phoretic pattern of the acid proteases in the grafts was identical with that in normal embryonic proventriculi. These results show that proventricular mesenchyme can direct gizzard epithelium to form proven- tricular glandular epithelium, and that it can induce in the gizzard epithelium pepsi- nogens which were identical with those found in the embryonic proventriculus, 948 Developmental Biology DB 122 INDUCTIVE INFLUENCE OF DIGESTIVE TRACT MESENCHYME ON PRIMARY HYPOBLAST CELLS IN AVIAN EMBRYOS. S. Yasugi and T. Mizuno. Zool. Inst., Inaiel, Sieulo np Wheaio Wolo, Wols7O> Primary hypoblast of chick and quail embryos, destined to differentiate into yolk sac endoderm and express cysteine lyase activity, was associated with pro- ventricular mesenchyme of chick embryo and cultivated in the coelom of chick embryo. Within 1 day after cultivation, hypoblast cells showed cysteine lyase activity, which persisted at least 8 days after the association. After 6 to 8 days, hypoblast cells became basophilic and began to form glandular structure, and at the same time, cells reactive to the antiserum against an embryo-specific pepsinogen appeared in some grattse | Atterr 0) top 2ndaysi asthe majority of the grafts possessed well- developed glandular structure, and the epithelial cells were reactive to the antiserum. These results indicate that the primary hypoblast differentiated initially into yolk sac endoderm with cysteine lyase activity according to its own developmental fate. However the hypoblast changed its course of develop- ment to differentiate into the proventri- cular epithelium, performing morphogenesis on the one hand, and expressing pepsinogen on the other hand, under the specific influence of the proventricular mesenchyme. DB 123 DISTRIBUTION OF DNA POLYMERASE OC AND/3 IN THE INTESTINAL EPITHELIUM OF CHICK EMBRYO. H. Kitani? A. Matsukagé? T. Moritd and Y. L@cige? Daye, Wilol,, wae, Sei?, Goll. Geia, Edu?, Osaka Univ., Toyonaka and Lab. Bio- chem?, Exp. Rad”, Aichi Cancer Center Res. Inst., Nagoya. Distribution of DNA polymerase & and/f3 in the developing chick intestine were investigated by indirect immunofluorescent methods with antibodies against chick em- bryo DNA polymerase & and #3. DNA polyme- rase & was detected (1) in almost all epi- thelial cells of the duodenum in the 8- day embryos, (2) in the epithelial cells located in both basal and middle part of the previllous ridges being to form the villi, but not in tips of the ridges in the 12-day embryos, (3) in the epithelial cells located in the lower half of the villi just formed, but not in the upper half of the villi in the 18-day embryos, and (4) only in the cells of crypts in 2- week chickens after hatching. On the other hand, DNA polymerase /3 was detected inthe cells of all parts of the duodenum at any stage of development. In addition, almost all epithelial cells detectable DNA poly- merase Q were also found labelled with 3H-thymidine, if administered. Correlation between proliferative ability and DNA poly- merase & distribution in the epithelial cells in the duodenum has been discussed. DB 124 PRESENCE OF SUCRASE IN THE CHICK YOLK SAC. S.Matsushita. Dept. Biol., Tokyo Women’s Medical College, Tokyo. The presence of sucrase in the yolk sac of the chick, which strongly resembled the sucrase of the chick intestine, was demon- strated biochemically and immunologically. The sucrase was partially purified from the particulate fraction of the homogenate of the yolk sac of 4-day-old hatched chicks by papain solubilization, ethanol precipitation and Sephadex G-200 chromato- graphy, and the properties of the enzyme was compared with those of the purified sucrase of the chick intestine. The immunodiffusion using the antiserum raised to the purified intestinal sucrase showed that the yolk sac sucrase was identical with the intestinal sucrase. PH optima, kinetic properties and substrate specifi- city of the two enzymes agreed well. How- ever, the size of the yolk sac sucrase was estimated to be slightly smaller than that of the intestinal sucrase by Sephadex G- 200 chromatography and SDS-gel electropho- resis. The immunofluorescence and immuno- electron microscopical studies using the antiserum raised to the intestinal sucrase revealed that the sucrase was bound on the luminal surface membrane of the endodermal cells of the yolk sac of hatched chicks and embryos. However, the unbound sucrase may also exist in the cytoplasm. DB 125 DEDIFFERENTIATION AND REDIFFERENTIATION OF PIGMENTED EPITHELIAL CELLS IN CELL CULTURE. Y. Itoh and M. Yasutomi, Biol. Lab., Aichi Med. Univ., Nagakute, Aichi. Synthetic activities of melanin were investigated throughout dedifferentiation and redifferentiation of pigmented epithel- ial cells (PECs) of chick embryos in cell culture. Dissociated PECS vigorously pro- liferated in the medium containing phenyl- thiourea and hyaluronidase, gradually lost melanosomes and finally dedifferentiated. In the early stage of dedifferentiation, tyrosinase activity rapidly disappeared. It seems that dedifferentiated PECs (dPECs) are in essentially neutral state, because dPECs can differentiate not only to pigment cells but also to lens cells depending upon the subsequent culture condition. When aPECs were transferred into the medium con- taining ascorbic acid, tyrosinase activity came to be detected again after about 24 hrs, in Goldi bodies histochemically and in cell homogenate biochemically. At about 60 hrs the formation of melanosomes was observed both under a light microscope and TEM and within 5 days almost all dPECs re- differentiated to pigment cells. In conclu- tion, the expression of melanotic phenotype of the cells was completely inhibited in the bipotent dedifferentiated state. We can now provide a homogeneous cell population, which can synchronously redifferentiate to pigment cells, for an analysis of molecular mechanisms of melanogenesis. Developmental Biology 949 DB 126 MSH AFFECTS MELANOGENIC DIFFERENTIATION OF AVIAN NEURAL CREST CELLS IN VITRO. M.Satoh and H.Ide. Biol. Inst., Tohoku Univ., Sendai. MSH is known to stimulate melanogenic differentiation of neural crest cells in amphibians and mammalians. But the effect of the MSH on avian neural crest cell dev- elopment has not been reported. In the present study, we report that &-MSH affects melanogenic differentiation of avian neural crest cells in vitro. Neural tubes with neural crest cells were obtained from H-H stages 14-16 quail embryos by trypsin digestion and cultured in a medium consisted of 81% MEM, 15% horse serum and 4% chick embryo extract. After 24hrs in culture, neural tubes were scrap- ed away and neural crest cells were subcul- tured in the medium with or without 0.lpg/ ml &-MSH. Alpha-MSH stimulated melanogenic differentiation but did not affect growth of neural crest cells. In clonal cultures, pigmented, non pigmented and mixed colonies appeared in treated and control cultures. The ratio of pigmented colonies to total colonies was increased by X-MSH. Thus K-MSH seemed not only to accelerate melanogenic development but also to affect commitment of neural crest cells to melanogenic dif- ferentiation in vitro. The morphology of the differentiated melanocytes was not sig- nificantly different between treated and control cultures. It was suggested that the action of K-MSH was mediated by cyclic AMP. DB 127 THE BEHAVIOR IN VITRO OF MOUSE NEURAL CREST CELLS. Rottco, Biol. inst. , Tohoku Univ, S., Sato Bike idkeuchi, Dept. of Biol. Sci., Tohoku Univ., Sendai. Although the differentiation of the neu- ral crest(NC) cells especially in quail and chick has been widely investigated in recent years, more detailed mechanisms controlling segregation of NC cells into different lineages remain to be unclear. In order to decipher these mechanisms, we utilized monoclonal antibodies to detect the stage- or cell-type specific markers in NC cells. The monoclonal antibodies(28B, 310E, 312C) against the cell surface of mouse melanoma cells(4CS5) were used in our study. 310E and 312C indicated the identi- cal antigen specificity. Although the reactivity of both 310E and 312C to NC cells after 3 days in culture was not ob- served, these antibodies bound to the catecholamine (CA)-producing NC cells cul- tured for 12-15 days. 310E also reacted with the morphologically undifferentiated CA-positive cells. When the reactivity of these antibodies to epidermal melanocytes was investigated, the same results were obtained. 310E reacted with either mature melanocytes or immature one. None of the tested cell types except for melanoma (4CS5) reacted appriciably with 28B anti- body. ~ DB 128 NEURAL-INDUCING CAPACITY OF MESODERMALIZED PRESUMPTIVE ECTODERM: A-S.suzuki. Biol. Dept., Fac. of Gen. Bduc. Univ. of Kumamoto, Kumamoto. The presumptive mesoderm exhibits two unique properties: it forms the primary mesodermal axis by its capacity to differ- entiate, and induces neural structures in the ectoderm by its neural-inducing action. However, it is not yet clear whether meso- dermalization is directly coupled with neural-inducing activity. Newly-mesoderma- lized ectoderm was used to analyze _ the temporal relationship between mesodermali- zation and neural-inducing capacity. Competent ectoderm of stage 11 was me- sodermalized by being brought into contact with swimbladder of Carassius for 3 hours. The mesodermalized ectoderm was combined with the reactor ectoderms of stages i2a, 12b, 12c, and 13b, respectively. The com- binants were cultured for one week. The mesodermalized ectoderm acquires high ability to induce neural structures in the competent ectoderm by its neural- inducing capacity. The neural structures were observed in 30 cases out of 34 combi- nants of stage 12a and the present study on origin of differentiated tissues defi- nitely showed that all the cells of the differentiated neural tissues originated from the reactor ectoderm. These facts suggests that the neural-inducing activity of the actor cells(organizer) appears in coupled with their mesodermalization. DB 129 NEUROGENESIS IN NORMALLY DEVELOPING EMBRYOS OF CYNOPS PYRRHOGASTER: OCCURRENCE OF CELL- DEATH AND A NEW THEORY FOR NEUROGENESIS. Be ino. Depts: of Biol., Fac..,of Educ, Miyazaki Univ., Miyazaki. The processes of neurogenesis in normally developing embryos of the newt were studied with their serial paraffinsections. During early stages of gastrulation, ectodermal cells fall on the bottom of the blastocoel. The number of falling cells varies among embryos, from zero to hundreds per embryo. In general, more cells fall in embryos with larger blastopore. These cells become situ- ated in the endoderm and are eventually disintegrated. Therefore, cell-death is involved to an individually variable degree in normal neurogenesis in the newt. At the end of gastrulation, the neural plate ap- pears occupying a large part of the dorsal and lateral regions of the embryo. The classical theory for neurogenesis or the induction theory, which has been devel- oped by experiments on isolated explants, is hardly compatible with the observed mode of cell-death. It seems also difficult to find topographical relation between the neural plate and some specific part of the archenteron roof. Thus, the observed facts in normal neurogenesis do not support the induction theory. To the contrary, observed facts are compatible with the idea that a mass of cytoplasm in the egg, the mesoplasn, establishes the clone of neural cells and these cells form the neural plate after eliminating cells without mesoplasm. 950 Developmental Biology DB 130 METHYLXANTHINES STIMULATE GEMMULE FORMATION IN FRESH-WATER SPONGE, EPHYDATIA FLUVIATILIS. Y. Watanabe!»*, C, Asano’ and S. Nemoto. 1Dept. of Biol., Ochanomizu Univ., Tokyo and 2Tateyama Marine Lab., Ochanomizu Univ., Tateyama. It was demonstrated that theophylline strongly stimulated gemmule formation in young sponges developed from gemmules (Rasmont, '74). Cyclic nucleotides are reported to induce gemmule formation in S. lacustris (Simpson & Rodan '76), but not in E. fluviatilis (Rasmont '74). In the present studies, the effects of some methylxanthines other than theophylline and cyclic nucleotides were examined on gemmule formation in E. fluviatilis. Newly hatched sponges were cultured in the M-medium containing methyl- xanthines (caffeine, isobutylmethyl- xanthine and theophylline) and cyclic nucleotides (cAMP, DBcAMP, cGMP and DBcGMP Ne Gemmule formation was also stimulated by both isobutylmethylxanthine and caffeine, though the rate of gemmule formation was less than that of theophylline. Both cAMP and DBcAMP were found to induce gemmule formation. DBcAMP was much more effective than cAMP. Effective concentrations of DBcAMP ranged from 0.001 to 10mM. These results make it highly likely that cAMP plays an important role in some processes of gemmule formation, DB 131 TISSUE COMPATIBILITY BETWEEN COLONIES AND BETWEEN PRIMARY POLYPS OF POCILLOPORA DAMICORNIS. Mo Hidakas Dept.) Of Biola Unaiven Ove tine Ryukyus, Okinawa. In scleractinian corals, intracolony grafts are compatible while intercolony grafts are invariably incompatible. On the contrary, when planulae settle close to each other, they fuse to form an aggregated colony. Grafting experiments with primary polyps of Pocillopora damtcornis were performed to examine whether the primary polyps lack a histoincompatibility system as shown by adult colonies or only primary polyps that are genetically close to each other can fuse. Planulae spawned by a colony were collected and allowed to settle On ya plastve sheet. | hen two) ipxaimainny, polyps were brought into contact and maintained in this position. When two primary polyps derived from different colonies were kept in contact, they fused with each other. Even primary polyps derived from colonies of different color morphs fused with each other. In adult colonies, however, branches of different colonies did not fuse. Fusion of the tissues was observed only in isografts. The present results suggest that the coral lacks a histoincompatibility system in the early stage of colony formation. DB 132 STUDIES ON THE ESTABLISHMENT OF RADIAL SYMMETRY TENTACLE REGENERATION IN AURELIA POLYP AND EARLY STROBILA T. K.>r Kato ‘and Ss voshaharal.. Depts bole Osaka Kyoiku Univ., Osaka If the polyp is transversely cut at any level of the body, the tentacle regenerati- on in the lower piece may occur first at 4 perradial sites, second at 4 interradial, and finally at 8 adradial ones. This means also that simultaneous regeneration takes place at a opposite site. Treatment with cell division blocking agents such as mito- mycin C, aphidicholin and colchicin before the cut brings about inhibition and/or dis- order of regeneration. Looking the course of regeneration, we can divide 3 types: the first one is that the first regenerating tentacle(s) appear solitary or at not oppo- site (not pajred), the second one is that they are at both paired and not paired Sites, although many of the specimens of these types show the forthcoming tentacles will trend to occupy the vacant opposite Sites. The third one is the same as in the control animals (no treatment with agent). The first type is recognized more frequent- ly in the case of prestrobila or early strobila than in that of polyp. These facts Suggest that during the furrow formation, the transverse pattern of each disk may newly reestablish somewhat apart from the previously existed organization of polyp. DBeSs STUDIES ON THE ESTABLISHMENT OF RADIAL SYMMETRY TENTACLE REGENERATION IN AURELIA POLYP AND EARLY STROBILA II. S. Yoshihara and K.-I. Kato. Dept. Biol., Osaka Kyoiku Univ., Osaka. As mentioned in the abstract given just before, the tentacle regeneration can be divided into 3 types according to the pro- cess. In order to know possible key how can the paired or symmetrical site regeneration of tentacles be achieved, we have examined tentacle regeneration in the polyp in which 1,2,3,0r 4 canals have been surgically re- moved before transverse cut. Although the paired tentacle regenera- tion in the polyp which begins at perradial Sites is hardly disturbed even when the animals are treated with drug, the removal of single or non-paired plural no. of Ca- nals may lead to the non-paired tentacle regeneration at the per- and interradial sites sometimes, although that of paired one (at the opposite site each other) does hardly exert on the order and the site of regenerating tentacles. This tendency was often found in the non-treated and mito- mycin treated animals, but not so much in the aphidicholin treated ones. It is known from these facts that canals have no di- rect relation to the tentacle regenera- tion, but in someway to the order and site of it. Role of canal in the horizontal or- ganization is worth to study further. Developmental Biology 951 DB 134 MEDUSA OF FRESH-WATER POLYP, ASTROHYDRA. H. Hashimoto. Dept. of Biol., Fac. of Educ., Shizuoka Univ., Shizuoka. a japonica Hashimoto, 1981, is a tiny fresh-water polyp with special tentacles. The polyp is always solitary and reproduces asexually by giving off a planula-like organism, frustule, but its sexual reproduction is so far not known. In 1984 (May-June), medusae unexpectedly appeared from Astrohydra cultured in the laboratory. The bud of this medusa does not form a basal stalk, which is present in Microhydra, so the medusa is directly released Foi the lateral of polyp body. The newly released medusa is only 0.5 mm in diameter and its umbrella is a normal bell-shape with four long and four short tentacles. The tentacles of this medusa bear many bristle-like papillae with one to two nematocysts on apices. A papilla consists of only a vacuolate single cell and sometimes reduces to a short process particularly when tentacle is retracted. This structure of tentacle is symbolical of the medusa of Astrohydra. The radial canals are mostly four in number and the manubrium forms a quadrangular prism, of which basal half functions as a stomach. The statocysts of this medusa are devoid of velar centripetal canals. One medusa grew to a diameter of 1.35 mm with about 15 statocysts and 29 tentacles, but died of disease. The gonads were not formed. The other medusae died in earlier stage. DB 135 MITOTIC DENSITY AND HISTOLOGICAL EVENTS OCCURRING IN THE FRESHWATER PLANARIAN. Y.Kishida and Y.Iwata. Biol. Lab., Sch. of Educ., Okayama Univ., Okayama. In the freshwater planarian, Dugesia japonica japonica, the cephalic piece was joined to the caudal one. Intercarary re- generation occurred between both pieces. The joined animals were cut transversely into six small fragments and each fragm- ent was fixed in 30% acetic acid and then stained by immersion in lactic fuchsin on Slide glass for 10 minutes at 20°C. Then a coverglass was put gently on the frag- ment. Mitotic density was calculated in each fragment. On the 3rd day after join- ing, mitotic density rose in the region at some distance from the suture in the caudal piece, and on the 4th day the peak of mitotic density shifted anteriorly to the region contiguous to the suture. High mitotic density in the intercarary rege- nerate was retained for two weeks after joining and dropped later gradually. His- tological observation showed that the ac- cumulation of the undifferentiated cells, blastema, was not formed in the interca- rary regeneration. It was suggested from these results that the contact between the different body levels stimulated the mitotic activity of the cells in caudal piece and afterwards the original propor- tion of the worm's body was regulated by means of morphallaxis in cephalic piece. DB 136 NORMAL DEVELOPMENT OF A FRESHWATER PLANARIAN, BDELLOCEPHALA BRUNNEA. T. Nakata and W. Teshirogi. Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. Planarians spawn the composite eggs by entosomatic fertilization, in which several egg-cells and numerous yolk-cells are enveloped with the hard egg shell. Because of this peculiarity, hitherto, on the embryonic development of planarians few investigations have been done. The reporters investigated the early development of Bdellocephala brunnea, from fertilization — cleavage — blastula — embryonic pharynx formation — tempo- rary intestine formation — permanent pharynx formation — differentiation of intestinal and nurvous systems — to hatching. Then, the post development where the hatched juveniles become adult with differentiated genital organs ( Ovary, testis and copulatory apparatus ) was observed. The table of " The normal stages in the development of Bdellocephala brunnea " was completed. DB 137 PRIMARY TISSUE CULTURE OF FRESHWATER PLANARIAN IN NEWLY DEVISED MEDIUM. K. Tohya and W. Teshirogi. Dept. of Biol., RacewOr Sci... Hurosak. Univ... Harosaki. Collected worms were cultured in the sterilized condition without feeding, rinsed with culture water containing anti- biotics at several days' intervals. As a pre-treatment, they were immersed in the culture water during a whole day, and sterilized by five minutes' radiation of ultraviolet rays from about 30 cmdistance. The tissue fragments of worm bodies*were cultured using the medium mentioned below in the flat dish at 17° C and pH. 7.4-7.6. We have modified the culture medium after referring to the data presented by the previous workers. Some available results for this culture of the cells could be obtained. Namely, neoblasts, intestinal cells, gland cells and other cells iso- lated from tissue fragments were adherent to the dish bottom, forming pseudopodia or ruffled membrane. And the cells mi- grated actively from the tissue fragments. These isolated cells or migrated ones sur- vived for 1-2 weeks. 952 Developmental Biology DB 138 SUPERNUMERARY SEGMENTS AND ABNORMAL SEGMENT DIFFERENTIATION ARE INDUCED BY INHIBITORS OF DNA SYNTHESIS IN THE HORSE- SHOE CRAB EMBRYOS. Tee EOWr Dept. of Biol., Fac. of Edu., Shizuoka Univ., Shizuoka. Embryos were treated by inhibitors for the purpose of analysis of mechanism of body segment- ation. Inhibitors increased the number of abdominal segments and changed the differentiation of each segment at the rate of 80-1003. The embryos with supernumerary segments were examined and the results induced the following presumptions. (1) The primordia of segments are formed one by one from the growth zone at the posterior end of embryonic area, and they are determined soon. (2) When DNA synthesis is inhibited, the forming primordium differntiates into the structure between the anterior segment and itself. (3) The abnormal differentiation is caused by the time lag between DNA synthesis and the other metabolism. (4) The differentiation of each segment succesive- ly determines ~ that of next o®@ segment in ® (oR Inhibition of @ c® both case of DNA synthesis normal DOO®D DOD® segments and abnormal ones. DOO®®D DOeaS® (5) The whole ae number of DOOOO®D DOOO® @® segments is © oe i determined as VOOO® DOOBOHO®D the results. DO@BHO::. ‘ REISE ee Embryo with Figure. » Normal embryo Coane See DB 139 STUDIES ON FREEZING OF DROSOPHILA EGGS. Y. Takada and Y. Kuroda. Lab. of Pheno- genet, Dept. of Ontogenet., Nat. Inst. Genet., Mishima. Drosophila is one of the materials which have been the most extenively used in various fields of genetics. All stocks of wild-type and mutant strains of Drosophila are Maintained at 18°C to°25 ¢€ ingthe most laboratories. In the present experiment, an attempt was made to freeze Drosophila eggs at -80°C in an electric freezer. Male and female flies of Oregon-R strain of D. melanogaster were kept in glass tubes at DI CeEOT hours to collect eggs. Eggs were dechorionated by treatment with 3% sodium hypochloride solution for 30 minutes. Eggs at the appropriate stage of develop- ment were selected under a binocular micro- scope. They were transferred to salt solu- tion containing 10% glycerol and frozen in small glass vials. After several days, eggs were rapidly defrozen, washed by salt solution, and incubated in salt solution at 25°C for 24 hours. Hatched larvae were scored and cultured on the standard food to allow them to proceed the further develop- ment. No reagents used to enhance the per- meability of vitelline membrane produced good results. Thus among 70 eggs at the stage 1, 2 or 5 used for freezing, 20 eggs hatched to larvae. Seveval of them devel- oped to fertile adults. DB 140 RELATIVE GROWTH BETWEEN FEMUR AND TIBIA IN THE GERMAN COCKROACH, BLATTELLA GERMANICA. ING UENO el, IDkejie4 Gis isnloOls> MESS Gr Sci., Nara Women's Univ., Nara. Femur and tibia length of fore, mid, and hindleg, and head width were measured throughout larval stages up to the adult in both sexes. All the leg segments showed positive allometric growth against head width. Postero-anterior and disto- proximal growth gradients along the body and leg axes were detected. Total length O£& femuG and tibia of forelleq anya particular instar was nearly equal to that of midleg in the preceding instar, and to that of hindleg in the instar before the preceding instar. Ratio of tibia length to femur length was high in hindleg, intermediate in midleg, and low in fore- leg. The ratio increased as the stage advanced in mid and hindleg. Correlation coefficients between any two of the 12 leg segments, femur and tibia of both sides of the body, were calculated in every instar up to the adult stage. In all the three kinds of legs, the values between femur and femur, and tibia and tibia, of opposite side of the body were higher than those between femur and tibia of the same leg. It was also revealed that foreleg correlated more closely to midleg than to hindleg, and midleg, more closely to hindleg than to foreleg. DB 141 ARRANGEMENT FORMATION OF SCALE PRECURSOR CELLS ON THE PUPAL WING BUD OF PIERIS RA- PAE. A.Yoshida and K.Aoki. Life Science Inst. Sophia Univ. Arrangement formation of scale precur- sor cells was examined of the upper sur- face of the fore-wing bud of white cabbage butterfly, Pieris rapae, during early pu- pal stage, using scanning electron and optical microscopes. 20h(after pupation): Polygonal pattern of epidermal cells, which formed the mono- layer sheet, with microvilli was seen on the surface. 24.5h: On the distal region, few cells without microvilli emerged. These were the scales precursor cells as they would form rows later. While, on the proximal region, such cells were not seen. So, the arrangement formation process ini- tiate earlier on the distal region than on the proximal region. The process on the distal region will be described as follows. 28.5-34.5h: Some cells without and with microvilli became long and slender proxi- modistally, and gradually arranged in the anteroposterior direction. 36-37.5h: Those slender cells, which were the scale pre- cursor cells arranged anteroposteriorly, swelled from the monolayer cell sheet. As described above, scale precursor cells are anteroposteriorly arranged in the monolayer sheet of the epidermal cells, before they swell from that sheet. Developmental Biology 953 DB 142 SEXUAL DIFFERENTIATION IN THE LAST ABDOMINAL GANGLION DURING POST EMBRYONIC DEVELOPMENT OF BOMBYX MORI. A.Nemoto, A.Yoshida, K.Aoki. Life Science Inst. of Sophia Univ., Tokyo. In the holometabolous insect, Bombyx mori, the last abdominal ganglion (LAG) innervates the genitalia. The ganglia XI (GXI), GXII and GXIII in the larva fuse into the adult LAG during metamorphosis. There are no significant sexual differ- ences in the gross anatomy in the larva. After metamorphosis, in the adult the LAG innervates the genitalia which have are strikingly different. We observed the changes that occur during metamorphosis in the LAG by the reduced silver staining method. In the male LAG fiber tracts from nerve bundles extended directly to the midline. Such fiber tracts were never ob- served in females. In contrast in the fe- male, the fiber tracts were irregular. We calculated the ratio of the area of the neuropile to the whole ganglion from the sections. This ratio was higher in females than in males. It is suggested that these sexual differences are associ- ated with the function of the genitalia of the two sexes. DB 143 EARLY EMBRYOGENESIS OF EMBRYONIC LETHAL, KIDNEY-SHAPED EGG IN BOMBYX MORT. K. Miya. Lab. Appl. Entomol., Fac. Agr., Iwate Univ., Morioka. Within the kidney-shaped egg laid by the homozygous female, the embryo develops only ectodermal organs, but no mesodermal organs. The early embryogenesis of this strain was examined with light and elect- ron microscopy. Until the early germband stage no remarkable differences were dis- tinguished between the normal and kidney- shaped eggs. Afterwards, in the kidney- shaped egg large amount of cytoplasm and several cells were extruded from the pre- sumptive head region into the yolk at the "pyriform”" stage, resulting in inhibition for the formation of a normal wide head lobe and then an invagination in its cen- tral region, the first sign of the meso- derm differentiation. This phenomenon proceeded gradually toward the posterior part and thus the differentiation of meso- derm became impossible throughout the en- tire length of germband. From the results it was suggested that in the kidney-shaped egg the excessive extrusion of the cyto- plasm and cells accopanying with the es- tablishment of antero-posterior organiza- tion might induce the inhibition for the normal formation of head lobe and meso- derm. DB 144 EFFECTS OF VARIOUS INHIBITORS ON EMBRYONIC DEVELOPMENT OF CRICKET. C.Tateishi, M.Koike, S.Yukawa and T. Nakazawa. Dept. of Biology, Faculty of Science, Toho Univ., Funabashi. To elucidate the molecular mecha- nism of the embryonic development in insect, effects of several types of inhibitors on the morpho- logical development of cricket (Gryllus bimaculatus) embryos were examined with a numerical estimation during development up to’ the hatching period. Inhibitors, colchcine, cytochalasin B, potassium cyanide, cycloheximide and cepharanthin were treated succesively for each 4 hr from just after oviposition to yolk cleavage stage (40 hr after oviposition). All of used inhibitors exhibited dose-dependent effect on the hatchability. Most sensitive stage against inhibitors was the period dumingue 2 has sb com Oviposit2on.. This result shows a possibility that change in the program of development occurs within 12 hr after oviposition. In the embryos developed within this period, there may exist chemical processes such as cell division, microtubule- concerned system, respiratory system, protein synthesis. These processes must play important roles in the proceeding of the program during the subseguent development. DB 145 LATERAL UV IRRADIATION TO THE STRATIFIED CONSTITULNTS OF CENTRIFUGED CHIRONOMUS BGG AND THEIR FHOTOREVERSION. H. Yajima. Dept. of Biology, Fac. of Sci. Ibaraki Univ., Mito. Developmental types expected by centri- fugation of Chiraonomus egg were modified by the subsequent uv irradiation. The modifed types change with the irradiat- ed stages. Late uv irradiation of the centrifuged egg in parallel to the long axis should damage the tip of redistri- buting cytoplasmic zone as well as the end of egg. To clarify the change of types with the development is ascribed to a simultaneous irradiation of the tip of redistributing cytoplsam with the end of centrifuged egg, the anterior yolky end or clear cytoplasmic zone of the ege centrifuged with below 3,000 g for 5 min were separately irradiated from the lateral side by shielding the remaining part with a piece of deck glass. The developmental results do not differ between the early and late irradiation of yolky end. The both damages do not photorecover. Larly irradiation to the cytoplasmic zone causes a precocious occurrence of effect of the "late" para- llel irradiation. The effect is photo- reversible. These facts show that the modified pattern changes depending on whether the cytoplasmic zone is irradiat- ed or not with the yolky end. 954 Developmental Biology DB 146 DORSO-VENTRAL PATTERN OF MELANOPHORE DISTRIBUTION IN BULLFROG TADPOLES. lilo UGA, EOL, WaASwo5 WOlaOkU WiaalwWo . Sendai. ie elucidate the mechanisms of dorso- ventral pigment pattern formation, the mber of cells in melanophore colonies was counted and the degree of melanin persion in the cells was measured 6 a 1g dorso-ventral axis in the skin of Frog tadpoles. The number of melano- S adm tie Colom, giac rae meaim caieme ter slanin-containing area in each melano- phore decreased toward ventral skin. How- ever, the melanophores in the ventral skin fully dispersed melanin by the addition of MSH. SAMS WAS jue@liweSwewuOia Ost weleimMo— phores and the degree of melanin disper- Sion are under the control of MSH, the effects of qa-MSH on cultured melanophores of taspole tail skin were examined in the SSSSaeS Cit ClOieSeil syacl Wweimwrel Sikkim x nese WSlsarachwiGecl GE joie@ilii ice voi and melanin dispersion were inhibited ony ila’ eielGlaglOm Ox wldle WeradwisgeIl Slim Cx IseAOw., Cilliplaouyvela ao SUE AMaAnlorTLOMS yes observed in the melanophores cultured Witla wae COieSail Slkiia SScwIeac wo Phus, swe Sugsses LNA avicmitsalas kernal environment reduces the responsiveness of melanophores to MSH which results in low proliferation rate and melanin-con- Centrated Stave of melanophores aim chile ventral regions. DB 147 INDUCTION OF MELANOGENESIS AND PIGMENT RESPONSE IN STEM CELL TYPE CLONES O§ GOLDFISH ERYTHROPHOROMA CELLS J. Matsumotol, T. Akiyamal, J. D. Taylor¢éand vs We Wee Wee, BIOs, KeLO Umar dle Dept. Cine aC, Seilo > Wesyne Stace: Unive » Detroité Inasmuch as autologous (carp) serum induces brightly colored, melanin and iridescent pigmentation at certain freq- ency in uncloned goldfish erythrophoroma (GEM 81) cell line, stem cell type clones were separated by picking up pigment-free colonies in its autologous serum-added soft agar suspension culture.Dual exposure of these clones to 1.5% DMSO or 0.4ug/ml1 tunicamycin (4 days) and 20% autologous serum (continuously thereafter) resulted in appearance of a variety of melanogenic and pterinogenic subclones. The morpholo- gical traits (cell size and shape), mode of pigmentation and physiological proper- ties (responsiveness, photosensitivity and contact behavior) of these induced melano- genic cells were variable between colonies but similar for all cells within a single colony. These findings indicated that the parent population of this cell line cont- ain stem cells or their like which are pluripotent for expression of pigment characters and that upon induction, such cells can manifest pigment response which is specific to terminally differentiated melanophores and which is heterotopic to the original tumor. DB 148 CHANGES IN CYTOSKELETAL PROTEINS DURING IN VITRO DIFFERENTIATION OF A GOLDFISH ERY- THROPHOROMA STEM CELL TYPE CLONE TO PIGMENT RESPONSE-COMPETENT MELANOGENIC VARIANTS. T. Akiyama and J. Matsumoto. Dep. Biol., Keio Univ. Yokohama. The peptide composition of a stem cell type clone of goldfish erythrophoroma cells and its derived melanogenic variants with or without responsiveness (activity of pigment translocation in response to certain neurotransmitters or hormones) were examined using modified O'Farrell's two dimensional electrophoresis. Particu- lar attention has been paid on peptides associated with cytoskeletons, actin and 57K dalton protein. The results indicated that the differentiation of a stem cell type clone to melanogenic variants with responsiveness accompany 1) formation of actin isoforms, causing its profile from a single spot (@ actin) toa group of isomers presumably @ or its like, @ and Y actin and 2) appearance of 57K protein. The latter protein was reported to be phospho- rylated at pigment translocation (ACTH- induced dispersion) of normal goldfish xanthophores. This would indicate that the formation of these two peptides were essential in acquisition of responsiveness (pigment translocation) in pigment cells of neo- plastic origin as in the normal counter- parts. DB 149 DEVELOPMENTAL CHANGE OF PROTEIN CONSTITU- ENTS IN CHICKEN HEARTS REVEALED BY TWO- DIMENSIONAL GEL ELECTROPHORESIS. M.Oh-ishi and T.Hirabayashi. Inst. of aLOdko S@ilo, Widsiwyo Or Wewlkuloe, Wioairales. - Developmental changes in major protein constituents of chicken cardiac muscle were examined by applying a computer sys- tem newly devised for image analysis to the respective protein spots separated by two-dimensional gel electrophoresis. The electrophoretic patterns through de- velopment (from 7-day-old embryo to 26- day-old chick) were alike to each other, suggesting the occurrence of differenti- ation at early stages. Quantitatively, the molar rates of myo- sin subunits to actin gradually increased during development, while those of tropo- myosin were rather static. The result im- plys that the number of thick filaments is smaller than that of thin filaments at early stages, in Spite of the fact, that heart beating at 1-day-old embryo. Tropo- nin T began to increase from 10-day-old embryo. As for intermediate filament (IF) proteins, vimentin increased slightly but desmin decreased as embryos developed . We also noticed the decreases of 61K and 56K proteins localized near an IF ISAO wala 1SfQaLOia Ehael Oi VHC joxeOwSalin ILoweil= ized near myosin light chain 1 region. Developmental Biology 955 DB 150 MYOFIBRIL FORMATION IN CHICKEN GIZZARD SMOOTH MUSCLE CELLS. S.Hirai and T.Hirabayashi. Inst. of Biol. Sci., Univ. of Tsukuba. Ibaraki. The intracellular distributions of muscle proteins in the developing chicken gizzard smooth muscle cells were investi- gated by immunofluorescence labeling of enzyme-dispersed cells. The fibrous dis- tributions of myosin, actin, and tropo- myosin were demonstrated in the myoblast of 7-day-old embryo. Double staining experiments showed that these proteins were incorporated into common fibers. d- actinin was distributed as a dotted line along the actin-containing fiber, and desmin usually showed fibrous distribu- tion which did not correspond to that of others. On the other hand, we demonstrated Cat+-dependent contractility in the myoblast of 7-day-old embryo by exposure of detergent-extracted cells or glyceri- nated tissues to Mg*tt-ATP solution. These results suggests that the syntheses of contractile proteins in embryonic gizzard myoblasts are follwed by an immediate assembly of them into myofibrils which are comparable to that of matured muscle. However, there was some difference between distributions of myosin and actin in these myoblasts: Most actin and tropomyosin seemed to be assem- bled into myofibrils, while a consider- able amount of myosin remained unassem- bled in cytoplasm. DB 151 EFFECT OF HALOTHANE GAS ON THE HATCHED CHICKEN. II. ON THE RECOVERY OF LIVER DAMAGE BY SHOSAIKOTO K.Nonoyama, H.Tanaka and H.Hasegawa. Biol. Lab., Aichi Univ. Educ., Kariya. Previously the authors reported the effect of Halothane as an anesthetic gas on the hatched chi- cken in order to clarify the influence on the hu- man body using the same gas. As a result it was found that the gas influenced the development of the chicken organ, especially the differentiation of the legs and the destruction of mitochondria in the liver tissue of the chicken. On the other hand, the liver damage of chicken owing to the in- halation of the anesthetic gas for long period continuously was found by electron microscopic ob- servation. Recently, it was clarified that the dose of Shosaikoto as an oriental medical agent effectively aids recovery in liver disease. From these facts, the authors examined the effect of Shosaikoto on the liver damage of hatched chicken treated with the anesthetic gas. The body weight of the anesthetized and drug-administered chicken was recovered almost as a control. And the LDH pattern obtained from anesthetized and drug-ad- ministered chicken liver was as almost same as a control experiment. On the electron microscopic photograph, in the liver of the chicken anestheti- zed and then given medicine for 20 days, the swe- lling of mitochondria was recognized, but the ly- bosome granules was almost normal as well as that of the control. From the results of pharmacologi- cal and clinical studies and this experiment using anesthetized hatched chicken, the medicine Shosai- koto was seemed to be effective for the cure of the liver disease. DB 152 DEVELOPMENT OF CEREBELLAR HYPOPLASIA IN HYPERBILIRUBINEMIC GUNN RATS H.Keino, S.Kashiwamata. Dep. Perinatol., CNStA Deven. NOSio., Adena Gunn rat is a mutant with heperbilirubinemia inherited as an autosomal recessive trait. Homozygous (jj) and heterozygous (j+) Gunn rat cerebella were examined histologically from postnatal day 5 to 60. The cerebellar vermis of jj rats showed that the anterior and medial lobes were Significantly smaller in area than those of j+ rats on and after postnatal day LO; However, in the nodulus no significant difference in area was observed between both rats. L and S$ cells which showed strong acid phosphatase activity were observed exclusively in the jj rat cerebellum. The appearance of S and L cells reached a miximum around day 15 and 30, respectively. On day 15, S cells were abundant in the anterior and medial lobes but scarce in the posterior lobe. S cells with lipid granules in their lysosomes were seen in the anterior and medial lobes after day 10 and gradually increased in number up to day 30. It is suggested that the bilirubin-induced severe hypoplasia accompanied by the appearance of S cells mainly occurs in the late- and intermediately-maturing lobes, but hardly in the early-maturing lobules such as the nodulus and uvula. DB 153 THE PRODUCTION OF CLONE FISH IN MEDAKA, O. LATIPES K.Naruse, K.Ijiri, A.Shima and N.Egami GO Ousee LOS tery Hie’ Chen OMe OIGall ety (UI ou POM OL ViOn Tokyo. To produce homozygous individuals, the eggs, which were activated by UV-irradia- ted sperm(UV-sperm), were exposed to heat shock(41°C for 3 min at 92 or 95 min after insemination-LHS) or hydrostatic pressure (700kg/cm for 10 min at 85 min after inse- mination-LHP). The haploid embryos were converted into diploid by the arrest of the first cleavage. The eggs from homo- zygous female were activated by UV-sperm and converted haploid into diploid by the arrest of second meiotic division by heat shock(41°C for 2 min at 2-3 min after ' insemination-EHS). Some hatched fries were fed methyltestosterone diet(40pg/g diet) to produce sex-reversal males. Sex-rever- sal males thus obtained were mated with original females and progenies can be ob- tained. According to DNA-microfluorometric me- thod, the embryos which were fertilized with normal sperm and exposed to LHS or LHP were 4C(tetraploid).While the embryos which were activated by normal sperm and exposed to EHS were 3C. These results in- dicates that LHS and LHP cause the arrest of the first cleavage and EHS cause the arrest of the second meiotic division. 956 Developmental Biology DB 154 INDUCTION OF METAMORPHOSIS IN THE LARVAL LAMPREY. Shintaro Suzuki. Dept. of Comp. Endocri- nol., Inst. of Endocrinol., Gunma Univ., Maebashi. In the larval lamprey, Lampetra reis- snerit metamorphosis was induced with po- tassium perchlorate. After treatment for 3 months, 11 of 29 larvae finished meta- morphosis and stuck to the wall of the aquarium. Such complete metamorphosis was induced only in larger larvae with a to- tal body length of more than 12.5 cm. In smaller larvae, metamorphosis was induced in part eyes, oral apertures, branchio- pores, and other parts of the body were at different metamorphic stages. However, in the untreated larvae metamorphosis did not occur during the experiment. Thyroid follicles were formed from the endostylar cells normally in the metamorphosed ani- mals, as in the spontaneously metamor- phosed animals. Animals in which metamor- phosis was partially induced had trans- forming endostylar cells which were not hypertrophied. With enlargement of the dorsal fins, the eggs(oocytes) greatly increased in size after the induction of metamorphosis. DB 155 CHARACTERIZATION OF THE EPIDERMAL FACTOR WHICH INDUCES THYROID HORMONE-DEPENDENT MESENCHYMAL TISSUE REGRESSION. K. Niki and K. Yoshizato*. Dept. of Biol., Schol. of Edu., Waseda Univ., Tokyo, and “ene, Oe Pilasicle Succ, Sclal, ox Mec, Kitasato Univ., Kanagawa. Epidermal tissues play a key role in the tadpole tail fin contraction. Contraction of the tadpole tail fin mesenchymal tissues requires both thyroid hormones and a factor released from a tail skin explant. We call the medium containing this factor(s) as a skin- conditioned medium (SCM). In the present report, we tried to characterise the BAGEOR GS) le eEnnGhems EM. Heating of SCM at 100 C for 10 min produced some white precipitates. Activity of the factor was not destroyed by this treatment and recovered in the supernatant. Digestion With trypsin or pronase E at 20 C for 30 min did not destroy activity in SCM. The activity was recovered both in the dialisate and in the retentate after dialyzing of SCM. Partial purification of the factor(s) was performed using a Shephadex G-10 gel. In conclusion, the factor(s) is heat resistant and has a relatively small molecular weight. DB 156 PRIMARY CULTURE OF ANURAN TADPOLE TAIL EPIDERMAL CELLS ON COLLAGEN GELS: INTERAC-— TION BETWEEN CELLS AND COLLAGEN GELS. A. Nishikawa and K. Yoshizato*. Dept. of Biochem. and *Develop. Biol. Lab., Dept of Plasele Swe. , Kitasato Univ. School of Medicine, Sagamihara. Epidermal cells were separated from the bullfrog tadpole tails and cultured on a reconstituted collagen gel in order to study the behavior of cells on collagen fibers. Cells were maintained in two lealinels} © E media to see the hormonal ef~ fects on the behavior, a normal medium with fetal calf serum (FCS) and a hormone (steroid and thyroid hormones) -deprived medium with charcoal-treated FCS (CTS). The cells spread well over a collagen gel, forming a typical epidermal sheet in the normal medium. On the other hand cells did not spread well over the gel, forming a scattered clump of cells in the C€TS- medium. Supplement of steroic or/and thyroid hormones to this medium did not improve the cell spreading, suggesting the presence of other factor(s) in FCS which is responsible for the spreading. Cells in the normal medium solubilized a collagen gel after a week in culture. The CTS- medium diminished the solubilization. A physiological meaning of these observa- tions are now being explored. DB 157 REGIONAL SPECIFICITY OF DERMIS IN DEVELOP- MENT AND HISTOLYSIS OF TRANSPLANTED SKINS OF ANURAN TADPOLE DURING METAMORPHOSIS. T.Kinoshita and K.Watanabe. Dept. of IBaL©@IL 6 5 School of Dent. Med., Tsurumi Univ., Yokohama. Back- and tail-skin were dissected from Rana japonica tadpole (Taylor and Kollros, stage 18) and separated into dermis and epidermis by treatment of 0.13% protease. Homotypically and heterotypi- cally recombined skins were constructed from these dermis and epidermis and trans- planted in tail of the original tadpole. Skim) Grabts, wsing dermis §& Gompeecnalesoicina degenerated simultaneously with resorption of the tail. However, skin grafts con- taining dermis from back-skin survived even after end of metamorphosis on the posterior end of juvenile frog. Further- more, all epidermis underlain with dermis from back-skin formed secretary glands and flattened epithelia characteristic in adult back-skin, regardless of region from which the epidermis came. These results suggest that the dermis is regionally specific and controls differentiation of the epidermal cells in back- and tail-skin of metamorphosing tadpole . Developmental Biology 957 DB 158 ALKALINE PHOSPHATASE ACTIVITY DURING DEVEL- OPMENT IN THE BULLFROG A. Kashiwagi. Lab. for Amphibian Biol., Fac. of Sci., Hiroshima Univ., Hiroshima. The changes in theisozyme pattern of al- kaline phosphatase(Aph) extracted from five organs including the skin, kidney, liver, pan- creas and intestine, at seven developmental stages from the tadpole(Taylorand Kallros' stage V) through the juvenile frog were ex- amined in Rana catesbeiana by the method of acrylamide-gel electrophoresis. In the elec- trophoretic patterns obtained fram the five Organs, a total of nine bands were recognized and were numbered 1~9 according to mobility from slow to fast. The results of analyses showed that the electrophoretic patterns remarka- bly differed by organ and developmental stage. Band 2 appeared in the kidney at stage X and remained later. Band 7 appeared in the pan- creas at stage XV and remained until one month after metamorphosis. Band 9 suddenly appeared one month later in the pancreas. Band 6 ap- peared in the liver at stage VIII and disap- peared at stage XXV. Bands 5 and 8 appeared in the intestine at stage V to remain until stage XXI and thereafter band 4 appeared. Band 3 appeared in the skin at stage X and band l appeared after stage XXIII. While the activi- ty of Aph remarkably increased at the late tad- pole stage,it decreased temporarily and in- creased again with the progress of develop- ment after completion of metamorphosis. DB 159 THE SEX OF AUTOTETRAPLOIDS IN RANA NIGRO- MACULATA. Y. Kondo. Lab. for Amphibian Biol., Fac. of Sci., Hiroshima Univ., Hiroshima. In order to elucidate the sex of auto- tetraploids(4n), the present author pro- duced many autotetraploids in Rana nigro- maculata. First, a mature 4n male was ob- tained from an unfertilized egg trans- planted with a triploid blastula nucleus. Eggs of a diploid female were refrigerated shortly after insemination with sperm of the 4n male. The result showed that 18 4n frogs were raised from these eggs. Of these 4n frogs, nine were females and nine were males. Seven of the males were used to produce 4n frogs by the same method as the above. When eggs of seven females were refrigerated shortly after insemination with sperm of the 4n males, 202 4n tad- poles were produced. Of these tadpoles, 80 were preserved within one month after metamorphosis and their sex was examined. It was found that 55 were females, 18 were males and seven were juvenile hermaphro- dites. The remaining 122 4n tadpoles were reared until sexual maturity. Of these ma- ture 4n frogs, 97 were females and 25 were males. When the number of the juvenile and mature 4n frogs was totaled, 152 were fe- males, 43 were males and seven were juve- nile hermaphrodites. If the latter are counted as males, as they are considered to become males soon or later, 75.2% were females and 24.8% were males. DB 160 A STUDY ON "GLOMERULOCYTES" IN THE COMPOUND ASCIDIAN, POLYANDROCARPA MISAKIENSIS. K. Hashimoto™®, H. Mukai’ and H. Watanabe’? l1Shimoda Mar. Res. Ctr., Univ. Tsukuba, Shizuoka. 2 Dept. Educ., Univ. Gunma, Gunma. 3Meiji Inst. Hlth. Sci., Kanagawa. George (1939) designated a strange blood cell type found only in P. tincta as "glo- merulocytes"(GCs). We found the similar cell in P. misakiensis, and examined its location, ultrastructure and origin by LM and TEM. In LM, the GCs were present in various lacunae in the connective tissue, most abundantly in the mantle. They were large biconcave disc-shaped cells, measur- ing 12-13 Mm in diameter and ca. 34m in thickness. A spherical nucleus was located in the central area. Most of the cytoplasm was filled with fine fibers arranged around the nucleus concentrically. The fibers were strongly stained with haematoxylin. There were large round cells which also contained the concentric fiber structure in the epi- dermis. Some of them had fewer fibers which stained less with haematoxylin compared to the GCs, suggesting that they may be imma- ture GCs. In TEM, the GCs mainly contained only a few mitochondria and lysosome-like organelles in perinuclear cytoplasm. The intracellular fibers measured ca. 0.1-0.4 dm in diameter. Their ultrastructure resem- bled that of the tunic fibers. The presump- tive immature GCs in the epidermis also contained fewer such fibers. These findings suggest that the GCs arise from the epider- mal cells. DB 161 SEPARATION AND NATURE OF THE LYMPHOCYTE- LIKE CELL OF A COMPOUND ASCIDIAN, BOTRYLLOIDES SIMODENSIS. K.Koyama, K.Hashimoto~and H.Watanabe% 1Dept. of Phsiol. Chem., Nippon Vet. Zootech. College, Tokyo.*M.I.H., Odawara. 3Shimoda Mar. Res. Ctr., Univ. of Tsukuba, Shimoda. A method for separation of the lympho- cyte-like cell (LLC) of a compound ascid- ian, Botrylloides simodensis, is de- scribed. The blood of B.simodensis was mixed with Ca-Mg free artificial sea water and centrifuged with the swing type centrifuge, and LLC rich fraction wag separated. When 4.010’ blood cells were separated, 1.95x10°cells were obtained as LLC rich fraction. The purity of LLC be- fore separation (ave. 43.4%) was in- creased to 86.1% after separation, and the average yield of LLC was 11.2%. Judg- ing from trypan blue exclusion, above 70% of LLC were viable after separation. The contaminating cells were amoebocytes, morula cells, granulocytes, compartment cells and their average purity were 6.0%, 2.7%, 4.2%, 1.0%, respectively. The sepa- rated LLC has a large nucleus, small amount of cytoplasm and the ultrastruc- tural feature of indifferentiated ele- ment. Con A-binding site was observed on LLC and all other types of blood cells. 958 Developmental Biology DB 162 FORMATION, ENLARGEMENT AND CALCITE-RESORP- TION IN THE PORE PLATES OF THE SEA URCHIN, ANTHOCIDARIS CRASSISPINA. OetOchhs Depo EE WOU. aHaC mm iOroCHacn, Ehime Univ., Matsuyama. The coronar growth of a sea urchin, Antho- cidaris crassispina, are investigated by LM, SEM and TEM. In all echinoids new coro- nar plates are added at the apical end of the corona throughout the life cycle. The plates are sifted toward the peristome and they grow peripherically. In A.crassispina ambulacral(A) plates are composed of 3-9 pore plates, a primary tubercle overspread on a part of these pore plates, and the number of the pore plates increase with the coronar growth. Resorption of calcite is known at the peristomical face, but the transitional curves of the pore plate numb- er of the A-plates suggest that the resorp- tion does occur in a part of the primary Single A-plate. The pore plate is perforated by two pores which belong to same tube foot. SEM photo- graphs and natural growth zones suggest that during growth the two pores are shift- ed toward the newly formed part of the pla- te. The calcite of the plate is resorped at the adradial walls of the pores, wherwas is added perradially. TEM photograph show that phagoctes contact on the calcite of the ad- radial wall. Not only does the resorption occur in the pore plate, but also is at the suture between the two pore plates. In the suture, many collagen fibers observed by TEM: DB 163 ULTRASTRUCTURAL LOCALIZATION OF Ag-NOR PROTEINS IN FULLY-GROWN OOCYTES AND PREIMPLANTATION EMBRYOS OF MICE. I.K.Takeuchi and Y.K.Takeuchi. Dept. Embryol., Inst. Dev. Res., Aichi Prefec. Colony, Kasugai. The nucleoli of fully-grown oocytes are observed as large spherical bodies consisting of compactly- aggregating fibrous materials (compact nucleoli), at the periphery of which a few fibrillar centers and dense fibrillar components are located. The Ag-NOR proteins are localized in the fibrillar cen- ters and the dense fibrillar components, whereas the compact nucleoli are devoid of these proteins. In the condensed chromosomes of the postzygotic one-cell embryos undergoing second meiotic division the Ag-NOR proteins are present in the small patches associating with the chromosomes. In the one-cell embryos of pronucleus stage, the fibrillar centers positive to the Ag-NOR staining are located at the periphery of compact nucleoli, but no dense fibrillar components are encountered. The dense fibrillar components and the granular components become numerous in the nucleoli of the embryos from four-cell stage onward. The number of fibrillar centers also increases from four-cell embryos on- ward, while the compact nucleoli become broken up into small spherical fragments and gradually disap- pear. In these embryos, the Ag-NOR proteins are most numerous in the fibrillar centers and in lesser degrees in the dense fibrillar components, but almost none in the granular components and the fragments of compact nucleoli. The presence of the Ag-NOR proteins in one-cell mouse embryos may chal- lenge the previous views that these proteins may have a principal role in the ribosomal RNA synthe- sis. DB 164 ASSOCIATION OF PERICHROMATIN GRANULES WITH THE NUCLEAR PORES OF THE CHINESE HAMSTER GROWING OOCYTES. Y.K.Takeuchi, S.Sonta* and I.K.Takeuchi*, Anat. Lab., Gifu Coll. Med. Technol., Seki, and *Inst: Dev. Res., Aichi Pretec. Colony, Kasugai Perichromatin granules (PGs), measuring about 35-50 nm in diameter and stained with the Bernhard's uranyl-EDTA staining, are especially numerous in the nucleoplasm of the Chinese hamster growing oocytes. In the early growing oocytes, a considerable num- ber of PGs are observed to associate with the nuclear pores (29.8 % of nuclear pores examined). No figures showing the passage of PGs via the nuclear pores are found, but some fibrous materials appear to emit from these PG-associated nuclear pores into the cytoplasm. In the middle-stage growing oo- cytes, the frequency of PG-associated nuc- lear pores increases to 35.5 %, but it declines into 27.9 % in the late-stage growing oocytes. In the oocytes of the ant- ral follicles, the number of PGs in the nucleoplasm greatly decreases, and only 3.3 $ of nuclear pores are associated with the PGs. The i.p. injection of actinomycin D (0.5 mg/kg) into the female animals consid- erably decreases the frequency of PG- associated nuclear pores. The function of PGs has been speculated to play a role in storage and transport of RNA transcribed in the nucleus. The present results seem to Support this view. DB 165 AGE EFFECTS ON THE MITOTIC ACTIVITY OF EPIDERMALMMELANOCYTES DURING THE HEALING OF SKIN WOUNDS IN MICE. T. Hirobe. Dept. \Bi0l., Fac. Educawuon, Iwate University, Morioka In the epidermis within 1 mm from the cut edge, the melanocyte population positive to the dopa reaction increased dramatically until the third day, then gradually decreased. In contrast, the population in the regenerating wound epidermis appeared on the third day, and increased until the seventh day, then gradually decreased. Pigment-producing melanocytes in mitosis were found immediately after wounding in the epi- dermis within 1 mm from the wound edge. Epidermal melanocytes in neonatal mouse skin can be stimulated to undergo mitosis immediately adjacent to a skin wound. Similar experiments were performed in the skin from 4.5-, 8.5-, 20.5-, and 60.5- day-old mice. The extent of the increase in the melanocyte population diminished progressively with age.Moreover, pigment- producing melanocytes in mitosis were less frequently found in the skin from the older mice than in younger skin. Therefore, it is concejvable that the proliferative activity of the epidermal melanocytes during the healing of skin wounds gradually declines with the stage of development. Developmental Biology, Endocrinology 959 DB 166 The namatode Caenorhabditis elegans: its life span and the failure of the pharyngeal pumping. Hirofumi Doi, Tokyo Metropolitan Institute of Gerontology, Sakae-cho, Itabashiku, Tokyo 173. The study of aging must be linked not only to the survival curve but also the failure of functions. Hosono et al (1978) reported on the survival curve of C. elegans. The survival curve at 25°C shows that: The worms begin to die at the fifth day from hatching. Up to the tenth day about two-third worms of the population died. From that day the death rate per day changes down. That is, the sur- vival curve is classified into two phases. I am studying the two phases from the failure of the pharyngeal pumping. Strain N, was used. I recorded the pumping worm by worm on the VIR tape every day from hatching. The worms were cultured at 24°C. I could not re- cord the pumping continually for a long time (one minute), because they locomote very quickly. So, I took for the representation of the function the pumping rate per 10 pulses. The pumping rate at L, stage is about 3.3 sec/ 10 pulses; at the egg laying stage (the second and third day from hatching), 1.75 sec/10 pulses. After the egg laying stage, the change of the pumping rate is classified into two types; sudden and slow. The former worms stop their pumping sud- denly at the fourth-sixth day after hatching. They die in the first phase of the survival curve (the mean life span is 8 days). The later worms change down slowly their pumping and stop at the 12-th - 15-th day. They live for a long time (the mean life span is 16 days) and die in the second phase. DB 167 ELONGATION OF DROSOPHILA VIRILIS LIFESPAN CAUSED BY CYCLOHEXIMIDE. S$.Ohba! and T.Aigaki2 Dept.of Biology, Tokyo Metropolitan Univ., Tokyo' and Dept. of Biology, Tokyo Metropolitan Institute of Gerontology, Tokyo. Effects of cycloheximide, an inhibitor of protein synthesis, on adult lifespan of the TK strain of D. virilis were examined on flies fed on the yeastextract-sucrose medium which was newly devised for the study of longevity and ageing (Ohba 1983). Preliminary tests in which flies were fed on 10% sucrose medium (no yeastextract) suggested that cycloheximide prolonged adult lifespan at the concentration of 5-50 éMg/ml culture medium. Inthis study three levels of cycloheximide concentration (0,10 and 50 pg/ml) were simultaneously tested. The content of yeastextract was 0, 0.5, 1.25 and 5% and of sucrose was 10%. Only in the case of 0.5% yeastextract medium striking elongation of lifespan occurred in cyclohe- ximide-added groups. Mean lifespan was 71.24, 92.18, 88.58 days in females and 33.50, 75.08, 80.24 days in males for media containing 0 (control), 10 and 50 wg cyclo- heximide/ml medium, respectively. The in- crease of lifespan was 25-30%in females and 125-150% in males. On the 0% yeastextract medium too, lifespan elongation was signi- ficant but the increase was always less than 10%. On media with higher concentration of yeastextract, 1.25 and 5%, cycloheximide had no significant effect on adult lifespan. EN 1 SUMMATION MECHANISM OF PHOTOPERIODIC COUNTER FOR PUPAL DIAPAUSE IN PAPILIO MACHAON. alone a K. Shimada. Inst. Low Temp. Sci., Hokkaido Univ., Sapporo. The larvae of P. machaon required successive short days for the onset of pupal diapause. Reciprocal transfer of larvae between short(LD10:14) and long days(LD16:8) revealed that the older larvae were more sensitive to photoperiod than the younger larvae. When the larvae were exposed to short days during the early instars, they required 12 successive short days to raise the incidence of pupal dia- pause to 50%. Whereas, a 7-short-day exposure during the late instars was critical for the diapause induction. The critical number of short days was changed with the abbreviation of the fifth instar produced by allatectomy. This was remark- able in the allatectomized larvae exposed to short days during the early instars. The critical number of short days reduced from 12 to 9.5 days with the decrease in the length of larval period. While, it was less reduced when the allatectomized larvae were exposed to short days during the late instars. The present results suggest that the larvae are able to add up successive light:dark cycles but the rate of increase in diapause-inducing effects per short-day is not constant. The critical number of short days depends on the larval develop- ment and the length of sensitive period. EN 2 HORMONAL CONTROL OF THE SEASONAL FORMS IN THE SMALL COPPER BUTTERFLY, LYCAENA PHLAEAS DAIMIO SEITZ. K. Endo and Y. Kamata. Environ. Biol. Lab., BLOM. Minst., Face ob Seis, Yahapuchi’ Univ. Yamaguchi. Spring and summer forms of this butter- fly are characterized by wing color of red- dish brown and red, respectively. Decapitation or decerebration made on 0- day-old LD-pupae (destined to be summer or intermediate form by long day) caused the ratio of intermediate form to rise,whereas, in O-day-old SD-pupae (predetermined to be spring or intermediate form by short day), these operations did not bring about any significant changes in the seasonal form. Brains of O-day-old LD-pupae transplant- ed into the abdomen of decapitated SD-pupae were found to change the most recipients into summer or intermediate form. On the other hand, when brains of O-day-old SD pu- pae were transplanted, no significant changes occurred in the seasonal form. Furthermore, when 20-hydroxyecdysone was applied to O-day-old LD- or SD-pupae, they produced more reddish wings as compared to those of the saline-treated control. These results indicated that the brain of LD-pupae secretes a factor causing the wing color to be brownish (summer form); in the absence of this factor, SD-pupae de- velop into spring form. Besides, haemo- lymph ecdysteroids may be involved in the control of seasonal-form development. 960 Endocrinology EN 3 PROTHORACICOTROPIC HORMONE BIOASSAY: LARVAL-LARVAL BOMBYX ASSAY. AsGeSuzukrvandehemSshwzakie BaoOlee lnSiteer. Fac. of Sci., Nagoya Univ. Nagoya. A larval assay method for the pro- thoracicotropic hormone (PTTH) has been established, using fourth-instar larvae of a racial hybrid Shunrei x Shogetsu of Bombyx mori. Larvae neck-ligated just before the critical period for PTTH secre- tion failed to undergo the next larval moult. Injection of Bombyx brain extracts caused such larvae to moult to the fifth- instar with a reasonable dose-response relationship. The PTTH unit by Bombyx larval assay has been defined from this dose-response curve. An acetone-dried powder of 2000 brains from Bombyx pupae 2-3 hr after ecdysis was extracted with 2% NaCl, heated, and subjected to Sephadex G-50 gel filtration. Each fraction thus obtained was assayed for PTTH using Bombyx larval assay presently developed as well as by Bombyx pupal assay and Samia pupal assay. A 4K-PTTH peak by Samia pupal assay anda 22K-PTTH peak by Bombyx pupal assay were detected as previously reported (Develop. Growth and differ., 25, 593) and the PTTH activity detected by the Bombyx larval assay exactly coincided with the 22K-PTTH peak. These results thus reconfirmed the presence of species specificity in Bombyx PTTH molecules but failed to show the stage specificity. EN 4 TDENTIFICATION QF THE JUVENILE HORMONE FROM THE STLKWORM, BOMBYX MORI. M.Kimura and S.Sakurai. Dept. of Biol., Fac. of Sci., Kanazawa Univ. Ishikawa. We identified the juvenile hormones from the extracts of both whole body and haemolymph of Bombyx mori. The JHs were purified by TLC and HPLC, and their chemi- cal structures were identified as JH I and II by mass-chromatograph. JH I was major JH in Bombyx. No JH III was detected. Then JHs secreted by corpora allata were examined. Corpora allata on day 0 of 5th instar were cultured with aaeemeenriton nine in Grace s medium. Ethyl acetate ext- ract of medium was co-chromatographed with standard samples. JHs produced by CA were identified as JH I and II as well, and the former was the major component. The JH species were further confirmed by epoxide hydration. EN 5 ECDYSONE SECRETING ACTIVITY OF RING GLA- NDS OF SARCOPHAGA PEREGRINA IN VITRO. K.Ogino and T.Ohtaki. Dept. of Biol.,Fac. of Sci., Univ. of Kanazawa. Kanazawa. The ecdysteroid species from ring gland of Sarcophaga peregrina was identified to be ecdysone by mass cultivation of ring gland,and separation with TLC and HPLC. The ecdysone secreting activity of ring gland was determined by RIA. The ring gl- ands were cultured in vitro for 4hr, and concentration of ecdysone in medium has been determined. Three peaks were record- ed. The first and highest peak occured at the stage of white pupae(w.p.), and level decreased till 20hr after w.p. The second peak occured around the stage of the cry- tocephalic pupa. The third increase began at 60hr after w.p., and high level was kept for hours. The first peak induced pupation. The third peak was thought to induce adult development. But the raison d'etre of the second peak is unknown. EN 6 COMPARISON OF ECDYSONE METABOLISM IN DIAPAUSING AND NON-DIAPAUSING PUPAE OF SARCOPHAGA PEREGRINA. A.Moribayashi!, H.Kurahashi2, and T.Ontaki3. Lab. of Technol.', and Dept. of Med.Ent2 ,N.1.H., Tokyo. Dept. of Biol.,Fac. of Sci.,Kanazawa Univ., Kanazawa. Adults of S.peregrina were maintained in two different light regime, 16L8D and 12L12D. Adults in 16L8D deposit larvae which are pre-programed for non-diapause development, whereas those in 12L12D deposit larvae pre-programed for diapause in the pupal stage. We examined ecdysone metabolites obtained after injection of 3H-ecdysone to mature larvae or pharate pupae pre-programed for diapause or non- diapause and tried to find the difference of the metabolic pathway between them. The radioactive metabolites were obtained from pupae 72 hrs after the injection of 3H-ecdysone into mature larvae and 48 hrs after the injection of it into pharate pupae grown in these two light regime and analysed by TLC or HPLC. The ratios of three metabolites, conjugated OA, OB and free ecdysteroids, differed from each other, and further there was a marked difference in the components of free ecdysteroids between the metabolites in diapausing and non-diapausing pupae. TherceEO re; senor peregrina, metabolic pathway of ecdysone in diapausing pupae is presumed to differ from that in non-diapausing one. Endocrinology 961 EN 7 MODE OF JUVENILE HORMONE ACTION ON PROTHORACIC GLANDS OF POST-WANDERING LARVAE OF SAMIA CYNTHIA RICINI. Yuko Fujiwara and Hironori Ishizaki. Brol. iInst., Fac. of Sci., Nagoya Univ., Nagoya. The prothoracicotropic hormone(PTTH) and juvenile hormone(JH) have been shown to activate the prothoracic glands(PGs) in the pupal cuticle formation of several insects. This study attempted to clarify the function of these two hormones on PGs activation. Samia larvae just after gut- purge were (A) allatectomized, (B) allat- ectomized and then given methoprene(MP), the JH analogue, and (C) sham-operated. Hemolymph ecdysteroid titers of (A), (B) and (C) peaked on GP-4 (four days after Gae-purge), GP-3.5 and GP-3, respec- tively. PGs of the three groups were incubated in vitro in Grace’s medium for 2 hr without PTTH as preincubation, then for 4 hr with or without PTTH. The PTTH was prepared from day-1 fifth-instar larval brains of Samia. PGs from sham-operated or MP-treated larvae cultured with PTTH were fully activated by GP-3 while PGs from allatectomized larvae did not become active until GP-3.5 PGs cultured without PTTH showed a low activity and no signifi- cant differences were observed among the three groups. These results suggest that JH acts on PGs to endow them with the responsiveness to PTTH during post- wandering stage of Samia. EN 8 ECLOSION HORMONE OF THE SILKWORM (BOMBYX MORI): ISOLATION AND DETERMINATION OF AMINO TERMINAL AMINO ACID SEQUENCE. H.Nagasawa’*, T.Kamito’, S.Takahashi’, A.Isogai*, A.Suzuki* and H.Fugo’. *Dept. of Agr. Chem., The Univ. of Tokyo, Tokyo. *Fac. of Agr., Tokyo Univ. of Agr. and Technol., Tokyo. Eclosion hormone (EH) is well-known as an initiator of a series of eclosion beha- vior in insects. EH was purified from 180,000 pupal heads of the silkworm, Bombyx mori, to a homogeneous state by a 16-step purification procedure, which consisted of heat-treatment, fractional precipitations, gel-filtrations, ion- exchange chromatographies, hydrophobic chromatography and high performance liquid chromatographies. Approximately 2 million- fold purification was attained to obtain 10 pg (1.2 nmol) EH with an overall yield of 3.3 %. EH activity was measured by an ability to elicit precocious eclosion behavior in a pharate adult Bombyx. The purified EH was active at a dose of 0.83 ng. EH was found to be a peptide with a molecular weight of ca. 8,400 and possess- es a disulfide bond, which is essential for hormonal activity. By automated gas- phase sequencer, the sequence of 13 amino terminal amino acid residues was determin- ed to be: H-Ser-Pro-Ala-Ile-Ala-Ser-Ser- Tyr-Asp-Ala-Met-Glu-Ile-. EN 9 CONJUGATE FORMATION OF STEROIDS IN THE SILKWORM OVARY. M.Ogiso, S.Y.Takahashi and E.Ohnishi. Biol. Inst., Fac. of Sci., Nagoya Univ. Nagoya. Accumulation of conjugated forms of ecdysteroids in ovaries and eggs have been reported in many insects. In cultured Ovaries of the silkworm, Bombyx mori, [7H] ecdysone and ['*C]-labeled vertebrate steroid hormones were transported from the medium and a part was transformed by both conversion and conjugation. Phosphatase digestion of the conjugated forms yielded free forms of steroids and of metabolites. Further characterization of the conjugated forms in cultured ovaries was performed by phosphatase digestion of double-labeled conjugates originated from %* Pi and ?H,or '*C-labeled steroids. [*H]Ecdysone tracer experiments in pupal isolated abdomen also showed the presence of several forms of conjugates with different susceptivity to enzymic hydrolysis. These conjugated forms were found in blood and fat body as well. Since these data suggest the presence of several forms of the conjugates,analysis on the nature of the conjugates by HPLC combined with enzymic hydrolysis is under investigation. EN 10 BOMBYCOSTEROL, A NEW TYPE ECDYSTEROID ISOLATED FROM BOMBYX OVARIES. i 5 Ohnishi ,T,Mizuno ,K.Watanabe ,N.Ikekawa ,Y.Fujimoto Testo. unSt., MaCule oC sly Nagoya Univ., 2. Dept.Chem.,Tokyo Inst. Technology. In order to know the role of ecdyster- oids in ovaries and embryos, we have been engaging in analyzing the nature of the ecdysteroids found in the ovaries of the silkworm. From the ovaries, we have found 5 ecdysteroids in free and conjugated forms. We have detected the presence of the 6th species, as a peak in HPLC profile This ecdysteroid was isolated from a’ large amount of materials by the methods reported previously (Ohnishi et al.,'81). The compound was analyzed by NMR,FT-IR, and mass spectrometry. The structure is quite peculiar, but it nevertheless be- longs to ecdysteroid and we named it " Bombycosterol".It has no keto-group at C- 6 position, instead has OH-group. No OH- group was found at C-14 position. Rng A and ring B are joined by trans-junction. Implications of this structure is dis- cussed in relation to the possible bio- synthetic pathway of ecdysteroids in the ovary. 962 Endocrinology EN 11 EFFECTS OF STEROID HORMONES ON SPERMATO- GENESIS IN THE FIREFLY, LUCIOLA LATERALIS. N.Takeda and M.Nakayama. Dept. of Biol., Hace OLTSC La, CLOhOmULEAVEy me nntclocS laste In the European firefly, Lampyris noc- tiluca, Naisse (1966) has shown that an androgenic hormone is secreted from the apical tissue of the testis. in the present study, we attempt to clarify the presence of the sex hormone and its role in reproduction by using the Japanese finery, LuUctola Watenalsts.) sbi Re Tent miaTOm the testis of L. noctiluca, the testis of L. lateralis is convered with a dense fat body and no apical tissue was found. By radioimmunoassay, progesterone and testo- sterone were detected in the testes. Testes obtained from larvae were cultured in medium CSM-2F with hormones for about 10 days to examine their possible action on spermatogenesis. In the medium containing progesterone and testosterone, spermato- genesis did not occur; however, the testes enlarged. In the medium containing andro- genic glands extirpated from the sow bug, Armadillidium vulugare, no spermatogenesis occurred. On the contrary, spermatogenesis was induced in the medium containing ecdy- sone as found in other insects (Takeda, 1972). Thus, spermatogenesis in L. lateralis is not induced by sex steroids but by ecdysone. EN 12 NEUROSECRETION AND ITS POSSIBLE ROLE IN REPRODUCTION IN THE LEECH, ERPOBDELLA LINEATA. K. Oka, N. Takeda and T.Hashimoto. Univ. of Toho. Tokyo. In the present study, we attempt to clarify the neuroseretion and its possible role in spermatogenesis in the ISEGin, “Iauge@jyeloille, Itin@eieeio Was meieve cells in the cerebral ganglion and anterior nerve cord are grouped in 21 cell compartments delimited by septa of connective tissue. They are arranged symmetrically to the median line.the neurosecretory system consists of aldyhyde fuchsin poitive @-cells and acidphilic8-cells. Spermatogenesis was divided into five stages. The testis in winter is in immature and contains four stages. On the contrary, the testis in summer iS in mature and contains all five stages, especially stage 5. Among the neurosecretory cells in each cell compartment,the eo -cells in cell compartment ILal varied in their stainability during a year. The stainablility increases to a maximum level during winter and gradually decreases to a minimum level toward summer. This variation in the K-cells correlates well with spermatogenesis. Thus, AF positive materials accumulated in the cytoplasm during winter are suggested to be released and act to concern spermatogenesis. EN 13 ISOLATION OF ANDROGENIC GLAND HORMONE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. Y.Katakura! , K.Haino-Fukushima? and Y. Hasegawa § |! Dept! of Biol.) KeromUnivar Yokohama, Depits On Biola |lackmotmsCilas Tokyo Metropolitan Univ. ,Tokyo. The androgenic gland hormone responsible for the development of all male sexual ch- aracteristics was extracted from the whole reproductive organs with the androgenic glands of Armadillidium males and partial — ly purified by a procedure including ammo- nium sulfate precipitation, gel filtration and ion exchange chromatography. The andro- genic gland hormone was a protein as judged by proteolytic digestion. Its molecular weight was estimated to be about 15,000- 17,000 by gel filtration analysis(Katakura, 1 Wy fig Y= 0 qu = oo INO) ob oo CWA” i We attempted further purification of the hormone from the extract of the androgenic glands and the testes of A) vulgare eine active substance after ammonium sulfate precipitation was applied to HPLC using a colum of Hitachi gel GOl3).. The ACH acittvaluy was concentrated in a single peak on HPLC. Its degree of purity was also ascertained by SDS-polyacrylamide gel electrophoresis. The molecular weight of the protein was estimated to be about 17,000 from the SDS electrophoretic mobility. Determination of the isoelectric point and amino acid com- position of the androgenic gland hormone is now in progress. EN 14 EFFECTS OF NEUROPEPTIDES ON THE HEART RATE IN MOLLUSCA. H.Takayanagi and N.Takeda. Dept. of Biol., Fac. of Sci., Toho Univ., Funabashi. (a The molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH,) has been known to affect smooth musclé or cardiac muscle (Greenberg and Price, 1979). The present work is undertaken to clarify the action of neuropeptides including FMRFamide and neurotransmitters on the heart rate, and the localization of FMRFamide in the central nervous system by immunocyto- chemistry. Animals used were the clams, Meretrix lusoria and Tapes philippinarum, the pond snail, Cipangopaludina chinensis and the African giant snail, Achatina fulica. FMRFamide and serotonin induced rhythmical activity in the heart rate. Immunocytochemical experiments using anti- sera to FMRFamide provide clear evidence for the localization of FMRFamide immuno- reactivity in the central nervous system. FMRFamide was detected in the neurons of the cerebral, visceral, parietal, pedal ganglia. Among them, the neurons in the cerebral ganglion stained strongly. Thus, FMRFamide is suggested to be released from the neurons in the central nervous system into the hemolymph to provide long term maintenance and regulation of tone, rhythmicity and excitability of cardiac muscle. Endocrinology 963 EN 15 THE FINE STRUCTURE OF THE OPTIC GLAND IN TERRESTRIAL PULMONATES. S. Ohtake, K. Sugiyama* and N. Takeda? Dept. Of Biol., Nihon Univ., Sc. of Med., Tokyo and *Dept. of Biol., Fac. of Sci., Toho Univ., Funabashi. The optic gland in terrestrial pulmo- nates secretes a gonadotrophic hormone ( Takeda, 1982 ). The study was undertaken to clarify the fine structure of the optic gland to know its regulatory mechanisms. Animals used were the slug, Limax margi- natus, and the snail, Euhadra peliomphala. The cytoplasm of the gland cells is filled with large granules about 750-1300 nm in diameter delimited by a thick cortex in E. peliomphala and 800-1700 nm with thin cortex in L. marginatus. Numerous dilated and irregular cisternae of the rough ER were seen. The lumens of these cisternae are filled with fine granular material. The method of formation of large granules is uncertain. At the breeding season, the medial neurosecretory cells of the brain appeared active in the synthesis and re- lease of the secretory material. Although no neurosecretory cell bodies occur in the tentacular nerve, neurosecretory axons penetrate into optic gland cells. Cobalt filling reveals that the axon of one medial neurosecretory cell projects to the optic tentacles, near the optic gland. The observations suggest that the optic gland is controlled by a neurohormone originating from the brain. EN 16 CHROMATOPHORES OF MIDGUT, HINDGUT, AND OF VENTRAL NERVE CORD OF THE FRESHWATER SHRIMP, CARIDINA DENTICULATA. M.Miyawaki and T.Tsuruda. Dept. of Biol., Fac. of Sci., Univ. of Kumamoto. Kumamoto. The chromatophores located at the dorsal surface of intestine and ventral nerve cord of a freshwater shrimp, Caridina denticulata were observed light and electron microscopically with some experimental treatments. The pigment cells, particularly red chromatophores, exibited dispersion after the eyestalk removal, which is compatible with many reports in epidermal chromatophores. Circadian rhythm was also observed in the pigment migration in the red chro- matophores, dispersion by midnight and concentration by daytive which might be due to the intermittent secretion of neurohormone, such as eyestalk hormone. Sexual dimorphism was assumed to be present in the chromatophore behavior, i.e., matured females usually possess dispersed red chromatophores in the midgut, while males have concentrated ones. By electron microscopy any conspicuous cell organelle such as microtubule or Golgi complex has not been observed in the chromatophores of these internal organs. EN 17 RELATION BETWEEN THE OOCYTE GROWTH AND THE Y-ORGANS IN THE ISOPODB CRUSTACEAN, ARMADILLIDIUM VULGARE, S,Suzuki, Biol, Lab Col,, Yokohama, Kanagawa Pref, “? The reproductive cycle in Armadilli- dium vulgare is concomitant with the molt cycle, The present work examined the effect of Y-organ ablation on the oocyte growth in intermolt stage during differ- ent seasons of the female annual repro- ductive cycle, Adult females (9,5-12,0 mm body length) were collected in pre- breeding season (February and March) and breeding season (May-September), They were kept in a petri dish at 25+2°C, Females were dissected 40 or 50 days after operation and their ovaries were examined for increase in oocyte diameter, The paraffin sections of the ovaries were also histologically examined, Normal and sham operated animals molted, and mature eggs were deposited in the oostegites during the experimental period, In Y-organ ectomized females, the maturation of the oocytes failed to occur and further growth of oocytes ceased, The denatured oocytes (pycnosis and irregular in shape) were observed along the inside edge of the Y-ectomized ovaries, namely the opposite side to the oviduct, These results may suggest that the oocyte growth is under the control of the Y-organs, EN 18 NEURONAL CELLS CONTAINING IMMUNOREACTIVE VASOPRESSIN AND OXYTOCIN IN THE GANGLIA IN THE EARTHWORM, Pheretima hilgendorfi. K. Kinoshita and S. Kawashima. Zool. Inst. Fac. o£ Sci., Hiroshima Univ. , Hiroshima. A number of vertebrate hormones have been reported in various species of inver- tebrates, The present study is aimed to report the presence of ganglionic cells containing arginine vasopressin (AVP)- and oxytocin (OXT)- like substances in the earthworm, Pheretima hilgendorfi, by im- munohistochemistry. ihecker these anti-AVP and anti-OXT reactive neurons are separate from classical aldehyde fuchsin (AF) posi- tive neurosecretory cells was tested in serial paraffin sections. In all ganglia groups of scattered neuronal perikarya and axons strongly reactive to AVP and OXT antisera were observed, but AF positive cells were predominantly present in the cerebral and subesophageal ganglia. In the cerebral and subesophageal ganglia anti- AVP and anti-OXT reactive cells were vari- ous in size, and they were generally larger than AF positive cells. Some AF positive cells were reactive to anti-AVP or anti-OXT serum, but some were not. The present histological observations clearly showed that AVP-like and OXT-like sub- stances were widely present in the gangli- onic cells and nerve fivers in the earth- worm. However, their physiological role is yet to be clarified. 964 Endocrinology EN 19 UROTENSIN II-IMMUNOREACTIVE NEURONS IN THE CAUDAL NEUROSECRETORY SYSTEM OF THE CARP AND THE DOGFISH. K. Owada and H. Kobayashi. Dept. of Biol., Fac. of Sci., Toho Univ. Funabashi. The localization of urotensin II(UII) neurons was studied in the caudal neuro- secretory systems of the carp(Cyprinus carpio) and the dogfish(Triakis scyllia) by peroxidase-anti-peroxidase method. UII antiserum was raised against synthetic UII of the goby, Gillichthys mirabilis. In the carp, three types of UlII-immunoreactive cells occur: small, medium and large. Small cells were located in the spinal cord dorsal to the urophysis, medium-sized cells occurred immediately anterior to the urophysis, and large cells anterior to the medium-sized cells. Axons from all three populations entered the urophysis and terminated near blood vessels. Each size group included a small number of non- reactive cells. UlII-immunoreactive cells occurred haphazardly in the dogfish pre- caudal spinal cord. Their axons terminated on the ventro-lateral surfaces of the spinal Corde Again, there were some nonreactive cells. EN 20 OCCURRENCE OF UROTENSIN I AND II-LIKE PEPTIDES IN VARIOUS TISSUES OF SEVERAL VERTEBRATES. 2 Y. Kobayashi” and K. Lederis’. 1. Dept. Pharmacol., Shimane Med. Univ., Izumo, 2. Dept. Pharmacol. and Therapeu., Fac. Med. Univ. Calgary, Calgary. ef Bite Fa EG SS Tver Be ee ree a The distribution of urotensin I (UI) and II (UII)-like immunoreactive sub- stances in about 20 tissues of rat, chicken, turtle, frog, 7 species of teleost (perch, sucker, canary rockfish, lingcod, hake, muggy and midshipman), 3 Species of the elasmobranch (ratfish, longnose skate and dogfish) and hagfish was investigated using radioimmunoassays FOE Ulan | Uiki. UI was found in brains, spinal cords and pituitaries of all teleostean fish, but not in other tissues or in other species. UII was found in brains and spinal cords of all the tested species except rat, but not in other tissues except in the pituitaries, red muscles and stannius bodies of some fish. Both UI and UII were observed in olfactory lobe, optic lobe, thalamus, cereblum, pons and medulla, and anterior spinal cord of sucker and the contents were ranged about 20-fold for UI and 4-fold for UII. These results suggest that (1) UI and UII-like substances have some role in the teleostean central nervous system and (2) UII-like substances are possibly neuropeptides distributed in various vertebrate species. EN 21 IMMUNODETECTION OF MCH-PRODUCING CELLS IN THE NUCLEUS LATERALIS TUBERIS OF TELEOST BRAIN, 2 NeNaitoe, Y.Nakail and H.Kawauchi. 1. Dept. of Anat., Showa Univ. Sch. of Med., Tokyo., and 2. Sch. of Fish. Sci., Kitasato Univ., .Iwate. Melanin-concentrating hormone (MCH) purified from the chum salmon pituitary is a peptide containing 17 amino acid residues and having potent melanin- concentrating activity in the fish melano- phore. To reveal the precise site of origin of MCH, immunostaining for MCH was performed in the brain and pituitary of the chum salmon and the rainbow trout using a specific antiserum raised against synthetic MCH. Immunoreactivity for MCH was detected in the neurons and neuronal processes in the pars lateralis of the nucleus lateralis tuberis, which is located in the ventral floor of the hypothalamus. Numerous positive-staining processes of these MCH-neurons projected to the pituitary, extending into neuro- hypophysial tissues with in the pars intermedia and, to a lesser extent, in to the pars distalis. No pituitary cells showed any cross-reactivity. These results suggest that MCH was biosynthe- sized in the neuron of the pars lateralis of the nucleus lateralis tuberis and released in the neurohypophysis. EN 22 IMMUNOELECTRON MICROSCOPY OF GONADOTROPHS AND THYROTROPHS IN TELEOST PITUITARY GLAND. H.Ueda!, H.Kagawa!, Y.Nagahama” and S.Fujimoto!. ‘Dept. of Anat., Sch. of Med. Univ. of Occupational and Environmental Health. Kitakyushu. *Lab. of Reprod. Biol., Nat. Inst. Basic Biol. Okazaki. By the use of the protein A-gold tech- nique, immunoelectron microscopic labelings of gonadotropin (GTH) and thyrotropin (TSH) were examined in amago salmon ( Oncorhynchus rhorurus). In the specimens fixed with periodate-lysine-paraformaldehyde (PLP) and embedded in epon, the hormonal antigenici- ties were well preserved. In the specimens fixed with PLP followed by osmium postfixa- tion, antigenicities were considerably re- duced resulting in a decrease in the number of gold particles. The ultrastructural preservation appeared much more satisfacto- ry with the latter technique. Using the specific antiserum against chum salmon GTH, the immunoreactive gold particles were preferentially located on granular inclu- sions of basophilic G cells and chromopho- bic V cells. The antiserum against human B-TSH reacted with secretory granules of basophilic B cells. Differential locali- zation of these two antisera was confirmed on the same ultrathin section by the use of different sized gold particles. These results suggest that in amago salmon pitui- tary gland, the granular inclusions of B cells are the source of TSH, and the G and V cells contain GTH. Endocrinology 965 EN 23 ISOLATION OF PROLACTIN SECRETED FROM CULTURED EEL PITUITARY. M. Kishida and T. Hirano. Ocean Res. Inst., Univ. of Tokyo, Tokyo. Prolactin (PRL) is an important hormone for freshwater adaptation in some euryhaline teleosts. As higher verte- brates, PRL secretion in teleosts is under inhibitory control of the hypothalamus. When isolated from the hypothalamus, secretion and production of PRL are known to be stimulated. 260 eel pituitaries were cultured in a defined medium (MEM with EARL's salt) which was made slightly hypo- tonic to stimulate PRL secretion. Three bands were seen when the culture medium was applied on PAGE at pH 9.5. The main band was assumed to be PRL, since the extract of rostral pars distalis containing mostly PRL cells showed a major band with a similar mobility. 730 ml of the medium was concentrated using Diaflo membrane (YM-5), and then subjected to gel filtration on Sephadex G-75 followed by a chromatofocus- ing column of PBE 94, thus yielding 2.0 mg eel PRL. The molecular weight was estimat- ed to be about 23,000 by SDS-PAGE, and isoelectric point about 6.0 by isoelectric- focusing. 40N-terminal amino acids were sequenced, with 50 $ homology with ovine growth hormone. The crude protein obtained from the medium was 10 times as potent as ovine PRL in Na-retaining activity using hypophysectomized Fundulus. EN 24 RADIOIMMUNOASSAY OF N-TERMINAL PEPTIDE OF SALMON PROOPIOCORTIN (NPP I) A.Takahashi, T.Hirano (Ocean Res. Inst., Univ. of Tokyo, Tokyo), H.Kawauchi (School SEyreshs Sci., Kitasato Univ., Sanriku), and N.Naito (School of Med., Showa Univ., Tokyo) Antiserum against chum salmon NPP I was raised in a guinea pig. Since NPP I has no tyrosine residue and its amino-terminus is pyro-glutamate, iodination was performed by LPO method using modified NPP I with N-ter- minal extention, i.e. H-Val-Lys-Gly/H-Lys- Gly-NPP I, after incorporation of 3-(4- hydroxyphenyl) propionate. Final dilution of the antiserum was 1:200,000. Poly- ethylene glycol was used for separation of B/F. Plasma and pituitary extracts of several salmonid species showed parallel dispacement of the labelled hormone, where- as those of other species as well as plasma from hypophysectomised rainbow trout showed no cross-reaction. Plasma levels of NPP in FW adapted salmonids were about 20 ng/ml. NPP content in the neurointermediate lobe (NIL) of trout and chum salmon was 10 and 20 times greater, respectively, than that of the pars distalis. Therefore, the plasma profile of NPP may reflect mainly secretion from the NIL, together with MSHs and endor- phin. Although we have previously suggested that NPP is associated with interrenal function in rainbow trout, no significant change in plasma NPP was observed when trouts were exposed to shallow water stress EN 25 DEVELOPMENT OF A RADIOIMMUNOASSAY FOR SALMON GROWTH HORMONE. J.P. Bolton, A. Takahashi, T. Hirano, Ocean Res. Inst., Univ. of Tokyo, Tokyo, and H. Kawauchi, School of Fish. Sci., Kitasato Univ., Iwate. Recently, the first salmon GH was purified from chum pituitaries and used to develop an RIA. Antisera were raised in rabbits and the GH iodinated with chloramine T. Monom- eric GH was used for the assay. Insolubili- ty of the monomer was overcome only when 4M urea and 4M guanidine hydrochloride were added to the buffer (pH9.0). Denaturing of the GH was also neccesary for successful iodination. The antisera were screened for sensitivity and specificity in a double antibody RIA under disequilibrium condit- ions. The most sensitive antisera was then used in the RIA. A standard curve was dev- eloped over the range 2-128ng. Extensive cross-reactivity was seen with a pituitary glycoprotein but was removed by affinity chromatography; there was also no cross- reaction with chum PRL and GTHs, ovine GH and PRL, and presumptive eel GH and PRL. With pituitary extracts, parallel curves were seen only with chum and rainbow trout, although slight displacement was seen with charr extracts. It is hoped to completely validate the assay by ensuring the absence of cross-reaction with hypophysectomised salmonid plasma, and to modify the protocol to increase assay sensitivity for measure- ment of plasma GH levels. EN 26 CHANGES IN PLASMA PROLACTIN LEVELS IN MATURE CHUM SALMON DURING FRESHWATER ADAPTATION. T. Hirano, S. Hasegawa and T. Ogasawara. Ocean Res. Inst., Univ. of Tokyo, Tokyo. The plasma prolactin (PRL) levels in mature chum salmon caught in the bay were below 1 ng/ml. When they were transferred to fresh water (FW), the plasma PRL in females increased gradually after 4-8 hrs, attained a maximal level of about 8 ng/ml after 24 hrs and high levels were maintain- ed after 3-7 days. In contrast, no change was seen in males transferred to FW. In both males and females, plasma osmolality decreased significantly 1 hr after transfer to FW, and a low FW level was attained after 4 hrs. On the other hand, plasma cortisol levels were higher in females than in males, and a significant reduction in the plasma level was observed after trans- fer to FW in both sexes. Plasma thyroxine levels were variable and there was no difference between males and females either in SW or in FW. Involvement of PRL in reproductive process is unlikely, since there was no change in plasma levels of gonadal steroids in both males and females after transfer to FW. Thus, the low PRL levels in males in FW may be due to an in- crease in secretion rate accompanied by an increased clearance rate. It seems to be also possible that PRL is involved in some physiological processes other than osmo- regulation in FW. 966 Endocrinology EN 27 ENDOCRINE CONTROL OF OSMOTIC WATER PERMEABILITY OF THE EEL GILLS T.Ogasawara. Ocean Research Institute, University of Tokyo, Tokyo. Although much is known about ion exchange mechanisms across the teleost gills and their hormonal control, less work has been devoted to regulation of the water perme- ability. When gills isolated from fresh- water(FW) eels were incubated in CaCl 5 at concentration of more than 0.5 mM, the osmotic water permeability became lower than those incubated in DW. Hypophysectomy resulted in a decrease in the water perme- ability of FW eels, and prolactin therapy restored the normal permeability. In sea- water(SW) eels, the gill water permeability was still greater than in FW eels and addi- tion of CaCloa to the incubation medium had no effect on the osmotic water influx. When SW eels were transferred to FW, the water permeability decreased to FW level within 1h. Prolactin was not involved in the acute reduction of the water perme- ability after transfer to FW. On the other hand, when the IXth and the xXth cranial nerves were transected in SW eels, signifi- cant reduction in the water permeability was observed, suggesting that these nerves are involved in keeping the gill water permeability at high level in SW. It is possible that the acute reduction of the water permeability after transfer from SW to FW is also caused by some neural mechanisms via the cranial nerves. EN 28 EFFECTS OF STANNIECTOMY ON INTESTINAL CA ABSORPTION IN THE EEL (ANGUILLA JAPONICA). S. Hasegawa and T. Hirano. Ocean Research Institute, University of Tokyo. Tokyo. Surgical removal of the corpusles of Stannius (CS) results in an increase in plasma Ca level in the eel, with the increase being more marked in seawater (SW) or in 10 mM, 1eaGip Jsolutiion than a nieerselsin— water (FW). Therefore, hypercalcemia fol- lowing stanniectomy seems to be dependent upon the availability of exogenous Ca ions, Among osmoregulatory surfaces of fishes, the gills and intestine are possible targets of the CS hypocalcemic factor. In present study, effects of stanni- ectomy on intestinal Ca absorption were examined in the eel. After stripping the outer muscle layers, the intestine wags everted and filled with Ringer solution containg 2mM Ca before incubation in the same Ringer solution or in 10 mM Ca-Ringer solution for 60 min at 18°C. Stanniectomy of both FW eels and SW eels had no effect on net Ca absorption in normal Ringer solution. Stanniectomy in FW eels caused an increase in net Ca absorption when the mucosa was bathed in 10 mM Ca-Ringer solu- tion, but a similar effect was not seen in stanniectomized eels in SW. Hypophysectomy resulted in an overall decrease in water and electrolyte absorption, but no specific effect on Ca absorption was seen. Intestine may not be the major target of the CS hypocalcemic factor. EN 29 EFFECTS OF STANNIECTOMY ON INTESTINAL CALCIUM TRANSPORT IN THE JAPANESE EEL N. L. Collie and T. Hirano. Ocean Research Institute, University of Tokyo, Tokyo. The intestine as a site of action of fac- tors secreted by the corpuscles of Stannius (CS) has received little attention. Calcium transport was characterized in vitro across eel intestines mounted in modified Ussing champers. The bidirectional and net fluxes of Ca were measured along with electro- physiological parameters (transepithelial potential difference (PD), resistance (R), and short-circuit current (Isc)). Intes- tines from sham and unoperated eels active- ly absorbed Ca at a small but significant net flux of 1.74 0.2 nmolles/ Joma hemes comparison, SCX eels showed a 12-fold higher net flux, principally owing to a greater mucosal-to-serosal unidirectional flux. The PD, R, and Isc values of SCX eel intestine were all greater than the values measured in sham animals, though the dif- ferences were not statistically signifi- cant. Addition of a saline CS extract ( 8 or 80 ug/ml) directly in vitro caused both a marked inhibition of Isc and R and a transient though significant decrease in net Ca flux which was dose-related. These results indicate that 1) FW eel intestine can absorb Ca in the absence of electro- chemical gradients, 2) CSX stimulates the net flux of Ca, and 3) addition of CS extract affects the intestinal transport of Ca and potentially other ions as well. EN 30 IMMUNOHISTOCHEMICAL DEMONSTRATION OF PRO- LACTIN RECEPTOR IN SALMONID KIDNEY IN FRESH WATER. J.Kubota, T.Hirano(Ocean Res. Inst., Univ. of Tokyo, Tokyo) N.Naito, Y.Nakai(School of Med., Showa Univ., Tokyo) A.Takahashi and H.Kawauchi(School of Fish. Sci., Kitasato Univ., Sanriku) Prolactin(PRL) is known to act on osmo- regulatory surfaces such as gills and kid- ney to regulate water and electrolyte bal- ance in freshwater teleosts. Immunocyto- chemical demonstration of PRL bound to receptors in freshwater salmonid kidney was attempted by PAP method. When anti- serum(X5000) against highly purified chum salmon PRL was applied to freshwater- adapted chum kidney, strong immunoreactiv- ity was observed exclusively in distal tubules. No reaction was seen with anti- serum absorbed with chum PRL. Cross-reac— tivity in distal tubules was stronger in freshwater fish than in seawater fish. Cross-reactivity was also observed in the chloride cells of the gills, but not in the liver. The kidney of freshwater rain- bow trout also reacted to the antiserum as in the chum kidney, only distal tubules showing strong cross-reactivity. No reac— tivity was observed in the kidney of sea- water-adapted trout. These results strong- ly suggest that distal tubules are the primary receptor sites in the kidney of chum salmon and rainbow trout in fresh water. Endocrinology 967 EN 31 SERUM CALCITONIN LEVELS OF FRESH-WATER FISHES AND SEA WATER FISHES. Y. Sasayama and C. Oguro. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama Calcitonin is a hypocalcemic hormone in mammals. In fishes, however, this hor- mone does not cause clear decreases in serum calcium levels. In the present stu- dy, serum immunoreactive calcitonin (serum icT) level was compared between fresh- water fishes (5 species) and sea water fi- shes (7 species) by using a radioimmunoas- say kit for human (Dai-ichi Radioisotope Laboratory). Also in one species of carti- laginous fishes (marine sting ray), its level was determined for reference. Fur- thermore, serum calcium concentration of those animals was measured. In fresh-water fishes, the average le- vel of serum iCT ranged in 150-200 pg/ml, although it showed a wide variation from 1 to 400 pg/ml. Also in sea water fishes, Similar values were obtained. The average extent was in 200-300 pg/ml. The lower level was around 100 pg/ml. On the other hand, the serum iCT level of the sting ray was higher than those of bony fishes. Most of the ray showed values between 300 and 1000 pg/ml. When making a check on the data of serum iCT levels and serum calcium concentrations, no mutual relationship was recognized between them. These facts suggest a possibility that in fishes calcitonin is not directly con- cerned with calcium regulation. EN 32 ENDOCRINE-REGULATED MORPHOLOGICAL CHANGES IN THE GONAD DURING SEX REVERSAL IN PROTOGYNOUS WRASSES M. Nakamura,* K. Yamauchi,** Y. Nagahama*** and G. E. Grau**** * Terkyo-Univ.; ** Hokkaido Univ., *** Nat. Inst. Basic Biol., *** Hawaii Univ. Two species of the wrasse, Thalassoma duperrey and Halichoeres tenuispinis, were observed histologically and ultrastructu- ally in order to study the endocrinologi- cal mechanism of natural sex reversal in £aSna En T. duperrey, the initial changes from ovary to testis were characterized by a decrease of mature oocytes, however, young oocytes in the peri-nucleolus and chromatin-nucleolus stages remained normal in appearance. On the other hand, presumed spermatogonia increased gradually in number on the periphery of the ovi- gerous lamellae. Clusters of steroid producing cells were found in the stromal tissue and they increased in number in parallel with the progress of sex reversal. The young oocytes eventually disappeared completely due to degeneration, but the Ovarian cavity persisted in the matured testis. In both species, we observed a marked increase in a variety of stromal cells in the central region of ovigerous lamellae. Our results suggest that endogenous steroids may play a role in the natural sex reversal of wrasses. EN 33 ROLE OF cAMP ON OOCYTE MATURATION AND OVU- LATION IN THE GOLDFISH (CARASSIUS AURATUS). H. Kagawa! and Y. Nagahama”. ' Dept. Anat., Sch. Med., Univ. Occupational Environmental Health, Kitakyushu. * Lab. Reprod. Biol., Nat. Inst. Basic Biol., Okazaki. Effects of dibutyryl cAMP (DBcAMP), 3- isobutyl-l-methylxanthine (IBMX), theophy- lline, and forskolin on oocyte maturation and ovulation in the goldfish were investi- gated using an in vitro incubation system. HCG (100 IU/ml), 17a-hydroxyprogesterone (17a-OHprog; 100ng/ml), and 17a,20g-dihy- droxy-4-pregnen-3-one (17a,208-diOHprog; 100 ng/ml) induced 67.8, 63.6, and 100 3% oocyte maturation, respectively. One mM DB- CAMP blocked HCG-, 17a-OHprog- and 17a,208- diOHprog-induced oocyte maturation (25.9, 0, and 18.2 %, respectively). IBMX and theophilline (1 mM) blocked HCG- and ste- roid-induced oocyte maturation. Oocytes that had undergone oocyte matu- ration and follicular detachment in vivo were incubated in the presence of FGF), and IBMX, DBcCAMP or forskolin. PGF», alone stimulated 82 and 70 % ovulation at 10.0 and 1.0 ug/ml, respectively. At 10.0 ug/ml PGFo,, 5.0 mM DBcAMP, 1.0 mM IBMX and 1.0 or 0.1 uM forskolin blocked ovulation. At 1.0 wg/ml PGFoq, 5.0 mM or 0.5 mM DBcAMP TORO. OL UanMeisMxeandadiO, Oecd -OWOL WM forskolin blocked ovulation. These results indicate that some point in the process of goldfish maturation and Ovulation is inhibited by cAMP. EN 34 THE EFFECTS OF PH ON IN VITRO OVULATION OF GOLDFISH (CARASSIUS AURATUS) OOCYTES. F.W.Goetz! and Y.Nagahama’*. ‘Dept. Zool., Univ. Notre Dame, USA and *Lab. Reprod. Biol., Nat. Inst. Basic Biol., Okazaki. Effects of pH on goldfish (Carassius auratus) ovulation were investigated using an in vitro incubation system. Oocyte final maturation and follicular detachment were induced in vivo in gravid goldfish by HCG injections and elevated holding tempera- ture. Females were biopsied to determine the time of germinal vesicle breakdown and when oocytes should be removed for in vitro incubations. Prior to ovulation, the Ovaries were removed, dissected and mature intrafollicular oocytes were incubated in goldfish Ringers at different pHs ranging from 7.3-8.9. Different pHs were obtained by mixing tris-hydrochloride and tris-base at specific ratios. There was a significant increase in ovulation as the pH of incubation medium increased. Indomethacin was capable of blocking ovulation that occurred in media at any pH. However, this inhibition could be reversed by prosta- glandin (PG) F2qin the presence of indomethacin, indicating that the mecha- nism of ovulation was not inhibited by indomethacin. Increased pH did not increase the ovulatory response observed with exogenous PGF2,. The combined results suggest that an increase in incubation pH stimulates prostaglandin synthesis that, in turn, stimulates ovulation. 968 Endocrinology EN 35 FUNCTIONAL RELATIONSHIP BETWEEN AXON TER- MINALS AND PROCESSES OF EPENDYMAL CELIS IN THE NEWT MEDIAN EMINENCE Oo. Biol, WaSca, FAC. Or Sis sg Shizuoka Univ., Shizuoka. Fine structure of the palisade layer of the median eminence was studied in the newt. Three types of nerve axons and their terminals are present in the layer. Most of these axon terminate on the basement membrane of the blood capillaries of the primary plexus. Frequently, the processes of the ependymal cells also terminate around the basement membrane region. Ep- endymal cells, which consist mainly of a Single cell layer facing the third ventri- cle, have long processes extending to the palisade layer of the median eminence. In order to analyze the functional rela- tionship between the axon terminals and processes of ependymal cells in the base- ment membrane region, the number and the length of endings in direct contact with Capillary surface are calibrated, respec-— tively. More than 6C% of axons are found E® IS Wid COMEACT Cliimeculy Walicin wae Cao lary surface. Moreover, topographical differences in the coverage cf the neuro- hemal contact zone by the axon terminals and processes of ependymal cells are obviously demonstrated. The diversity of axon-ependyma relationship may reflect the functional state of the median emi- nence. EN 36 ONTOGENY OF LHRH-ERGIC AND VASOTOCINERGIC NEUROSECRETORY SYSTEMS IN THE BRAIN OF TOAD, BUFO JAPONICUS H. Nojiri and A. Urano, Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa Development of LHRH-ergic and Vasoto- Ccinergic (AVT) systems in the toad brain was studied immunohistochemically by the ABC method. The immunoreactive(ir) AVT ap- peared first in the neurohypophysis of tadpoles at stage 36 of Ichikawa-Tahara. Ir-AVT perikarya were demonstrable after stage 40. They were localized in the pre- optic nucleus pars magnocellularis dorsa- lis (MGCD) and ventralis (MGCV). Since the AVT neurons in the MGCV were located along the sulcus preopticus, they may be derived from neuroblasts in the germinal layer of this locus. The dispersed AVT neurons in the MGCD, which characteristically include melanin granules, seem to have different origin. They may be derived from neuro- blasts located along the sulcus ventralis thalami. On the contrary, ir-LHRH neurons could not be demonstrated in the tadpole brains. One month after the metamorphosis, the presence of ir-LHRH was detected in the median eminence of juvenile toad. Ir- LHRH perikarya were not demonstrated even in the brain of the four months old juve- niles nonetheless the presence of ir-LHRH fibers. These results indicate that the AVT system which concerns osmoregulation develops earlier than the LHRH system which regulates reproductive activity. EN 37 EXTRAHYPOTHALAMIC PROJECTION OF LHRH AND VASOTOCIN IN THE BRAIN OF TOAD (BUFO JAPONICUS). Y.Jokura, S.Takami, and A.Urano, Dept. of Regul. Biol. ,Fuc. of Sci., Saitama Univ., Urawa. Extrahypothalamic projection of LHRH and Vasotocin (AVT) fibers in the toad brain were examined immunohistochemically by the avidin-biotin (ABC) method. LHRH perikarya are localized in the nuclei (n.) medialis septi and of the diagonal band of Broca, while AVT perikarya in the n. pre- opticus pars magnocelluralis. A part of LHRH fibers project anteriad to the me- dial and dorsal septum, and further some of them reach the olfactory bulb. Dorso- laterally LHRH fibers project to the optic tectum and posterior mesencephalon via the habenular and posterior thalamic regions. The AVT neurons send fibers postero-dor- sally to the n. posterocentralis thalami, posterodorsalis tegmenti mesencephali and isthmi. A dence network of AVT fibers were found in the n. lateralis septi and medi- alis septi. In addition, both LHRH and AVT fibers which run through the dorsal infun- dibular region reach the medulla oblonga- ta. These findings suggest that LHRH and AVT act as neurotransmitters or neuromodu- lators in the various regulatory procesg of reproductive behavior in the central nervous system. EN 38 THE EFFECTS OF MESOTOCIN ON VASCULAR AND RENAL FUNCTIONS IN TWO ANURAN AMPHIBIANS. M.Uchiyama and T.Murakami. Dept. of Oral Physiol., Nippon Dental Univ., Niigata. The vascular and renal responses of two anuran amphibians, Rana catesbeiana and Bufo bufo japonicus, to mesotocin (8-Ile-oxytocin) have been examined. Arterial blood pressure, measured in the dorsal aorta, decreased with this hormone in the bullfrog, while no obvious depres-— sor effect was observed in the toad. However, a high dose of mesotocin (over 200 ng/kg) was vasopressor in some toads. Under the condition that the conscious animal was placed in a plastic box con- taining tap water, urine was collected directly from the ureters. The rate of urine flow in the bullfrog was signifi- cantly lower than in the toad. In both anurans mesotocin was a diuretic agent and there was a dose-response relationship between the increase of urine flow and the dose of mesotocin (5 to. 100 ng/kg). In some cases, however, at high doses (over 200 ng/kg) the diuretic response was con- siderably reduced. Diuresis was accompa- nied by increases in GFR, Na excretion and free water clearance. On the other hand, mesotocin did not give distinct influence on the urine-to-plasma inulin ratio or the relative free water clearance. The present experiments suggest that mesotocin play a role in controling GFR and Na excretion in these anurans. Endocrinology 969 EN 39 PROLACTIN SYNTHESIS IN THE ANTERIOR PITUITARY GLAND OF BULLFROG LARVAE DURING METAMORPHOSIS. K. Niinuma, K. Yamamoto, and S. Kikuyama Dept. of Biol., School of Education, Waseda University, 160, Tokyo. According to our previous study the level of immunoassayable prolactin in the plasma and pituitary gland of bullfrog tadpoles goes up at late climax stage. To confirm this, the following experiments were carried out. Anterior pituitary glands from various metamorphic stages were incubated in a medium containing 3H-leucine. Prolactin was separated by subjecting the medium and pituitary to disc gel electrophoresis and its radio- activity was measured. Prolactin synthesis goes up gradually as metamorphosis pro- ceeds and reaches maximum at late climax stage with a little decline at the end of metamorphosis. On the other hand, pituitary glands from the tadpoles of various developmental stages were stained with an antiserum to bullfrog prolactin by PAP method. The cells stained with the anti- serum are relatively few during prometa- morphosis and they increase in number during the climax period. The results obtained in the present experiments are in good accord with the above-mentioned immunoassay results. EN 40 EFFECT OF VASOACTIVE INTESTINAL PEPTIDE ON THE RELEASE OF PROLACTIN FROM THE BULL- FROG PITUITARY GLAND IN VITRO. K. Koiwai, T. Seki and S. Kikuyama. Dept. of Biol., Sch. of Educ., Waseda Univ., 160, Tokyo. Prolactin(PRL) release from the bull- frog pituitary gland is under the hypo- thalamic controls of both stimulatory and inhibitory nature. At present TRH is pre- sumed to be one of the factors stimulating PRL release(PRF). In the present experiment, vasoactive intestinal peptide(VIP) which is known to Stimulate PRL release in mammals was test- ed for the prolactin liberating activity in amphibians. Bullfrog pituitary gland was incubated in 67% Eagle MEM containing various concentration(10-8-10-5M) of VIP. Amount of PRL released into the medium every 4 hr during 16 hr of incubation period was measyred by RIA. At the concen- trations of 10°°-10-6M, VIP stimulated PRL release dose-dependently. 10-®M VIP en- hanced PRL release upto 227% of the control In the Sjmilar experimental condition, TRH (2.8x10°°M) stimulated PRL release upto 315% of the control. The stimulatory effect of VIP as well as of TRH was partially suppressed by dopamine(10~°M). On the other hand, the presence of VIP-like substance in the bullfrog brain has been demonstrated by radioimmunoassay, although the exact location of the immunoreactive VIP is not known. It remains to be clarified whether endogenous VIP is a PRF in amphibians. EN 41 IN VITRO STIMULATION OF BULLFROG PROLACTIN RELEASE BY HYPOTHALAMIC EXTRACT. T.Seki and S.Kikuyama. Dept. of Biol., Sch. of Educ., Waseda Univ., 160, Tokyo. The effect of hypothalamic extract (HE) on prolactin(PRL) release from bullfrog pituitary gland was studied in vitro. Pituitary glands were incubated in 67% Eagle MEM at 25°C for 8hr. PRL content in the medium was measured by an homologous radioimmunoassay for bullfrog PRL. Bullfrog hypothalami were homogenized in 0.1N-HC1l and centrifuged. The supernatant was neu- tralized and served as HE. Extract from 0.01-1 hypothalamus in 1 ml of medium stim- ulated the release of PRL dose-dependently. This activity was nullified by incubation with bullfrog plasma at 37°C for 3” hr . Next, we investigated whether the PRL re- leasing activity is derived from thyro- tropin-releasing hormone (TRH), since TRH exists in bullfrog hypothalamus at high concentration and has a potent PRL releas- ing activity in vitro. When the extract was chromatographed on a Sephadex G-25, only the fraction which contained TRH pos- sessed the PRL releasing activity. IgG fraction in anti-TRH serum purified by a protein A-Sepharose partially blocked the stimulatory effect of HE. These results indicate that HE contains factors which stimulate release of PRL and that one of the factors may be TRH. EN 42 PLASMA PROLACTIN LEVELS OF BUFO JAPONICUS BEFORE, DURING AND AFTER BREEDING IN THE POND H. Yoneyama, S. Ishii, K. Yamamoto, S&S. Kikuyama. Dept. of Biol., school of Education, Waseda Univ., Tokyo 160. Blood samples were collected at Nobi- dome, Saitama-ken in March, 1983 by car- diac puncture from toads of the following conditions: (A) resting under the ground 10 days before the breeding migration, (B) migrating to a pond for breeding, (C) moving around the pond, (D) swimming ac- tively in the pond, (E) moving on _ the ground soon after breeding. Prolactin in plama was measured by a _ radioimmunoassay using anti-bullfrog prolaction serum. Plasma prolactin levels were low and at about the basal level of the annual cycle in Group A and B. They were signifi- cantly higher in Group C and D. The _ val- ues of Group E flactuated widely, whereas its mean level was about the same with that of Group A. Prolactin can not be a humoral factor which induces migration of toads to water, since its plasma level was low in the migrating toads. The high prolactin levels of toads found around and in the pond are supposed to be a re- sponse to the low osmotic pressure envi- ronment, as prolactin is Known to be necessary for adaptation to such an eén- vironment in some fish. 970 Endocrinology EN 43 VARIATION OF PLASMA THYROID HORMONE LEVELS IN TOADS DURING BREEDING SEASON Y. Tasaki, M. Inoue and S. Ishii Dept. of Biology, School of Education, Waseda Univ., Tokyo Plasma thyroxine (T4) and triiodothy- ronine (T3) levels of Bufo japonicus of a feral population in Sayama City, Saitama Prefecture showed distinct annual changes. Plasma T4 concentration was low in summer and autumn (0.09 to 0.24 ng/ml), and increased markedly from January to March, the maximam being (0.57 ng/ml) in March. We performed more detailed observation of plasma T4 and T3 levels around the breeding season at Nobidome, Saitama Prefecture. Blood samples were collected from,toads found under the ground just before the migration (A), actively moving toward a pond (B), swimming in the pond (C) and having come out from the pond (D). The mean plasma T4 level of Group A (0.37 ng/ml) was close to the level which was observed in March in the annual cycle study. The mean T4 level was higher in Group B to D, and the highest observed in Group C (1.88 ng/ml ). T3 levels showed Similar changes, although they were far lower than the T4 levels. These findings Suggest that thyroid hormone may play roles in the regulation of the energy metabolism under a low temperature environment in winter and intiation of the locomotor activity for the breeding migration in March. EN 44 PURIFICATION AND PROPERTIES OF 3,5,3'- TRIIODO-L-THYRONINE BINDING SITE ON PLASMA MEMBRANES FROM BEEF AND BULLFROG TADPOLE LIVER. K.Yamauchi, R.Horiuchi, S.Koya and H.Takikawa. Inst. of Endocrinol., Gunma Univ., Maebashi Recently, it has been demonstrated that 3,5,3'-triiodothyronine (T3) receptor on the plasma membrane regulates Tg uptake to the nuclei. To clarify the characters of T3-receptor on the plasma membrane, T3- binding site was partially purified from beef and bullfrog tadpole (Rana catesveiana) liver plasma membrane. la binding site was solubilized by 0.5% CHAPS and purified by T3-Sepharose and anion exchange resin (Mono Q). Ta-binding site was purified more than 1,000-fold by these steps. The molecular weight of T3-binding site of solubilized receptor was 80K dalton by gel filtration or 55K dalton by SDS-PAGE. Isoelectric focusing revealed one major peak vastepe “49. sMolecu Mar) swiziemiort Tabinding site from tadpole liver was Similar to that from beef liver. However, peptide mapping of 55K dalton protein by limited proteolysis by V8 protease in SDS showed some differences in amino acid compositions between beef and tadpole. EN 45 PURIFICATION OF BULLFROG GROWTH HORMONE. T. Kobayashi, H. Takasu, K. Kawamura, K. Yamamoto and S. Kikuyama. Dept. of Biol, Sch. of Educ., Waseda Univ.,160, Tokyor During the isolation of prolactin from the bullfrog pituitary gland, we obtained a fraction which was not adsorbed when acid acetone extract of pituitary powder dissolved in 0.01M Tris-HCl buffer(pH7.4) was applied to DEAE cellulose column. This fraction was found to contain growth hormone(GH) since it had a prominent GH activity. Attempt was made to purify GH further from this fraction. The fraction was dissolved in 20mM citrate buffer (pH5.7) and applied to CM-Toyopearl column. A fraction eluted by the buffer containing 80-130mM NaCl had a potent GH activity. Then this fraction was dissolved in 100mM sodium citrate in 50mM phosphate buffer (pH7.0) and applied to HPLC(TSK G2000SW). GH activity was localized in a fraction with a MW of about 20,000. The final product exhibited a single electrophoretic band on SDS polyacrylamide gel electro- phoresis with a mobility of polypeptide chain of MW of about 21,000. The iso- electric point of bullfrog CH yehusmobmatie ed was 7.2. From 570mg pituitary powder 0.6mg GH was obtained. Bioactivity of bull- frog GH was 1.1 times as potent as bovine GH when tested in hypophysectomized Xenopus using uptake of 35S-sulphate by xiphisternal cartilage as criteria. EN 46 EFFECT OF GROWTH HORMONE AND SERUM ON THE UPTAKE OF 3°S-SULPHATE BY XIPHISTERNAL CARTILAGE IN XENOPUS. J. Okuma and Si) Kikuyama. Deptymoe Biol. Sch omercuicrs Waseda Univ., 160, Tokyo. Injection of ovine growth hormone (GH) into hypophysectomized Xenopus juveniles accelerated the body growth and enhanced the in vitro uptake of 5S-sulphate by the xiphisternal cartilage dose-dependently Addition of GH to the incubation medium failed to stimulate the uptake of the labelled sulphate by the cartilage. On the other hand, when the xiphisternal cartilage from the hypophysectomized Xenopus was incubated in the medium containing 5-30 % serum obtained from the hypophysectomized specimen which had been treated with GH, the uptake of the labelled sulphate increased by 25-50 % % as compared with the cartilage incubated in the medium contain- ing the serum from the hypophysectomized Xenopus which had been injected with Saline The results indicated that GH is not a direct stimulatory factor of sulphate uptake by the cartilage but it may induce a factor(s) which enhances the sulphate uptake. Endocrinology 971 EN 47 PRELIMINARY ACCOUNT ON THE EFFECT OF THYROXINE AND ACTH ON METAMORPHIC TRANS - FORMATION OF DIGESTIVE TRACT IN HYPOPHYS- ECTOMIZED BULLFROG TADPOLES. C. Fujimoto, K. Kawamura and S. Kikuyama. Dept. Biol., School of Education, Waseda University, Tokyo. During metamorphosis of anuran tadpoles, a considerable shortening and a neogenesis of the intestinal villi and the stomach occur at climax stage (st. XXF-XXIV of Taylor § Kollros). However, it has not been determined if they are induced by thyroxine alone without intervention of other pituitary hormones than TSH. Late- prometamorphic tadpoles (st. XVIII) of Rana catesbeiana were hypophysectomized, maintained in tap water with or without Gest & 10.M of T4.and.sacrificed at st. XXIII-XXIV. Some tadpoles were treated with porcine ACTH (100 mU/day)in addition to Tg. Regression of the small intestine and morphogenesis of the adult-type intestinal epithelium along with the gastric mucosa were induced by T4 treatment, while those reared in tap water were in a metamorphic stasis at st. XX. Peptic activity of the gastric region was 5-10 times increased from st. XXIII on in Ty- treated animals as well as in intact animals, as measured by hydrolysis of haemoglobin. Effect of ACTH was not conspicuous. [In conclusion, both morpho- logical and functional development of the digestive tract can presumably be evoked by thyroid hormone alone. EN 48 ISOLATION OF GONADOTROPINS OF THE TOAD, BUFO JAPONICUS. Etandaa “and “ScJishii: Dept. of Biol., School of Education, Waseda Univ. Tokyo. Gonadotropin-like substances were isolated from a glycoprotein fraction of about 5,800 toad pituitary glands by means of combinations of the following column chromatographies: ion exchanges with CM cellulose and DEAE Sephacel, chromato- focusings and high performance gel permeations with G3000SW or G3000SWG. Gonadotropin activities of chromatographic fractions were monitored by two different radioreceptor assay (RRA) systems, one using toad testis receptors and radio- iodinated bullfrog FSH and the other using bullfrog testis receptors and radioio- dinated bullfrog LH. The RRA activities were detected in both acidic and basic fractions after the CM cellulose chro- matography. Only the acidic fraction was further purified with DEAE cellulose and an active fraction was recovered. In the chromatofocusings, the activities were eluted at pH 9.4 and a basic pH lower than 8.0 in the basic substance, and at pH 4.5 and a lower pH than 4.0 in the acidic substance. These fractions were finally purified by the gel permeations. Toad gonadotropin-like substances are similar to gonadotropins of bullfrog and other tetrapod vertebrates in their chromato- graphic behaviors, but FSH and LH were not discernible at the moment. EN 49 HEPATIC GONADOTROPIN BINDING SITES OF THE BULLFROG. K. Kubokawa and S. Ishii. Deptartment of Biology, School of Educaion, Waseda University, Tokyo 160. Highly purified gonadotropins of the bullfrog, Rana catesbeiana, bound specifically to a crude plasma membrane of the bullfrog liver. These hepatic binding sites had a high specificity and affinity to gonadotropins which are Similar to those of testicular gonadotropin receptors of the same species. We may suppose a possibility that our gonadotropin preparations were contaminated largely with other peptide hormone such as growth hormone or prolactin, although chemical purity of hormone showed that this could hardly occur. As purified bullfrog prolactin could not replace the gonadotropins in liver, only growth hormone would remain as the possible candidate. In order to exclude this possibility, we designed a binding experiment in which radioactive hormone was incubated with a constant concentration of the testicular receptor and various concentrations of the liver receptor. The results showed that our gonadotropin preparations contained substances which could bind specifically to both the testis and liver, and these Substances are supposed to be gonadotropins and not growth hormone. EN 50 SEXUAL DIFFERENCE IN THE LARYNGEAL REGION OF THE TREE FROG HYLA JAPONICA: OBSERVA- TION AND EXPERIMENTAL STUDY. Hisaaki Iwasawa and Yasuko Nakayama. Biol. Inst., Niigata Univ., Niigata. The developmental process of sexual dif- ference in the laryngeal region was studied with relation to the development of other male sexual characters and testicular dif- ferentiation. No sexual difference was found in the size of the arytenoid cartilages in newly metamorphosed frogs (July), but in frogs at one month after metamorphosis, the carti- lages were slightly larger in males than in females. At this time the testes were notab- ly increasing in size and spermatogenesis was taking place. Sex can be discriminated externally from the laryngeal size in these first-year autumn frogs. In November the space occupied by the left and right arytenoid cartilages in males was about 14 times as large as that in females. Among similar- sized frogs,the postero-medial process of the hyoid, especially the cartilaginous por- tion of this bone, was definitely longer in adult males than in adult females. Newly meta- morphosed frogs were injected intraperito- neally with 50 pg testosterone every 3 days. After 30 days the development of the arytenoid cartilage and the ampulla of the Wolffian duct was significatly stimulated regardless of genetic sex, but no melanosis was seen in the throat skin. 972 Endocrinology EN 51 STIMULATION OF PROTEIN PHOSHORYLATION IN PRIMARY CULTURES OF XENOPUS LAEVIS HEPATO- CYTES BY ESTRADIOL-176. K.Sato, A.Kawahara and M.Amano. Cell and Dev. Biol. Lab., Fac. of Inte. Arts and Sci., Univ. of Hiroshima, Hiroshima Estrogen induces the synthesis of egg yolk protein precursor vitellogenin in the liver of oviparous vertebrates. Vitellogenin is highly phosphorylated protein; there is a possibility that enzymes which phospho- rylate proteins are stimulated simultane- ously by estrogen. To determine this possi- bility, the effects of estrogen on the activ- ity of protein phosphorylation was examined. Furthermore, the phosphorylated proteins were analyzed by the two-dimensional gel electrophoresis. Protein phosphorylation increased gradually with time of estrogen treatment. On day 5 of estrogen treatment, estrogen stimulated the incorporation of POA AnEOmEhe ssecneitedmandmthessimeracelm an lar proteins by 3- and 2.5-fold, respec- tively. The distribution of 32P-labeled intracellular proteins on the gel showed few change but individual proteins were more phosphorylated as compared to that without estrogen, indicating that phospho- rylation stimulated by estrogen modified not only vitellogenin but also the estrogen- unstimulated proteins. The protein kinase activity measured in cell-free extracts was 5 times higher than that of the control cells. These data indicated that estrogen enhanced protein phosphorylation by ele- vating protein kinase activity. EN@S2Z EFFECTS OF LIGHT-CYCLE AND MELATONIN ON THE HYPOPHYSIS-TESTIS SYSTEM IN HYLA JAPONICA. Yoko Takeuchi and Hisaaki Iwasawa. 13) oy) Pan ok cara \\ ss Ke f- hh ot WO) os ae | se fo a Effects of melatonin on the hypophysis- testis system under 12L-12D, 24L-OD and OL-24D light cycles were studied. Adult male frogs were injected intraperitoneally with melatonin (10 ywig/g body weight/day) every 3 days for 30 days in May. The frogs were kept at 20 + 2°C. The hypophyses and the testes were fixed with Bouin's solu- tion, embedded in paraffin and sectioned elie 5) jbhulc The hypophyseal sections were stained with Alcian blue-PAS-Orange G, and the number of gonadotropin-producing cells was determined. In the 12L-12D- treated groups, the number of the gonadotropin- producing cells was much smaller in the melatonin-injected frogs than in the con- trol frogs. The results of histochemical examination showed that the activity of A°-38 hydroxysteroid dehydrogenase in the interstitial cells of the testes was sig- nificantly reduced in the melatonin-in- jected frogs of the 12L-12D and 24L-OD groups. The results obtained in the pre- sent experiments suggest that melatonin has an inhibitory effect on testicular function through a hypofunction of the gonadotropin-producing cells. EN 53 EFFECTS OF FAT BODY EXC TS TON ON SrESieiGuianE ULTRASTRUCTURE IN YOUNG FROGS OF RANA NIGROMACULATA. Tohru Kobayashi and Hisaaki Iwasawa. Biol. IMSibss Niigata Uinhiiwes S Nigigiaiear Second-year frogs collected in the middle of May were divided into the following groups and the _ physiological role of the fat body on testicular function was examined electron-micro- scopically: As initial’ “cont rolleeeprme tame control, C; excision of fat body (ERB) masuE EFB + testosterone. The frogs were kept at 21 + 2°C, and the frogs in group D were intraperitoneally injected with 0.5 mg testosterone every 3 days. The experimen- tal frogs were fixed 30 days after the first injection. The “tes ticul dyeisn ema groups C and D was significantly smaller than that in group Be Mostegenncelmes were at the primary spermatogonial stage in group A, and all spermatogenetic stages were observed in’ group B. No spermatids Or spermatozoa were seen in group C, and primary spermatogonia, spermatids and spermatozoa were observed in group D. The interstitial cells were well developed in group C compared with those in group B, TWaAes a number of lipid droplets, mitochondria with vesicular cristae, and a few s-ER were observed, though the so-called Sertoli cells were poorly developed. The results described above suggest that the fat body plays an important role in’ tesitiicultad Shuniciaane EN 54 CALCIUM KINETICS IN URODELAN AMPHIBIANS UNDER THE CONTROL OF CALCEMIC HORMONES. C. Oguro, Y. Sasayama. Department of Biology, Toyama University, Toyama. Calcium content in tissues of urodelan amphibians was determined and compared with that of anurans. Ca content an the urodelan skin was much lower (1x10 ug/g) than that in the anuran skin) (@xl0gue/oor Then Ca kinetics was studied using 45Ca. Comparison of 45Ca activity in tissues of Hynobius nigrescens was made between 2 hours and 1 week after *°Ca administration. All tissues except bones contain less 5Ca 1 week after than 2 hours after. On the contrary, bones accumulate much 495Ca 1 week after than 2 hours after. In Cynops pyrrhogaster, parathyroidectomy (PTX) re- sulted in a marked decrease in serum Ca concentration 5 days after PTX. However, serum Ca concentration increased to nearly normal level 20 days after PTX. Five days after PTX when hypocalcemia occurred, Ca content in all tissues was not changed. However, 20 days after as serum Ca re- covered, Ca content in the femur decreased Significantly. In PTX-sham operated C. pyr- rhogaster, Ca content in the femur de- creased markedly 5 days after operation when serum Ca concentration was normal. These facts suggest that the femur sup- plies Ca into serum under the control of PTH and the femur loses Ca without exoge- nous Ca supply. The vertebra is not much involved in Ca kinetics in relation to PTH. Endocrinology 973 EN 55 PLASMA CALCIUM AND PHOSPHORUS IN THE OVI- PAROUS SNAKE DURING REPRODUCTIVE PERIOD. M.Yoshihara, M.Uchiyama, T.Murakami, H. Yoshizawa’, M.Fujimori2? and C.Oguro? Dept. of Oral Physiol., Nippon Dental Univ., Niigata, 1. Dept. of Oral Anat., Matsumoto Dental Coll., Shiojiri, 2. Dept. of Biol., Toyama Univ., Toyama. In the oviparous snake, Elaphe quadri- virgata, plasma Ca and inorganic phospho- rus (Pi) during reproductive period were studied. Plasma Ca in female snakes before Ovulation (May) was higher than that in male snakes (15-30 vs. 13-15 mg/100 ml). At the beginning of ovulation (early and mid June), plasma Ca in female snakes showed a remarkable increase (30-130 mg/ 100 ml) and it decreased after ovulation (12-14 mg/100 ml). This level was main- tained till 1-2 weeks before oviposition. However, plasma Ca began to increase again and the level at oviposition (late June and early August) was higher (20-40%) than that in pre-oviposition. Plasma Pi in fe- male snakes increased at ovulation (from 3-6 mg /100 ml in May to 6-18 mg/100 ml) and decreased thereafter (3-7 mg/100 ml). Plasma Pi at circa-oviposition showed low level (2-4 mg/100 ml), however, the level increased after oviposition (3-7 mg /100 ml). These results indicate that the pat- tern of changes of plasma Ca and Pi in re- productive female snakes is similar with laying hen. However, the changes are more drastic in snakes than laying hen. EN 56 AN EVIDENCE AS A CIRCULATING AGENT OF MULLERIAN INHIBITING SUBSTANCE. A.Ohuchi and T.Noumura. Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa. Mullerian inhibiting substance(MIS), which causes regression of the embryonic Millerian ducts(Md), has been regarded as a locally acting agent in mammals and birds. This study was designed in chick embryo to determine whether MIS could cause regression of Md at a site distant from testes; (1)the effect of embryonic testes on female Md grafted onto the chorioallantoic membrane(CAM), and (2)the effect of testes grafted onto CAM on the Md of host female embryo. In the first series, Md from 7.5-8-day-old female embryos were grafted onto CAM of 9-day-old embryo. After 3 days, the grafted Md con- tinued to develop in female hosts, whereas those in male hosts showed clear signs of regression which were similar to those in the male embryo during normal development. In the second series, implantation of two 10-day-old embryonic testes onto CAM of 7- day-old female embryos caused various degrees of regression in host Md. A cor- relation existed between the degree of regression induced in host Md and the degree of development of the testicular grafts. These results suggest that the circulating level of MIS in chick embryo may be sufficient to cause regression of a Millerian duct at site distant from its Origin, as a circulating agent. EN 5/7 ONTOGENIC CHANGES IN TESTOSTERONE METABO- LISM BY THE BURSA OF FABRICIUS T. Hariya and T. Noumura. Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa The bursa of Fabricius is a lymphoepithe- lial organ in the birds, of which the nor- mal development is inhibited by androgen, especially testosterone. Before testoster- one binds to receptors in target cells, it can be enzymatically transformed into a number of metabolites with diverse hor- monal activities. The present study was undertaken to describe ontogenic changes in testosterone metabolism by the bursa in the chick and duck embryos in relation to the quantitative changes in tissue com- ponents. The bursa was incubated in vitro with *H-testosterone at four stages of bursal development: of plica formation, of epithelial bud, of lymphoid coloniza- tion, and of follicular maturation. In relative contents of tissues, the mesen- chyme was more at early stages but de- creased with age, the epithelium was near- ly constant, and the lymphoid cells in- creased rapidly during late stages. Seven metabolites in the duck and eight in the chick were identified. The production of each metabolite changed during organo- genesis, but main metabolite was 5a-andro- stane-3a,178-diol trough the bursal devel- opment. The results suggest that the quan- titative changes of metabolites with age may reflect the relative contents of bur- sal tissue components. EN 58 EFFECTS OF CASTRATION AND GONADAL HORMONES ON BURSA OF FABRICIUS AND THYMUS IN JAPANESE QUAIL. Y. Mase and 1. Oishi, Dept. of Biol., Fac. of Sci., Nara Women's Univ., Nara. We, previously, reported that the deve- lopment of the bursa of Fabricius and the thymus in long photoperiod could be divided into two stages, i. e., the early stage in which faster growth in both organs than in body weight occurred, and the later stage in which involution of both organs occurr- ed. In this report, 1) effects of gonadal hormones on the later stage of the develop- ment of these lymphatic organs in male and female guail and 2) effects of castration on the early stage of the development in male quail were investigated in long and short days. Testosterone implantation su- ppressed the bursal weight at 6 and 8 weeks of age and the thymus weight at 6 weeks of age in both long and short days. Estradiol implantation showed suppression of each organ in females at 6 or 8 weeks of age under short days. Castration at 2 weeks of age suppressed the bursal growth at 5 to 6 weeks of age, but showed no effect on the thymus growth. Thus, testosterone in male (and probably estradiol in female) induces involution of the bursa of Fabricius and the thymus in the later stage of their development. However, the testis seems to be necessary for the bursal growth (but not for the thymus growth) in the early stage of its development. 974 Endocrinology ENF 53 ANDROGEN RECEPTORS IN THE CLOACAL GLAND CYTOSOL OF THE MALE JAPANESE QUAIL: PARTIAL PURIFICATION AND EXCHANGE ASSAY. M.Aihara!, Y.Jokura2and S.Ishii® ‘Dept. of Buel. 5 IMaASs OI Seil,, Wol@ Windy, Wines basha,, —cDepte pofs Regqulls sBiolm shack alot Sci, oateamnal Unive, sUrawal, anc. ss) epitessolr Bole Sch. of IACWIC 5 p Waseda Univ., Nishi-waseda. Our previous studies have shown that cloacal gland cytosol from male quail contains specific androgen receptors (dis- sociation constant, 0.42 nM; number of binding sites, 70 fmol/mg protein). In the present work, cytosolic dihydrotestoster- one (DHT)-receptor complex was partially purified by chromatography on columns of heparin-agarose, Sephacryl S-300 and DEAE- cellulose in the presence of sodium molybdate. The molecular weight of par- tially purified material, as estimated by gel filtration, was 250,000. In addition, an exchange assay method for determing cytoplasmic androgen recep- tor in cloacal gland was developed using 3H-methyltrienolone (R1881) as the labeled ligand. Non-labeled, receptor-bound DHT was exchanged with °H-R1881 during a 2-h WANN Bie QS C Ox Clweime 8 in ate 5 Co At 4 C, less than 20% exchange occurred overs an 18h) periods Using, sehus assay, procedure, the concentration of androgen binding sites can be evaluated as a function of either endogenous androgen or non-labeled androgen administered by in- jection. EN 60 RECEPTIVITY OF QUAIL ADENOHYPOPHYSIS TO VARIOUS LUTEINIZING HORMONE- RELEASING HORMONES (LHRHS) iio ietsteoiet UF Se iteinisl _ ik, Mien? Y. Hasegawa? , M. Igarashi2 , M. Wada? . lWaseda Univ., Tokyo, 2Gunma Univ., Maebashi,?Tokyo Med. Den. Univ., Ichikawa. LH and FSH releasing potencies of chicken LHRH II (His® ,Trp’,Tyr®-LHRH), a newly identified chicken LHRH, were compared with those of chicken LHRH I (Glré -LHRH), mammalian LHRH (Arg®-LHRH) and analogues of the latter in in vitro and in vivo conditions. The chicken LHRH II was almost equipotent with chicken LHRH I and mammalian LHRH in both potencies in both conditions. The increment of LH release by LHRHs was higher than that of the FSH release. One of the super-agonist for gonadotropin release in mammals, [D-Ala®, des-Gly"?] -LHRH-ethylamide, showed the same potency as chicken LHRH II in stimulating LH and FSH releases from the quail adeno- hypophysis in vitro. However, an antago- nist of LHRH in mammals, [DPhe2,Pro?,DPh ]-LHRH, showed no activity in inducing gonadotropin release. The quail gonado- tropes have fairly less specific recep- tivity for LHRHsS than mammalian ones. This conclusion may be generalized to the relation between mammals and birds. EN 61 PHOTOPERIODIC CONTROL OF LH SECRETION IN MALE JAPANESE QUAIL—DETERMINATION OF PHOTOINDUCIJBLE PHASE P-Hatanaka and M.Wada“~. WaSeda Univ., Tokyo, Tokyo Med. Den. Univ., Ichikawa’. In male Japanese quail, long photo- periods (longer than 12.5 hr light/day) cause increased secretion of gonado- tropins. However, continuous lighting is not required throughout a long day to stimulate gonadotropin release. In the first experiment, a 15-min (0.25L) light pulse was given to quail kept under a Short day (8L:16D) during the dark period. LH increase was detected after 2 days of treatment only when the pulse was given during 12.5-14.5h after dawn. This result suggests that a rhythm of a photoinducible phase for LH secretion exists setting the phase at from 12.5 hr to 14.5 hr after dawn. Next experiments, blood samples were taken mainly every 2 hr in the first 2 days after transfer from a short day (8L:16D) to a long day (16L:8D) or toa skeleton photoperiod (8L:4.5D:1L:10.5D) in which an 1L pulse was given during the photosensitive phase. In both photo- periods, plasma LH concentrations changed in a similar pattern; LH increased slight- ly during the first dark period and it increased again during the second dark period. The results suggest the increase of LH under long days are accomplished by coincidence of the internal photoinducible phase and the external lighting. EN 62 ANNUAL CHANGES IN PLASMA GONADOTROPINS AND SEX STEROID IN THE WHITE IBIS IN CAPTIVITY S. Ishii, A. Hattori, H. Sakai*, S. Waka- bayashi* and M. Wada**. Dept. Biol., Waseda Univ., Tokyo 160, Dept. Biol., Sch. Dentistry, Nihon Univ., Matsudo-shi 271* and Dept. Biol., Tokyo Med. Dent. Univ. Ichikawa-shi 272** Three males and three females of the white ibis (Threskiornis melanocephalus), kept in large flying cages, were used as a model of the most rare avian species, the Japanese crested ibis (Nipponia nippon). Blood samples were collected twice a month from the wing vein. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) or estadiol-1l7 beta (E) in plasma of the samples were measured by radioimmunoassays. FSH levels were high in spring and early summer and low from late summer to winter in either sex. LH also showed a similar change in its plasma levels. T and E levels changed rather irregularly. In males, the peak of T was observed in May or June, when gonadotropins also had their peaks. However, E levels in females showed a peak in December, when FSH started to increase. We also examined FSH and LH concentrations in plasmas collected in October, 1982 from two females and amale of the Japanese crested ibis, which are only living individuals in Japan now. Their levels were almost idintical to those observed in white ibises. Endocrinology 975 EN 63 NEURAL CONNECTIONS BETWEEN THE SUBFORNICAL ORGAN, PARAVENTRICULAR NUCLEUS AND LATERAL HYPOTHALAMIC AREA IN THE JAPANESE QUAIL (Coturnix coturnix japonica). T. Tsukahara and H. Kobayashi. Dept. of Biol., Fac. of Sci., Toho Univ., Funabashi. The subfornical organ (SFO) and preop- tic area (POA) are receptive sites for the induction of drinking by angiotensin II (AII) in the quail. The paraventricular nucleus (PVN) produces antidiuretic hor- mone and the lateral hypothalamic area (LH) is a drinking center in mammals. We have investigated neural connections among these four areas in the quail brain by using the horseradish peroxidase (HRP) technique. By injections of HRP into the SFO, it was found that labeled fibers de- scended toward the LH along the stratum cellulare externum through the PVN and that labeled perikarya were present in the POA and PVN. Injections of HRP into the PVN showed labeled perikarya in the POA and LH and fibers projecting to the SFO. Injections of HRP into the LH revealed labeled perikarya in the SFO, POA and PVN. It is concluded that the SFO has an efferent projection to the LH and receives afferent fibers from the PVN, and that there exist reciprocal connections between the PVN and LH. Those observations sug- gest that the LH may receive information from the SFO, POA and PVN. EN 64 WATER BALANCE IN THE JAPANESE QUAIL AND THE BUDGERIGER, WITH SPECIAL REFERENCE TO ANGIOTENSIN II. H.Kobayashi, T.Karakida, -Kasuya, Ves Okawara and kK. Yamaguchi’. ept... “of Biol., Toho Univ., Funabashi and “Dept. of Physiol., Niigata School of Medicine, Niigata. The Japanese quail (Coturnix coturnix japonica) kept under a 12L12D photoperiod showed two peaks of water intake: one hour (07:00-08:00) after the light was turned on and one hour (18:00-19:00) before the light was turned off. Plasma angiotensin II (AII) concentration also showed two peaks one hour before each peak of water intake. The budgeriger (Melopsi- ttacus undulatus) showed a peak of water intake around 19:00 when the light was turned off. Plasma AII concentration did not increase at this time. Injection of AII induced drinking in the Japanese quail and young budgeriger, but not in adult budgeriger. Captopril (SQ14225), an inhib- itor of the converting enzyme; inhibited drinking in the Japanese quail, but not in the budgeriger. Water deprivation for eleven days induced an increase of plasma osmolarity in the Japanese quail, but not in the budgeriger. In conclusion, AII appears to have some connection with drinking habits in the Japanese quail, but not in the budgeriger. The budgeriger seems to retain body water better than the quail during periods of dehydration. EN 65 EFFECTS OF HOUSING CONDITIONS ON AGE DIF- FERENCES IN PLASMA GONADOTROPIN LEVELS IN MALE MICE. T. Machida, K. Tsutsui and S. Kawashima. 460 «dnsc., ac, Gr Sei?» Hiroshima Univy, Hiroshima. In order to elucidate the influence of social environments on age changes in re- productive functions, 3-month-old CD-1 mice were housed either singly or in groups of 1642, 362%, 56 and 36, respectively, and plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured in male animals at 4 or 13 months of age. At 4 months, plasma LH levels were highest in the 164? group and in isolated males, while the ventral prostate weight was heaviest in 164? males and least in i- solated males, High testicular activity in 164? males seems to be the result of ele- vated levels of plasma LH. However, in iso- lated males, testicular response to like- wise elevated plasma LH levels seems to be less than in 164? males. On the other hand, in the 3¢ group, plasma LH level was higher at 13 months of age than at 4 months, whereas in isolated males plasma LH level was not different between 4 and 13 months of age. Regardless of the housing condi- tions, plasma FSH levels were consistently higher in 13-month-old males than in 3- month-old ones. These results indicate that the aging process of reproductive functions in male mice is influenced by the housing conditions. EN 66 GONADAL CHANGES IN MOUSE OFFSPRING OF MOTHERS GIVEN A CONTINUOUS INTRAVENOUS INFUSION OF HUMAN CHORIONIC GONADOTRO- PIN (hCG) DURING GESTATION. N.Takasugi, T.Iguchi, J.Kurihara, A.Tei and M.Takase. Biol. Dept., Yokohama City Univ., Yokohama. Early postnatal hCG treatment induces persistent-estrus syndrome in female rats, but effects of prenatally administered hCG on gonads are unknown. In the present study, testes and ovaries of offspring (hCG-mice) of ICR/JCL mice given a contin- uous intravenous hCG infusion of 24 or 120 IU hCG/day on days 15-19 of gestation were examined at different ages. Male hCG-mice at 15 days had testes with Leydig cells Similar in number to those at 10 days, whereas the controls showed a decrease in the number at 15 days. In comparison with the controls, mitotic rate of the inter- stitial tissue was lower in male hCG-mice at 30 days; intratubular spaces were form- ed as early as at 15 days without sperma- togenesis; various disorganization of the germinal epithelium was found in some of the seminiferous tubules. In adult female hCG-mice, a slightly longer estrus, a higher frequency of estrus and larger number of total estrus days were observed; Ovaries at 160 days contained more follicles and fewer corpora lutea, sug- gesting a slight degree of persistent- estrus syndrome. 976 Endocrinology EN 6/7 VAGINAL ADENOSIS AND UTERINE METAPLASIA IN MICE EXPOSED PERINATALLY TO DIETHYLSTIL- BESTROL. M.Takase, T.Iguchi and N.Takasugi. Dept. Biol., Yokohama City Univ., Kanazawa-ku, Yokohama 236. Pregnant ICR/JCL mice were given either S.c. injections (inj) of diethylstilbestrol (DES) in daily doses of 20, 200 or 2000 ug/ day, (d)Nor wee usaonis (Ginko eZ Oo Z100 ug/d DES for 4 days starting on day 15 of gestation. Newborn female mice were inject- ed with different doses of DES, 0.01 to 50 ug, for 5 postnatal days. The present study was aimed at examining the frequency of vaginal adenosis (Ad) and uterine metaplas- ia (UM) following the exposure to DES, all mice were killed at 30 days of age. Neona- tal inj of DES (0:1-50 wd/d) induced Ad in the fornical (F) region in frequency of 36-70 %, but not UM in doses used. A high incidence (88-91 %) of Ad was observed in the F and middle (M) regions in offspring of mothers given 200-2000 ug/d DES inj, but not in those of mothers given 20 ug DES/d. UM was encountered in 38-46 2 in offspring of mothers given DES inj. Ad (11 3) and UM (22 %) were induced in offspring of mothers given DES inf even in the dose Cie AO WE/Cla shut Ox ZOO We/el DES WMmME© js1eSCj= nant mice caused a higher incidence of Ad (71 %) and UM (86 %) in their offspring. Sequential examination of vaginal epitheli- um in offspring of mothers given inj of 2000 ug/d DES revealed Ad appearance in the F and M regions at 15 days of age. EN 68 GROWTH OF NORMAL ENDOMETRIAL EPITHELIAL CELLS IN SERUM-FREE COLLAGEN GEL CULTURE. T.Iguchi*, F.-D.A.Uchima, P.L.Ostrander, S.T.Hamamoto and H.A.Bern. *Dept. Biol., Yokohama City Univ., Yokohama 236 and Dept. Zool. & Cancer Res. Lab., Univ. California, Berkeley CA 94720. Endometrial epithelial cells isolated from ca. 35-day-old BALB/cCrgl mice, 6-7 d after ovariectomy, were grown in primary collagen gel matrix (CGM) culture ina serum-free (SF) medium composed of a basal solution of Dulbecco's modified Eagle's me- dium and Ham's F-12 medium (D:H; 1:1; v/v), supplemented with insulin (I, 10 ug/ml), epidermal growth factor (10 ng/ml), cholera toxin (10 ng/ml), transferrin (10 ug/ml), and bovine serum albumin V (BSA, 5 mg/ml). Three dimensional growth with sheet-like and duct-like structures occurred in the CGM. The sheet-like outgrowths disappeared after 5 days of culture. Duct-like out- growths were often found to contain a lumen surrounded by a single layer of epithelial cells which resembled uterine epithelium in vivo, or several layers of epithelial cells with superficial keratinization. EM of these cells revealed apical tight junctions Two-fold growth of endometrial epithelial cells occurred, judged by increase in DNA, when cultured in SF medium. However, growth was not observed when these cells were grown in D:H medium supplemented only with I and BSA. (Supported by NIH grants CA05388 and CA09041) EN 69 ULTRASTRUCTURAL CHANGES IN UTERUS DURING DEVELOPMENT OF EXPERIMENTALLY-INDUCED ADENOMYOSTIS IN MICE. Y.Ohta’, T.Mori*and H. Nagasawa’. Biol: Dept., Tottor: Unive, Tottori, —Z200lemmmcee Fac. of Sci., Univ. of Tokyo, Tokyo, “Eso Animal Res. Lab., Meiji Univ., Kawasaki Ultrastructural changes of the endome- trium and myometrium during the develop- ment of adenomyosis were examined in SHN mice with ectopic pituitary grafting. Early sign of the adenomyotic changes was the invasion of stromal fibroblasts into the inner myometrium. The invasion was observed mainly along the blood vessels throughout the myometrium. Marked deve- lopment of blood vessels was also observed in the myometrium of the pituitary grafted mice as compared with controls receiving isograft of submaxillary gland. Smooth muscle cells of the inner myometrium be- came parallel to the long axis of the ute- rine horn unlike the controls in which the muscle cells were arranged concentrically. At advanced stage of adenomyosis, the inner and outer myometria were loosely arranged, consisting of degenerated cells. Associ- ated with these changes in the myometrium, the endometrial stromal cells were also proliferated and hypertrophied in the pitu- itary grafted mice. Disintegration of the myometrium and proliferation of the endometrial stroma are responsible for invasion of endometrial tissues into the musculature. EN 70 DELAYED IMPLANTATION IN THE JAPANESE LONG-— FINGERED BAT III. CHANGES IN PLASMA PRO- GESTERONE CONCENTRATION. Ke Keimuiraa As Dakeday and i Am Uichricdaiar FILA, Ose JNgaling IRGINCOCIS5 WEO4 OI wexG. Sei. & WeClac, Siaibnisian Winiwo, Weca, @ZOO1. ILald. ; Fac. of Agr., Kyushu Univ., Fukuoka. In the Japanese long-fingered bat, Miniopterus schreibersi fuliginosus, copu- lation, ovulation and fertilization occur in quick succession in autumn and the fe- males enter into hibernation in a pregnant condition. Embryogenesis proceeds very slowly because there is a delay of implan- tation and delayed development after im-— plantation during hibernation. Plasma progesterone levels were meas- ured by the RIA method. The mean value was high at the delayed implantation stage just before hibernation in early November (11.1 ng/ml), and then decreased at the early im- plantation stage in hibernation in late January (4.7), but it increased at the ear- ly placentation stage just after arousal in early April (8.0), and hightened further at the middle placentation stage in mid-May (11.8). Maintenance of gravid females at 25 C for 2 weeks in winter resulted in a Significant increase in the progesterone level (10.5). These facts suggest that the retarded embryonic development is a direct and pas- sive response to the cold and depressed me- tabolism, as indicated by Kimura & Uchida (1983) and Uchida et all. (1984). Endocrinology 977 EN 71 EFFECTS OF COMPENSATORY TESTICULAR HYPER- TROPHY AND CHANGE OF HORMONAL LEVEL IN HEMICASTRATED RATS. T.Furuya, J.Kohno and M.Hokano. Dept. of Anatomy, Tokyo Medical College, Tokyo. The hormonal and testicular effects of hemicastration were examined using rodent models. Wistar strain rats were hemicas- trated at 5,10,15,20,30 and 40 days of age, and autopsied 30 days after surgery. Tes- ticular hypertrophy in rats hemicastrated at 5,10 and 15 days of age exceeded that of control rats by 44%, 33% and 9% respective- ly. Hypertrophy decreased as the age at hemicastration approached 15 days and did not occur in rats 20 days of age or older. Serum gonadotropins were measured by RIA, using a double antibody method. A signif- icant 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. Plasma testosterone and DHT were measured by RIA. These androgens increased during the sexual maturation of the testes until 60 days of age. A higher concentration of plasma testosterone was detected in 15 and 20 day-old control rats than in hemicastra- ted rats. On the contrary, in 40 day-old hemicastrated rats, plasma testosterone was higher than in control rats. In the same Manner, plasma DHT was lower in 10 day-old hemicastrated rats than in intact rats, but in 30 and 40 day-old hemicastrated rats, plasma DHT was higher than in control rats. EN 72 INDUCTION OF MALE SEXUAL BEHAVIOR IN CAST- RATED MALE RATS: ADMINISTRATION OF TESTOS- TERONE IN SILASTIC TUBE S. Kusaka, H. Nagasawa, K. Yamanouchi* and Mie ets = Experimental Animal Reserch Laboratory, Meiji University and * Dept. of Anatomy, Juntendo University School of Medicine. Male sexual behaviors ( mount, intro- mission and ejaculation ) are dependent on the blood level of androgen. Castration Suppresses sexual activity and the _ sup- plement of androgen recovers it. However, long-term administration of androgen is requiered for complete recovery of the activity. In this study, effect of administra- tion of testosteron (T) in different lengths of silastic tubes ( 3 cm x 2 or 5 cm x 2, # 602-285, Dow-Corning ) implanted sc on the male sexual behavior was ex- amined in castrated or non-castrated male Wister rats. Behavioral tests were carried out 5, 10, 15 and 20 days after T implan- tation. None of males with tube alone showed male sexual behaviors. Males with Sx2 T tube showed mount and intromission behaviors more frequently than males with 3x2 T tube. Ejaculation frequency in 5x2 T tube group also was higher than that in 3x2 T tube group on day 20. Sexual activi- ty of normal males was similar to that of males with 3X2 T tube EN 73 EFFECTS OF DIFFERENTLY-SIZED POSTWEANING GROUPING AND PREPUTIALECTOMY ON SERUM TESTOSTERONE LEVELS IN MALE MICE. S. Koike and T. Noumura. Dept. of Reg. BLOw.s, fac. OL SCi., Saltama Univ., Urawa. The influence of crowding in different group sizes on serum testosterone (T) lev- els was compared between the intact and the preputialectomized male mice of the ICR strain during maturation from 21 to 200 days of age. At weaning on day 21 when the removal of preputial gland was per- formed, intact and preputialectomized male mice were randomly housed in group of 1,2, 5,10 per cage, respectively. Serum levels of T were determined by RIA. The levels of circulating T reduced and the onset of puberty delayed with increas- ing group size. There was a striking indi- vidual variation with T values ranging from 0.33 to 20.5 ng/ml in mice of the same age, due to social ranks and housing conditions. Crowding had no uniform influ- ence on animals in a cage. Suppression was observed topically ina part of popula- tion, and was intensified with an increase in group size. However, isolation (indi- vidual housing) appeared qualitatively different from group housing. Circulating T levels in preputialectomized males were lower than those in intact males. This result suggests that the removal of prepu- tial gland, a pheromone secretory organ, May induce a decrease in aggressive beha- vior in male mice. EN 74 AUTORADIOGRAPHIC STUDY ON THE INTERNAL- IZATION OF FSH-RECEPTOR COMPLEX IN THE MOUSE TESTIS in vitro, A. Shimizu, K, Tsutsui and S, Kawashima. ZO0L). inst, ; Fac. of Scei., Hiroshima’ Univ: Hiroshima, With the aim to obtain morphological ev- idence for the internalization of FSH-re- ceptor complexes, light- and electron-mi- croscope autoradiography was carried out in the present study in testicular pisces in- cubated with radioiodinated FSH ( I-FSH), In light microscope autoradiograms, silver grains were preferentially present on the seminiferous tubules.About 60% of the total number of grains was present on Sertdéli cells. These grains correspond to the spe- cific FSH binding sites, because very few grains were left when the tissue were incu- bated with an excess amount of cold FSH. In electron microscope autoradiograms, about 50% of the grains were observed on the plasma membrane and basal lamina after 10 min of incubation. After longer incubations (20 and 60min) the number of grains in- creased in Golgi apparatus, lysosomes and endoplasmi¢- SEeecul ian. These results sug- gest that 3lt-FSH binds first to the plas- ma membrane of Sertoli cells, then incorpo- rated into the cytoplasm by the process of internalization. The increase in grains on the lysosomes after longer incubation may indicate that the internalized FSH-receptor complex or FSH dissociated from the complex may be subjected to degradation, 978 Endocrinology EN 75 AUTOREGULATION OF TESTICULAR FOLLICLE STIMULATING HORMONE RECEPTORS IN THE MOUSE. K.Tsutsui, A.Shimizu and S.Kawashima, Zool. Inst.) face Of Sen.) Hiroshima Wniwva Hiroshima. In the mouse testis a decrease in the number of follicle stimulating hormone (FSH) receptors was noted when plasma FSH concen- tration reached a maximum level immediately prior to puberty. The adult levels of FSH receptors were lower than the level of FSH receptors around puberty. Hypophysectomy at adulthood induced a significant decrease in the testicular weight, but the binding of I-FSH per unit testicular weight was increased markedly 16 and 40 days after op- eration. The total binding of FSH per two testes in mice 16 days after operation was 2.2 times that of the intact controls. The Scatchard plot analyses of the binding showed that an increase in binding was due to an increase in the number of the binding sites. Injections of NIH-FSH-P-2 to hypoph- ysectomized mice (100 pg/day) for 10 days increased the testicular weight, but sig- nificantly decreased 31]-FSH bindings both per unit weight and per two testes as com- pared with those in non-injected hypophy- sectomized mice. These results suggest that a high level of circulating FSH reduces its own receptors from around the puberty and through this downregulation the adult level of FSH receptors is maintained in the mouse. EN 76 CHARACTERIZATION OF OVARIAN LUTEINIZING HORMONE (LH) RECEPTORS OF CYNOMOLGUS MON- KEYS DURING MENSTRUAL CYCLE. IW YOSINGEL, Ko WwOkocey W,Cla@ excl S.klomyO- Tsukuba Primate Center for Medical Science N.I.H. and *The Corporation for Production of Laboratory Primates, Ibaragi. Specific LH-receptor have been ideti- fied in the ovary in cynomolgus monkeys (Macaca fascicularis) during menstrual cycle. Duplicate aliquots of 100 pl (con- taining the equivalent of 7 mg of ovarian tissue) were incubated for 3 hr at 37°C with approximately 125 fmol of labeled LH (LER-907). Separation of bound and free hormone was performed by centrifugation. Then the radioactivites bound to pellets was determined. Nonspecific binding was determined by including 20 pg of the cynomolgus pituitary extract in the incu- bation mixture. The Scatchard plot was used for analyzing the data. The number of binding sites was con- stant regardless of the phase of the men- strual cycle. The dissociation constant (Kd) was low both in the follicular phase and in the periovulatory period, increas- ing markedly in the luteal phase. However, this increase of the Kd was observed only in the ovary of one side with a large corpus luteum. Our results demonstrate that the ovarian LH-receptor changes its characteristics with the change in the phase of the menstrual cycle. EN 77 TRANSPLANTATION OF NEWBORN NEURAL TISSUE INTO AGED RAT BRAIN : EFFECT ON OVARIAN FUNCTION. S.Kobayashi’, A.Matsumoto?, S.Murakami’, Y.Arai 7; and M.Osanai-. 1)! Depteseaone Tokyo Metropol. Inst. Gerontol., 2) Dept. Anat., Juntendo Univ., Sch. Med., Tokyo. Recently, many reports have shown that the impaired brain functions could be recovered by neural tissue transplanted in the brain. In this study, we trans- planted neural grafts of the medial basal hypothalamus (MBH) or cerebral cortex from newborn rats into aged female rats whose gonadal functions decline and ova- ries show atrophy. These grafts of the MBH or the parietal cortex were stereo- taxically implanted into the 3rd vent- ricle of the aged Wistar females (21-30 months of age). Three weeks after trans- plantation, the brain and ovaries of each host were examined histologically. The ovaries of 6 out of 8 MBH-transplanted rats showed varying sizes of follicles and healthy-appearing corpora lutea. The mean ovarian weight of MBH-transplanted females was significantly greater than that of intact and cortex-transplanted females (p<0.05). These results suggest that the MBH grafts from newborn rats may recovered the declined ovarian function in aged females. In addition, the ultra- structural observations suggest that the graft may have a functional connection with host brain. EN 78 ESTROGEN STIMULATES THE GROWTH OF THE NEO- NATAL HYPOTHALAMIC TISSUE TRANSPLANTED INTO THE ADULT BRAIN. S. Murakami, A. Matsumoto, and Y. Arai., Dept. Anat. Juntendo Univis Sch{Med=y tokyor The preoptic (POA) or mediobasal hypo- thalamic (MBH) tissue taken from newborn female rats was transplanted into the TaAdieGl wWeImMipieaele of the ovariectomized adult rats. From the day of grafting, estradiol-178 in silastic capsules were placed subcutaneously to 21 out of 30 host animals for 1 or 4 weeks. Four weeks after brain transplantation, all animals were perfused with 10% formalin solution. The brains were removed, and the serial sec-— tions at 10 pm were stained with cresyl- violet. The outline of the graft was traced out in every fourth section of each brain. Then, the volume of the graft was computed. The volume of the POA grafts in host animals treated with estrogen for 4 weeks (0.07+0.02 mm3 ) was significantly larger than that of the POA grafts in non- estrogenized host animals (0.02+0.01 mms , Pre On©5))ie However, estrogen exposure for 1 week did not increase the volume of the POA grafts. The volume of the MBH grafts was not significantly different among all groups of the animals examined. These data suggest that the treatment with es-— trogen through the host animal influences neuronal development in tne POA graft, increasing its volume. Endocrinology 979 EN 79 EFFECTS OF MET-ENKEPHALIN AND CHOLECYSTO- KININ (CCK4) ON SERUM LH AND PRL CONCEN- TRATIONS. 2 2 H. Uemura,, M. Wada’, A. Hattori’, and H. Kobayashi . 1piol., Lab., Kanagawa Dent. Coll., Yokosuka, Dept... of Gen. Educ., Tokyo Med. Dent. Univ., Ichikawa, 3Dept. Biatapokac. Ot Sci:, Toho Univ., Funaba- shi. Pellets of CCK4, Met-enkephalin (MEN) or naloxone (opiate antagonist) mixed with cholesterol were implanted into various anatomical sites in the rat hypothalamus; serum LH and PRL concentrations were meas- ured by radioimmunoassay about one week after the implantation. Intact animals and those implanted with only cholesterol pellets served as controls. CCK4 reduced serum LH concentration when it was im- planted into the median eminence (ME), but it elevated serum LH level when implanted into the nucleus ventromedialis (HVM). CCK4 implanted into the HVM increased serum PRL, but CCK4 implanted into other Sites did not alter serum levels of LH or PRL. MEN implanted into the nucleus dor- Salis (HD) increased serum PRL level. Naloxone implanted into the HD decreased serum PRL. MEN seems to stimulate PRL release through action on the HD. Implan- tation of naloxone into the ME decreased serum PRL, but MEN did not, suggesting that opioids other than MEN stimulate PRL release at the level of the ME. EN 80 STIMULATION OF NEURONAL PROCESS GROWTH BY ESTRADIOL-178 IN FETAL RAT HYPOTHALAMUS - PREOPTIC AREA IN CELL CULTURE. M.Uchibori and S.Kawashima. Zool. Inst. Fac. of Sci., Hiroshima Univ,, Hiroshima, The aim of our study is to clarify the morphogenetic effects of sex steroids in cultures on neurons from perinatal rat hypothalamus-preoptic area(POA). Dissoci- ated cells of the hypothalamus-POA and the cerebral cortex from fetal rats were grown in primary culture, the effects of added estradiol-178 (E72) on the growth of neuro- nal processes were studied. In cultures of the hypothalamus-POA of 20-day-old fetuses E2 significantly increased the total neu- ronal process length after 2 or 4 days of culture as compared with control cultures. The stimulatory effects of Ej were not different between male and female fetal preparations. While, in the hypothalamus- POA preparations from 16-day-old fetuses E7 failed to show any stimulatory influ- ence. None of the cerebral cortical cul- tures were affected by Ez. The present findings in the fetal hypothalamus-POA are consistent with the report by other inves- tigators on the ontogeny of estrogen re- ceptor in the hypothalamus-POA that the concentration of the receptor was very low on 16 days of gestation, then it in- creased drastically on 20 days. The present findings indicate that the neuronal compe- tence in response to E72 during fetal life is related with the ontogeny of receptor. EN 81 AGE-RELATED CHANGES IN THE MITOTIC POTENCY OF PROLACTIN CELLS IN THE RAT. S.Takahashi and S.Kawashima, Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. Prolactin (PRL) cells undergoing mitosis were immunohistochemically identi- fied in colchicine-pretreated rats. In adult female rats the mitotic index of PRL cells was higher at estrus than at other stages of the estrous cycle and than that in male rats of comparable ages, If adult female rats were ovariectomized on the second day of diestrus, the mitotic indices at estrus were less than those in sham-operated controls at estrus. As estrogen increased the mitotic indices of PRL cells, we studied the mitosis of PRL cells in response to estrogenic stimula- tion in non-gonadectomized rats at 30 and 60 days of ages. After estrogen treatment, the number of mitotic PRL cells were more in females than in males at either age. In ovariectomized females at 60 days the responsiveness of the pituitary to estrogen was lower than in intact females. At 30 days of age ovariectomy failed to decrease the responsiveness of pituitaries. These findings clearly demonstrate that PRL cells can undergo mitosis and that a sex difference in the mitotic potency of PRL cells is present. The higher respon- siveness of adult female pituitaries may be due to the additive effect of exogenous estrogen with their ovarian estrogen. EN 82 THE LOCALIZATION OF TRH IN THE ANTERIOR PITUITARY OF THE NEWBORN RAT. T. Shinkai and H. Ooka. Dept. of Biol., Tokyo Metropol. Inst. of Gerontol., Tokyo. The immunoactivity of thyrotropin-releas- ing hormone (TRH)was detected in the anterior pituitary of the newborn rat with the pre- embedding peroxidase-antiperoxidase (PAP) complex method. The PAP reactivity was observed in secretory granules of TSH-, LH-, FSH- and PRL cells. Previous works have shown that hypothal- amic TRH concentration is low at birth and increases to the maximal level within 2-3 weeks. Our fine-structural studies showed that the secretory granules of TSH-cells were weakly stained with PAP during the period between the day 1 and 4, and the PAP reac~ tivity varied inintensity among granules in the day 6. The granules were fairly stained between the day 8 and 12. The intensity of staining in the rat older than 16 days is similar to the adult level. The result suggests that morphological TRH immunoactivity in TSH cells of the newborn rat reflects the content of TRH in the hypo- thalamus of the rat. 980 Endocrinology EN 83 EFFECTS OF NEONATAL Ty- AND MONOAMINE TREATMENT ON THE CONCENTRATION OF Ty, IN THE BLOOD OF THE RAT. H.Ooka and T.Shinkai. Dept. Biol., Tokyo Metropol. Inst. Geront., Tokyo. The concentrations of thyroid hormones in the blood of the rat are very low at birth, and they increase with the development of brain-pituitary-thyroid system. The administration of serotonin or N-acetylserotonin (NAS) for the first 10 days of life suppresses the increase in the concentration of T,. The level of T, in the neonatally NAS-treated rats is normal when they become adult, in contrast with the permanent hypothyroidism in neonatally T,y-treated rats. TSH secreting cells in anterior pituitary proliferate intensively in the first 10 days of life, and this proliferation of TSH cells is completely suppressed by exogenous T,. No inhibition of TSH cell proliferation is observed under neonatal NAS treatment. It is possible that hypothyroidism induced by neonatal T, treatment is due to the delay in the period of multiplication of TSH cells to later stages of postnatal development. EN 84 EFFECTS OF SUCKLING ON THE FINE STRUCTURE OF THE PITUITARY PARS INTERMEDIA IN INFANT MICE. A MORPHOMETRIC STUDY. Yo Kobayashay Depts bilo ia Cops SiCaler Okayama Univ., Okayama. Our previous studies suggested that the pituitary pars intermedia hormone might be involved in the regulation of aldosterone secretion in sodium depleted mice(1974, 19 76). Recently, in vitro studies have con- firmed our results indicating the stimula- tion of aldosterone production at physio- logical doses of a-MSH(Vinson et al., 1981) and of §8-MSH(Yamakado et al., 1983) in isolated rat adrenal cells. Therefore, the present study was designed to search for a physiological condition in which the pars intermedia hormone would be released in normal mice. Male and female ICR/JCl mice, at 7 days of age, were allowed to be nursed for 1 hr after being isolated from their mother for 5 hrs(control). The pituitaries of infants were processed for the electron microscopy. The endoplasmic reticulum increased in % volume of the cytoplasm from 19.5+1.5% to 42.0+0.5%, and the number of Golgi granules increased from 1.2+0.1/yu? to 2.1 +0.1/u? While the secretory granules de- creased in number from 2.9+0.1/y’?to 1.6+ 0.1/u% These data indicated a new evidence that the secretion of pars intermedia hor- mones was activated by suckling in infant mice. EN 85 IMMUNOHISTOCHEMICAL AND ELECTRON MICRO- SCOPIC STUDIES ON THE DEVELOPMENT OF THE MOUSE POSTERIOR LOBE. K.Kawamoto, Y.Yamada and S.Kawashima, Zool. Inst, , Hac. of scr... HinoshimamUneave Hiroshima, The interactions between pituicytes and neurosecretory axons of the posterior lobe during embryonic development in the mouse were studied by means of immunohistochemis- try and electron microscopy (EM). Vaso- pressin-immunoreactive material (VP) was first detected in the supraoptic nucleus and the external layer of the median emi- nence in 15th day embryos, In the posterior lobe (PL) anlage and the paraventricular nucleus VP was not detected until 16th day, The initial localization of VP in the PL was in the peripheral area, but on 18-19th day VP was distributed over the entire PL. While, oxytocin-immunoreactive material was detectable in the hypothalamo-neurohypo- physeal system after 5th postnatal day. In EM observations, the PL anlage in 13- 14th day embryos was composed mainly of un- differentiated pituicytes having poor cyto- plasm, and the axons were very few. On 15th day, the number of axons penetrated to the PL anlage was increased mainly in the peripheral area, Concomitantly, the pitui- cytes showed morphological signs character- istic of increased activity. These results suggest that the pituicytes exert a stimu- latory influence on the growth of neuro- secretory axons. EN 86 SYNAPTIC PATTERN IN THE BED NUCLEUS OF THE STRIA TERMINALIS OF MALE AND FEMALE RATS. M.Miyakawa and Y. Arai. Depicts yAnaier Juntendo Univ. Sch. Med., Tokyo. The presence of sexually dimorphic Synaptic pattern has been demonstrated in certain brain areas where sex steroid receptors are rich during neonatal period. The bed nucleus of the stria terminalis (BST) receiving afferents from the amygdala has also been shown to contain sex steroid receptors in newborn rat brains. Since the BST is considered to be involved in regulation of gonadotropin secretion, a semi-quantitative study was made to ultrastructurally examine whether sexual dimorphism in synaptic pattern was present. Axodendritic synapses made on dendritic shafts (shaft synapses, SHS) and spines (spine synapses, SPS) were counted diftterentialiy, sper) S,O0OeSsG i. sum i otmauine neuropil in the medial and lateral parts Om was esi) ain imele eincl wemele ies. Synaptic pattern was found to be different between the medial and lateral parts, the SPS/SHS ratio being significantly larger in the medial part than in the lateral part. However, the numbers of SHS and SPs and SPS/SHS ratio were not different between male and female rats in both parts of the BST. Thus, sexual dimorphism in Synaptic organization is not necessarily present in all the areas where receptors for sex steroid are abundant. Endocrinology, Morphology 98 | EN 87 THE ROLE OF SEPTUM IN LORDOSIS INHIBITING MECHANISMS IN THE FEMALE RAT K. Yamanouchi and Y. Arai Dept Anatomy, Juntendo Univ. Sch. Medicine inorder Go elucidate the role of the septal area in the lordosis inhibiting mechanisms, efferent and afferent fibers of the septum were cut in four different portions in ovariectomized rats. Ventro- medial or bilateral ventrolateral cuts were made by 1 mm L-shaped Halasz knife (VMC or VLC) at the ventral border of the septum. Dorsal (DC) or posterior fiber connection (PC) was also transected by means of 2.5 mm L-shaped knife or curved knife (1.4 X 2 mm). All females were treated with estradiol benzoate (EB) daily for 3 days and O.5 mg progesterone on the fourth day. Three behavioral tests were carried out at 1 or 2-week interval with MMGnmeasimp Gose of EB from Q.2, 0.5 to 2 pg. Females with VLC showed markedly high scores of lordosis quotient, compared to other group in the tests primed with 0.2 and O.5 pg EB. Soliciting behaviors such as ear wiggling and hopping were observed more frequently in the VLC group than in other groups. These results suggest that the origin of the forebrain lordosis inhibiting signals is in the septum, presumably descending from or through the ventro- lateral part of the septum. EN 88 EFFECT OF DEAFFERENTATION OF DORSAL PRE- OPTIC AREA(POA) ON LORDOSIS REFLEX IN NEONATALLY ANDROGENIZED FEMALE RATS. Y. Kondo, A. Shinoda, K. Yamanouchi*, and Wee Aad = & Dept. GL YEsychol:,; Fac. ovon Letters, Gakushuin Univ,. Tokyo aud’ “Depu. Of Anat., Juntendo Univ. Sch. of Med., Tokyo. It is well known that neonatal androgen treatment suppresses female sexual behav- iors in the female rat. Previous studies reported that there is an inhibitory sys- tem of lordosis behavior in the forebrain. In this study, effect of the dorsal POA deafferentation on lordosis behavior was examined in female rats injected with various doses of testosterone propionate (TP) on day 4. After maturation, the animals with or without dorsal POA deaffe- rentation were castrated and implanted Silastic tubes containing estradiol-178. Behavioral tests were carried out 5, 10, and 15 days after implantation of estrogen with progesterone pretreatment prior to each test. Neonatal TP treatment effectively sup- pressed the display of lordosis. The dor- sal POA deafferentation reversed it. The degree of recovery of lordotic activity following the dorsal POA deafferentation is negatively correlated with the dose of TP given neonatally. It suggests that neonatal androgen also influences’ the inhibitory and/or facilitatory systems of female sexual behaviors in the brain. EN 89 SEX DIFFERENCE AND LEFT-RIGHT DIFFERENCE IN THE HYPOGASTRIC GANGLION OF MALE, FEMALE AND ANDROGEN-EXPOSED FEMALE NEWBORN MICE. YWevUZukKL ends y. Aral. Lab. Biol... Asia Univ., Tokyo and Dept. Anat. Juntendo Univ. Sch. Med., Tokyo. In the previous reports, we have shown a sex difference in number of neurons of the hypogastric ganglion which innervates the internal genitalia and a significant increase of neuron numbers in the ganglion following prenatal exposure of sex horm- ones. In the present study, we calculated the volume of the ganglion of male, female and prenatally testosterone propionate (TP)- exposed female newborn mice. 2 mg TP was injected to pregnant mothers from days 14 to 16 of gestation. The volume of the ganglia at both sides in males was Significantly larger than those in females. The volume of the ganglia in TP- exposed females was significantly larger than those in intact females, but did not reach the male ievel. In males, the ganglion at the left side was Significantly larger than that at the mpi o Sue. On chen otien hand, ithe) lert— right difference in the volume of the ganglion was not recognized in the intact or TP-exposed females. MO 1 WOUND CLOSURE IN PLANARIAN EPIDERMIS. 1. SCANNING ELECTRON MICROSCOPIC EXAMINATION OF THE NORMAL EPIDERMIS. S. Ishii. Cent. Res. Lab., Fukushima Med. Col., Fukushima. A comparative SEM examination was made for the normal skin of 5 species of pla- narians, which consisted of epidermis, a basement membrane and subepidermal muscu- latures. The epidermal cells, polygonal and 10 pp in average diameter, had 2 kinds of surface specializations, the cilia and the microvilli. In the ventral epidermis ciliation was consistently heavy through all the species examined, while in the ”’ dorsal side it showed a high degree of variation in density, depending upon the difference of species; sparse in P. teshi- rogi, moderate in B. brunnea, and dense in P. vivida, P. auriculata and D. japonica. The microvilli seemed to vary in size and density according to the age of individual epidermal cells: they were the most numer- ous and the longest in a small juvenile type of cells, but with advance in age they became less remarkable, often involv- ing their ultimate disappearance in the oldest cells. Thus the mode of cell renew- al of planarian epidermis was now eluci- dated. Observations were further extended to a special type of epidermal cells at the marginal adhesive glands, which was of insunk type and perforated by glandular secretions of parenchymal origin. 982 Morphology MO 2 CULTURES AND COMPARATIVE MORPHOLOGY ON THE SYMBIOTIC DINOFLAGELLATES FROM THREE ACOEL FLAT WORMS, AMPHISCOLOPS SPP.. T.Yamasu. Div. of Gen.Educ., Univ. of Ryu- MAbs Ele MIG NC, Wishes Cue toil, Coll, oO Sci., Univ. of Ryukyus. Okinawa. Three Amphidinium-type and a Prorocentr- um-type dinoflagellates were cultured from three acoel flat worms, Amphiscolops spp. and were studied morphologically. The algae from the brown species collected from Amami in 1971 at first and later also from Okina- wa had single pyrenoid into which lamellae of the chloroplast penetrated in a radial arrangement as Similar as in A.klebsii. The other algae from the small green species collected from Chatan(Okinawa)had four py- renoids as same as in A.chattoni. The third algae from the large yellow-green species collected from Zammpa(Okinawa) in 1984 were smaller than the second ranging from 18 to 27 om in length. They had a short epicone, two pyrenoids and large spherical bodies more than two. Fundamentally, details of various organelles were fairly similar am- ong species. Another symbiotic dinoflagel- late from the small green species, reported by the author in 1980 as a member of Exuvi- aella(=Prorocentrum), ranged from 44 to 59 um in length and from 38 to 54 am in width. Surfaces of both the right and the left thecae were provided with numerous tiny concavities and trichocyst pores. Anterior part of the right theca was concaved much than the left theca.This species seems to beyellosely sre lateds tome. concayumy. MO 3 ELECTRON MICROSCOPIC STUDY ON THE ZOO- CHLORELLAE IN GREEN FRESHWATER SPONGES. Y.Masuda. Dept.of Biol., Kawasaki Med. SEhool si Kunvasiiaskaes {ONE MICAS UNAS Bucs seOUhnGl ais wie reeSel Ilu= lar symbionts in some freshwater sponge Species. lJexamined the annual changeof the Zoochllonrelilae im) sponcaiilay lacus secon. lected from a pond near the lake Biwa in Shigasprerectunc. | Ine themsamnplicsn collect. ed in February and April, a few zoochlorel- lae having no starch grains were observed in both archaeocytes of the sponges and Ehnesocytes of the; gennules.. Une June-s many, zoochlorellae having starch grains were observed. However, only a few zoochlorel- TIEMWEGE Sees anche cg pS mand larvae wor the sponges. In November, many zoochlorel- lae in the archaeocyte which were at the beginning of the gemmulation contained the starch grains. Two different types of zoochlorellae (large and small) were observed in both archaeocytes in Radiospongilla cerebellata and R. sendai collected from a pond in Okayama prefecture. R. cerebellata con- tained mainly the large zoochlorellae (Ca. 4um in diameter), but the small zoochlorel- lae (Ca. 2um in diameter) were mainly ob- served in R. sendai. Small zoochlorellae were as large as those of S. lacustris. The large zoochlorellae had pyrenoids in their body, whereas no pyrenoid was en- countered in the small zoochlorella. MO 4 MECHANISM OF CHANGES IN THE NEMATOCYST COMPOSITION DURING FORMATION OF SWEEPER TENTACLES IN THE CORAL GALAXEA FASCICULARIS. I. Miyazaki, M. Hidaka and K. Yamazato. Dey 5 Ose soils | Wins Ose telne KOvWSwWS - Okinawa. The coral Galaxea fasetcularis develops highly extensible tentacles with nematocysts different from those of ordinary tentacles when it was kept in contact with other corals. To understand the mechanism of changes in the nematocyst composition during transformation from an ordinary into a sweeper tentacle, histological sections of ordinary, intermediate, and sweeper tentacles were examined. Microbasic b-mastigophores (MbM) characteristic to sweeper tentacles first appeared at the peripheral region of the acrosphere and then gradually replaced the cnidae of the ordinary tentacle, microbasic p-mastigophores (MpM) and spirocysts. In some intermediate tentacles, MpM and spirocysts were observed in the gastrodermis. This may indicate that these cnidae were removed from the epidermis and disintegrated in the gastrodermis during the transformation process, though extrusion of these cnidae from the epidermis may also occur. MbM in a fully developed sweeper tentacle is much thicker and has a less conspicuous shaft in an undischarged capsule than the MbM found in ordinary tentacles. MO 5 HISTOLOGICAL STUDY ON THE INTESTINAL TISSUE IN THE EARLY REGENERATION OF THE LAND PLANARIAN, BIPALIUM NOBILE Y. Shirasawa and N. Makino. Dept. of Biol., Tokyo Med. Coll., Tokyo. Histological transformation and cyto- logical changes in the intestinal tissue and the surrounding parenchyma, attended with early regeneration, were examined in the prepharyngeal region of Bipalium nobile. Epithelial cells facing intestinal lumen of intact worms were well-stained by AF (Gomori's aldehyde fuchsin), while basement cells were not stained. In re- generating worms, the diminution of AF- stainability of epithelial cells and the increase of the stainability of the intestinal lumen were observed. The increase of AF-stainability was also recognized in ventral parenchyma contain- ing nerve cells. Nerve fiber in the ventral parenchyma was well-stained by copper sulfatesilver nitrate, both in intact worms and regenerating ones. Electron micrograph shows that RER of granular cells are more active in intact worms than regenerating ones, and that granules of the cells decrease in the latter. Morphology 983 MO 6 THE POLARITY ABOUT THE ARBYORMAL TYPE OF THE ROGENERATION IN BIPALIUM NOBILE, N,.Makino and Y,Shirasawa, Dept. of Fiol., Tokyo Med. Coll,, Tokyo, Authors have studied the rerfeneration in the lard planarian, Bipalium nobile of giant land planarian is a species’ that carries out the fragmentation, and Bipa- lium nobile regenerate normally from arti- ficial cut piece i~ every level of the body, But sometimes, a cut piece o* them was linked together and made a ring, then the pola~ity in the piece was lost, The weight of a ring niece decreased little by little, but the case in the regenerating piece be-ame lean rapidly, Perhans this shows that the regeneration from a piece will exhaust the energy in tiss‘i1cs for the morphological formation, Similarly, ina regenerate study of senior author, Makino's experiment in thirty years ago, in Polycla a, Stylocus ijimai was observed that the animal regenerated considerably, but nost-= erior transverse cut piece made a cone by immediate agglutination of the wound, and the polarity was lost without normal re- generation, Anyhow, it never lives to ring formation, soon it will die, althourh it has the ability to the normal regeneration by its physiological sradient, MO 7 ULTRASTRUCTURAL STUDY OF GONADAL DEVELOPMENT IN THE FRESH-WATER LEECH, ERPOBDELLA LINEATA M. Shirasawa and N. Makino. Dept. of Biol., Tokyo Med. Coll., Tokyo. The development of larval gonads and the differentiation of reproductive cells were observed light and electron micro- scopically in Erpobdella lineata. Testis sacs develop almost in line on both sides of the posterior nerve cord. Sac wall cells and male cells differentiate from a specific cell with a large clear nucleus. Primordial male cells in the sac have small dark nuclei, which become large and clear later. The centrioles appear in the Golgi zone at this stage. A part of the sac wall cells remain undifferentiated form and construct the thick portion of the wall where new male cells are produced. A pair of ovisacs develop near the end of the oesophagus. At first, female cells in the sac have spindle nuclei, then gradually, their nuclei become large and globular. At this stage, dense inclusions appear in mitochodria. Among the female cells, specific cells with small dark nuclei were observed. They differentiate into nurse cells and follicle cells. The origins of female cells, the specific cells, and also ovisac wall cells are thought to be the same undifferentiated cell. MO 8 FINE STRUCTURE OF BOTRYOIDAL TISSUE IN THE LEECHES, HIRUDO MEDICINALIS (2) H.INAMURA.DEPT,.BIOL. , TOKYO MED.COLL. , TOKYO The prosperity and decay of granules of botryoidal tissue(cytoplasm) were examined. It was observed that according to a light microscope,this tissue showed a granular structure and according to an electron mic- roscope,granules in mature leeches (200-1000 mg) consisted of three different types; type 1(L1i-granules) wes uniform,not clear a limiting membrane and was about 1.0-2.0 pm in diameter,type 2(L2-granules) consisted of many particles,a limiting membrane and was about 1.0-2.0 pm in diameter and type 3 (P-granules) consisted of dense bodies,a limiting membrane,and was about 0.5-1.0 pm in diameter.L2-granules had a positive rea- ction for acid phosphatase.In larvae(35-50 mg) ,many of the granules were L2-and P-gra- nules,and with Li-granules few in number. It was observed that according to a fluore- scent microscope with B-excitation and a 530 absorbing fiiter,granules in mature leeches starved during 6 months were disti- nguished yellow fluorescence from fluoresc- ent loss.After 5 days of eating,all of the granules exhibited yellow fluorescence. Granules of larvae were similar to those of mature leeches. It is suggested that metabolism has an effect on fluorescent granules which have a relation to L2- and P-granules and there is the relation between nucleus and ER because of after 5 days of eating,nucleus was surroumded by developed ER. MO 9 BKIOLUMINESCENCE OF CYPRIDINA IL, INTERPRETATION OF LUMINOUS SUESTRATE SITE IN LABRUM GLAND. T.Saito, M.-Fukuda* and S.Taguchi** Eiol. Inst.,School of Med.,Kyorin Univ. *Lab.for Electron Microscopy Kyorin Univ. **Biol. Inst.,School of Med.,Keio Univ. We have reported the morphology of lumi- nous organ of Cypridina hilgendorfiiji in the 52nd and 53rd meeting of this society - The labrum gland has three types of cells; the cell with small tut electron dense granules, with large and homogene- Ously electron light granules, and with large granules including small electron dense granules, respectively. Which type of the cell is responsible for luminous substrate or enzyme, is our present subject. The material Cypridina hilgen- dorfii was fixed and imbedded in epoxy resins in the same way as for electron microscopy. The samples were prepared from the region of labrum gland by cut- ting in 2um thickness. By exciting with U.V.(max.420nm) using fluorescence micro- scope, luminescence was photographed. The luminous area was corresponded to the cell, which has small dense granules. We concluded that this type of cells, which have small dense granules, contain lumi- nous substrate, luciferin. Other types of cells are supposed to contain luminous enzyme. To obtain a direct evidence, some experimente are now undertaken. 984 Morphology MO 10 OBSERVATIONS ON THE KIDNEY OF THE LAND- LOCKED SAND LAMPREY, LAMPETRA REISSNERI (DIBCWSKI1). Mizuho Ogawa, Dept.Biol.,Saitama Univ.,Urawa, Saitama The kidney structures of the landlocked sand lamprey, Lampetra reissneri (Dibowski) at two life stages, late ammocoetes and adult were examined. The paired kidneys of the adult lamprey locate on dorsal wall of the body cavity, while the tissue is more ventrally positioned than that of the common river lamprey,L. japonicus. The adult kidney of the sand lamprey has a single elongate complex renal corpuscle which extends almost its entire length. The nephrons of this fish are divided into four main segments: neck, proximal, distal and collecting tubules. This classification was done on the basis of cell structure, height and stain differentiation. By the late ammocoetes, only a few of the anterior renal tubules opening into the coelom by a ciliated funnel remain. The other renal tubules, however, become Surrounded by phagocytic cells in adipose tissue as part of the processes of degeneration. The newly developed adult kidney is seen at the ventral tip of the adipose tissue stated above. Therefore, the kidney of the ammocoetes does not contribute to the formation of the adult opisthonephric kidney. MO 11 THE NEURAL GLAND AND DORSAL STRAND IN RELA- TION TO THE NERVE GANGLION OF THE ASCIDIAN, HALOCYNTHIA RORETZI. ) Z.Hoshino, T.Numakunai and S.sSuzuki. Dept. of Biol., Fac. of Educ., Iwate Univ., Nero, i) Were, WHOL, Sweso, WOMoOku Wadyao o Aomori. In order to understand the function of the neural complex of the ascidian, H. roretzi, its cellular composition was observed with the light and electron microscope. The neu- ral complex of ascidian consists of the nerve ganglion, neural gland and dorsal strand. The nerve ganglion, from which the unpaired visceral nerve arises posteriorly, lies in the mantle wall between the bran- Chial and atrial siphons. The neural gland, the upper portion of which is adja- cent to the nerve ganglion, extends close above the dorsal wall of the pharynx as a rather thick strand of tissue. The duct of the neural gland extends posteriorly as a fine strand, dorsal strand, which leaves suddenly the viscera and turns toward the body wall. The dorsal strand terminates ab- ruptly after running down in the body wall for some distance. In addition to the vis- ceral nerve, some thin bundles of fibers containing nerve cells arise in places from the nerve ganglion, and are distributed around the neural gland. Frequently, the epithelium of the neural gland forms thin tubules in the visceral nerve and the thin bundles of fibers. The function of the tu- bules is obscure. Further histological and embryological investigation is necessary. MO 12 A COMPARATIVE MORPHOLOGICAL STUDY OF GALL- BLADDER(IV) FINE STRUCTURE OF POLYPTERUS SP. GALLBLADDER. K.Higashi. Dept. of Histol., Kanagawa Dent-— al Coll., Yokosuka. The fine structure of gallbladder epithe- lium of polypterus sp. which has lungs were compared with that of lung fish(protopterus sp.). The gallbladder epithelium were com- posed of simple columnal cells which were subdivided into five zones. I.Cuticular zone: numerous microvilli which were longer than those of lung fish were projected into the lumen. Sometimes, apocrine processus were present in this zone, and small ellip- tical, electron-dense granules were present in the apical cytoplasm. II.Dark zone: ecl- usters of mitochondria were present in the center of this zone. PAS-positive granules which were seen in this zone of lung fish gallbladder cells were not presented. III. Supranuclear zone: well-developed Golgi ap- paratus and large lysosomes were seen. IV. Nuclear zone: V.Basal zone: the basal mar- gins were irrgular in outline. The large numbers of empty pinocytotic vesicles which were shown in the peripheral cytoplasm were observed to open to the basal surfaces. So- metimes, a part of cytoplasm of basal port- ion appeared to penetrate the basement meb- rane and to touch with cell in the lamina propria. From those findings, it was sugg- ested that structures of gallbladder cells in this study were more smillar to those of amphibian rather than those of lung fish. MO 13 LIGHT AND ELECTRON MICROSCOPIC OBSER- VATIONS ON SHE INTESTINAL WALL OF THE LARVAL MEDAKA, ORYZIAS LATIPES Io LO¢ASiqnal eincl WoOOw> BiOils WaAStG., HAC. Or SCs, Slalzuokea Winthv., Sialwzuoka. The intestinal wall of the medaka has been studied by light and electron micro- scopy. The animals used in this study were obtained daily after hatching for the first week. For the light microscopy, paraffin sections were stained by Gomori's paraldehyde fuchsin and Glimelius' method. On the 1-day larva, goblet cells and ar- gyrophil cells are found in the mucosa of the intestine. By 2-4 days after hatching, small cell groups containing argyrophil cells are frequently observed. By electron microscopy, columnar epithelial cells having numerous microvilli differentiate at the anterior region of the intestine, whereas the cells of this type are still not established in the posterior region of the 1-day larva. Some endocrine cells containing basal granules and goblet cells can be found in the mucosa of the 1-day larva. Occasionally, aggregation of un- differentiated cells surrounded by some amorphous substance are found in the mucosa of the 4-day larva. The basal gra- nulated cells are often found near the cell aggregation. These observations clearly show the early differentiation of the intestinal endocrine cells in the larval stage of the medaka. Morphology 985 MO 14 MODIFICATION OF THE BARAIN PATTERNS BY DOMESTICATION IN THE CARP. H. Masai and Y. Sato. Dept. of Anat., Osaka Univ. Med. Sch., and Dept. of Anat., Yokohama City Univ. Sch. of Med. The brain patterns of some varieties of domesticated carps were studied macroscopically and microscopically, in comparison with those of the Japanese wild carp. The pattern of the optic tectum varies prominently among the major brain subdivisions in nishikigoi carps. The tecta are displaced laterally, and their medial margins are divergent post- eriorly in a V-shape. Such variation is found in other domestic carps, and seems to show atrophy of the tectum. During domestication the pattern of the optic tectum was modified as an effect of artificial selection of target characters for human food and enjoyment. MO 15 STUDIES ON THE GRANULOCYTOPOIESIS IN THE KIDNEY OF THE GOLDFISH. H.Tsuchiya, T.Gomi, A.Kimura, T.Hashimoto* Ganirahaca, and H. ishizuka**#*, *lst. Dept: Bt eAidien. soos Dept. of int..Med.. Toho Unive open. of Med., Tokyo, ***1ist Dept. of Oral Anat. Tokushima Univ. Sch. of Dent. We have been studying on the granulocyt- opoiesis of the Cyprinidae with the view of comparative hematology. Present observati- ons were refered to the kidneys of the gold fish. The inter tubular connective tissues were actively granulopoietic organ similar to other Cyprinidae. However only granulo- poiesis of hetelophils were observed. Their granules have a medium degree of ele- ctron density, characterized by having var- ious shaped core of high electron density which have lamellated structures. These structures were found in the immature cells moreover these cells have two stages for mitosis throughout their life, and the oth- er characteristic structures were the exis- tence of a markedly developed Golgi appara- tus. Mature cells are only seen in eosino- phils which is the same to crucian carp. Then we observed the cells of various Stages that included fine fibrous and cris- talline in the granules, its looked like young basophils of crucian carp. We found granulocytes of all types contained well- developed Golgi apparatus in their cytopl- asm. We have confirmed that the granulo- poietic organ in goldfish have the same characteristic of crucian carp. MO 16 ELECTRON MICROSCOPIC OBSERVATION ON THE BLOOD-AIR BARRIER OF THE LUNG IN THE LOWER VERTEBRATES. A.Kimura, T.Gomi, H.Tsuchiya, T.Hashimoto and ) she hug ical. Department of Anatomy, *Laboratory of Electron Microscope, Toho University School of Medicine, Tokyo. The= authors have been studying the lungs of animals, ranging from the Lungfish to Mammals, from the viewpoint of comparative anatomy. In the present study, the blood-air barrier which were formed by the three-layers, which AC met thin cytoplasmic projection, the basement membrane and endothelial cell, these were observed in the lower vertebrates. 1) Lungfish and Amphibia (Urodela) These alveolar epithelial cells were not differentiated. These cytoplasmic projections were thicker than upper vertebrates and some of them included the Coll Organedsvesl. Ui elas alinhe reer hilbulele substance, we could observe some of the well-developed COmmMagenousmes Lubbers, The blood-air barrier showed from two to four times as thick as Mammals. 2) Amphibia (Anura) and Reptile These alveolar epithelial cells were already differentiated. In the cytoplasmic projection of Type LI cell, many pinocytotic vesicles were observed, but cell organelle was hardly discernible. The blood-air barrier had the midthickness between Lungfish and Mammals. MO 17 ABSENCE OF BLOOD VESSELS IN THE BRAIN PA- RENCHYMA OF HYNOBIID SALAMANDERS. K. Tsuneki!, M. Oujil, H. Akiyoshi? and K. Ichihara2. Dept. Biol., Shimane Univ., Matsue, Shimane! and Dept. Pathol., Totto- ri Univ., Yonago, Tottori. The serial sections of the brains of hynobiid salamanders, Hynobius nebulosus, H. naevius, and H. kimurai, were studied with a light microscope. Blood vessels were found in the meninx and the choroid plexus. They were also located in the olfactory bulb and the neurohypophysis. In the bulk of the brain parenchyma, how- ever, blood vessels were not found. These results were confirmed by India ink-perfu- sion study. The brain of H. nebulosus was also studied with an electron microscope, but blood vessels were not found in the brain parenchyma. In addition, the brains of various caudates other than hynobiid salamanders were studied with a light mi- croscope, but their brain parenchyma was well vascularized. Hynobiid salamanders may be the only vertebrate group that lacks parenchymal vascularization in the brain. In these salamanders, oxygen re- quired for neuron activity may be supplied by diffusion from meningeal capillaries and the choroid plexus bathed in the cere- brospinal fluid. The reason why only hynobiid salamanders lack parenchymal vas- cularization in the brain is unknown. 986 Morphology MO 18 CHEMOARCHITECTONICS OF THE CENTRAL NER- VOUS SYSTEM IN XENOPUS LAEVIS. Y. Sato. Dept. of Anat., Yokohama City Univ. Sch. of Med. Yokohama I studied the chemoarchitectonics of the CNS in Xenopus laevis using adults and tadpoles at various stages of deve- lopment. The animals were staged accord- ing to the normal tables of Nieuwkoop & Faber (1956). Frozen unfixed brains were cut into serial,frontal, and sagittal Sections Obes Une lhe Gilenneremetinod was used for monoamine oxidase (MAO), and Nachlas' method for succinic dehydrogena- se (SDH). There was moderate to strong MAO activity in the strial complexes, the septal nuclear area, the dorsal habe- nular nucl., the preoptic area, the hypo- thalamus, the interpeduncular nucl., and the nucl. raphes. There was moderate to strong SDH activity in the medial pall- ium, the habenular complex, the isthmi, the corpus cerebelli, the torus semi- circularis, and the nuclei of the lateral line lobe. In stage 49 to 51 larvae the medial pallium, the torus semicircularis, the cerebellum, and the lateral line lobe showed SDH activity. The striatal comp- lex and the hypothalamus showed a con- spicuous MAO reaction. All these results were typical of the conditions fround in vertebrates in general. MO 19 MORPHOLOGICAL ALTERATION OF XANOPUS SKIN GLANDS AFTER KEEPING IN THE AIR AND INJECTING ISOPROTERENOL. K.Fujikura and S.Inoue. Inst. of Endocri- nol., Gunma Univ., Maebashi. As we have reported, there are four types of skin glands in the adult Xenopus laevis; the granulated, mucous and the small granulated glands, and the tentatively named SPK gland which is exclusively in the nuptial pad. To see changes of these glands upon various stimuli, we have tested the effects of (1) isoproterenol which is an B-adrenergic agent and (2) dry milieu. By injecting isoproterenol, only the granu- lated gland excreted viscid white material, while the other glands were unconcerned to this drug. The skins of some anuran species and the newt did not change much as of Xenopus upon injecting. Histological obser- vations of the Xenopus skins which were kept in the air up to death, were made regarding the thickness of the skin and the areas of the glands using image analyzing apparatus. The mucous glands shrinked much, indicating 29%54% of the normal values, while the values on the skin, epidermis and the granulated gland were 61183% of the normal. These results imply that only the mucous gland is responded to the dry milieu to secrete watery content, and causes shrinkage of the structure. However, the mucous gland in the skin piece which was excised and kept in the air did not show such changes. MO 20 THE EFFECT OF NERVE ON REGENERATIVE CAPA- CITY OF FORELIMBS OF RANA BREVIPODA POROSA DURING POSTMETAMORPHIC GROWTH. S.Kurabuchil, S.Aiyama! and S.Inoue2. Ipept. of Anat., Nippon Dental Univ., Tokyo and “Inst. of Endocrinol., Gunma Univ., Maebashi. We have previously reported the limb re- generative capacity of the several anurans (Ranidae, Hylidae and Rhacophoridae). Among them, Rana brevipoda porosa and Hyla japo- nica, showed SOEE heteromorphic regene- ration only when they were in very young froglets. In this report, the relation of the limb regenerative capacity and nerve quantity (number of nerve fibers and area of nerve per cross sectional area of limb) during postmetamorphic growth of Rana brev- ipoda porosa was examined. As the body length of the froglets increased after met- amorphosis, there was inversely a decrease of the regenerative capacity coming to loss of regeneration. The level of nerve quant- ity was high in the younger froglets and it rapidly decreased as the body size increas- ed, when the nerve level was 1/5 (area) or 1/10 (number) of the younger froglets. The adult limbs whose nerve quantity was sur- gically augmented by receiving the sciatic nerve, were able to retain regenerative capacity in which the nerve quantity was almost the level of the younger froglet. The results indicate that the nerve qua- ntity in the limb is also deeply involoved in the anuran heteromorphic regeneration. MO 21 FORMATION OF CARTILAGE IN THE NERVE-INTACT AND DENERVATED BONE-EXTIRPATED FORELIMBS OF ADULT ANURANS. S.Inoue, Y.Shimoda and M.Tabuchi. Inst. of Endocrinol., Gunma Univ., Maebashi. As has been reported in the regeneration of the urodele limbs, the regenerates have bones differentiated even if the limb ske- letons have been removed at the time of ampu- tation. We have tested this phenomenon using adult anurans, Xenopus laevis and Rana rugosa. In Xenopus, the ventral sides of the limbs were surgically opened and the humeri, radii and the ulnae were remo- ved. Then the operated limbs were amputated at the zeugopodia. Although the limb stumps decreased in length after the operations, they produced small regenerates which grew slowly. In the histological sections, inde- pendently differentiated cartilages were recognized in the connective tissue at the bases of these small regenerates. This cartilage formation in Xenopus is striki- ngly contrasted with the bone-removed Tris turus regenerate in which the cartilage formation occured in the regenerated bla- stema. A few non-regenerative experimented limbs also showed independently differen- tiated cartilages in the connective tissue. The denervated limb did not usually form cartilage, or rarely showed weak cartilage formation. In the radii and ulnae-extir- pated regenerates of Rana rugosa the car- tilage formation was also occured in the specimens on day 57. Morphology 987 MO 22 OBSERVATION OF EARLY PHASES OF LIMB REGENE- RATION OF XENOPUS LAEVIS AND TRITURUS PYRR- HOGASTER WITH SPECIAL REFERENCE TO MUSCLE. M.Tabuchi,! S.Inove! and S.Kurabuchi.? linst. of Endocrinol., Gunma Univ., Maebashi and 2Dept. of Anat., Nippon Dental Univ., Tokyo. The early phases of regeneration of Xenopus laevis after amputating the limb at stylopodium was histologically examined com- paring it with that of Triturus rrhogas- ter. Muscle regeneration 1S very poor in the limb regenerate of Xenopus. In the injured muscle, the formation and disappea- rance of the retraction clot lasted for 4 day after the operation in both species. However, the twisted appearance and dis- appearance of the myofibril in Xenopus did not retrograde so deep as of Triturus, and several ovoidal muscle nuclei were often gathered in a muscle cell of which sarco- plasm was hyaline. The lesion in Xenopus muscle may not be so profound as in Tritu- rus and its restoration was more rapl than in Triturus. The rate of labeling of 3H thymidine by Xenopus muscle during 3 weeks after amputation was not much differ- ed from that of Triturus. But in Triturus there was aggregation of the mesenchymal cells around the tip of the regenerating muscle on day 18 which had a large number of labeled nucleus. While in Xenopus there was accumulation of fibroblast-like cells containing fairly number of labeled cells which was not joined on the injured muscle. MO 23 MORPHOLOGICAL CHANGES IN THE REGENERATED TAIL OF Rana japonica TADPOLES DURING METAMORPHOSIS. H.Takahama and K.Watanabe. Dept. of Biol., School of Dental Medicine, Tsurumi Univ. Yokohama. Regenerated tail was investigated mor- phologically in larvae of Rana japonica during metamorphosis after tail amputation at Taylor and Kollros stage XIII. By meas- urement of the regenerated tail tip and tail stump during metamorphic stages, the rate of shortening was less in the regen- erated tail than in the stump. Muscle fi- bres in the regenerated tail were immature even at stage XXI. These fibres had small- er diameters compared with the muscle fi- bres in the stump. The muscle fibres in the regenerated tail contained abundant free ribosomes and rough endoplasmic re- ticulum but poor myofibrils. Centrioles were frequently seen. Fusion of these fi- bres were observed. No degeneration occur- red in the muscle fibres in the regener- ated tail while some fibres in the stump degenerated. At stage XXII, degenerating muscle fibres were visible in the regener- ated tail. It seems clear that muscle fi- bres in early myogenesis in the regener- ating process do not degenerate. MO 24 LOCALIZATION OF FIBRINOLYTIC ACTIVITY IN THE TAIL OF ANURAN TADPOLE DURING METAMORPHOSIS. F. Sasaki. Dept.of Biol. School of Dental Medicine, Tsurumi Univ. Yokohama. The present study has been performed to eluicidate the mechanism of fibrinolysis in the tadpole tail of Rana japonica and R.catesbeiana during metamorphosis. Fibrinolysis autography(Todd'58),Gram- Weigert staining for fibrin,histochemical observation of ACPase, immunohistochemical of plasminogen activator(PSMG-Act) and electron microscopy were applied. At the prometamorphic stage,the tail epidermis showed scattered foci of fibrino- lytic activity,especially in parts of the fin. In desquamated epidermal cells,no fibrinolysis was found in the superficial epidermis, however, it was observed in the secondary layer of the epidermis. Macro- phages,lymphocytes and other cells were also observed in the secondary layer of the epidermis. During the advanced stage of tail atrophy,intense fibrinolysis was found in the notochordal cells. ACPase also displayed particularly intense activity in same area. PSMG-Act was mainly localized in the epidermis, layer of mesenchymal cells under the collagenous lamella and notochord cells. Features of the varied profile of notochordal cellular necrosis included myelin figures,autosome,autophagia vacuoles,dense granular bodies and fibrinous bodies were observed. MO 25 FINE STRUCTURE OF THE SO-CALLED GILL REMNANT IN ANURA. Y. Sasaki and N. Makino. Dept. of Biology, Tokyo Med. Coll., Tokyo. The so-called gill remnant in the cervical region of anura was studied by electron microscopy. Materials are Rana nigromaculata and Rana rugosa. There's no difference be- tween these two species by the histologi- cal observation. The small gill remnant named by Makino is the same structure with the gill remnant by electron microscopic studies. Both the gill and the small gill remnant have a well-developed blood vessel in this tissue. And they chiefly consist of lym- phocyte-like cells and cells seem to macrophages. The former has cells of vary- ing sizes, and very irregularly shaped, extremely dense granules are often found in the cytoplasm of the latter. The rate of cell division in these organs is high. Heterophilic leukocytes, plasma cells and the mast cells are sometimes observed. The cells that have similar to C-cell granules of mammalian thyroid were not observed at this time. And figures of the degenerate cells in the macrophage and deformed elythrocytes were found. These organs seem to perform the lym- phoid function from these cell structures. 988 Morphology, Behavior Biology MO 26 FINE STRUCTURE OF THE LINGUAL DORSAL SURFACE IN TWO SPECIES OF FROGS. S.Iwasaki and K.Kobayashi. Dept. of Oral Anat., Nippon Dent. Univ., Niigata. The scanning electron microscope was used to investigate the ultrastructure of the lingual dorsal epithelial cells in two species of frogs, Rana ntgromaculata and Hyla arborea japonica. The specimens were prepared by a method involving osmium post-fixation and acid-treatment to remove extracellular material adhering to the surface of the tongue after the prefixation in Karnovsky's fixative. The lingual surface structure showed almost the same figure in both species. Namely, in the dorsal wide surface except for the anterior margin and the posterior protrusions, filiferm papillae consisted of a large number of non-ciliated cells with microridges on their surface and a small number of ciliated cells were distri- buted compactly, among these filiform papi- lae. A round sensory disk was located on the top of each fungiform papilla. The surface of the disk was widely occupied by the cells with honeycomb structure on their surface, and very small number of cells with microvilli on their surface were scattered among these homeycomb structure. Each sensory disk was encircles by the cabliltatedmcelalmibanders MO 27 AUTONOMIC INNERVATION OF CEREBRAL ARTERIES IN THE LEOPARD FROG. (Rana nigromaculata) T. Tagawa, A.ishikawa, K.Udatsu and K.Okamoto. Dept. of Anat., Sch. of Med., Fukuoka Univ., Fukuoka. Histochemical and immunohistocnemicai techniques for demonstration of catechoi- amine, acetylcholinesterase and VIP have used to study the distribution of adrener- gic, choiinergic and VIP positive nerves on the cerebral biood vessels of leopard frog. The adrenergic and cholinergic nerve fi- bres on the cerebral arteries of leopard frog were iess dense than those of mammals. Those nerve fibres on the extra-cranial ar- teries were more denser than those of intra -craniai arteries. Cholinergic nerve bun- dles ran alongside of the ophthalmic artery. It seems that adrenergic nerves enter the cranial cavity along the internal ca- rotid mainly and the occipito-vertebral ar- teries, and cholinergic nerves enter there along the internal carotid and the ophthal- mic artery. Also, adrenergic, cholinergic and VIP positive fibres were observed in close contact with intra-parenchymal arterioles and capiliaries suggesting the possible ex- istense of central innervation. This fea- ture, however, waS by no means common. MO 28 ULTRASTRUCTURAL CHANGES INDUCED BY LOW TEMPERATURE OF THE CELLS OF PROTHORACIC GLANDS AND CORPORA ALLATA IN THE SILKWORM, BOMBYX MORI Tonal Tamana High School, Kumamoto Keeping the larvae of 3rd premolt rest- ing period and those of 4th in a cold chamber(10C) for 10 days produced the fol- lowing changes in the cell structure of prothoracic glands and corpora allata of the silkworm. (1) Prothoracic Glands: Lipid droplets and rough ER were observed in the cytoplasm in the 4th premolt resting periods, whereas these changes could not appear in those of 3rd. In contrast to that reported previ- ously, there were very scarce deformed mitochondria in the case of cold treat- ment. Almost all mitochondria were rod— shaped. (2) Corpora Allata: The intercellular spaces between corpora allata cells en- larged by the cold treatment. These spaces seemed to be made by shrinkage of sur- rounding corpora allata cells. At the pe- ripheral region of cytoplasm showed fin- ger like projection. The nerve fibers originated from nervi allati became re- duced in number. The results obtained by the cold treatment suggests some relation- ships between corpora allata cells and the supplying nerve fibers. BB 1 HORIZONTAL MOVEMENT OF DAPHNIA MAGNA IN LOW OXYGEN CONCENTRATION. M. KOBAYASHI. H.GONOI. Dept. Biol., Faculty Sci., Niigata Univ., Niigata. The phototactic swimming activity of hemoglobin-poor (6.3 mgHb/g dry wt) and hemoglobin-rich (115.5 mgHb/g dry wt) Daphnia in a low oxygen concentration at 20°C was studied to evaluate the role of hemoglobin in low oxygen concentration. The swimming speed of pale and red ani- Mals was 52.6 #220) and 5305 £0437" cem/mamn respectively. The swimming distance per sec waS approximately three times the body length and did not significantly differ between them. The total swimming distance of pale animals after exposure to a low oxygen concnetration until final standstill of antennal and pedal movement toward the light source was constant, approximately 10 m, in very low oxygen concentration and was free from the effect of oxygen concentration. It, however, increase in oxygen concentration and even exceeded LOO m ge OWoa5 wMlOs /o Red animals even in very low oxygen concentration and rapidly gained a more distance with a very slight increase in oxygen concentra- tion, totalling to above 100 m at 0.15 mlOj/1. These results indicate that high concentration of hemoglobin plays an important role in the swimming activity under low oxygen concentration. Behavior Biology 989 BB 2 SYSTEM THEORETICAL CONSIDERATIONS OF CLAS- SICAL CONDITIONING OF CRAYFISH. H .Yokoi and M.Saito. Institute of Medical Electronics, Faculty of Medicine, Univers- ity of Tokyo, Tokyo. Dark stimulus is presented as CS simul- taneously with an electric shock as US. After the CS-US trial is repeated approxi- mately 10 times, CS-alone trial is perfor- med as a test trial. The crayfish showed three kinds of behaviors. First, sensitiz- ation was observed immediately after an el- ectric shock was presented. Secondly, cla- Sssical conditioned response was observed in test trials when the interval between trials was varied randomly. Thirdly, rhy- thmical response was observed regardless of dark stimulus when the interval between trials was constant, that is, 60 seconds. From the viewpoint of the system theory, the crayfish was modeled to describe these three kinds of behaviors. In addition, to investigate the model, a system analyti- cal method was considered. BB 3 ANALYSIS OF MATING BEHAVIOR IN DROSOPHILA AURARIA COMPLEX. Y.Oguma, H.Kurokawa, S.Akai*, H.Tamaki and J.Kajita. Inst. Biol. Sci., Univ. of Tsu- kuba., Ibaraki. *Yamanashi Gakuin J. Col- lege., Kofu. We attempted to clarify what consists of components in mating behavior and how it genetically relates to species discrim- ination among the four siblings of D.aura- ria complex. A serial studies by means of observation for successive mating behavior were conducted by using a videocorder with a small observation chamber (¢15mm, 5mm in depth) under a light condition (1500 lux). The principal results obtained are as fol- lows: 1. We recognized 13 different but consecutive components belonging to mating behavior. All or almost all of male flies of D.auraria and D.quadraria similarly did not represent wing iastay while those of D.biauraria and D.triauraria did it. At the stage of attempted copulation, males of all four species consistently showed wing display and simultaneously females spread both wings, following copulation. 2. Another experiment using a larger ob- servation chamber (50x50x4mm) in which 10 females and 15 males were placed together revealed that male flies behaved to show orientation to females by means of their "sight". Furthermore, we could examine a critical distance which they could notice females was only 20mm. BB 4 SEXUAL ACTIVITIES IN DROSOPHILA AURARIA COMPLEX. [A saene ha: ee H.Kurokawa, Y.Oguma, S.Akai*, J.Kajita and H.Tamaki. Inst. Biol. Sci., Univ. of Tsuku- ba., Ibaraki. *Yamanashi Gakuin J. College. We attempted to study the sexual activ- ities of flies of D.auraria complex by the accounts of the observation of sexual be- haviors. Flies were reared on the Bowling Green Univ. Medium seeded with live yeast at 25t1°C and 60% R.H. in an artificial light condition (L:D=14:10 hr), being the light period between 5:00 and 19:00. I. The test for sexual maturation: It was found that there were a little but critical differences in sexual maturation between the four species. D.triauraria ma- tured somewhat faster, conversely, D.biau- raria did slower than the others. The male flies of the four species, in any case, all matured sexually by 4 days after eclo- sion. II. Sexual activity depending time of day: No mating pairs were detected with- in a 30-min observation time in a dark pe- riod (19:00-4:00). Mating index (Spiess et al., 1966) of D.triauraria was constantly high in a light period, and those of D.bi- auraria and D.quadraria were high at early morning (about 4:00) and at noon. In D.au- raria, mating index was also highest at 4:00 and at noon but decreased thereafter. The flies tested, however, came to mate when they were shifted from a dark to a light condition with only 3 lux illumina- tion. BB 5 QUANTITATIVE ANALYSIS AND COMPUTER SIMU- LATION OF FEEDING BEHAVIOR OF THE FRUITFLY, Drosophila melanogaster. Ichiro Shimada? and Yoshiyuki Kawazoe~’. Dept. of Biol. Sci., Tohoku Univ.’ and Edu- cation center for Information Processing, Tohoku Univ.”, Sendai. In order to analyze quantitatively the feeding behavior of Drosophila in the micro test plate, we measured colorimetri- cally the ingested volume of sucrose so- lution from the wells and counted the dwelling time at the wells by video tape recording. When the two sorts of sucrose solutions (10mM and 5S5mM) were put into symmetrically either sixty wells (high distribution density) or eight wells (low density 4:4), the numbers of visits on both sucrose solutions were approximately the same. The result indicates that the movement of the flies could be regarded as a random walk. At both distribution densi- ties the flies showed the clear preference to 10mM sucrose to the same extent. We have performed a Monte Carlo simulation for the two-choice feeding behavior of Drosophila. The simulation suggests that the two different mechanisms with and without memory might lie in the two-choice process. 990 Behavior Biology BB 6 VARIATION OF PAUSE DURATION AND ABDOMEN PUMPING IN "PHASE II(P-II)" BY COPULATION AND PGE,INJECTION IN VIRGIN CRICKET, GRYLLUS“BIMACULATUS. N.Ai and M.Ishil. Dept.Biol., Tokyo Gakugei Univ., Tokyo. In oviposition behavior of cricket, the time course of P-II in mated females is clearly shorter than that of virgin females, Copulation and transplantation of haemolymph of mated females are very impor- tant to make short of the time course of P-II (N.Ai,N.Yamaguchi and S.Ohyama,1983). In addition to these observations, it was able to measure clearly the female abdomen pumping movement which appeared just before the end of P-II. And its num- ber of rhythmical pumping movement seemed to correspond to the time course of P-I1. In virgin female, number of pumping was large(5-7 times more than mated female's), but after copulation it became apparently small. "Treatment On RGA sain) cron mviol the virgin female(inside of Body cavity) indu- ced the decrement of the time course of P-II and number of pumping. Otherwise, in- hibitor substance of synthetase of PGE., NAPAP being injected to virgin female just before copulation, the time course of P-II1 and number of pumping were still remaind the same as that of virgin females. In this experiment, it was found that the role of PGE,is primary thing to make egg oviposit smoothly to the substrate. BB 7 WHEN DOES AN EGG PROGRESS INTO THE GENITAL CHAMBER IN THE OVIPOSITION BEHAVIOR OF THE CRICKET ? T.Sugawara. Dept.Biol.,Saitama Med.Sch., Saitama. The transparent window made on the 7th sternum of crickets(Teleogryllus commodus ) ensured the direct observation of an inter- nal genital area. The egg-laying process of such operated females was filmed by 8mm cine camera then was analysed. When the animal penetrates her ovipositor into the substrate, which is moist sand in the experiment, she extends her abdomen and makes the valvulae glide rapidly back and forth on each other, with her legs holding firmly the substrate. At the end of the penetration, the retraction of the subgen- ital plate and the short withdrawal of the Ovipositor were observed. These actions were proved to correspond to the retraction of the genital chamber subsequent to the progression of an egg into it. In rare cases where virgin females laid eggs, the process was the same as that of mated females, except that the resting phase following the egg progression was prolonged greatly in the former. It seems reasonable to take the resting phase as the fertili- zation phase. The females whose oviducal nerves had been sectioned usually could not transfer the eggs into the genital chamber in spite of their long efforts. Obviously the nerves command the progression of eggs. BB 8 FACTORS REGULATING URINATION PATTERNS IN MALE AND FEMALE MICE. M.Daumae, N.Matsuzawa and T.Kimura. Dept. of Biol., Coll. of Arts and Sci., Univ. of Tokyo, Tokyo. A marked sex difference is known in mouse urination patterns. Male mice produce large numbers of spots which are dispersed through the arena and often small, while females urinate rather infrequently. In the present study, it was found that castration of males did not reduce the frequency of urination. Females treated with neonatal androgen also frequently urinated and ovariectomy significantly increased their rate of urine deposition. In the study of ontogeny of urination patterns, the earliest difference in urination patterns between females and males or neonatally androgenized females was observed in 20 days of age. These results suggest that neonatal androgen plays an important role in establishing the male urination patterns, and after the establishment, the pattern persists regardless of hormonal conditions in adulthood. To investigate the function of the urine deposition in social interaction in mice, we deprived olfactory bulbs of male mice. Bulbectomy increased rate of urine deposition. It is possible that olfactory feedback system is working in the regulation of urination patterns. BB 9 SOCIAL DOMINANCE OF MALE MICE IV:AGONISTIC TENDENCIES OF JUVENILE AND MATURE MICE. Solsehvasinsl. Wee. BitOile, wWac, Educ. . Kagoshima Univ., Kagoshima. Agonistic behaviors of male mice were observed to approach social structures. Three or 6 male mice were cohabitated at 4 weeks of age. The observations were con- ducted immediately after renewal of a cage every 7 days from 6 to 12 weeks old. So- cial activities(aggressions & mounts) of 6-male colonies were declined with weeks, As for 3-male colony, a male was apt to become dominant (39/61), but in a certain number of cages 2 (12/61) or all (4/61) males became aggressive. A given male of 16 cages and given 2 males of 5 cages wene always dominant. The reversals of order are rare occasions. Social interactions between 2 mature males were also observed. Each subjected male mouse was cohabitated with a strange male 3 times. The agonistic strategies shown by mice could be classi- NEC! see S teyoeso Nimeile Ose ey7oe 1 (4'9)/ 120) become aggressive against any partner. A male of type 2 become dominant or sub- ordinate according to the partner (54/120) There are few males (type 3, 17/120) which show no aggressive behaviors. A male which had been dominant in a 3-male colony showed the type 1 strategy. The agonistic tendencies of male mice are mainly decided before 6) weeks of age): Behavior Biology 991 BB 10 ROLE OF SEPTAL FIBERS IN THE ONSET OF ARTI- FICIALLY INDUCED PARENTAL BEHAVIOR IN RATS. BH. (Koranyi, K. Yamanouchi, Y. Arai. Dept. of Anat. Juntendo Univ. Sch. Med. Tokyo. Experiments were designed to study the involvement of septal area in the develop- ment of artificially induced parental be- havior in virgin female and male rats. Under ether anaesthesia anterior roof deafferentation (ARD) was made by Halasz- type L-shape micro-knife. Sham-ARD, cin- gulate cortex lesioned and intact groups of rats were used as controls. Rats were allowed 3 weeks to recover from brain surgery before they were exposed to the continuous presence of 3 standard size pups for several days. In 24h intervals, when pups were changed for freshly nourished young, behavioral tests were made. Each session lasted for 1h and was Supplemented by additional two spot-checks to record the occurence‘of cnaracteristic forms of behavior (at first the avoidance of pups, and later retrieving, nursing and crouching). ARD did not result in any modification of the initial avoidance of pups indicating normal sensory information processing. However, it was found that ARD Significantly reduced the incidence of different forms of parental behavior in female, and almost completely blocked its development in male rats. It is concluded that septal influences contribute to the CNS mechanisms underlying the development of parental behavior. BB 11 INVOLVEMENT OF DOPAMINE SYSTEM IN THE MECHANISM OF MEMORY FORMATION OF MICE. N.Mishima, K.Teraoka and R.Yoshioka. Biol. Lab., Fac.of Pharm., Univ. of Tokushima-Bunri. Tokushima. Cell bodies of Dopamine or Norepineph- rine neurones of mice were chemically le- sioned with 6-hydroxydopamine injection into substantia nigra or locus coeruleus, or electrically lesioned with electrodes inserted into each locus, immedeately after training of T-maze avoidance task. After body weight and locomotor activity in home cage were fully restored, memory retention was tested. Mean retention levels of SN-lesioned groups were signifi- cantly lower compared to each sham-control (electric lesioned mice : 53%, chemically lesioned mice : 65%). On the contrary, imparement of norepinephrine system did not affect the retention level. When the mice received SN-lesion or LC-lesion after establishment of memory formation, task performance of those mice was not impaired. These results suggest that SN-lesion impairs the step of formation, but not storage or retrieval of memory and only dopamine system in catecholamine systems plays important role in the mechanism of memory formation. BB 12 AGGRESSIVE BEHAVIOR AND DIFFERENTIATION OF CATCH TENTACLE IN HALIPLANELLA LUCIAE J.Mizunoya and Y.Kakinuma. Dept. of Biol., Fac. of Sci., Kagoshima Univ., Kagoshima. Catch tentacle is used for aggressive interaction in acontiate anemones. There are some cases that H.luciae has catch tentacles(C.t.) in addition to feeding tentacles(F.t.). Exclusively intraspecific aggressive behavior was found in this species. The following behavior was observed in detail ; An aggressive individual extended and brandished C.t. in the direction of an opponent. When the C.t. touched the opponent, nematocysts were discharged and C.t. adhered to the opponent. This caused the opponent to be injured. Ihe ditierentiation of C.t. from F.t. was found in the portion injured by EeoRtOlm © sibien Ol Ges Obes. The following was also observed ; C.t. were mainly differentiated from the primary tentacles. Tentacles on the principal radii were more easily diutirerentiated into C.t. than those on the interradii. The differentiation from F.t. to C.t. was proceeded from the base to the top of the tentacles. The function of the C.t. started to work when two-thirds of tentacle were dubiterentratved into C. vc. BB 13 ANALYSIS OF VOCALIZATIONS IN THE LEACH'S STORM-PETREL (OCEANODROMA LEUCORRHOA) M.Taoka, T.Satol, T.Kamada2, and H.Okumura? Life Sci,Inst.,Sophia Univ,,Tokyol, Dept.of Oral Physiol. ,Hokkaido Univ,School of Dent. ,Sapporo2. Marine Biol,St.,Fac,of Sci. ,Hok- kaido Univ, ,Akkeshi3, The vocalizations Of a nocturnally active seabird, the Leach's Storm-Petrel, were studied at their breeding colony in Daikoku Island, Hokkaido. Three distinct vocaliza- tion patterns were recognized, namely short call, duet call and threat call, The short call usually made up of a series of ca. 10 notes and was vocalized by both aerial and grounded birds, but most frequently by the birds in the burrows. This call was nbdt associated with specific situations in the burrows. Distinct sexual dimorphism was found in the fundamental frequencies of the short calls. The duet call consisted of a rapid series of chirps which was regularly intercepted by a long wheeze, and lasted continuously up to many minutes, This call was given in the burrows, usually when the both members of the pair were present, and they duet in most cases, An egg or a chick was not found in the burrows of duettists, suggesting the close association with the courtship behavior, The threat call was a short series of almost unstructured noise and vocalized together with short call during aggressive encounters. The vocaliza- tions of the chicks and intermediate calls between chick and adult calls were also described, 992 Behavior Biology BB 14 PHONETIC CHARACTERS AND SOUND PRODUCING MECHANISMS OF THE CALLING IN THE JAPANESE TOAD. M.Kusunoki, M.Satou and K.Ueda. VAIS WASiEG, PACS GIF SEilo, Wialw. Oi WOKVO, Tokyo. The phonetic characters and the sound producing mechanisms of the calling were examined in the Japanese toad. Frequency structures and temporal characters of the mating call (MC) and release call _ (RC) were analyzed. MC and RC had the same fun- damental frequency of about 700 Hz and pulsated at the same frequency of about 100 Hz. The distribution of the length of the call envelope as well as that of the call interval considerably overlapped be- tween MC and RC. These results suggest that the toad does not discriminate these two calls as different sound signals. Next, we tested the sound producing abili- ty of excised larynx by sending air from the trachea. The larynx from the male toad but not from the female toad produced pulsated sound resembling natural calls. The movement of the excised larynx during sound production was analyzed using high speed cinematography. From the results it was concluded that the pulses of the call sound are produced by the vibration of the arytenoid and that the characters of the sound are determined passively by the structure of the larynx. BB 15 ULTRASOUND VOCALIZATION ELICITED BY NECK- STROKING OF RATS. R.Yoshioka, N.Oshima, Y.Nishitani and N. Mishima. Biol.Lab.,Fac. of Pharm., Univ. of Tokushima-Bunri., Tokushima. When dorsal neck of the rat was gently stroked by experimenter's fingers, ultra- sound(U.S.) vocalization was elicited. This stroking-U.S. appeared in older rats than rats in eye-opening age(14-16 day rats), whereas the well-known U.S. elici- ted with isolation from their nest or rolling in hand disappeared almost by the eye-opening age. Stroking-U.S. of 14-16 days old rats was hardly distinguished from handling-U.S. by the sonagram analy- sis. However, the frequency of stroking- U.S.fell down gradually with age(50 kHZ in 14-16 days old pups and 25 kHz in adult rats). The stroking-U.S. is pure-tone like ultrasound having narrow bandwidth and its duration was long (500-1500 msec in adult). This type of U.S. was also emitted by defeated rats which received violent attacks in fighting behavior or freezed rats which were exposed to foot electric shock. These results suggest that emission of stroking-U.S. manifests some emotional state in anxiety or fear. These anxious state against experimenter's stroking was evoked after eye-opening in rats. BB 16 CHARACTERISTICS OF SLEEP-WAKING RHYTHM IN DOMESTIC CAT. A N.Kuwabara, K.Seki and K.Aoki. Life Sci. Inst., Sophia Univ., Tokyo. *JAMSTEC, Yokosuka. Intrinsic sleep-waking rhythm in the domestic cat has been investigated by mea- suring EEG. Under a LD12:12 conditions, the sleep-waking rhythm shows a nocturnal almost synchronized in light-dark cycle, showing that there shared total sleep time more in light phase than in dark phase. It can also synchronize to a LD15:9, noting ultradian component is included in a cir- cadian pattern. While in the constant darkness and lightness sleep-waking cir- cadian rhythm altered into remarkable ul- tradian rhythm as days go on. When the preparation was re-exposed to a LD cycle, sleep-waking rhythm resynchronized immedi- ately after LD cycle set again. In subse- quent experiment a LD cycle was advanced for 6 hours, and about two weeks later, delayed for 6 hours. Under these condi- tions, resynchronization to the shifted LD cycle was observed at a few days, which showed one of the characteristics of the circadian rhythm. These above mentioned restults suggest that intrinsic sleep- waking rhythm is ultradian rhythm under constant dark condition. In the absent of light-dark timing cue remarkable ultradian rhythm is dominant. In the face of LD cycle, sleep-waking rhythm synchronizes to a LD cycle showing circadian pattern which include an ultradian component. BB 17 THE EFFECTS OF THE PINEAL COVERING AND OF P-CHLOROPHENYLALANIN ON THE CIRCADIAN CHANGES IN PINEAL ULTRASTRUCTURE IN THE JAPANESE COMMON NEWT. M.Kikuchi, A.Chiba and K.Aoki. Life Sci. Inst.,; Sophia Univ., Tokyo. We have shown that the daily changes in the number of synaptic ribbons(SR) and dense-cored vesicles(DCV) in the pineal organ of the Japanese common newt are en- dogenous circadian rhythms. To study the causes of these rhythms, an electron mi- croscopic investigation was undertaken to examine changes in the number of SR and DCV under a light-dark cycle of 12:12 when (1)the pineal organ was covered with alu- minum foil and (2)PCPA(a serotonin deple- tor) was injected into the abdominal cavi- ty. The results were that (1)the number of SR showed a freerunning rhythm although the eyes were intact and Oe eee daily changes in the number of SR and DCV were not observed. This suggests that there is an oscillator entrained to the light-dark cycle via the pineal organ, not via the eyes, and that melatonin synthesis plays an important role in the expression of the circadian rhythm. The oscillator controlling the circadian changes in num- ber of SR and DCV may be one of the oscil- lators that are implicated in locomotor activity rhythm. Behavior Biology 993 BB 18 LOCOMOTOR ACTIVITY RHYTHMS OF THE MEDAKA UNDER LIGHT-DARK CYCLES AND CONSTANT CON- DITIONS. T. Yokota and T. Oishi, Dept, of Biol., Nara Women's University, Nara. Locomotor activity rhythm of the medaka was recorded by a phototransister system under natural condition and various light- ing regimes. A shoal of the medaka under natural condition during summer indicated a typical diurnal pattern with the activi- ty onset at dawn and the activity end at dusk. The peak of the amount of activity was at the middle of day and it was seve- ral hours ahead of the peak of water tem- perature. When artificial light-dark (LD) cycles were applied to the medaka, most of the fish subjected to LD 12:12 or LD 14:10 showed diurnal activity. Phase angle di- fferences between activity onset and light on (Wonset) corresponded nearly to light on and those between activity end and light off (fend) showed mostly negative values. An anticipatory behavior was ob- served in only a few fish. Transient pe- riods were observed in only one fish out of 7 when LD cycles were shifted 6 hours. Distinct free-running rhythm was not observed under DD nor in dim LL (3 lux). In consclusion, locomotor activity rhythm of the medaka seems to be either weakly coupled to the circadian oscilla- tor(s) or composed of several components which behave differently under constant conditions. BB 19 CIRCADIAN RHYTHMS OF OPTIC LOBE AFFERENTS IN THE CRICKET GRYLLUS BIMACULATUS. K.Tomioka and Y.Chiba. Biol. Inst., Yama- guchi Univ., Yamaguchi In insects, two kinds of tissues have been focussed on as a pacemaker site: the optic lobe (cockroaches and crickets) and the cerebral lobe (silkmoths, mosqui- toes and house flies). However, neither tissue has been demonstrated to house the self-sustained oscillator. To examine whether the cricket optic lobe contains circadian pacemaker, long-term recordings of afferent neuronal activity of the optic lobe were performed using a suction elec- trode. The multiple unit activity of optic lobe afferents exhibited two types of circadian rhythms. Type-I formed an un- roofed trapezoid lacking its roof in the night time of LD and sinusoidal wave form peaking in the subjective day under DD. Type-II showed, in the night time of LD, a characteristic trapezoid persisting in an ensuing DD. Under LL both types made larger trapezoid in the subjective night. Removal of the subesophageal ganglion and/ or cerebral lobe did not disturb these rhythms. Thus, the optic lobe can maintain its periodic motion without any neural input from the other part of the central nervous system, suggesting that it contains the circadian pacemaker which controls the overt rhythm via the afferent neural path- way. BB 20 EFFECTS OF OPTIC LOBE ABLATION ON LIGHT DEPENDENCY OF MOSQUITO CIRCADIAN ACTIVITY Y.Chiba and M.Kasai. Biol.Inst., Yamagu- chi Univ.,Yamaguchi. The mosquito Culex pipiens pallens is highly active under DD, showing a circadian rhythm, But under LL, the activity is considerably lowered and there is no statistical detection of the rhythm. We proposed views previously that (1) a crucial part of the central nervous system controlling the rhythm may be located in the cerebral lobes (CL) and (2) an extraocular photorecep- tor presumably located in CL, rather than the compound eyes, plays an essen- tial role for the rhythm to be entrained to LD cycle. The views are based mainly on the fact that the rhythmic activity occurred under LD and DD even after the ablation of the greater part of the optic lobes. We found recently that the post- operative mosquito was highly active and rhythmic even under LL: the additional fact supporting the above-itemized view (1). This suggests that the low activity of intact mosquito under LL may be due to an inhibitory effect of photorecep- tive information processed in the "compound-eye"-"optic-lobe" system (CE- OL). This inhibition does not occur in C.p.molestus, suggesting the subspecific difference in the function of CE-OL. BB 21 CIRCADIAN CLOCK CONTROLLING THE ECLOSION RHYTHM OF THE SILKWORM, BOMBYX MORI. I. Shimizu and K. Miura. Labo. of Plant Ecological Studies, Fac. of Sci., Kyoto Univ., Kyoto. In order to find out the dynamics of the eclosion clock of the silkworm, systematic experiments manipulating the light and dark conditions were carried out. In the photoperiod of LD 8:16, the eclosion peak in the scotophase occurred 4 to 5 hours before the onset of light, and a light-on peak was also observeq- In LD 16:8 the eclosion peak occurs only at light-on. With photophase in excess of 20 hrs, minor peak was observed in the photophase before, the light-off, and major one at light-on. An insertion of the light-pulse in DD pro- duced a circadian rhythm(c.a.24hrs) and that of dark-period in LL also produced a particular persisting free-run rhythm in LL(c.a.18hrs). Phase shift experiments showed that off-transition in the early subjective night determined the phase-delay and the on-transition in the late subject- ive night determined the phase-advance. That is, off-signals in the first half of the night simulated a new dusk and on- signals simulated a new dawn. The light pulse at 8 hrs after the end of the photo- phase of LD 8:16 produced a delay by off- transition and an advance by on-transition. A scheme of the dynamics of eclosion clock was proposed. 994 Behavior Biology, Ecology BB 22 THE EFFECT OF BRAIN ABLATION ON CIRCADIAN RHYTHM IN EPTATRETUS BURGERI. S.Ooka!, H.Kabasawa* and S.Kinoshita?®. Atomi College’, Tokyo; Keikyu Aburatsubo Marine Park Aquarium’, Miura; and Misaki Marine Biol. Station*, Univ. of Tokyo, Miura. The activity patterns in Eptatretus burgert were recorded by an infrared- light sensing system set up outside the aquaria. The animal showed nocturnal activity in 12L-12D, and had clear circa- dian rhythm in constant darkness. When the light-dark cycle was reversed, the animal adapted to the new one within 2 weeks. The individuals, which were removed their eye- balls, showed almost the same manner in activity as the intact animals, both in 12L-12D and in constant darkness. Brain ablation (except medulla oblongata) affected the activity patterns as follows: 1) In light-dark cycles the activity was seen only an light period. 2) im contant darkness, sporadic signals were found in GHEE tel (SsbuS5 3})) In OUR GD, ela AGieswaltey was observed only in light period. 4) When light-dark cycle was reversed, the animal adapted to the new one promptly. 5) Active signals were constantly maintained in light for 77 hours. Thus the brain-ablated individuals lose their circadian rhythm, and the activity seems to be the direct response to the light. BB 23 FACTORS FOR ENTRAINMENT OF STRIDULATORY ACTIVITY IN LONG-HORNED GRASSHOPPER. Pe Nakataniy, la OrSuUmand Hemp EtOrnm De peErmor Baell oy, eK, Cie Seino, Wins, Ose WeneIGEEelS Yamagata. Stridulatory activities of adult male long-horned grasshopper, Hexacentrus japonicus, were recorded under LD12:12 CWE GSO) 1b /O) Gob SO) inje P. (S.) iwayamaensis > P. sinensis nona > . Imes Ws Secon, ~ eG 7 THE DIURNAL ACTIVITY OF THE SEA-ANEMONE, PARASICYONIS ACTINOSTROIDES AND ITS RELATIONSHIP WITH ZOOXANTHELLAE. K. Ozaki and Y. Kakinuma. Dept. of Biol., Fac. of Sci., Kagoshima Univ. Kagoshima. The sea-anemone lives together with much Zooxanthellae(the symbiotic algae). We found the diurnal change of the form of sea-anemone's tentacles, such as swelled in the daytime and became thin at night. In order to investigate the relationship between the change of the tentacle's form and the amount of algae, the individuals of sea-anemone with much algae and those with a little of them were kept in water under the following conditions; i.e., in the day light, in the constant illumination, and in the constant dark. Meanwhile, the fluctuation of the quantity of pH(index of CO,), DO(Dissolved oxygen), and NHy in the environmental water was examined as indices. We observed the Giurnal activity in the change of the form of the tentacles as an index, by checking the diameters of the thickness of them. The results indicate the exchange of the form was occurred by the stimulation of light, regardless of the amount of algae. Especially, in the case of the sea-anemone with much algae in the light, such a symbiotic relationship was found that the photosynthesis was performed by algae, which absorbed CO, and NHy, probably metabolic products of the sea-anemone, and emitted O2. EC 8 CORAL ESTABLISHMENT ON A SUBMERGED CONCRETE PILLAR IN SINGAPORE. L.M.Chou §& T.M.Lim. Zoology Dept., National Univ. of Singapore. 1. Sesoko Marine Science Center, Univ. of the Ryukyus, Okinawa (until 19 Dec. 1984) A study of the coral community on a 10-year old submerged vertical concrete pillar at Pulau Hantu (lat.1°13.6'N,long.103°45'E), Singapore was made and compared with that on the adjacent reef slope. Grids containing squares of 10 cm were used to estimate areal cover of individual colonies. The community on the concrete pillar was richer in terms of species diversity, coral cover, colony number, size range and depth range. Twenty- seven species occurred on the pillar compared with 12 on the reef slope. Coral cover on the pillar was 30.91% (19.05% on the slope) while the number of colonies was 128 (33 on the slope). The size range of 2 to 1599 sq.cm on the pillar (20 to 1295 sq.cm on the slope) showed that while young colonies were being formed, the more established ones were reaching larger sizes than those on the slope. The depth range of 0.5 to 6.5 m on the pillar (1.0 to 5.6 m on the slope) indicated that it is a better substrate for coral settlement. Six species, Acropora aculeus (Dana), A. cytherea (Dana), A. valida (Dana), Agaricia incrustans Bernard, Psammocora digitata Milne-Edwards § Haime and Montastrea curta (Dana) which are recorded for the first time in Singapore waters, were all present on the man-made concrete pillar. BERS LIFE SPAN,GROWTH AND FOOD HABITS OF THE FRESHWATER FISH,ORYZIAS LATIPES UNDER NATURAL CONDITIONS. O} Lemalo NeEgambivens Zool inisitrcn tic) Cen olCeiaame Univ. of Tokyo. a In laboratory,the Medaka lives about 3 years and lays eggs during the whole year. The present study shows that under natural conditions its ecology is quite different. Regular samplings were made in a rice- field near Lake Imba(Chiba) since October 1983. The analysis of the otolith proves that the Medaka lives only one year. They hatch in spring,grow until the late summer, do not increase in size during the winter and grow rapidly in the early spring to reach the sexual maturity. The breeding season begins in mid-April and runs until the extinction of the adults in late June. The cause of the sudden disappearance of the adults is unclear. But this might be due to their large size which makes them more vulnerable against the predators. On the other hand,the rapid drop in the planktonic biomass,which occurs naturally at this moment,added to the inability of the Medaka to exploit the benthonic resour- ces can also partially explain their extinction. The most important foods in automn are the Collembola and others ter- restrial insects fallen on the surface of the water,while in winter and the early spring they are the Collembola and unicel- lular organisms like Navicula or Fragilaria Ecology 997 EC 10 ALLOPATRIC TWO TYPES OF DEVELOPMENT IN THE POLYCHAETE, NEANTHES JAPONICA. M. Sato° and M. Tsuchiya°®. °Dep. of Biol. Fac. of Sci., Kagoshima Univ. and °°Dep. Gt Biol. Fac. of Sci., Ryuku Univ. Egg size and developmental pattern after artificial fertilization were examined in the brackish-water polychaete, Neanthes japonica collected at three estuaries in Hiroshima, Miyagi and Aomori Prefectures in Japan. Diameter of eggs in Hiroshima, Miyagi and Aomori populations was about 140, 160 and 220 pm respectively. Embryos were enveloped by jelly layer which had been formed just after fertilization. Free swimming life of larvae out of jelly layer began at trochophore stage in Hiroshima and Miyagi populations, but at 3-setiger- ous nectochaeta stage in Aomori popula- tion. Setae of nectochaeta of the former populations were longer than those of the latter one. These results show the existence of two types of development in N. japonica, i.e., longer-pelagic development from smaller eggs and shorter-pelagic development from larger eggs. Optimum salinity for the latter type of development was lower (12- 18 % than that for the former type reported by Kagawa (1955) (22-24 2%). The shorter-pelagic development may result in limited larval dispersal within the upper estuary. Fe SEXUAL DIMORPHISM AND PROTOGYNOUS HERMAPH- RODITISM IN LEPTOCHELIA NEAPOLITANA (TANA- IDACEA, PARATANAIDAE). S.Ishimaru. Zool. Inst., Fac. of Sci., Hokkaido Univ., Sapporo. A rearing experiment was conducted in a small tube-dwelling tanaid Leptochelia neapolitana to know whether or not a fe- male has the ability to change her sex to male. Copulatory females bearing first brood were reared separately in different containers, and their developmental stages were followed. After releasing manca 1 instars, fe- males molted to a preparatory stage and once again developed their ovaries gradu- ally. Those with full-mature ovaries then molted to a copulatory stage, but their eggs were not layed in their marsupia and gradually degenerated. Two males appeared after the next molt. A female, which once failed to mate and has very little chance to reproduce if she remains a domicolous and passive female, can raise her fitness when she reverses her sex and actively walks about searching for mates. This situation would be ful- filled when male mortality is selectively higher than female and population sex- ratio is consequently skewed to female, or when population density is so low that a female cannot be effectively searched out by males, EG a2 EOLIS-HYDROZOA ASSOCIATIONS II. SPECIALIST AND GENERALIST. Y. Hiranol and Y. Hirano2. Mukaishima Mar. Biol. Lab., Fac. of Sci., Hiroshima Univ., Hiroshimal, Zool. Inst., Fac. of Sci., Hokkaido Univ., Sapporo?. During the study of eolis-hydrozoa associations in Oshoro, Hokkaido, the eo- lis Coryphella athadona was found to be a quite generalist species for food habit. Choice experiment was conducted with two hydroid species Eudendrium boreale and Orthopyxis platicarpa on which Coryphella densely inhabit. The results showed that there is innate difference of preference for food even though the eolis is a gen- eralist, and that the eolis is attracted to the hydroid species which it has eaten up to that time. The other choice experi- ment was made with Orthopyxis platicarpa and Sertularella miurensis which usually co-occured on seaweeds in the field. Coryphella did not show significant dif- ference of preference between the two hydroid species in the experiment. In the field, however, very few of Coryphella appeared on Sertularella, to which the other eolis Cuthona sp. (specialist) swarmed. It is thus possible that the distribution of generalist may be re- stricted by presence of sympatric special- Sits EG i3 GEOGRAPHICAL DISTRIBUTION OF MOUNTAIN-— BROOK=DWELLING AMPHIPODS IN KANSAI AREA M.Hiroki. Dep.of Sci.Ed., Kyoto Univ. of Ed., Kyoto. Field investigations are being conducted to disclose the geographical distribution of freshwater amphipods in Japan. Until the present, some results mainly on mountain-brook-—dwellers in Kansai area have been obtained as follows: (1) Three gammaridean species — Gammarus nipponensis, Paramoera japonica and Anisogammarus annandalei were found. (jv phavordsks except one where G. nipponensis was found are connected finally to the Seto Inland Sea or to the Pacific Ocean. The exception was the Fukui-gawa River that flows into Wakasa Bay. (3) P.japonica was found in brooks north of lat.35°N, which are connected to the Japan Sea or to the Seto Inland Sea. (4) All the brooks where A.annandalei was found are located around and above Lake Biwa (This lake itself is a well- known habitat of this species). A.annan- dalei was found also in the waters in rather flat areas near the seas. Studies on what factors bring about the differences in the geographical features between these species should be expected. 998 Ecology, Taxonomy and Systematics EC 14 ADAPTATION AND EVOLUTION OF A BIOLOGICAL TIMING SYSTEM. M. Saigusa. Coll. of Liberal Arts & Sci., Okayama Univ., Okayama ee ————E————E—————eee In my hypothesis about the timing mech- anism of Sesarma larval release rhythm, the light cycle directly entrains the A oscil- lation, and the tidal or moonlight cycle entrains the B oscillation whose phase im- mediately controls the release of larvae. B is, as a driven element, coupled to and phased by A. As the high water moves to the right relative to the light regime, the tidal component follows it up to about 8-9 h from sunset. There would be a limit to the phase-angle difference between A and B, so that when that limit is reached the driven system should be forced to leap back to the original phase, resulting in a solar day component. Larval release rhythm by the Izu popula- tion also can be accounted for by this mod- el, if one would assume that the phase con- Ecol) of Avon) BetsantunctilonmspecmintcEeoma local population: the limit of phase-angle difference between A and B is reached much earlier than the case of Kasaoka population rhythm. In addition, the field data suggest that the |period Jo Behas ay Varee winter individual variability ranging from the value very close to 24 h to about 24.8 h for this population. In other words, Izu population involves females in which the period of Bas) close to) that vor a cimcadian rhythm. EGHS DISTRIBUTIONAL MODES OF STAUROMEDUSAE IN MOBA (SARGASSUM REGION) . VY. Hirano. .Z0ol, insta, .tac. OL, SCitey Hokkaido Univ., Sapporo. Stauromedusae have unique habit in the respect that they never swim freely but attach to seaweeds. Four species occur at Oshoro in Hokkaido; Haliclystus steine- geri, Haliclystus auricula, Stenoscyphus inabai and Sasakiella cruciformis. Comparative investigation was made on the distributional mode of each species. O£ four species, He) auniculaymct ina. bai and Sa. cruciformis occurred sympat- rically in midsummer. H. auricula was predominant at higher part of seaweeds, while St. inabai and Sa. cruciformis were dominant at lower part of them. The major components of preys were identified from gut contents as follows: amphipods and harpacticoids in H. auricula; only harpac- ticoids in St. inabai; gastropods and bi- valves in Sa. cruciformis. It is consid- ered that coexistence of these three species is ensured by the different re- quirements of food and microhabitat. Although H. steinegeri is similar to H. auricula in characteristics described — above, there were seasonal and spatial segregations between the two species. EC 16 THE COMPOSITION AND THE SEASONAL CHANGES OF THE PHYTAL ANIMALS IN THE POTAMOGETON MALAITANUS REGION IN LAKE KITAURA, INFOMMUEISES “OCILG Aliisieg 5 Wele5 Om SCiAo5 blO= kkaido Unive, sapporos) i. Morinoes Depemsen BLOM. 5 Hace Of (SCA, Lbarralsian Uinaiviermel aor A quantitative investigation of the phytal animals living on freshwater sub- merged plant, Potamogeton malaianus was carried out from Dec. 1980 to Dec. 1982, Total individual number of the phytal animals except for Protozoa, Rotatoria, Turbellaria and Aeolosomatidae (Oligochae- ta) fluctuated between 0.5/cm*leaf in Sept ember to 20.4/cn? leaf in January. It kept high level in winter, and rapidly decrea- sed in early spring when old leaves were replaced by new ones. Uherearver.secome low peak was found in summer. Among phy- tal animals, both nematodes and oligo- chaetes predominated in most seasons and had over a half of whole animals in number except for April. The fluctuation of the total individual number of the phytal animals correlated with the abundance of attached matter on leaves which was mostly composed of algae and detritus. In winter, the attached matter on the upper side of leaves formed a thick and loose mat with various spaces, whereas on the under side, it was thin and dense, This mat of attach- ed matter seems to supply not only food but also living space to phytal animals. tS 2 DISTRIBUTION(%) OF LORICA MORPHOTYPES IN ROTIFER,B.PLICATILIS AND ITS RELATIVES. M.Sudzuki.Biol.Lab.,Nihon Daigaku,Omiya Possible morphotypes have been analysed basing on more than 3,000 specimens,i.e. 670 from Estonia(E) ,239 Bangkok(B) ,168 Sin- gapore(S),199 & 166 Nagasaki(N1,N2) ,173 Ka- gawa(K) ,243 Owase(O),151 & 160 Matsuzaka(M 1,M2) ,591 Shizuoka(Z) ,234 Shonai(A)& 222(H anaoka(H) .As regards pectoral margin, they are:hepatotomus Type;98-91% in N2,N1,M2,H, Z,O & Ml1,76-69% in K & S,36% in E,4% in B, mulleri Type;35% in A,8-2%3 in K,E,M1,Z2,H,M 2,B & N2, 13 in S,N & O,decemcornis Type; 3-1% in E,K,O & M2,41% in H.spl Type=tria- ngle laterals + low medians;94% in B,64-56 % in A & E,27% in §,6-2% in O,M1,K & H.sp2 Type=Koste('80)'s fig.3;11%3 in K,4<1%3 in NI & Z. As regards occipital margin:typicus T- ype;100-94% in N1,N2& M1,61% in H,20% in O, «1% in S&E,rotundiformis Type;100-79% in E, B,K,S,M2,0 & Z,38% in H,<1% in N1,sp3=regu- lar triangle;100% in A,10-6% in M2,M1.Such types as found by Murray('13,fig.47a) ,Nog- rady('83,fig.3) were not detected. From the result above,following are considered-:1) specimens reported by Murray,Koste,Nogrady should not be included in plicatilis s.st. 2) rotundiformis could be treated as a goo- d species,3) Such specimens provided with shared characters as mentioned in types sp 1 & sp 3 could be regarded as the one new to science. Ridder's('67,fig.3) &Rodewald's ('37,fig.7) specimens may be considered as aberrant forms of rotundiformis. Taxonomy and Systematics 999 T$.2 ON THE HYDROID, PLUMULARIA FILICAULIS, WHICH FOULS THE BODY SURFACE OF "KOMBU" IN HOKKAIDO, M.Yamada and E.Kobatake. Zool. Inst., Fac. Sci., Hokkaido Univ., Sapporo, and Nat. Inst. of Genetics, Mishima. In these years the body surface of com- mon kelps, Laminaria spp., was fouled by a hydroid along the coasts of Hokkaido. The hydroid was identified as Plumularia filicaulis which was known from southern Japan, and it has been clarified that a northern species, P.undulata, is synonyn- ous with the above species. The specimens from different localities around Hokkaido showed more or less local varieties. Pla- nula larvae were obtained from the colon- ies in Oshoro and others, and the planula attached on a substratum in laboratory and developed to a primary polyp within seve- ral weeks. In some cases the primary polyp gave buds to form stolons or upright stems. Seasonal variation of the trophosome and gonosome and the life-cycle was found. It seems that the change is due to some en- vironmental conditions at the locality. This hydroid species shows a different se- lective preference for the substratum for different locality, but in general it at-— taches on the algal body surface, especi- eiky ion the kelp if it exists ‘there. 1s 3 CHROMOSOMES OF NERITIDAE (MOLLUSCA). H.K.Nakamura. Seto Mar. Biol. Lab., Kyoto Univ., Shirahama, Wakayama. Neritidae is cytogenetically important as the sole archaeogastropod group in which a sex-determining chromosome has been found. Chromosome numbers examined in neritids from Hong Kong(the first 5) and Japan(the last 2) by the air-dry method are as follows(for males, & for females as well; x, sex-determing chromosome; h, he- terochromatic chromosome): Nerita squamu- lata, 2n=22+x/n=llt+th; N. lineata, 2n=23 (probably 22+x)/n=l1l+h; N. polita, 2n=22+x /n=llt+h & 2n=24(=22+xx); N. yoldii, 2n=22 +x/n=ll+h & 2n=22+xx; Dostea violacea, 2n= 20+x/n=10+h & 2n=22 (=20+xx); both Hemine- rita japonica and N. albicila, 2n=22+x/n= lil+h & 2n=22+xx. An allocyclic chromosome is observed in all 7 species in male mei- osis; it reaches the maximum condensation at some stage other than late metaphase, shows heterochromatic staining, and must correspond to the single x-chromosome in male mitotic complements. Besides the Ne- ritidae, a chromosomal sex-determining me- chanism in Prosobranchia has so far been reported only from the more advanced order Mesogastropoda. Non-archaeogastropod cha- racteristics shared by the Neritidae lead some taxonomists to insist on separating this group from the order. Examination of the chromosomal sex determination may afford a clue to the taxonomic and phylo- genetic status of this group. TS 4 TARDIGRADES FOUND IN THE MOSSES OF CITIES OF JAPAN. K.Utsugi. Biol.Dept.,Tokyo Women's Medical College, Tokyo. To examine the presence and the distri- bution of tardigrades, the mosses were collected from 332 locations in 27 cities of Japan. In one city, the collection of mosses grown on soil, rock, concrete or tree trunk were made from 4 to 12 loca- tions, except 131 locations in Tokyo. After macerating these mosses for 3 to more hours, samples of the precipitation were microscopically examined. Several species of tardigrades repre- senting one genus or more genera (Hypsi- bius, Macrobiotus and Milnesium in Eutar- digrada or Echiniscus and Pseudechiniscus in Heterotadigrada) were found in 116 out OEtgs”2 locations an ald. @f: 27) cities. It appears that most of these tardig- rades prefer to mosses grown on neutral or weak alkaline substrates (pH 6.5 to B60) is TS 3 Phylogenetic Analysis of Tropomyosins from Skeletal Muscles of Horseshoe Crabs. Junichi Miyazaki and Tamio Hirabayashi. DHS teow uO Cdn Uns 6 Ol Stk bal, Ibaraki. Skeletal muscle tropomyosins of extant horseshoe crabs, Tachypleus tridentatus (T.t.), Tachypleus gigas (T.g.), Tachypleus rotundicauda (T.r.), and Limulus polyphemus (L.p.), were examined by peptide mapping and amino acid analysis in order to obtain some new data which may be useful for elucidating phylogenetic relationships of the four species. Peptide maps with chymotrypsin, trypsin, cyanogen bromide, and Staphylococcus protease, and amino acid compositions were compared among four species. The results showed that tropomyosins of horseshoe crabs were very similar to one another. Patterns of T.t. and T.g. had especially large similarity, but differed slightly from the pattern of T.r. or L.p. On phylogeny of horseshoe crabs, various studies have shown that American horseshoe crab, L.p., is disparate from three other Asian species. Present results are compatible with those of previous studies. On phylogenetic relationships of Asian species, the present data have suggested that T.t. and T.g. are more similar to each other than to T.r. This seems to be in accord with the relationships indicated by morphological study. 1000 TS=G OCCURRENCE OF TETRAPLOID BISEXUAL SPECIES (PRESUMABLY FACULTATIVE THELYTOKE) IN HAR- VESTMEN (ARACHNIDA, OPILIONES) AND ITS PROBABLE ORIGIN. N. Tsuxrusaka. Zool Gnsit a, Hokkaido Univ., Sapporo. OOo Ox SOleo Leiobunum platypenis and L. globosum are thelytokous (presumably facultative) harvestmen and are widely distributed in northern Japan (Tsurusaki, unpubl.). Based on chromosomal survey of 13 populations, chromosome numbers of these two species were determined as follows: platypenis (6, 2) 2n=24, (22 only) 2n=4x=46-49; globosum (GG) BISen~52, (2) AastbeAoode 7 iG ws remarkable that both male and female of globosum were tetraploid, since polyploid bisexual species are extremely rare in an- imals. Both sexes of the two species show- ed virtually identical sex chromosome com- positions, being XY for the diploid form ial VOM asOie lace) wEimesyollOwcl s@imns5 Wags strongly suggests that the sex determinant in these species is not present on the sex chromosomes but on one (or a pair of) autosome(s). Various kinds of circumstan- tial evidence suggest that globosum has evolved from the tetraploid thelytokous line of platypenis through the change of gene regulation caused by the chromosomal rearrangement. The supposed loss of sex delerminins tune Tlon om isiexerchizomosomes ain the present two species may have been an important prerequisite for the evolution Gil EWES OLLOAGCl loISExMal SpCCLOS o Is 7 ON A FAUNISTIC SURVEY OF THE FRESHWATER HARPACTICOID COPEPODS IN THE TOHOKU DIE SABRC ay Yo Kikuchi Leake Hydirobwole (Stats, hace of Sci., Ibaraki Univ., Itako-machi. In September 1983, a faunistic survey was carried out for the freshwater har- pacticoid copepods in the Tohoku district. Materials were collected from interstitial waters of holes in sand and gravel bars in rivers, and among mosses and water weeds in or nearby stream waters. The rivers, in which a large number of harpacticoid copepods were collected, are as follows: Ohtsubo-gawa (Aomori Pref.), Sukui-gawa (Iwate Pref.), Mabuchi-gawa(Iwate Pref.) and Akiyamasawa-gawa(Miyagi Pref.). The following rivers, where the collected materials involve Parastenocaris sp. are: Ohtaru-gawa(Yamagata Pref.), Kizura-gawa (Yamagata Pref.), Ohyu-kawa(Akita Pref.), Kotorise-gawa(Iwate Pref.), O-kawa(Iwate Pref.) and Akiyamasawa-gawa(Miyagi Pref.). The harpacticoid species collected by the present survey are as follows: Canthocamptidae Canthocamptus mirabilis Bryocamptus hiemalis Bryocamptus zschokkei Epactophanes richardi Attheyella sp. Parastenocaridae Parastenocaris sp. Taxonomy and Systematics IS & NAUPLIUS Y AND CYPRIS Y (PROBLEMATIC CRUSTACEAN LARVAE) FROM JAPAN. T. It6. Seto Mar. Biol. Lab., Univ., Shirahama, Wakayama. Kyoto Three types of nauplius y and three types of cypris y are reported from the North Pacific. Two types of nauplius y are similar to the types I and IV des- cribed by Hansen (1899) in the patterns of the mesh-like structure of cephalic shield. The other type of nauplius y has been previously unknown. A nauplius which was similar to Hansen's type IV molted into a cypris y in the laboratory, with leaving a series of naupliar exuviae. This cypris obtained was of the type des- cribed by It6 and Ohtsuka (1984). A cypris y of a previously unknown type had a pair of frontal filaments, each asso- ciated with a compound eye. Similar fron- tal filaments associated with compound eyes have already been known in an asco- thoracid larva from St. Croix (Grygier, 1983). This fact supports the view that Hansen's y crustacean relates with Asco- thoracida, though clear differences are still present not only between them but also between y crustacean and Cirripedia. The carapace of the known cypris y is not bivalved, and the cypris y with frontal filaments had also some ribbon-like fila- ments near its oral pyramid. These characteristics have been found in neither ascothoracid larvae nor cirriped cypris larvae. Is-g TAXONOMY AND GEOGRAPHICAL DISTRIBUTIONS OF SEASHORE INHABITING PHILOSCIIDS (CRUSTACEA ; ISOPODA) IN JAPAN. N.Nunomura. Toyama Science Museum, Toyama. About 80 specimens of red-coloured sea- shore inhabiting philosciids from 15 loca- Iiigies in Japan were collcctiedrasUinvaslmmnenE only 3 species of this genus have been known from various parts of the world. At closer examinations, they proved sto represent two species of the genus Litto- rophiloscia. Specimens collected from Osaka are obviously separated from the others by their paler colour, having a small concavity at the tip of pennes, shape of pleotelson and so on. This spec- ies seems to occupy a rather peculiar position. Another species can be divided into 3 types by, thes number Of iteevh won mt hcmetemmoie maxilla and presence of spines on the male first pleopod:10+type is distributed from Niigata to Oki Island and Hachijo Island; Q+type is distributed at Wakayama, Miya- zaki and Miyake Island; 9-type is distri- buted from Aomori to Kanagawa. From the results obtained it is assumed that they were Specialized into 3 types from the same ancestor, and 10+type is most primi- tive and 9-type is most advanced. This species is considered to be most closely related to L.richardsonae from the west coast of North America. Taxonomy and Systematics 1001 TS 10 DEEP SEA AMPHIPODA OF THE GENUS LEPECHINE- LLA (CRUSTACEA) FROM JAPAN. Se) Gam6. Dept: of Biol:,; Fac. of Educ., Yokohama Natl. Univ., Yokohama. In the genus Lepechinella (Dexaminidae) about thirty species have been described. All the species are collected from the abyssal depths and their body is measured about 5 - 10 mm long, very fragile and easily broken. Only one species, L. saga- miensis Gam6, 1981, has been recorded from Japan (704 m deep). By careful examination of the specimens taken from the abyssal depths in Sagami Bay (950 m deep, by the survay Of the Kanagawa Pref. Fish. Exper. Sta., 1980, and 1680 m, by the cruise KH- 80-1 of R/V Hakuho-Maru of the Ocean Res. Inst., Univ. of Tokyo, 1980), Suruga Bay (700 - 740 m, by the cruise KT-83-18 of R/V Tansei-Maru of the Ocean Res. Inst., Univ. of Tokyo, 1983) and off the Sanriku coast (1890 - 5370 m, by the cruises KH- 81-4 of R/V Hakuho-Maru, 1981, and KT-84-9 of R/V Tansei-Maru, 1984), eight species were newly found. These species may be distinguished from the related species by having the different armatures on the body and the characteristic shape of the coxal plates. The species may be new to science. TS 1l LARVAL DEVELOPMENT OF THE STONE CRAB, HAPLAOGASTER DENTATA (DE HAAN, 1844)(CRU- STACEA: ANOMURA: LITHODIDAE) REARED IN THE LABORATORY. K. Konishi. Z00i1. inst., Fac. .of .Sci., Hokkaido Univ., Sapporo. The larvae of lithodid anomurans, espe- cially in the members of the subfamily Hapalogastrinae are not well known. The complete larval development of Hapalo- gaster dentata, a stone crab occurring on the southwestern coast of Hokkaido, is described based on larvae reared in the laboratory. This species passes through four zoeal stages and one megalopa ('glau- cothoe'). At temperature of 15°C and sali- nity of 35 °/oo, the development through four zoeal stages to the first crab takes at least 30 days. The zoeal and megalopal stages of H. dentata are compared with all known cultured species of the Lithodidae and morphological differences between two subfamilies are noted in zoeal instar and carapace armatures of megalopae. The zoeae of the present species resemble those of Dermaturus mandtii, but they are distin- guished by carapace length and setation of antennal scale and uropods. Main larval characteristics of the present zoeae are very much similar to those of "Species C", an unidentified planktonic lithodid zoea described by Kurata (1964). In view of larval evidence, taxonomic and phylo- genetic relationships between the Lithod- idae and the Paguridae are also discussed. TS 12 HERMAPHRODITE SPECIMENS OF THE FRESHWATER CRAB, GEOTHELPHUSA DEHAANI (WHITE). S. Gam6* and K. Sugawara’. 'Dept. of Biol., Fac. of Educ., Yokohama Natl. Univ., Yokohama and *?Yugawara High Sch., Yugawara. Three anomalous male with ordinary male external characters, female genital pores and some rudimentary female pleopods were found in a collection of the freshwater crab, Geothelphusa dehaani (White), exam- ined for the study on the intraspecific geographical variations in the southern part of Japan. No parasite could be found in these specimens. One of them was dis- sected. The gonads on each side contain both male and reduced female portions and are connected to the gonopores in the usu- al way. The ovotestes, which are found in a specimen bearing the external male char- acters in the normal condition by Matsu- moto (1955), could not be observed in the present specimen. The reason for the her- maphroditism is not clear. The other two specimens are reserved in further studies. 1S 13 LARVA OF THE AUSTRALIAN SHORE CRAB, CYCLO- GRAPSUS GRANULOSUS H. MILNE-EDWARDS (CRU- STACEA, GRAPSIDAE, SESARMINAE) K. Muraoka. Kanagawa Pref. Museum, Yokohama The author had an opportunity of resea- rch on the larvae of the Australian ende- mic shore crab, Cyclograpsus granulosus during the cruise of R.V. Hakuhd Maru of the Ocean Res. Inst. Univ. of Tokyo. The ovigerous females were collected from the upper intertidal region, at Hoba- rt, Tasmania, in January 1984. They were kept in an aquarium with until the larvae hatched. The major characteristics of the first zoea are as follows. The cephalotho- rax has a dorsal and rostral spines, and a pair of lateral spines. The bisegmented endcepodite of maxillule bears 1 seta on the first segment and 5 setae on the dis- tal segment. The bilobated endopodite of maxilla bears 2 setae on each lobe. The setation of endopodite of the first and second maxillipeds is 2,2,1,2,5 and O,1,6 respectively. The abdomen has 5 segments and telson. A pair of lateral knobs is present in the second and third abdominal segments. The larvae of the genus Cyclograpsus have been described for the five species: C. sinereus, C. insularum, C. lavauxi, C. punctatus and C. intermedius. The first zoea of the present species differs in mi- nor features in comparison with the larvae previously described. 1002 Taxonomy and Systematics TS 14 BIOCHEMICAL PHYLOGENY OF THE SEA-URCHINS OF THE FAMILY TOXOPNEUSTIDAE, N. Matsuoka, Dept. of Biol.,Fac. of Sci., Hirosaki Univ., Hirosaki, (1) The family Toxopneustidae in the seas around Japan is represented by four species of sea-urchins, They are Toxo- pneustes pileolus, Tripneustes gratilla, Pseudoboletia maculata and Pseudocentro- tus depressus, (2) The phylogenetic relationships among these four members of this family were investigated by electrophoretic analyses of 13 enzymes, (3) The biochemical phylogenetic tree for these species constructed from Nei’s (1972) genetic distances suggests that Pseudoboletia maculata is closely relat- ed to Toxopneustes pileolus, and that Pseudocentrotus depressus is strongly differentiated from the other three species, (4) These results agree with the phylogenetic relationships of these species inferred from morphological, zoogeographical and immunological evi- dence and show the phylogenetic value of electrophoretic data at the genus level, TS 15 THREE NEW SPECIES OF THE GENUS BOTRYL- LOIDES FROM THE VICINITY OF SHIMODA,. Y. Saito and H. Watanabe. Shimoda Mar. Res. Ctr., Univ. of Tsukuba, Shimoda. Most of Japanese botryllid ascidians seem to resemble one another in morphology of their colonies and zooids. Therefore, it is difficult to distinguish respective species exactly on the preserved specimens that are devoid of gonads in zooids, Thus, we cultured several botryllids collected from the vicinity of the Shimoda Marine Research Center all through the year in the cove near the Center, and made observations on the details of their life history. This work enabled us to classify them into some distinct species inclusive of new species. This time, we presented three new species of the genus Botrylloides, and reported their morphology and life history in detail. Further, comparing these three new species and the two already estab- lished species, B. simodensis and B, violaceus, one another, we pointed out the taxonomic significance of the manner of the sexual reproduction in botryllids. Additionally, from the results of the observations on the details of the sexual reproduction in the above five species of Botrylloides, the outline of the evolution of the manner of the sexual reproduction in this genus became clearer. That is, the manner may be progressing gradually from ovoviviparity to viviparity. TS LO PHYLETIC LINE AND KARYOTYPE EVOLUTION OF THE THAI MEDAKA, ORYZIAS MINUTILLUS. W. Magtoon’ and H. Uwa’,*Fac. of Sci., Sri- nakharinwirot Univ. at Bangkhen, Bangkok and ’Fac. of Sci., Sinshu Univ., Matsumoto. The karyotype of O. minutillus (Bangkhen) revealed 2n=34 chromosomes consisting of 4 meta-, 1 submeta-, and 12 acrocentric pairs (NF=44). It was characterized by having "large" 4 metacentric chromosome pairs which seem to have occurred through Robertsonian centric fusion. The small submetacentric pair manifested satellites on the short arms. O. minutillus is widely distributed in central and northern Thailand along the Chao Phraya river. Karyotypical variation was detected between specimens from Bangkhen and Chieng Mai. Genus Oryzias so far studied have been karyotypically divided into three groups: Monoarmed (O. melastigma and O. javanicus), biarmed (O. curvinotus, O. luzonensis and O27 latipes), and fused (Oe wcellebensits) chromosome type, which are distributed in southwest Asia and the west side of south- east Asia, east Asia, and the Sulawesi, respectively. O. minutillus was judged to be of fused type and its karyotype was similar to that of O. celebensis. Interspecific hybridization studies also revealed that O. minutillus was more compatible with O. celebensis than the geographically neighbouring O. javanicus (Jakarta) and O. sp. (northeast Thailand). US iy MORPHOLOGICAL STUDY OF JAPANESE STILL TYPE SALAMANDER(HYNOBIUS). H.Nambu. Toyama Science Museum, Toyama. Morphological features of H.lichenatus from Aomori, Yamagata, Miyagi, Niigata, Gunma and Tochigi, Hynobius sp. from Toyama, H.n.tokyoensis from Ibaragi and Aichi, H.n.nebulosus from Okayama, Tottori and Nagasaki, and H.abei from Kyoto (Sato, 1934) were studied. H.lichenatus from various localities shows slight variation of skull and body features, and is distinguishable from other species. H.abei is distinguishable from other species by its characteristic skull feature. H.n.nebulosus from 3 local- ities shows a variation of skull and body features. H.n.tokyoensis is distinguish- able from H.n.nebulosus by its skull fea- ture. Hynobius sp., having an unique skull feature, is distinguishable from other species. This species is considered to be most closely related to H.n.tokyoensis.The position of lumbar vertebra of H,.lichen- atus, Hynobius sp, and H.n.nebulosus from Tottori is at the 17th vertebra, but that of H.n.tokyoensis and H.n.nebulosus from Okayama and Nagasaki is at the 18th. It is considered that the position of lumbar vertebra is influenced by their locality. From the results obtained,it is assumed that the Japanese still type salamanders were specialized into separate form in 5 areas from a common ancestor. Taxonomy and TS 18 BIOCHEMICAL DIFFERENTIATION OF THE TREE- FROG GROUP DISTRIBUTED IN THE FAR EAST M. Nishioka and M. Sumida. Lab. for Amphibian Biol., Fac. of Sci., Hiroshima Univ., Hiroshima. In order to elucidate the evolutional relationship among the members of the tree- frog group distributed in the Far East, 14 kinds of enzymes extracted from skeletal muscles and liver and four kinds of blood proteins were analyzed by the starch-gel electrophoresis in a total of 168 frogs in- cluding Hyla arborea japonica from Sapporo, Tsushima and Hiroshima, Japan, and Suigen, Korea, H. hallowelli from Amami-oshima, Japan and H. chinensis from Taiwan. The results showed that these enzymes and pro- teins are controlled by 26 loci. Of the lat- ter, AAT-A locus is occupied by two alleles, while MPI, ADA, Pep-D and Ab loci are occu- pied by six to eight multiple alleles which reveal 10-14 phenotypes and show that the tree-frogs are polymorphic at ahigh level. The rate of heterozygotes, rate of loci with multiple alleles and number of alleles per locus in the tree-frog group were 12.0%, 39.8% and 1.53 on the average, respectively. When the genetic distances are calculated according to Nei (1972), it was found that H. chinensis is more remotely separated from H. a. japonica than H. hallowelli is, while H. chinensis is closely related to H. hallowelli. The four populations of H. a. japonica are very closely related to one another, as expected. TS 19 CESTODE FAUNA OF CAVE BATS IN NANSET SHOTO. I. Sawada. Biol. Lab., Nara Univ. of Educ., Nara. Cave bats in Nansei Shoto were col- lected to classify the systematic posi- tion of chiroperan cestodes. Myotis macro- Ss dactylus in Tanegashima was infected with Vampirolepis tanegashimensis, R.ferrumequ- inum nippon in Yakushima with Hymenolepis nishidai, R.cornutus orii in Amamioshima and Tokunoshima with V. isensis peculiar to R.c.cornutus, R.perditus in Ishigaki- shima with V.isensis and V.iriomotensis, R.imaizumii in Iriomoteshima with V.isen- sis and V.iriomotensis. Miniopterus schre- ibersii blepotis in Ishigakishiam and Iri- omoteshima were infected with V.hidaensis peculiar to M.s.fuliginosus. — From the standpoint of bat's cestode fauna the following distributional conne- ctions among bats are presumed: (1)M.mac- rodactylus in Tanegashima has intercommu- nications with M.macrodactylus in Fukui Pref., (2)R.ferrumeguinum nippon in Yaku- shima with R.f.nippon in Shikoku, (3) R. rditus in Ishigakishima and R.imaizumii in Iriomoteshima with R.c.cornutus in va- rious places of Japan, (4) M.s.blepotis in Ishigakishima and Iriomoteshima with M.s.fuliginosus in various places of Japan. Systematics 1003 S520 KARYOSYSTEMATICS OF THE SORICINE INSECTI- VORES OF JAPAN, WITH SPECIAL COMMENTS ON THE PHYLOGENETIC POSITION OF THE JAPANESE WATER SHREW, CHIMARROGALE P. PLATYCEPHALA. Ye Obara “and Ts Tada. “Dept. of°8Biol. f Fac. Of oCi., Hirosaki Univ., Hirosaki. Karyotypes of seven taxa of the Japa- nese shrews (Soricinae: Sorex gracillimus, S. shinto shinto, S. s. saevus, S. ungui- culatus; Crocidurinae: Crocidura dsinezumi chisai, Suncus murinus riukiuanus, Chimar- rogale platycephala platycephala) were comparatively analyzed in order to inquire into their systematic relationships from a karyological standpoint. The karyotypes of the Japanese water shrew, Chimarrogale p. platycephala, were presented for the first time in the present report. They were quite heterogeneous, in both numerical and constitutional aspects, as compared with those of either subfamily studied, and closely resembled those of the European water shrew, Neomys fodiens, studied by Fredga and Levan (1969). These findings strongly suggest that both Japanese and European water shrews are closely related each other, and hence their relationship could be well explained by not "polyphyle- tic" but "monophyletic" differentiation. Therefore, the current taxonomic system in which the former is included in Croci- durinae and the latter in Soricinae should be revised. Ts 21 PHYLOGENETIC RELATIONSHIPS OF SIX TAXA OF THE JAPANESE VOLES REVEALED BY KARYOLOGI- CAL AND BIOCHEMICAL TECHNIQUES. I. Yoshida, Y. Obara and N. Matsuoka. Dep ol Biol, hac. Of Scie, Hirosaki Univ., Hirosaki. Chromosomes and 15 different enzyme sys- tems of six taxa of the Japanese voles ( Clethrionomys rutilus mikado, C. rufocanus bedfordiae, C. montanus, C. andersoni an- dersoni, C. a. niigatae and Eothenomys smi- thi) were examined to establish their phy- Togenetic relationships, making use of dif- ferential staining (G- and C-banding) and electrophoretic techniques. While interspecific variations in the size of C-bands were observed only in the Y chromosomes, almost perfect G-band homo- logy as well as the karyotypic similarity were found among six species studied, indi- cating that all of the microtine species studied here are regarded as descendants from a common ancestral form, hence being closely related each other. The biochemi- cal dendrogram based on the Nei's genetic distances supported the taxonomic system presented by Corbet (1978) at the genus level, but not at the species level. The dendrogram obtained here practically dif- fered from any of the taxonomic systems so far proposed. Thus, we propose here, from both karyological and biochemical stand- points, a revised system of taxonomy of the Japanese voles. 1004 Taxonomy and Systematics -, C- AND N-BANDING ANALYSES IN THE CHRO- MOSOMES OF THE JAPANESE GRASS VOLE, MICRO- TUS M. MONTEBELLI, WITH SPECIAL ATTENTION TO THE KARYOTYPIC COMPARISON WITH THE ROOT VOLE, M. OECONOMUS . 3 K. Yamakage, N. Nakayashikit J. Hasegawa Brae) Wa Cloaitag IDs OP WiGilles HAG s Oi SCie 5) Hamosakes Umi.) eheaOsa Len (Gk Dept. of Legal Med., Sch. of Med., Iwate Med. Univ., Morioka, ey Deo Ow lays WiScl, 5 Cieaclo SElac of Envy. Sci., Hokkaido Univ., Sapporo) The chromosomes of the Japanese grass vole, Microtus m. montebelli, were examined in detail by G-, C- and N-banding methods, paying special attention to the karyotypic comparison with the root vole, Microtus oeconomus studied by Fredga et al. (1980). jInterspecifie comparison of G—, C- and N-bands confirmed that (1) the Japanese grass vole and the root vole show almost perfect G-band homology in all of their chromosomes, excluding the pericentromeric region of the X, and this is also the case with the C-bandang, spavvernus. —§ (2) vine x chromosome of the Japanese grass vole is io] ines “AS Neieee As wie Of alas 2OOW WOLS Aiacl tiais As AwinelioMealole wo wae GClilitq ference in the size of C-band, and (3) the GUSTING LOM PACESION OW NOMS @vElieS Gla icieeies) in these two species and this discrepancy seems to be caused by the translocation and inactivation of NORs. These findings strongly suggest that the Japanese grass vole may share a common ancestor with the root vole as closely related species. Is COMPARISON OF BLOOD PROTEIN BETWEEN KOREAN WEASEL (MUSTELA SIBIRICA) AND JAPANESE WEASEL (MUSTELA ITATST). S.Watanabe. Dep.Zool.Fac.Sci.Kyoto Univ., Kyoto and Y.Kawamoto. Primates Res.Inst. Kyoto Univ., Inuyama. Horizontal starch and polyacrilamid gel electrophoresis was practiced to detect the differences in blood protein between Korean weasel and Japanese weasel. The number of samples examined was 8 (M. sibirica, from Kyoto) and 12 (M. itatsi, from Ishikawa) respectively. Each sample was identified with one of the two species according to the coloration of fur and the tail ratio. These species differ from each other in Hemoglobin (Hb), Albumin, Post and Pre-Albumin, Transferrin, Leucine Aminopeptidase (LAP). Only LAP showed intraspecific variation, and Hb offered the most distinct interspecific differ- ence; there was only one band in Hb of Korean weasel while two bands were ap- peared in that of Japanese weasel. Further analysis in A and B chain of Hb revealed the possibility of gene dupli- cation, and these results didn't agree with the opinion to assign Japanese weasel to a subspecies of Korean weasel (Imaizumi, 1960). TS 24 MORPHOGENESIS AND SUBSTRATUM ENVIRONMENT OF THE POLYP AND THE MEDUSA OF THE CYTAEIS SP. Y. Kakinuma and K. Yano*. Dept. of Biol., Fac. of Sci., Univ. of Kagoshima and *Kagoshima Junshin Girls' H. S., Kagoshima. The Cytaeis sp. live symbiotically on the host shells of the Niotha livescens. By examining the interspecific relation be- tween Cytaeis sp. and N. livescens, it was concluded that the morphogenesis of polyp, stolon and medusa among the Cytaeis sp. depended upon the physiological conditions of living N. livescens and was accelerated or controlled by the changed of the substratum environment. Especially it was observed that the formation of the polyp closely related to groups of microorganisms which exuberated characteristically in a secretion of the living N. livescens before the breeding season. The existence of microorganisms of type A bacteria had an effect on the increase of the polyp. In the breeding season, the secretion was restrained. This caused the formation and swimming out of the buds of medusa, while the number of the polyp decreasing when the condition of substratum was changed by removing part of soft body of Ne Eve's Cems) arg itastelc itallelaye We found the budding formation of medusa was from ten to twenty times greater than under the normal condition. Such an ab- normal generation seemed to be occurred by the abnormal condition of the substratum. IS 2 DIFFERENTIATION OF PULMONARY ALVEOLAR EPITHELIAL CELLS IN SOME LOWER VERTEBRATES. T.Gomi, A.Kimura, H.Tsuchiya, T.Hashimoto and *H.Fujita. Department of Anatomy, *Laboratory of Electron Microscope, Toho University School of Medicine, Tokyo. 1) Lungfish and Urodela The cells in these animals are of only one type, with the characteristics of Type 1 cells and Type II cells, and no morphological and functional specialization has yet occurred. 2) Anura and Reptile In these animals, a clear differentiation into Type I cells of the squamous lining type, and Type II cells of the cuboidal secretory type is observed. al) IOS IF CSllss Hasse cells are comparatively small, and thin cytoplasmic projections covered capillaries. The blood-air barrier is composed of the epithelium, basement membrane and endothelial cell. Organelles are hardly observable in the cytoplasm, and there is no osmiophilic lamellated body. id))) UNWjexer de ColIlse Waese Collis ise welaeie large. Mitochondria, r-ER, multivesicular body and Golgi apparatus as well as osmiophilic lamellated body specific to these cells exist in the cytoplasm. It is considered from these findings that alveolar epithelial cells begin to differentiate phylogenically in Anura. Taxonomy and Systematics, Miscellaneous 1005 TS 26 VARIATION OF AYU, PLECOGLOSSUS ALTIVELIS (TEREOSTEI, OSMERIDAE), FROM VIEWPOINTS OF TERRITORIALITY, MORPHOLOGY AND ISOZYME PAT- TERNS. H. Kawanabe and M. Nishida. Dept. of Zo- ol., Kyoto Univ., Kyoto, and Dept. of Mar. Sci., Univ. of Ryukyus, Okinawa. The variation of territoriality among Ayu populations was firstly recognized by the senior author in 1970, and he distin- guished into Ryukyu-, Kyoto- and Biwa-ty- pes. The first one is restricted to live in Okinawa and Amami Islands (26-28.5°N), the third only in/around Lake Biwa (35- 35.5°N), and the second is widely distri- buted from Yaku to Hokkaido Islands as well as the southern part of Korean Peninsula (30.5-40°N). Recently the junior author examined some morphological characters and isozyme patterns by electrophoretic analy- Sis for the all three types. He has dis- covered that Ryukyu-type is completely dif- ferent from the other two types in both morphology as well as isozyme patterns and Biwa-type is genetically quite similar to Kyoto-type. Ryukyu-type of Ayu should be put in different subspecies, and will be descrived as P. altivelis ryukyuensis by the junior author. MI 1 OSHORO MARINE BIOLOGICAL STATION, HOKKAIDO UNIVERSITY, IN VIEW OF ITS HISTORY. F. Iwata. Zool. Inst., Hokkaido Univ., Sapporo Two remarkable events occurred at this station have enforced us to inform a nec- essity of prosperity for future develop- ment: one on the publication of a list of 79 contributed papers, of which 60 were offered within the last 10 years (1974- 1983); the other on the record of some 2800 users in yearly average for the same period. This station was founded as an affiliated laboratory of the Agricultural College, Tohoku Imp. Univ. in Sendai in 1908 and controlled until 1918. The mana- gement was succeeded as follows: Agricul- tural College, Hokkaido Imp. Univ. in Sapporo (1918-1935); Executive office (1935-1940); Faculty of Agriculture (1940- 1949); Faculty of Fisheries, Hokkaido Univ. (1949-1971); five departments of the Hokkaido University for common usage (1971-). The 79 papers, however, include 35 for taxonomy and morphology, 22 for reproduction and development, 17 for phy- Siology and ecology, and 5 for biochemi- stry. In conclusion, it is expected to reinforce this station in management and equipment for the education and research on marine biology. Request for a list of the contributed papers is addressed to the Director of the Oshoro Marine Biological Station, c/o Faculty of Science, Hokkaido University, Sapporo 060, Japan. 1006 Physiology, Cell Biology PH 118 Y + MRAM GS RCIRL CF ME OBR. MAK RE SRM (ARK: £%) RELATIONSHIP HSETWEEN CFF—VALUE AND COMPONENTS OF RECEPTOR POTENTIAL IN THE MANTIS SHRIMP COMPUND EYE. Y. Okuno and T. Yanase. (Dept. of Biol. Osaka Kyoiku Univ. Osaka ) YY IB—-RMMK, MRK SATHSHOSRAMAMR (CFF) it 20~SOHZOMCRHARSHS. ARRCIM—HARHOLSR BOWSSHVGBS COM (CF Ftransient)¢, TOROLHERROK (C F Fplateau) XBXZUE. CFF (t)lt#IV75HzT, CFF (p) KEVZ2Z29OHzMVMCHOE. CFF(t)/RUCFF(P)OML, M—H* MRI HTARBRCO 6 RREOMOMBRRELOOMKODWTRD te. GBRiId, onFF(a) te plateaums (b)OMY (VI RUEDH (b/a)ze, onFOWSH MY (c), of fF¥O HM (d)RUtransientH 5 plateausB~ DBA (e) ORE (aV/sec)e CHS. CEFF (t)ttb/a, dic, CF F (p) lid, b/a, b KRMVAMe mL, 8s RU e LEDKHAMBMRD SHED ask. CFF(t)MACKF(P)KVAMOMWOlcT, COMlEdb, d, b/aC bok. a : On transient (mv) b: Plateau (mv) bi c: Attack Speed (mv/s ) d e : Recovery Speed (mv/s) : Bent (mv/s) CF F( transient) eee ae CFF (plateau) + |++1 |+4 ——— (0.70) | (0.80) | (0.30) | (0.83) PH 119 = Se %lJS manubrium hair OMPMiEMSA. skal - BAR te (ESK- ee at) BAM i (AbXK- = - BS AB) MECHANORECEPTION OF MANUBRIUM HAIR ON PRETASUS IN THE HONEY BEE. S.Shigeta, K.Aoki and ‘H.Okumura. Life Sci. Inst., Sophia Univ., Tokyo. ‘Akkeshi Marine Biol. Station, Hokkaido Univ., Akkeshi. SY NF EMIT HT UC RIET SCEDBAMSNTWS. PANS, FAIS SP EICIOO~1 KHz OFFA SFRSEY, DY INF DS ARMECKRRIC TES CER ABUTWS. BAB —mICeEST ST HEHO manubrium hair (CHE LC, BRHEDRZAOBEIC DWC, BABCTERH DS aaN7C ) PASTA ERRME DO DAINY TGS eA T SE, WL DDOGSRR-VUBBSNkK. Arr FIGESISES REO fea Ros Cle. RUBS AT BG LI, EI, 100Hz DL FO RISRE TIS, KOBMSIGANR-LVe ZN UTE o CORSEOM Mme =SBSFAMAmli ko CHMNK.A aOFZRR IS. ITO RR SAAD 5 KO. EDORED 10T DOMES (9+2 OMNES) PP. TEDEMDOMEAX BRT ACeEDCSEK. CNODKERI2, (HO Rw eee, Midi -BTS,. CNHECIFITIIAH¢CORSAMSew ULTWTEWD. CORRS AIE ODO Femmes. JFT7DWICAO AAT RRR CBORAMCHRUTWSOHDEFZAZAS5NSG. CB 47 Euplotes woodruffi (#Em) CBI AZRKRRWOR SI PKITT HORRORS . BR RAN (CREA > He Wi) EFFECT OF FOOD CONDITIONS ON THE LENGTH OF IM- MATURITY IN EUPLOTES WOODRUFFI ( CILIOPHORA ). T. Kosaka, Zool, Inst., Fac. of Sci,, Hiroshima Univ,, Hiroshima, HEKT-6O APES RW OAKILEREMWMOA CHS. 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T, Murakami, Zool, Inst,, Fac. of Sci,., Hiroshima Univ,, Hiroshima, MEMILAKEOR REM (BM) 7U-Ticesy ARYA-44 AO? Bodice Mi e23C SC 3ADR St, U7 Mia~z30, 25, 23, 20, 18, loeor BEICBMLK. S24MH CHHBEBAL, UFO RAS Mie zr-—Y (6.°S-D)O HSI, AROBRIHEC, FAM BRPOMM MBE DoORD?e. 30C Clete NH ld 8. 6hr (6, - SO Decay ome) oo Gy 5 yo Wate reles Obie (ug) © NOS ey Pere eeu ie CH onto 3 Yh) 4 20 Oeil, Wing (CLR) oto WD) US) Me Te lesa, One \ 40 -49-11%) , 15 Gld28. Thr (38 - 50-12% ) CHS, KiB CIHRBH OS °C HRL, SHR ODWMOMMTHHGREB< 24. SHECORMO FRMMls, ENSHNIIMDR (148), 41 (18) , MA (BO) 5 Ce (BO) 5 BO Ce), SY (oe) eGiaD . IIS EHMER LTS, LDL, BHtTI7UYO Flew ZIAD A4ON, @70YHRICIITS ARH It, BRMIUDRARMCHMET, 20. 1- 23.7% GC—-HEUTWSA, 1RU1IBC CH, Balsa BOA CH ( 190H DR) , MHTHRECLASG MxHniW, CNMS#BCKBTE, 0O-1LEDR Ric BECAMIKAS. SRHOERMTLTADOEF ls (82%) © Cell Biology, Genetics, Biochemistry 1007 CB 49 Hit Euplotes harpa OB MHALVYALBRMRIE 0 o G@ABZ(KBA:H- wD) SELFING AND ENCYSTMENT IN EUPLOTES HARPA ( CI- LIOPHORA ). H. Kanemoto, Zool, Inst,, Fac. of Sci., Hiroshima Univ,, Hiroshima, AkmMmioORMLeRRI, YARBRL, BK HSATATIAC EMH Ds *CKOT, FORWUEOS EMOWTHRETS. (DBRBLKIIROSTCEAR BAMBMRAN*?. 4HESWCHKHSANTUO Mls, SZRBACHLIUKeEHMAKR. BKHEE STRAOLESI, 60,77, 80, 100% THK. HZRDOOZSBKHAGHEIT7Z0UVAERWT, AR W(BRHCHH) CBKHEAHETANK. USE, ERBEICCOHABLRH 1] CHS. RUOBK #Si2. 266 (BKRHSHI22%) , 286 (10%) . 326 (44%) PDRKRICRMBSHNK. COFFKI, #A295,90. 100% CHsk. COCetKUIFOUYRM HPI OMMDROKAMMAEROCLERT. BK HSHit, MBLC OIERHTS. (2)Mfalt, RP DIHBPDSCAAMKHBEMORWOKREAEFKS MnNSZt, 2~3 BRK YAKABCRMISZ. YAP i. PUOMRRMICBMAT AL RP HI CRMY AST Bo CHECOMRTEH, YAUMHAELALG WYKACHS. (3/3, 10. 18, 24, HADRRKOM BAYT AURBEBRA FRSC EL, BHRETCE $213, 3,2, 3, 84OMMCYALERMS on, Br zt iz, 100 . 83, 100. 100, 92% CHak. CDC LWABRARM CYA} ome HEATAILCLRRT. CB 50 CAY OV LY ORCC SU ZRARROMMBE . NB #— GFRK: RR tH) The fine structure of the contact area during Conjugation in Paramecium tetraurelia. Y. Yashima. Dept. of Biol., School of Lib. Arts & Sci., Iwate Med. Univ., Morioka. ROADRARERKASNAET EROS We +m ARR FLDGR AE CHEMI NART EHR LK. BHis Paramecium tetraurelia, &51 (RAH, WD +/e LK. BRE HE RADABESSonneborn(1970) DHE ZBALK. RADOASRBMARDPOSRACK SUCH TIDES AECOM(G~5.5 BHD OY OV Eve SOsBERVYUGLA. MEK LAA 7CL HH. AK. Spurr SABA SAY. MMU LEY. DFY CBO_—BRALTH. BAVEABCHELK. RAB %#1.5 RHORACCORARER CABBAD BAK BEnree, tOElz 0.lus tHok. LMLETE EOE PAI LALRB ANZ k. PRB BlsHeDECKONTKSE (RY. BARR 4 CRAKSEL 0.748 tOHoKk. EF ERO RM wWAls MRRAKAELK. tOR. BABAOMTAZET. GRBANECETFEED RA WHAls 4H CIZLEALE BD5@pok. ULDRRDS,. ETEROR wA eDe sx 5nd. ARR OMB P. multinucieatum> P. caudutuam lum KE ATFOLAEFEMIAD, Tetrahysena DM 0.2ue FEY SKFMOK. GE 16 ASS KMYOCPRMERPREE (W.A.) EMRE. BARR + WAG 14 Fe CUBA + BE EB) THE ISOLATED MITOTIC APPARATUS AND CHROMOSOMES OF TUBIFEX EGGS (AQUATIC OLIGOCHAETE, ANNELIDA). M. Matsumoto and Y. Abe. Dept. of Biol., Fac. of Sci., Univ. of Yamagata. Yamagata. ELTNEBDIADRMOMIF CEM FAL, MAL SMORENMA DHL ZAI De SEABRARD REMY & BIC, EAR TOMDS 104K MA CINE OE LK. SRBELHOI DSM) MLRIOLMME &) BAH DS EFIRCLAENY RCIERVINF FORM CREM LB SRD > SNR & hh LR ERO RH, IED "TE)ICB LR. ROARED ARR MAR ATH File DFNMINT NWT & AERA tv CHELK. IF % FEARD 1B] BO fe Bi BEA S RIA DAKAR AES thal f- bivalentis#/160THS. OFFDINAINADMABRMCAN (£2.30 ACH FP RIDE RRO 2A DRT LC OFFM SND 2A CH NIL2.50 Hic Aa RAK LR E BD BREADS MEINS. PATH) LROKREEAH DH CTIUCHMA RE RAEIL LEVI AE (SS. PORE BRE td LCE ORR IE MS 3 HEH 1D3.40' ~ 3.50 OPM HC EEC ME DS REL PRADA d ME HHILR PRLS. LHS MRLEKRMOMRBAD Oi senes JRA AAA, VACHMENADIKR > RMOM ME CHEISIATS. HBKAROM.A. SC OPP HON. A. NMELDRC, COBRA O B1LIAMD 5 ERDOM MAMAS AEDERMHHNS. UELEDI EDS BIMMOBAT MEISHHDIOTC, TOMICREOKKEKOMIC—Alst Bee HAs BS tHe RPM PMOLERRDEL FREaCTBMEN, SHCRMC lL 7 MAREK ILI RIRBOBRUMRSRE THSRICBdHNS. 6.30’'~ 8.20’ DR 2SNM Mir lk, AB MRD 5 PHAADA, CLARI|RAS — MNOBKE LEMMAS LUBED MENK. BI 61 FT PUD BBALRBRISBIS B- FTI=VITIONT HIG OA - eK (RA: B- £M) THE FORM OFB —~ ALANINE INCORPORATED INTO THE PALE YELLOW PIGMENT OF PAPILIO XUTHUS Dept. of Biol., Fac. of Sci. , Univ. of Kanazawa. Kanazawa. FLT PND ED BS & KF GAS (cK OKynurenine ene alanyldopamineDHGMWTHSCeMMSDADINC Ahi, (DSBEOECMMBLASKROHO, S75 FOKynu renineéG- alanineds#~ 5 dkynureninels hahha THES TERE CARER TSDC AD BREST SCEMMSMITSN 7c —F7Z - alanine [a] BCA IC ERED KS ISE — 7 BHA SPAGICDAORADMETELTC. CDREHOG alanine## 3M T Hac. (AVR Ae BOTCATHRAVISTL Rio- gel P-2 DHFGAFUV EF IIA -K DIKE DGAaDFET S fraction® A DY BSS Lv TLC (Cellurose) C4SEL ZtELH 3c HER N-G -alanyl dovamine CHDK, COUWAMisis TICoEEL, GH BROMB Cis GBOBET SBZELOHTWSCEMS (KW DUELED/B - alanine (3 N-G ~ alanyldopamine C7E HASittavalcCAcT Kynurenine CHMAGUBBRARCIS BtLBASNSG 1008 Developmental Biology DB 168 HRYLrOMMHLCRBRCORE. He RES (BTV YARRA) —- RREB CRA: te 7 OF) THE RELATIONSHIP BETWEEN SPERM MOTILITY AND FERTILITY. 'S.Morisawa and *M.Morisawa ‘Biol, Lab,, St, Marianna Univ,, Kawasaki, “Ocean Res. Inst., Univ. Of Tokyo, Tokyo. —PICHHEOKRE LEM PFIU RB HADHS. wi CHHHEOBWOBLTU BY SBSH. Hit SHO BON M ICEL KM PRE RIC BRT 6SOHRHD 5 MFOBMAHERMAOM ICME AMM RASS EL BASNTWS,. UMLHFOBD tH li Bis 6B He KU CEC MPFAERAORREERRLOVAZ LEA ZN, MARMICMADAHN ANKE DP OSLBS > CH EHNICBR OMMBDHAAZALUASVOINKV ADH 4. ABE F ld KA 28 D> 5 EA LOC HB EH a) & AB OC AE BY) A 0S kK BPICH 57 CWZLM-OMPF-C, BDH ¢ SHO RI a AA ICT AICHHOMBCHS. =~VVARBT BHLHHSA THE LCOZERK CHT EH*E IES HCHAMBT ZL RS RRA LS BY. BR te REECH DED WERBT CORR 6 SZ HERD BAAS ILL CWUAZRBFOMFIN DO EAU BAL R 5 NEW VEE UOMMCHHFOBRHE TD bOBS MIT AAKREHAATERMHSDPCHS0 DB 169 MRE ST 77 = Dili e 4 SOMES C FEE ESR fill - #rtewe mE ( HA + HE HED) STUDIES ON THE FERTILIZATION AND DEVELOPMENT IN THE TWO TYPES OF ECHINOMETRA MATHAEI FROM OKINAWA. T. Uehara and M. Shingaki. Dept. of Biol., Fac. of Sci., Univ. of Ryukyu. Nishihara, Okinawa. WREST ADVANI, AACTHICBUZERGMOS PVICKEsC, IRNICBRENERANT 24 TRHS (FE~AMS) . Gellt. BEPH RMD ome 4 TOE Peels seaecOyNA f Ya, PAWS, go 7wee DKALZIBERAI. CORR, BRP VIVADAE DECAMAS—-ADERCHAGA (B-44 TERD) E-KOBHCHIENZAD (A-24 FERTF) DWH Ie (EU DIPS Ie CBE, INGA Gree, joes, BRICTH ARMED ECAECALRKORMED SHO, B24 7Cl4, ZEAE-KMOBHCHS. (hh, WA HOA Laka lt BOM FRE CIS EA ERDAS CZ &, (BED RZBITHWICH, BA FORFIZHUH CHNICML CARL FOYER ED SREB AWCER, WAP DAK FISLAKA TIELO BAY TICRBLPT WCERE, MA4 FTO PHF ICH SDITCEDASHN A. RAAB NS 2 n= 42 CHAPHRAOUE FICZRBRDASHNA,. UMLEDPSNBMDYSBALTES BALL FTORBZ-BRIT6O6HS,. UEDTED 56, FHVABOMBALZAOZLEC, BBUEAATE 5), BANCRADHEG ANS, DB 170 iE Hv HU OSE LR fil - Sintat(s - SME CHEK - E - em) STUDIES ON THE DEVELOPMENT OF DIADEMA SETOSUM FROM OKINAWA. T. Uehara, T. Miyagi and M. Shingaki. Dept. of Biol., Fac. of Sci., Univ. of Ryukyu. Nishihara, Okinawa. HOY ARITDVVT, 6A~IA (1984) IcB OGoExBC AS MOT RAAC, mAR2~3 AAD SPHOILEOICPIC, BELKEBRDSOn, FF NCO IM, FAKED COMBDSAR Sn. I ld rea nyHel CBO, MILE s ¢ UICUKBEL CWS, SHINS, Bim26>~27 CRC, H7 MRICS MEL, R22 ITIL AT VT DV AICESS. MSM Roi 4H (BLE OAR) OR, ARMOSADS|& ie & Ee LBA O AV CHT 4. FHI=ACORK< Kh RL KORBITHSNAT y b BEDE CH Z DIT MULT, HYACOVE, REAOWMEC, MORAD K) 6 mit SEL, H 260ONAZATy b}MSBA,. HE DLE CHRO A BBNID 5 to EIROREMA Bn, IT CX 4 “Posterior transverse rod” & 34] L CHV RO MPIC > CWS¢RbNS. 8 ARITMOREO NATTY bP SBEKE 100MIZTEO, AM BO Poway SOBRSH, AMR O Time COOTW<, 549 ECMCA, FAVA bEL PUB Fae PIEEREO, PRUNDEARSBObOb45n4, FIC, PHID OBURIC DO COLREARITHW Clad, Mi ik De th (kK D BA PRB HOREAMASLH, Zn6OeR D> 6 REARS LOB RAOMWEE EDR CS iz. <2 OFR, PEAKMIS 7 y NSDSERICBTSYVIETD = 36 25, SUISSE ERAS, MSG AIS MDAC, wv tS Hp KES < fo CWS. FHVARICEST AZFHVA DREAMS 424, NSA FV= 42K CHO DMZ IN fh 2 DAA ASGS Dy 1G ON Ge y DNipe FRED ISA PDEs DADE W7WED BE (a (iy oi BAS << FH TVS Developmental Biology, Morphology 1009 DB 172 Xenopus laevis 32 Alfa WIC Si S HR AMAII EF 47HRF conse — BeAL (KKK > +4) DEVELOPMENT OF DEFECT EMBRYOS AFTER REMOVAL OF THE BLASTOMERES OF B AND C TIERS AT THE 32-CELL STAGE. IN XENOPUS.. H.Takasaki. Dept. Biol., Osaka Kyoiku Univ., Osaka, Xenopus laevis 32M fa MILO HF MICHA FT SFI ( BR SIUC ROMM) SBRALICEOILFSEEMERLT VSZHheERFERRCLoTHN ce. FHEPRLELCMATS 4§OMRABRAIN ARH, ABBMAAK TRICK oD SBEKCKAMTELAZRD, MEBOFKRAKRRTS. TO 4SOSODEPROR MOMMKO TREAT ZH, KAA BORDA KIND. FRAAOMRARAKASNKATE RAMOKRMARBLLSD. TOBB, F° HHARMOHMR RACHA MFERORMIC, KR: MHAMORATHHMEY MBORMICHREDABOSNA. KKMOKMBAAER MOD N¢ERSEVAD, MMREICWLHPAKREAMHDSNSA!. WE OGRDSL I) KER ARREST EA O4F40 HRI ABU RICAAKTHS, (2)K HM 2 MARAIS NCIB HHOICRBLV MASTS, VAS ILUKAOMRLME MOPKReEBAL, SHHRACIDDRALEAATS, ttmeina. DB 173 Mo ZSRORKEMROD CL +a. PRM: - WARS (MAK PlLEB) NUMBERS OF GERM CELLS OF GONADS IN MALE QUAIL EMBRYOS DURING DIFFERENTIATION. Y. Ito and N. Yamamoto. Dept. of Physiol.. Gifu Univ. Med. Sch., Gifu. Mo AF MO+MRTBE LEGvVE MSH Bicste.. KMS BU CHHKHE Ric & 4H DS BERL EMR KBETSZ. AZRRSESRMBROKBEMAAMHRICBETS PEF PRBX4ZAroOKAOK. BEN7%IO4-15SBBReAOCKHK* BOBARMMARRe FLU 745 7—-RAOTHAML Zr. FX COFMBRDH OS MM eH HR DELCTHMABHL 1 BRE KO, BE Hvicront, MR. MMe tb AL. DOMRCISREKRERMBOARBE MASH, MMicCuwALEDEURBYDIA, Eh, RB MROBRZOKRDYS. KMBLbOt-emowe HeuRmMic err. DitOB@Byse,. DRROF MORK LE* MMMRH Ic HML OBA WM, RMic ws E-ROGMBKCUPICLAEMMORBEMEBO BFEROEMBL OG. £€roes2zs., MO 29 FESS MON RARIC RO D PRED AICS ZAR ARE GRA « HT ¢ 4H) A HISTOLOGICAL STUDY ON THE NERVE INNERVA- TION IN THE MANTEL OF SOME PELECYPODA. H, Ishihara. Biolog.Lab.,Fac.of Sci. & En- gineer., Aoyama Gakuin Univ., Tokyo. BORN LRKCRRMRS AY, PUM SAY Ys en? AH eA THY ON BARUCH A PRE A IE UT BR RE RK ECO Vo THRE L 7e. WFAA A ° WHERE ¢ RE GHB e MMA Lb RO, ER ICILARANHE © ME KO ETEL, TAR HEIL EI, (CRCBRL, FFT YY OW RAR © HII (tH Otero. MPAA IC RRA L fe A & BR RE RR ME AE Ut OF f AH RE SR ME OS 99 OF SAE PRES we CO HRD, THAR MEIC HE LO AT RMit Ae RL THE D, FMRI IE eM ta % (CAH OX Bh RE MRED, KK MRALETAALT, HC, Hike tram Mt HO eM MEH CMRA et HICH AHL eH, CT DIE KK (at Hh MEIC RRA. CO ARERR ME Ut PT x IC BAR a (ct BH Ga ROBARLAT. HM, MRRKRESLOS Le FIC Hoe KE RU SA 7c A PARE ES BLE Sh Ze. PREM HE HE © F ME BIC (d PERE TT A AHORA MBO. YY ONMRG VC Va HE A PH RE A HOS TR SRE ACB oH. AF TY OF RR RUE OM RA © PARED FR BE Ih EOE FT FEC RRR BC SRR EB LER ICO TH AGCRTHEOKS Be ORE RHE CBD ie. RRA K-— 7 Re WALK « HR * LEME RAAICKASHXORKBESC AL THR. MH EBRICHO CAA RRM ROTEL, ER FICBADA tt REMMELZNTA. CURICFAFTVV(CHECH OR. nF AA OMRRMISWO2MOBRDZnHE LH TMM THOR > X, BARGMRAIC Ste PREM AIC L BROS RM oFEL, TCHORAFMAMRRHIC, LR, HAS Dis RHC HHL TrHAD4ABwvwonze. MO 30 AR FS cE 0 ABBA D BAD Ee aC A RY 9 - AP Re: Rea: eR Re ARMA: OF ) COMPARATIVE AND HISTOLOGICAL STUDIES OF TEETH IN JAPANESE Teleostei. H. Tanaka, K. lide and Y. Higashi., Dent. Anat. Osaka Dental Univ. Osaka. SORA SMORRNBKe AA LOCA EA #4 10 B® 40 Ee AIC A OB Oe a a FRR SPECIE RORRSBA CLEP TAT. 1) Enamel (Enameloid) : M&O HOH IH Ic tt Cap K EUR e STS EnamelOFERAGNSZ. TH 5 is RE IC ERM DD oD 6TH SL TCXAHMHEnameloid CENHONTWIAZAMAKILE SisMick ,- CHBDBAGH, BAKIEU 7: Enamel SERARHODEMAMAHe ALTOS. ELS FF #€ id Ename | JB & Ic #€ A LU Enamell @ % 1 KU TWH EDSBBAV. 2)Dentin: RMOMOBRF ALARM L TOMBRF AQRA’ DAR” ICKH SNA. 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BA CIORN 1 ~ 2 pOMBD AB Hic nai UME AR ICR ARF RLFEL EW, Fi eG ld BEIT HA 4S 4 BEE D A Ka Hl 7 EB 7 DS BRAGN, COMM L DDT HOICRAME 2 ew LtW4, MA 6 ICHH BRO & TC ld HB A HMChHADMBMLEAICL KD 5s TARILe BU PaRBRE SRE < 75 TATE DMD ENK. BB 25 FT DIND tise - ESI USERS OWT. FSHh6—:8His (EBA: Lars) BEHAVIORAL ANALYSIS OF VISIUAL INPUT AND MOTOR OUTPUT IN PAPILIO XUTHUS. Y.lyobe and K.Aoki. Life Science Inst. Sophia Univ. Tokyo ROWARRT SYD NCHA AMICBRMAT SEX OK ¥7S LE BRR ZS RITBKEUTFASECHNNCGEU CT RIGARS FRX (st thee Ke UERROIB, BRA, Bem, Sa $418, RUMEO 5bDOBARAICAUIHADCH 4 a8, fA, (C4 4G BEUCH RSC UI. HIKEDE = DHE ROA TOBE CH Ati D NWN EBHERED RAAT D 5 fF RCOMRILBW CEBU. LERPROI, BRE, BMARMICDDDOoa FT, eR PADS 7K-EGH D5 FA6 OR ORMICSRAaANSR, ga. A, D#ARICDSSOWUVAE CHAS DNAZBDS 77, DWLEDS Biz 23 FACT. HEAT OBE CH AED NEB RE Nic ‘SIRS eICMt UC 6.39 ESB 7, eC UT ER RV ols THIN 1 5.4BEDOUAACWHAZSBDS, FS YF \\S GaGa RAC MRR eB ST St BAS5NSA, SC gas D5 MET Ot BES AS ISAKERETHN (a EbHls eo UT 2 9 BRITA NMIBACWHS. LIED 3 CH FMS AER & BIE ON SBIERED BBD Ot TTI, PSA KAR D 5S OD Herts RA EMIT EVO A WOCWALFASBNS., SR, UE DIE IED AMIDST BORBRM,. RULE BROBBDARMECSK=32—UOLO tS#8 UE RRC DWC REMEDSHAD « BB 26 AZTKRTOMABLI XRERGEMI TREO WT He tf OSk = GIT eas Eh Gaiew CIRCADIAN ECLOSION RHYTHM AND LOCOMOTOR RHY~- THM IN THE HOUSEFLY, MUSCA DOMESTICA L. M.Ai- zawa. Furuka Girls’? High, and H.Yoshino,. Furu~ kawa High, Miyagi. AZTKRKRUIEOMAALI’ KABPLORABHI ZX Keril@lo CW\4RAOBRe HOMER TFAR DH, PF RRMB LHRH T 7+} FITFTHKHEREBFEL, A LKRIO2Z2 Ki xe AW CRBEUE. ATR UGUHMCBO AA CHEF Ze Oo,. Ait YARIS FA FINOBBe REO RW, £k Hit FECOMB RMR MAMET Ly CHRSE, EES CC. MPRRE+1I9C~BCOMMACHEELAHEER DE-FTHMUIOR (Db, HY KA HPL RR. ARBDOMARIZ., Hie -7eILDER th 2ORHOKRU GC. VKFAOBeRUTHRWD, WA RAeeSR EL CAR EL HY -7HRWAA HNC HMbNZACLEMbDA KE. Fhod, KE Zi P — 9 | seis 26.5058) |) UPB Wala teecomoeas ChHhok,. EZATCT., COKRKOMKOKTEH zm. HBA DEFT 7 eA eo = Cs 2ee DDARKFFCHRL, 2veFFLEUECAME RR Cee ese 12 = OCR 26,8825 fF mB Ww I coles Osos ree Y) 5 Bela Wize IS 2 aa apis iy) DK A A. HBOR RABE CHMANTWSA BE tee RR TARR BK, IS 2 AAV VIATLYVOPAHOHEB. BAB (WHA: -RAB- 4H) SPREADING OF THE DISTRIBUTION OF PECTINATELLA MAGNIFICA, A FRESHWATER BRYOZOAN, IN JAPAN. S. Oda, Biol. Lab., Fac. of Gen. Edu., Rikkyo Univ. , Tokyo. AAPYVIAZT AY Pectinatella magnifica (Leidy)ié, 19 2%, MOM (URI) CRMSKARMCUTHR, #1973 FicdeEM IC BAR ARM EG TSERWRLE. SATCH, Wa MIC DMRL CWS. 1974 SUA) (RU BAG), HIR( IRB ), 1976F DA OUFRI (ENB + HEM > th ARORA - KBBORRAK BB), 1982 Sik ( BAM ), ME (1983) DH (de oRIR ( 7K HE PA th - HARB + BT AAI), SN Se (1984) BR (MM: +l) CORK. MNASNFTFHOVOHBELTAMASRTWSHECS CHS. CLTHMKESIENADATFEROCRKEHEIKEB HISZCERMVKE. NFTIFHOCKMOEC EK ES LTWS., CBULEHRAKAZZLIFIALANAL, Ram Oss Htc ko Chom MliN>S. ZAbTIAbL RARBORBICMHASNA}!( Oda, 1979). LEMT, C DESDERKRCAZEKIFFIALMKAFK £9 THE GIEN, AAVVIATFLYOFL BWMRACAM2TREEBASN . UML, PAV ARMREOAAVVUIAZTAYRAALK BRUKBBIOWTH, WEKICHRAL TWAS. Miscellaneous MI 2 RAS MRR RA OD SRR AS. BHAA ORGK + +) SUMMER SCHOOL FOR MARINE ZOOLOGY AT THE MISAKI MARINE BIOLOGICAL STATION. N. Isono. Dept. of Biology, Keio Univ., Yokohama, STAMSASP ROM, RRAS = te LRA cis PFRRACWRLETS (HwreBEHAs cVvsew BBARHOCMONCKR. MERA MRA REE GAHAPWe Ke Pry» 7ADFstve-7RBACH BLARBERD5, HACTEMRZBBASOMBHETRC, LRASMSk BRLALBAO1 89 8 (HH3 14) KBLEORBSALCMELAD CHS. 4“MREAI3IS (M6204%¢HAK) . BHRSAL ADS 3 wit, LRAORRTBOMMLRS1919% (KE8) = CBF DN, PRCONFRRAbBSMeZISKSER chk. ROAR. OW CMEM. SHAK + LH SL. REDRMORR. DM. MA. RECHBALA. 18HN19I23% (KE1 2%) VRRREHBSLERI, HRbH 2 MMK HMMS NAD, 193 9% (HAI 44) ORB2 4H FcCHMHRLEA. LOL. 19414 (AMIE Se) EPESNCORB2 SHMAREORSK HE SH, DRAB LRPok. SMHREN3284K OR). SRERCbE-KLBdDNS|. 1011 ZOOLOGICAL SCIENCE 1: 1012 (1984) [Book Review | © 1984 Zoological Society of Japan Animal Behavior. Neurophysiological and Ethological Approaches KiyosH1 Aoki, SUSUMU IsHII and HIROMICHI MoriTA, editors. Japan Scientific Societies Press, Tokyo and Springer Verlag, Berlin. 1984. 264 pp., index. This is an interesting little book that resists evaluation. It is based on a special funding program sponsored by the Ministry of Education, Science and Culture of Japan whose praiseworthy goal was to. stimulate interdisciplinary ap- proaches to the study of animal behavior. Under these auspices five symposia were held between 1979 and 1982 under such varied titles as ‘Neurons and Behavior’ and ‘Genetic and Developmental Analysis of Insects’. From these symposia twenty papers were selected for inclusion in Animal Behavior. Surprisingly, despite the breadth of topics, a certain amount of unity was achieved in the first part of the book by selecting a group of papers which together describe the morphologic basis, electrophysiology, genetics, and of course descriptions of particular behaviors in arthropods. Vertebrate behavioral analysis is treated in a less organized way. The other recognizably organized section, in the latter half of the volume, considers endocrine influences on behavior. Three papers deal with the role of the pineal and its nervous and hormonal influences on behavior, and especially behavioral periodicity. One must conclude from examining this book —or indeed, the field of behavior itself— that the multidisciplinary approach to understanding behavior has not yet produced truly basic unifying concepts. Electrophysiologic analysis still tells us little about the neurophysiologic basis of behavior. Descriptions of behaviors and _ their ‘releasers’ in general still cannot be analyzed in terms of specific neural pathways or components. The mode of action of hormones on behavior can be analyzed only in the most superficial sense. An example of the coming together of the morphological, developmental, general physio- logical and endocrine approaches can already be seen in such restricted sub-fields as J. W. Truman’s research on behavioral development in Manduca. Thus, these separate approaches are beginning to meet at isolated and specific points and, as this volume shows, we can expect more from the multidisciplinary approach in the future. AUBREY GORBMAN University of Washington Seattle, Washington, U.S. A. ZOOLOGICAL SCIENCE 1: 1013 (1984) [ Book Review | © 1984 Zoological Society of Japan Structure and Function of Peptidergic and Aminergic Neurons YUTAKA SANO, YASUHIKO IBATA and E. A. ZIMMERMAN, editors. Japan Scientific Societies Press, Tokyo and VNU Science Press BV, Utrecht. £963. 351 pp. Some of the releasing and inhibiting factors produced in the hypothalamus, which control the secretion of pituitary hormones, have un- expected effects at extrahypothalamic _ sites, unrelated to their primary activities. Conversely, it is now evident that the central regulation of the hypothalamus is affected by neurotransmitters. Progress in the search for the understanding of such neuro-endocrine integration has been greatly advanced in the last two decades by the application of immunocytochemical techniques for peptide hormones and for enzymes that have a role in synthesis of amines and acetylcholine. This multiauthored book is based on papers presented at the Fifth International Symposium, held at Otsu, Japan in 1981, under the auspices of the Taniguchi Foundation on the subject of ‘Structure and Function of Peptidergic and Aminergic Neurons’. It contains 23 papers by contributors, most of whom working extensively in the field of neuro- endocrinology. Therefore, although each article has its own intrinsic value as an important reference, the book as a whole is so edited that broader examination of closely related . articles will give the reader more general concepts con- cerning the architecture of peptidergic and aminergic neurons and their interrelated pro- jections. Of course immunocytochemistry is not a faultless method, but it has proven an extremely useful exploratory approach, as presented in this book. Several of the many valuable findings and suggestions brought out by this volume are as follows: (1) The neurons of the paraventricular nucleus and the lateral hypothalamus project down to the sacral segment of the spinal cord, suggesting that these nuclei can influence the spinal autonomic preganglionic neurons. (2) Other extrahypo- thalamic projections of magnocellular neurons and the presence of various peptides and mono- amines have been detected in the hippocampus, substantia nigra, circumventricular organs etc. (3) Peptidergic neurons producing thyrotropin- releasing and luteinizing hormone-releasing factors were suggested as potential mediators for other neuronal functions. (4) Ontogenetic changes of the levels of somatostatin suggest its possible function in the development of the brain stem aside from its primary inhibitory action for growth hormone (GH) release. (5) Two articles on the effects of neuropeptides and monoamines on prolactin and GH secretion represent physi- ological studies and link with other morphological studies. One is reminded of such pioneering work as that done by Loewi (1921-1924) on chemical mediation in nervous stimulation, by Lewy (1929) who declared that the vegetative nuclei of the hypothalamus form, with the pars nervosa, a single consecutive system, and by Harris (1955) who defined the relationship between endocrine activity and the development of the nervous system. Berta and Ernst Scharrer in their book ‘Neuroendocrinology’ (1963) a milestone of this field, have summarized the great strides made This volume shows even greater progress in the field, in neuroendocrinology since those days. and may inspire not only scientists working in the field but also non-specialists to feel an interest in the area encompassed within the framework of the general zoological sciences. SEIICHIRO KAWASHIMA Hiroshima University Hiroshima, Japan. 1014 ANNOUNCEMENT The editors express their gratitude to the following reviewers, who evaluated papers for ZOO- LOGICAL SCIENCE from May 1, 1983 to August 31, 1984. Without their assistance the journal could not function. Aida, K. Inoue, S. Murakami, A. Akino, M. Ishida, K. Muramatsu, S. Amano, M. Ishihara, K. Aoki, J. Ishikawa, M. Nagahama, Y. Aoki, K. Ishizaki, H. Nakano, E. Arai, Y. Ito, S. Nakazawa, T. Asami, K. Ito, T. Noumura, T. Tuchi, I. Baba, K. Iwasawa, H. Obika, M. Baba, S. A. Iwata, K. S. Oda, S. Bern, H. A. Ogawa, M. Kayjishima, T. Oguro, C. Chiba, Y. Katagiri, Ch. Ohba, S. Chino, H. Katakura, Y. Ohnishi, E. Kato, K. Ohtaki, T. Daikoku, S. Kato, Y. Oikawa, T. Kawamura, T. Okada, M. Eguchi, E. Kawanabe, H. Okajima, A. Ehara, S. Kifune, T. Okazaki, K. Kikuyama, S. Oksche, A. Fujii, R. Kimura, T. Osanai, K. Kinoshita, S. Osanai, M. Gamo, S. Kishida, Y. Gorbman, A. Kobayashi, H. Sakai, H. Kobayashi, M. Sato, T. Habe, T. Kobayashi, Y. Satoh, N. Hanaoka, Y. Kuroda, Y. Sekiguchi, K. Hayashi, K. Kurokawa, H. Shibuya, T. Hidaka, T. Kuwasawa, K. | Shigei, M. Hihara, F. Shigenaka, Y. Hill, R. B. Machida, M. Shima, A. Hiramoto, Y. Matsumoto, A. Shimada, H. Hirano, T. Matsumoto, K. Shimazu, T. Hisada, M. McGaughey, R. Shirai, H. Hiwatashi, K. Mizuno, T. Shiroya, T. Honma, Y. Mohri, H. Sugi, H. Hon, He: Mori, T. Sugiyama, M. Morita, H. Iga, T. Moriwaki, K. Tachi, C. Mitek, 1 Muneoka, Y. Taira, T. Takahashi, K. Takahashi, S. Takasugi, N. Tateda, H. Tazaki, K. Teshirogi, W. Tominaga, Y. Tomita, H. Tomonaga, S. Toyoda, Y. Uchikawa, K. Ueda, K. Uéno, S. -I. Urano, A. Wada, M. Wakabayashi, K. Watanabe, H. Watanabe, M. Watanabe, T. K. Watanabe, Y. Yamada, K. 1015 Yamada, M. Yamagami, K. Yamaguchi, Takao Yamaguchi, Tsuneo Yamamoto, M. Yanagimachi, R. Yasumasu, I. Yoneda, M. Yoshida, M. Yoshimura, F. Yoshizato, K. 1016 ANNOUNCEMENTS THE 56TH ANNUAL MEETING OF THE ZOOLOGICAL SOCIETY OF JAPAN The 56th Annual Meeting of the Zoological Society of Japan will be held at the College of Arts and Sciences (Kyoyogakubu), the University of Tokyo, from October 10 to 12, 1985. Further information and application form will be sent to the domestic members in April issue of ‘Bio- logical Science News’ (No. 161). The deadline for application is July 15, 1985. For application from foreign countries, please contact: Prof. HipEo Mouri Organizing Committee of the 56th Annual Meeting of the Zoological Society of Japan c/o Department of Biology, College of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153, Japan. Phone: 03-467-1171, Ext. 316. 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 (about US $ 2,000) will be awarded annually at the Annual Meeting of the Zoological Society of Japan to a few papers published in the journal ZOOLOGICAL SCIENCE during the preceding calendar year. Every original papers published in ZOOLOGICAL SCIENCE will automatically be candidates for the award. The aim of the award is to encourage contributions to this international journal. Selection Committee for the award will be organized every year. The Zoological Society of Japan 1017 AUTHOR INDEX A ee aw cucu cuiauanaanes sthuemeiine 905 se Ge eee 5 Mer ere 995 No oaoe ib vcad'avicwussgnohaen GMOS 1007 i uile's angcnsndo apts 865 : 883, 990 ee ae 911, 959 EN rena nscdannindhaesnseansstande as elt Sess 974 cee wpa ninas cannes deo Sew aes 986 a a cen ncn nn nk ec ereten 1010 oa weep a ne cncepn,cniny,nne ie aahteSlakee 989 ee ae 930, 938 8 0 918, 930, 935 nn, ace uenddesaneeehines 942 0 Se erene eee? 74, 915 I in cu din cc awinurw eden TORI E 954 EN ae erie. BA ewalamtica 985 — 0 es ee 895, 972 os oS ee errr ae 924, 942 os nea cnsncenen ddan desiebh 928 i ns at ened nd das 132 0 ee 187, 701, 882, 952, 953, 992, 994, 1006, 1010 NE aaah ciesanaceessar 126, 854, 977, 978, 980, 981, 991 PIE Moiese ond ccc spsaotaapinnntecaathe 864 ls ec casesnxiuinponnncanapaasemucetas 737 0 8 ener cere. Ieee ee 833 NN ei ha mative amelsuumen dab Os 125 Sr isnt ona socal nt pandbiepkanepdonasanaganthay 950 oo a eee ae 921 Ms en ah ecns asaaeucvesasecdenanetnied 910 eg ee a ne, eT 916 B oo ay ee nen Aer 870 RNS TUE cc wo caunkdanenccbensip lo adivwinemlezmakda 905 eo Se oe er rrener 100, 976 Co AL ee ee eee ae 965 CG CLCT Leet ky bi © ane a ee eee 935 CTRL Ta ae. Se Coe eee EER ARETE Es 873 RONCLII EIU ER St ck cuce co nny cs tanainicacuavarsidek ReatTt 471 OUT CITE 17 OO C7 ORR Se ee ED © Me ee ee 349 COMI AGS, 5 AURA et ee ce 992, 994 OUEVED Eh Ep ah ssa n ec a an a a $F 941 SU. SE > RS SS er 375, 993 COUPTHAT Os 1 (oes See na ae ais eg Ee 909, 910 ROSES Ne Me sarc is a to cinta seca qas nse tenw vtec, Meee 978 Clore TES) Cet See 2 sist ee ee 996 MMPI Cele acca becRai uns cuss ss cnaGisnaccsSniaslnee ys 966 D Pei eek cee ciatigs scienc cules sssuasam kins ime mpiets ates 151 PAUL Maly le ears Sac cnc acai is ale nlcs e's a SCD 990 RIND IS IN iors = cnt caaictsen cake eiuoebammekes fea 237 IDE Ran oe eee nen Ma Pree 959 BLY ee ne de cmece -eecbasdiécouencsene euvmanclt 868 PATRAS ISN MUN aN cue du etes vanes oe de mbuoe Cowen AN 1741 E SAR eM e o er desu e a) vic cowemats.sne US pnb utes see eto ase 874 PALIN eS Seb iccle cetera tnadeabesuup sean ses Meena tnuil 894 52707 1 [Sai eee ee 1, 892, 955, 996 MICE oh ao hovsicse ogncadlgsdeunacundun vet n then teats 864 ELS FENZL 12 SA oe Ae Seine ee a On, © eS 871 eli 0 Rg | Sea ne ene eae Weer. 959 Ein istatheh | (gs eek ape Cee eanmne aap 2 889 Re OE ge ie 0 a ees ae 925 Te OD. INR ee 2 Se a eee a 922 AVS TAEE | ia dct ay nents teens twsecseneuaerisbleeasns 881 F oS EB ae, a ae err eee mF 905 Rae ices oa nankacnnatecdseccedazascd codes 477 ee cn as dinpicinen scons nascckepboe~aclhisene 961 EO vasacunccasteseccsccenandasdhoiftahiien 927 So 545, 553, 711, 856 Phas ctcnascvcnd nan asecksecareustoashapQhteiyee 893 ees noid ss vasennsinaansinspqeeees nee 901 RA NE Loon ansnceansecschanesscsncehusttasaeeien 986 1018 Pujimoris. Mi... see steeteeaceencsceersce ecco 82, 973 Eujimotoy Ci xcactasnseeh cota gittcec eae ee eee 971 PUpMOtos IR) eticco eestor coco earner 860 Puyimoto Mir occacencceecncee cn hore eee 866 IBUJIMOLOs, Ses csacedecscietarsrrdae eee 964 PUFIMOLOS. Yi scaowcrawe anne tasedantaceneee eae 961 FUpINO, Xi GsduaRentowrat ene eee 923, 935, 936 Uisawas H..ssccavomuatenctoarateeen ern eeer 928 Pujishimea),. 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To ..ck co bsssccseedessadasnceeeeeeee 868 Patton As, 2° hss. is od ois ooinces be nee eRe 974, 979 Hattori, Te. Ge PA sok eee 945 Hayakawa, Y. ...sisecsccscnsetience sone cee ee ReEeRenEe 910 Hayashi, A. ni... 0..s00.sc20s.056000es eee eee 928 Hayashi, K.:. «..sscsssveeesatseae eset 2e ee eRe eeeeeeee 947 Hayashi, Ky. , .dcaaasctiosnnviontmnaeimiceseot ater 918 INODUOK a Tie ssacdctisanincisacctrmmaibucet on neaeeeree 887 INO da YDS, wc ccritarsodnacamnolcaee eee 926 IN@mawale | cissssicaseniaserasssaneenide ener eeee 841 ING GIET: @. c isasasacdiounddianeiecionsaasces eee eee ee 873 ING G@UGHIE Mie ic dosscnsimsnen@onaiioctions Are eeeomeee 865 INOJiTis AS. ascsivncscenidornawoanecaoedore eee 968 INomacuchi. Te. Ati sicccacsensncseesteoreee 895, 901 Nomina. Ken yo atssiciianh nanceniencicanetaotera eee 913 INonakas Mi osc scsssdenconacanaioosassuuse serene 922 INOnOyaMas Ko. iscsssasaceimmenicacomnescnee eee 955 INGUMUTATIS. Oxo cimiccameesaeaeeen 13, DUT Numan. 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Meee ene nee eee 939 Obi Kas Mee oe cis ansc erence rane ee oe 891 Obinatarscc3sseetaeeesecacnes 855, 904, 905, 906 DSO «sie. ciscssnreainnSeeeehatina eee Cree eee 956 Oe MAIO oe. cicceisssewedaosboceknwadocde. ee ee 877 (OXGE EY UR tera aes eae ne PMSA Thee 2 1010 aa S ee aa Fires ctl onan Coe ee 1010 Oda Se foc Sniss scarean aeons ene eee 995 Osasawatay Vs. | scscccawasundeconse ee eee 965, 966 Oe ata Koc orcs dicwevnacemaneecuonanaeen eat eee 862 (OPEN EI) <2 Se ene M eR mmN Rm ce 891 Draw has Mis, 2 sc esie sratiarnccevione eee ee ROL ee 984 Oe A siecccnwecnnsiecea Reece sce eee 960 DSO IV oes aucdrdeannsccede ee 961 GSU aS AV 5 28 sanitation es coa eo ee 989 (URSTI OS Gata ane aae eee rae a ia, Cail, SO, 972, 973 Oita tee es ae eo aie ee 946 OAS IIS eas pie ee ae recy ce 906, 907 Ojal oe al 5 A Cee er Pn ee ee eer re 914 ONG aS wae ree eect 631, 641, 959 ON=iSHH Miss sevcasc ce veceewasteweo seen beac 954 Oinmoredes Ak. sngssteben sie clen vep eonasorenes eee 914 QRMORIEIKG. iad cesses tees Ge ee 896 Ohnishvo, bc artic eae 114, 845, 961 OWMISHUAIK:. ses wscvascasseeneianemnede oer eee 911 OHM ISHS is..:hcs scsiscensloracstereiceoneence eee eee 801 OHOK A) Ts oasat daconasiwasweasetvomaens Gee enereeeeenenee 908 ONSUG I, Ree s..siiseteisiereicratalerotoretenotestelgiattloicklonl bee eee eee 945 @) 5c: Fad Deen MNES AP EMME MESO FRR ce coccoc 919 Ohta UY.) cctiossasecasdsaintrontiaenenenteeeeee pete eeeee 976 Oita kay Ave io sicaisieiajslnetorneidsaiatesfetclatroieretn rs Rea 998 ONtAKES; S.-i ssississcierdTovrctoretareiorrciercteroerehetole tee eee ee 963 Ohtaki, T. 2. oo ccccsicasicsiciesoll scectacn dee eee 960 ORT, Fle ised scscssiecsterectescteiolels/oretetol oles eee 291 OUCH, Av. ciecisicice se ceiseisineciedees odes ee ae 973 OZ) Be on. ciswsscasadawdieleiesceecod gee 673 OikaAWa Teo oso sseie sisicisieieleieieisteicieieloreletele eee 932 OISh VT.) s..casasaseracincssasientoeeseeeeeeeeee 973, 993 OiWa, Koss ceca) ces cncssdcassoends seeds eee 870 Optima. 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He. .o.:c.dcscsisaicionnlscese ee eee eee 858 Takahashi, K.. , . .sisscwicesaaveeksceene sheer Renee eee 870 Takahashi,,.K... 899 Cons eee eee va ee 934 Re adeiscaniunceseckacecakce tip ssipuaen 872 1027 AT REAM Bed in sonic chwc da siccdusin an cidseaksmarenaeaha 925 MRM EPEE TANI ne otoe is ays vine daniwakivis SobRELaeENRS 893 US EM eROME AMG ci5, vosnn am nudaaedanxensivacuacad 985, 1004 iiss ech sbciaass > nonvaanvaedessheeevRen 997 MUSIC HOU EN os ccctiesins enisadan uatitiows 872, 885 EUs REE UMNO Fos ok naples bin ticiaeeiam pninwc dows anseete 875 MSM NG ota vs ts sasnannvmanacicaswogesbas vette 837 MRE aeRIE oc sipnisuasseuldsoancandact 926, 931 SSP CUEI AN OE a cncrinnamosnannance ner ®benad 975 UPR YO, 0 oan c cemmninuakadamncisamnntice 861, 863 SSN Re MRIA UO 0 tao cmn sieeaacieccneucnn scaled ag Cheese 883 MTP MIRAE OY. 5 ais buie.n visits pnb nioeaiemaiivinrs Mb SAKE METE 859 CELE OY CR EEE ST pe TOR 50 GME erg: 885 PAA i ss cas cc cun Gob eek cenversdetss nck Outen 914 USL EE 6 ee oe eee ee een Ma Pots 875 PIC KHON tet ces ctuancntcuineaiasncaath vide vam eRe 985 SP SEPUUISANCIASING, (92) 3c. sat ccc os taecnceeaveds % «peta 1000 TTS) USERS ed NS Ce ro ee a a 963 PS SIESEPIE ISG OR. LO vic. coa dmcuas Gansmannisin 975, 977, 978 PRSUIUSUGUIN: tevsceeoac sa kaccaca ene counseve hake eee 882 REG bth Arm Pre-ccte sui Petetw eds cboaetrund yoaueuatdewens 935 U MUR OTI Wy cova cus cue tdaceen ss vanssakeneasesMocphtiekts 979 JOT Daas CA oo ee ne 940, 976 rien. Es “I Aes canaka reascunccenetas tual dee 976 CUTSUDIE Te a AY (Ra are a eo 968, 973 DCE a Ge 4 See ee ae neem ROE A 5S 902 UPC PLES RAD Cees aeons AO aE ARES 50 988 MEA OE rer Se ab ayy vew was cache douse oohs tebe 964 ROAM Bev dutc oakaegccckuwannacsaas 829, 881, 882, 992 Ts st ee ed Dd ee Se a pea AE ee AM 1008 TES hacia hen aces ese isbaess sons enna tee 861 Tier oc Sel = COE Cert Son ee eee ene mmr = hye t: 979 SS) el SE ae ee ey. wee, FYE yet 927 SIRS ER tcl secu vrar te peleca asus skhess holes ptlteu sabes 874 NOURI Cee g oe rags gh uinwihsenedne nade asl bedi eb 905 NE 2, el Ge Sor 911, 1007 ROA AI aa ce abe scp eanvpeessscntoatlenyeleue 882 na eee eee iran sr iaeces evsnzyevccd bephces 988 ERED es ee otis coven inesinpnande 89, 759, 968 TERN esos spp ssascisnvendvagiashsefadignees 929 NMRA io ok pac uvdeusessrdseveevirnahakesRelseeMes 876 se iics rebbinesescnvontesvneyieodteadsieheadne 927 I es see nvanpppscsvnizesvoshosennteaseialpieeib 999 Ree sense ancora namhunnbandeananaes 653, 1002 1028 Ww Wada WME 2h ow csBoanacosaussannoncsaneeeeetee 974, 979 Wakabayashi, S...cccocs. snwkeacntonchsicocs menace 974 Weakaliaraa Me. vctecnicngawescnrronnneckennenceae serene 920 Wakamatsu, Y..)caikcicnduncnatieiemerkenmaeeeeeeeee 891 Washes Fi cick succtdaccaccucsins comtatenetiene renee 884 Wratabes Fla ccs cceuiinnkhateiantnaencmencdeneneemeeee 994 Wyck tama be «Ell teccerasteeinaenircaieet 229, 957, 995, 1002 Watanabe: Ki.) sassiosnonaccocuidansasaccnenecee 114, 961 Watanabe Kw. da cco ea Shauneneccanenan seer 956, 987 Wratamabess M... cscissrcrsiccciacnysstaseintinatosedcokee eneee 787 Watanabe. IM lscy sasnnuweceaataitnmnraaaceeeneern mene 878 Watanabe. 2S). eccsccicustestuanseeweesan se cenoeeeee 1004 Watanabe iD. sissnacuiiidiosasonanaatees dae aS 897 Watanabe oil. IK... cuinnaasadiadoaadrinss ano anes 801 Watanabe say es iscsehaians aoceerasinons 50, 62, 911, 912, 914 Wie Fa DSS NG ararsscrsptensiclcratelororetranever 561, 892, 930, 950 Watanabe: Wis Gis. sseddsasnasaxsdtunsieaa en eee 601 Y DVS IEE S Bue tid is a cteliaidioe cence OE OE 944 GA SUCH Me: issu cacnccaheansaceasanasanoe oe ore eee 904 BYGAV TTT A DE dsc se ctmate seamen eee een eee 953 CATIA AI ies, werctacceasnisigavennce season e VAR eee 890 Wana dae SA: p vssse docerssanhavweneeeenaeeneleeeu sere 939 Rian ad AB HKG: cot atenientiroreutchbeekoier 856, 857, 858 Byamald al Mi» vscciecaibodaensdeatacnnssen scans. eee 999 (ANI 0E 102 DUG [A ne RnR EC APTS MME CA 333 PESTA a YORE Meret sarah nates ements: 930, 935 BYAAEM ACD IY 6s tet). ress arsterainsiacerdtvetardl tea ee sis Bee 980 WATIASAT AIKG wicerecahientericaci eee 148, 932 WAIN AR Abas ls « accesncencekih bantaneuenc teen 933, 946 NAIA GUC Ken sicd saisiovs sein aaseiov nee teaateneseneenee 591 Mama cuchiy. Ks. sjeassci sesso eeeeeee seer 892 Nana RUC I Ke snes sec ce dancasenescaesee coe eee 975 NAMA RUC I Mares weccacseaaeccneaonne 169, 867, 883 BY fil IVEY ss Ser cee Picadas Sosa anttescaree eee 917 (219) GT Ae rr ne nr mee he 856 SCENT CN ANU ey, by RN OURS Ae Se Soa aA ROR TD 1004 AMATO tO cAis\.cceceee cer een een eens 631, 641 NY ain amoto,c1D 2 ccsseehaeeiecwncs aehecnes eee 883 WAIN AIM OO. Elis esac ctor ees eee ween ee 900, 904 BAIN AT OtO mK i ocean rere ce eeeeR ore ee eco ae 894, 901 NAIAMOO, IK .s.saaseasdooaensatsetr eek ee 926 Mamamotos. Kos acdsee eee ee .. 969, 970 Yamamoto,’ M.. wvsicadisnwisiece cossiosecetaeeecee eee eee 501 Yamamoto, Mis .cccnncscecennnns ee ee eee 894, 901 Yamamoto, Nis cnciocesieeiisadesaada a. eee Eenee 1009 Yamamoto, T.S.s ascc00i80600050%08000000 eee eee 931 Yamanas: K,.. ossscscscastensanacdeccneh ee ace eee 869 Yamanaka, H.... ..0:0.800.00s0005000% 5002 eee 858 Yamanobe, T.. ~:0i0.4%.:.000%00nse0cen tee Re REE .. 871 Yamanouchi, KG. .ojssssaes ene 126, 977, 981, 991 Yamaoka, 1) « ccissssnnnnennsninieesankhhin ee eeeeeeeeee 889 Yamaoka, Ko. o3.ssenes00%snasiie picket eee 873 Yamasaki, Fi. .sc.ccsecseissersiesientere aceite eee 908 Yamasaki, Koo. sccsisiossersertaistoitevtetel te dee eee eee 908 Yamashita, Ko.» siiiiesristeiocdertelonencts etek ee eee eeneeee 995 Yamashita, Mois icc scccsossossccacacaacee eee eee 923 Yamashita, S.. «cs. cssisstessistsrieetentlenon eee eee 863 Yarmasus To ae ccsiccstoviereietetereletdieleteislelel ee S eee RE ERE 982 Yamauchi, Koo. sicssicisiseisiersiecietciels owtetststetclels see een 967 Yamauchi, Koo. ccsasscissnnsvoseddodddas eee eeeeeee 970 Yamazaki, Hi 1s. -sscéscasasescdccacdeneee eee eee 911 Yamazaki-Yamamoto, K. ..............cc00eee eee 943 Yamazato, Kit .scscscossesssadcanssseeeeeeeee 982, 996 Vana, As cocci cscnavaviccasanecandoe dese eee eee 886 Yanai Nis cccccccadsassccsnosoncedahadeeee eae eeeeeeeee 891 Van ase eT sii ccccccncccacsdodecce stade agen eeeeeee 1006 Yano Je sectesi sscssedassncacaaacdde scene eceeeeeee Penne 887 YVanoy Koo ovtensevenissvnav abe chee see e ee SOC eee EEE 1004 Yarnoy Mee avec ddancdeaessccsaasndncdo sane eeeeennee 882 Yashima,: ¥ << o...c%nisesneh sions a eka eee 1007 Yasuda, To: « ..n:cs.nnusestetuaeeanntinttiteltee epee ee EERE 62 Yasui, Bo& iccsoscassoocavasensos5dcahs eee 908 Yasueiy Si KF 4 > = a 5 \> & Ss - Uf mM el m n m Y wo S w = 27) ee a) N NOILMLILSNI_ NVINOSHLINS S3IYVYEIT_LIBRARIES) SMITHSONIAN INSTITUTION NOILN 5 Z = 3) gee = ft ae : 6 =u a. Z z Ae wn a ay fs 5 oe S 2 = = a S = 2 GY S a ee | B 1 2 = en a 7_ LIBRARIES SMITHSONIAN INSTITUTION NOILALILSNI NVINOSHLINS Sa1uvugi7 LIBR 2 “ 2 i s ” ie al SS ome, Ue a a Ee fe — , is 1 = ed, 5 < Sj a a ie S « Og Ss c = Ps c We, = = | 2 ay 2 wet = ae N_NOILNLILSNI_NVINOSHLINS S3IYVUSIT LIBRARIES SMITHSONIAN -NOILN : See : Se 5 — o : — ow = 5 oo = SS E x Leg 5 7 = Paine ps [sh i" - Ww - = =. ne - > Hs \ Se ee > oh 2 = 1_LIBRARIES SMITHSONIAN INSTITUTION NOILOLILSNI NVINOSHLINS S3J1YVUGIT_ LIBR = = Zs w z 77) aan zhi = + a = \z = Wy, = . 9 z 2 NN GS A 3 zh GAG ~ 5 5 e) 2 8 SENS fe) Hap re) Sp fi) ae A ie = ee 7 ae. N NOILALILSNI_NVINOSHLINS S31YVYaIT LIBRARIES SMITHSONIAN NOILE w z uw - tu 2 Ms: ai ra ” wa >, oa a ae + SAS om a c ~ we en a /26N7 = - > ; a > 7 —— = = ae = a a Zz ys z 2 We o - N_ NOILALILSNI pS TIUVUSITLLIBRARIES, SMITHSONIAN INSTITUTION | NOIL z = Pe = << =e fe) O 4 = 6 Oo GF, 2 j < = = = 2 3 > ; 2 . 2B 2 7_ LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI NYINOSHLIWS saiuvugi7_LIBR NOILONLILSNI NOILNLILSNI LIBRARIES ay Xv N_NOILNLILSNI NVINOSHLINS S3!Y¥vVYUSIT LIBRARIES SMITHSONIAN INSTITUTION NOtLi Gil LIBRARIES SMITHSONIAN Pas YVUYGIT LIBRARIES SMITHSONIAN ITUTION NOILNLILSNI ITUTION NOILNLILSNI ITUTION ITUTION Pr yvVUaIT ie ; Y 2: . z LO Asn” (SON Li? NVINOSHLINS S31Y4¥V¥dII LIBRAR NOILNLIL NVINOSHLIWS INSTITUTION SMITHSONIAN NVINOSHLIWS LIBRARIES 17 “ SMITHSONIAN * NVINOSHLIWS LIBRARIES SMITHSONIAN < a < ot = _ a re) z 2 INSTITUTION NOILNLILSNI = z —_ Sc = = > x 7 oa a a “4 z 1O SHLIWS = S3 'P- a 2-R:| Piss BRARIES = - 2 7) _ INSTITUTION NOILNLILSNI 2 e 2 a” en w” 3 0 = < = ~ = ; re) a Oo a = -! z ISHLINS SSZIYVYUSIT LIBRARIES : ; : 5 ED) 3 amy = = > = a — ow HSONIAN INSTITUTION NOILNLILSNI | wh te “ z < = | < = \ 4 = ro) r Lo Ee Wea Zz. = ee | IOSHLINS S3ZIHYVYSIT | ~ = ul an fe 2. SS. < anf ANY < 5 . > = oO 7" 2 THSONIAN” INSTITUTION NOILNLILSNI | | z | o fe) >] la > = ~ * o Zz Se ot! B RAR IES = 4 = NS 5 2s ~ a re) NS Ww Ss Oo ? x z oa S = : ’ a w” . SONIAN _INSTITUTION NOILNLILSNI - & uu FA of oa fond = i pe. < | : : : Zi ro) pe ro) = z | me FS u@ IBRARIES a — : a > II ND Oo % = 0, w A — = ERE ws) > mc] Ee NVINOSHLINS S3IYVYSIT_ LIBRARIES NOILNLILSNI NVINOSHLIWS INSTITUTION SMITHSONIAN NVINOSHLIWS ~ SMITHSONIAN 171 LIBRARIES SMITHSONIAN Ss Wug SMITHSONIAN NVINOSHLINS S31NVUg LIBRAR Salu¥vugil LIBRARIES SMITHSONIAN INSTITUTION. NOILNIL1 oc ee ac reel ae re. - wo mee x 2 > > za aw m i m wn ” INSTITUTION NOILOALILSNI NVINOSHLIWS hs ” ~ “ oe z = = ® RS ts, Lo Oo m 6 oT fae ea 2 g mee? Vil Ee z > AZ = > 4, = w” za Saiuvugi7 LIBRARIES SMITHSONIAN _ z i z = = eke - a = fe ee Lp = - a 7 a 5 9 GQ 3 Zz a ¥ z INSTITUTION | NOILALILSNI_NVINOSHLIWS aa ‘) - * Oo ha me YX = — > = ie F = W's z ao SSIY¥VYUEIT LIBRARIES SMITHSONIAN | ‘ ra ‘: w FA = = < ae am - 5: j : = Z = = > = ”Y ro ”~ INSTITUTION NOILNLILSNI_NVINOSHLIWS | rr) — a. ” uJ rT WwW ai ar bo ox — oc < <= << oc = oc a O = “TIBRARIES~ ef S31uVvuag rT nA ¥ 5 2 o pe) 5 ol : - 2 on e z | wn NOILNLILSNI NVINOSHLIW INSTITUTION, MOPSEAILSINI_ NVINOSHIING a. = = = \. = z = > - re) I \ 7) ” ” re) > O 2 ve z > = > , «& ” 2 saiuvugi7_LiBRARIES SMITHSONIAN _ z 7 z = a aes ae re = < if Lp 3 Ge ID & =.